Microbiological diagnosis of pulmonary tract infections: diphtheria, whooping cough. Prophylaxis and treatment

Microbiological diagnosis of tuberculosis, leprosy and other mycobacteriosis. Prophylaxis and treatment

Microbiological diagnosis of anaerobic infections: gas gangrene, tetanus and botulism. Causative agents of nonclstridial anaerobic infection. The anaerobic nonsporeforming gram-negative microorganisms – causative agents of mouth cavity soft tissues diseases .

Diphtheria

Causative Agent of Diphtheria. Extensive clinical, pathoanatomical, epidemiological, and experimental investigations preceded the discovery of the agent responsible for diphtheria. They paved the way for the discovery of the organism (E.Klebs, 1883), its isolation in pure culture (F. Loeffler, 1884), separation of the toxin (E. Roux and A. Yersin, 1888), antitoxin (E. Behring and S.Kitasato, 1890) and diphtheria toxoid (G. Ramon, 1923).

Morphology. Corynebacterium diphtheriae (L. coryna club) is a straight or slightly curved rod, 1-8 mcm in length and 0.3-0.8 mcm in breadth. The organism is pleomorphous and stains more intensely at its ends (Fig.) which contain volutin granules (Babes-Ernst granules, metachromatin). C. diphtheriae frequently display terminal club-shaped swellings. Branched forms as well as short, almost coccal, forms sometimes occur. In smears the organisms are arranged at an angle and resemble spread-out fingers. They are Gram-positive and produce no spores, capsules, or flagella.

Figure. Corynebacterium diphtheriae

C. diphtheriae may change into cone-shaped, thread-like, fungi-like, and coccal forms. In old cultures the cytoplasm of the organisms acquires a zebra-like appearance with unequally stained stripes. On ultrathin sections the cell wall has two layers, an inner osmiophilic layer and an outer layer forming a microcapsule The cytoplasmatic membrane is composed of three layers. During maximum exotoxin liberation membrane structures are seen as 'organelles', ovals, and rings. The cytoplasm is granular. The nucleoid is filled with fine osmiophilic fibrils. The metachromatic granules appear as dense granular structures surrounded by a membrane. A correlation has been revealed between the development of the membrane and the production of exotoxin. The G^-C content in DNA ranges from 51.8 to 60 per cent.

Cultivation. The causative agent of diphtheria is an aerobe or a facultative aerobe. The optimal temperature for growth is 37° C and the organism does not grow at temperatures Below 15 and above 40° C. The pH of medium is 7.2-7.6 The organism grows readily on media which contain protein (coagulated serum, blood agar, and serum agar) and on sugar broth. On Roux's (coagulated horse serum) and Loeffler's (three parts of ox serum and one part of sugar broth) media the organisms produce growth in 16-18 hours The growth resembles shagreen leather, and the colonies do not merge together.

According to cultural and biological properties, three varieties of C.diphtheriae can be distinguished, gravis, mitis, and intermedius, which differ in a number of properties

Corynebacteria of the gravis biovar produce large, rough (R-forms), rosette-like black or grey colonies (Fig.) on tellurite agar which contains defibrinated blood and potassium tellurite. The organisms ferment dextrin, starch, and glycogen and produce a pellicle and a granular deposit in meat broth. They are usually highly toxic with very marked invasive properties.

The colonies produced by corynebacteria of the mitis biovar on tellurite agar are dark, smooth (S-forms), and shining (Fig. 2, 2). Starch and glycogen are not fermented, and dextrin fermentation is not a constant property. The organisms cause haemolysis of all animal erythrocytes and produce diffuse turbidity in meat broth. Cultures of this biovar are usually less toxic and invasive than those of the gravis biovar.

Organisms of the intermedius biovar are intermediate strains. They produce small (RS-forms) black colonies on tellurite agar. Starch and glycogen are not fermented. Growth in meat broth produces turbidity and a granular deposit.

Fermentative properties. All three biovars of C. diphtheriae do not coagulate milk, do not break down urea, produce no indole, and slowly produce hydrogen sulphide. They reduce nitrates to nitrites. Potassium tellurite is also reduced, and for this reason C. diphtheriae colonies grown on tellurite agar turn black or grey. Glucose and levulose are fermented whereas galactose, maltose, starch, dextrin, and glycerin fermentation is variable. Exposure to factors in the external environment renders the organisms incapable of carbohydrate fermentation.

Toxin production. In broth cultures C. diphtheriae produce potent exotoxins (histotoxin, dermonecrotoxin, haemolysin). The toxigenicity of these organisms is linked with lysogeny (the presence of moderate phages-prophages in the toxigenic strains). The classical International standard strain, Park-Williams 8 exotoxin-producing strain, is also lysogenic and has retained the property of toxin production for over 85 years. The genetic determinants of toxigenicity (tox⁺ genes) are located in the genome of the prophage, which is integrated with the C. diphtheriae nucleoid.

In the commercial production of diphtheria toxin for vaccine, the amount of iron present in the growth medium is critical. Good toxin production is obtained only at low concentrations of iron (2 mcmol/L). At concentrations aslow as 10 mcmol/L, toxin production becomes negligible. Evidence suggests that, normally, the bacterium forms are presser which prevents the expression of the phage tox⁺ gene, and that this represser is an iron-containing protein.Thus, when the concentration of iron is abnormally low, the complete represser is not formed, and the tox⁺ geneis transcribed, ultimately yielding toxin.

The diphtheria exotoxin is a complex of more than 20 antigens. It has been obtained in a crystalline form. C. diphtheriae also contain bacteriocines (corynecines) which provide these organisms with certain selective advantages.

The diphtheria toxin contains large amounts of amino-nitrogen and catalyses chemical reaction in the body. The toxigenic strains of C. diphtheriae are characterized by marked dehydrogenase activity, while the non-toxigenic strains do not possess such activity.

Diphtheria toxin is excreted from the bacterium as a single polypeptide chain of about 61,000 daltons with two disulfide bridges. Although highly toxic for cells or animals, the pure, intact toxin is inert in cell-free protein systems, even when NAD is present. Thus, the secreted toxin is actually a proenzyme which, in cell-free systems, must be activated before it can function as an enzyme. This activation, as shown in Figure 3, is accomplished in two steps: (1) treatment with trypsm hydrolyzes a peptide bond between the disulfide-linked amino acids; and (2)reduction of the disulfides to sulfhydryl groups using a reducing agent such as mercaptoethanol yields two smaller peptides, which have been designated fragment A (21,150 daltons) and fragment B (40,000 daltons).

Figure. Sequence of events in the expression of enzymatic activity (ADP nbosylation of EF-2) in diphtheria toxin. Fragment A is nontoxicbecause it cannot cross the cell membrane, except when it is linked tothe fragment B portion of the molecule.

Fragment A is active in cleaving the nicotinamide moiety from NAD and in catalyzing the transfer of ADP-ribose from NAD to EF-2 when added to cell-free, protein-synthesizing systems, but it has no effect when given to animals or to intact HeLa cells. Thus, although fragment A is the activated enzyme (and hence contains allthe toxic properties), it cannot get into intact cells.

Fragment B, on the other hand, has no enzymatic activity, but it is needed for attachment of the toxin tospecific receptor sites on cells. Cells possess specific glycoprotein receptor sites for the diphtheria toxin, as suggested by the following observation: Rats and mice areover 1000 times more resistant to the intact toxin thanare other susceptible animals, but their cell-free protein-synthesizing system is equally sensitive to the enzymaticaction of fragment A. Moreover, toxin that is defectivein its A fragment (and is, therefore, nontoxic) but retains a normal B fragment, will competitively inhibit the actionof normal toxin on HeLa cells.

The question of whether the phage genome itself codes for the toxin or merely derepresses a bacterial gene, which could then synthesize the toxin, originally was solved using a series of mutant phages that induced the synthesis of mutant toxins. Moreover, the tox gene has been completely sequenced and unequivocally shown to exist in the phage genome.

Also, different toxigcnic strains of C diphtheriae vary considerably in the amount of toxin produced under identical conditions. This is, in part, because of subtle differences in the regulation of the tox gene expression, but amore obvious explanation for this observation was shown by Rino Rappuoli and his colleagues. Using specific DNA probes, they conclusively demonstrated that high-toxin-producing strains had two or even three tox genes inserted into their genome. Thus, the quantity of toxin produced was correlated to the amount of tox DNA within thetoxin-producing strain of C diphtherias.

In summary, the usual series of events leading totoxin action is as follows: (1) the toxin binds to specificreceptor sites on susceptible cells; (2) the toxin enters the cell (perhaps through a phagocytic vesicle that can then fuse with a lysosome), and lysosomal proteases hydrolyze the toxin into fragments A and B; and (3) reduction ofthe disulfide bridges (perhaps by glutathione) releases fragment A from fragment B; and (4) fragment A canthen enzymatically inactivate EF-2.

The diphtheria toxin is unstable, and is destroyed easily by exposure to heat, light, and oxygen of the air, but is relatively resistant to super-sonic vibrations. The toxin is transformed into the toxoid by mixture with 0.3-0.4 per cent formalin and maintenance at 38-40° C for a period of 3 or 4 weeks. The toxoid is more resistant to physical and chemical factors than the toxin.

Because diphtheria toxin is effective against many cells, the use of tissue cultures provides a model for studyingits mode of action. Early studies reported that, although toxin had no effect on the respiration of HeLa cells (human cervical carcinoma tissue culture cells), all protein synthesis stopped about 1 to 1.5 hours after the additionof the toxin. Surprisingly, dialyzed, cell-free, protein-synthesizing systems were entirely insensitive to the action of the toxin, unless oxidized nicotinamide-adenine dinu-cleotide (NAD) was added to the reaction.

Subsequent research has shown that the toxin possesses enzymatic activity that cleaves nicotmamide from NAD and then catalyzes the ADP-ribosylation of elongation factor 2 (EF-2). EF-2 is required for the translocasc reaction of polypcptide synthesis, in which the ribosome is moved to the next codon on the mRNA after the peptide bond is formed to the most recent aminoacid to be added to the chain. When EF-2 is inactivated by the addition of ADP-ribose, the ribosome is frozen, and protein synthesis stops. Insofar as is known, EF-2 from all eucaryotic cells (those studied include vertebrate, invertebrate, wheat, and yeast) is inactivated in the presence of diphtheria toxin and NAD, whereas the corresponding factor, EF-G (which occurs in bacteria), or the analogous factor from mitochondria, is not affected. The ADP-ribose is transferred to a histidine modified residue on the EF-2 molecule. This modified ammo acid (commonly called diphtheramide) does not exist in bacterialor mitochondnal elongation factors.

Antigenic structure. Eleven serovars of C. diphtheriae have been deter-mined on the basis of the agglutination reaction. They all produce toxins which do not differ from each other and are neutralized completely by the standard diphtheria antitoxin. A number of authors have confirmed the presence of type-specific thermolabile surface protein antigens (K-antigens) and group-specific thermostable somatic polysaccharide antigens (O-antigens) in the diphtheria corynebacteria.

Classification. The genus Corynebacterium comprises a species pathogenic for human beings and several species which are non-pathogenic for man and conditionally designated as diphtheroids. The majority of diphtheroids occurs in the external environment (water, soil, air), some of them are present as commensals in the human body. Properties of differentiation between diphtheria corynebacteria and the diphtheroids are given in Table 1. Japanese scientists isolated Corynebacterium kusaya from brines used in cavalla canning; it does not form volutin granules. Its presence in brine prevents spoiling of fish products during salting and drying.

There are 19 phage types among C. diphtheriae, by means of which the source of the infection is identified The phage types are also taken into account in identificaition of isolated cultures.

Table 1

Differential Characteristics of Corynebacteriun Species

Species	Exotoxin production	Erythrocyte haemolysis	Saccharose fermentation	Reduction of nitrates to nitrites	Urease production	Pathogenicity for humans and animals
C. diphtherias	+	—	+	—	+	Pathogenic for humans, causes diphtheria
C. pseudotuberculosis	+	d	d	d	+	Pathogenic for sheep, goats, horses, and other warm-blooded animals, sometimes causes in fection in humans
C. xerosis	—	+	+	—	—	Non-pathogenic for humans, dwells on eye mucosa
C. renale				+		Induces pyelitis and cystitis in experimental animals and pyelonephritis in calves
C. kulschen	—	+	—	+	—	Parasitizes in the body of mice and rats
C. pseudodiphtheriae	—	–	+	+	—	Non-pathogenic for humans, dwells on the mucous membrane of the nasopharynx
C. equi	—	—	+	—	—	Detected in pneumonia in animals, weakly pathogenic for experimental animals
C. bovis	—	—	—	+	—	Causes mastitis in animals, found in milk

Note: “d” – some strains are positive, some negative

Resistance. C. diphtheriae are relatively resistant to harmful environmental factors. They survive for one year on coagulated serum, for two months at room temperature, and for several days on children's toys. Corynebacteria remain viable in the membranes of diphtheria patients for long periods, particularly when the membranes are not exposed to light. The organisms are killed by a temperature of 60° C and by a 1 percent phenol solution in 10 minutes.

Pathogenicity for animals. Animals do not naturally acquire diphtheria. Although, virulent diphtheria organisms were found to be pre-sent in horses, cows, and dogs, the epidemiological significance of animals in diphtheria is negligible.

Among the laboratory animals, guinea pigs and rabbits are most susceptible to the disease. Inoculation of these animals with a culture or toxin gives rise to typical manifestations of a toxinfection and the appearance of inflammation, oedema, and necrosis at the site of inoculation. The internal organs become conjested, particularly the adrenals in which haemorrhages occur.

Pathogenesis and disease in man. Patients suffering from the disease and carriers are the sources of infection in diphtheria. The disease is transmitted by an air-droplet route, and sometimes with dust particles. Transmission by various objects (toys, dishes, books, towels, handkerchiefs, etc.) and foodstuffs (milk, cold dishes, etc.) contaminated with C. diphtheriae is also possible.

Carriers play an essential part in the epidemiology of diphtheria. The carrier state averages from 3 to 5 per cent among convalescents and healthy individuals.

Diphtheria is most prevalent in autumn. This is due to the fact that children are more crowded in the autumn months and that body resistance is reduced by a drop in temperature.

Histotoxin plays the principal role in the pathogenesis of diphtheria. It blocks protein synthesis in the cells of mammals and inactivates transferase, the enzyme responsible for the formation of the polypeptide chain.

C. diphtheriae penetrate into the blood and tissues of sick humans and infected animals. The diffusion factor due to which these organisms are capable of invasion is formed of a complex of K-antigen and lipids of the wall of bacterial cells. The lipids contain corynemicolic and corynemicolenic acids, the cord factor (trehalose dimicolate), and mannose and inositol phosphatides. The cord factor causes the death of mice, destroys mitochondria, and disturbs the processes of respiration and phosphorylation. The necrotic factor, alpha-glutaric acid, and haemolysin are considered to be factors of invasiveness.

Clinical studies and experiments on animals have provided evidence of the influence of pathogenic staphylococci and streptococci, on the development of diphtheria, the infection becoming more severe in the presence of these organisms. Hypersensitivity to C. diphtheriae and to the products of their metabolism is of definite significance in the pathogenesis of diphtheria.

In man, membranes containing a large number of C. diphtheriae and other bacteria are formed at the site of entry of the causative agent(pharynx, nose, trachea, eye conjunctiva, skin, vulva, vagina, and wounds). The toxin produces diphtheria! inflammation and necrosis in the mucous membranes or skin. On being absorbed, the toxin affects the nerve cells, cardiac muscle, and parenchymatous organs and causes severe toxaemia.

Deep changes take place in the cardiac muscle, vessels, adrenals, and in the central and peripheral nervous systems.

According to the site of the lesion, faucial diphtheria and diphtheritic croup occur most frequently, and nasal diphtheria somewhat less frequently. The incidence of diphtheria of the eyes, ears, genital organs, and skin is relatively rare. Faucial diphtheria constitutes more than 90per cent of all the diphtherial cases, and nasal diphtheria takes the second place.

Immunity following diphtheria depends mainly on the antitoxin con-tent m the blood However, a definite role of the antibacterial component, associated with phagocytosis and the presence of opsonins, agglutinins, precipitins, and complement-fixing substances cannot be ruled out. Therefore, immunity produced by diphtheria is anti-infectious (anti-toxic and antibacterial) in character.

Schick test. This test is used for detecting the presence of antitoxin in children's blood. The toxin is injected intracutaneously into the forearm in a 0 2 ml volume which is equivalent to 1/40 DLM for guinea pigs. A positive reaction, which indicates susceptibility to the disease, is manifested by an erythematous swelling measuring 2 cm in diameter which appears at the site of injection in 24-48 hours. The Schick test is positive when the blood contains either no antitoxin or not more than0.005 units per millilitre of blood serum. A negative Schick reaction indicates, to a certain degree, insusceptibility to diphtheria.

In view of the fact that the diphtheria exotoxin produces a state of sensitization and causes the development of severe reaction in many children, it is advisable to restrict the application of the Schick test and conduct it with great care.

Children from 1 to 4 years old are most susceptible to diphtheria. A relative increase of the incidence of the disease among individuals 15years of age and older has been noted in recent years.

Diphtheria leaves a less stable immunity than do other children's diseases (measles, whooping cough). Diphtheria reinfection occurs in 6-7per cent of the cases.

Laboratory diagnosis. Discharges from the pharynx, nose, and, some-times, from the vulva, eyes, and skin are collected with a sterile cotton-wool swab for examination.

The material under test is seeded on special media, e. g. coagulated serum, Clauberg's II medium, blood-tellurite agar, serum-tellurite agar, etc. Smears are examined under the microscope after 12-24-48 hours' growth, and preliminary diagnosis is made on the basis of microscopic findings.

C. diphtheriae does not always occur m its typical form. Short rods arranged not at a particular angle but in disorder and containing few granules are found in a number of cases. Diagnostic errors are made most frequently when investigations are confined to microscopical examination. Other bacterial species and non-pathogenic corynebacteria which are morphologically identical with the diphtheria organisms maybe mistaken for the diphtheria corynebacteria (Plate VIII). It must also be borne in mind that formation of volutin granules is variable, and therefore, this is not an absolute property. For this reason, contemporary laboratory diagnosis comprises isolation of the pure culture and its identification by cultural, biochemical, serological and toxigenic properties.

The toxigenic and non-toxigenic strains of diphtheria corynebacteria are differentiated either by subcutaneous or intracutaneous infection of guinea pigs, or by the agar precipitation method, the latter being relatively simple and may be carried out in any laboratory. It is based on the ability of the diphtheria toxin to react with the antitoxin and produce a precipitate resembling arrow-tendrils.

The agglutination reaction with patient's sera (similar to the Widal reaction) is employed as an auxiliary and retrospective method. It is performed with 5 serovars of C. diphtheriae; the reaction is considered positive beginning from 1 :50-1 :100 dilutions of serum.

To detect the sources of infection, the isolated cultures are subject to phagotyping. There are 19 known phage types.

Treatment. According to the physician's prescriptions, patients are given antitoxin in doses ranging from 5000 to 15000 units in mildly severe cases, and from 30 000 to 50 000 units in severe cases of the disease. Penicillin, streptomycin, tetracycline, erythromycin, sulphonamides, and cardiac drugs are also employed. Diphtheria toxoid is recommended in definite doses for improving the immunobiological state of the body, i.e for stimulating antitoxin production.

Carriers are treated with antibiotics. Tetracycline, erythromycin, and oxytetracycline in combination with vitamin C are very effective.

Prophylaxis. General control measures comprise early diagnosis, prompt hospitalization, thorough disinfection of premises and objects, recognition of carriers, and systematic health education.

Specific prophylaxis is afforded by active immunization. A number of preparations are used: the pertussis-diphtheria vaccine, purified adsorbed toxoid, pertussis-diphtheria-tetanus vaccine All preparations are used according to instructions and directions.

Reports show that only antibodies to the fragment B portion of the toxin molecule are capable of neutralizing the toxin, supposedly by preventing the attachment of toxin to the specific receptor sites on the cell surface. Treatment of the toxin with formalin, however, both detoxifies the toxin and protects fragment B from the action of proteolytic enzymes, resulting in better protective antibody production than that obtained by using untreated fragment B or defective toxins possessing anormal fragment B

It should be noted that not all immunized children acquire resistance to diphtheria. An average of 5-10 per cent of them remain susceptible or refractory (not capable of producing antibodies after immunization).Such a condition is considered to be the result of tolerance, agamma-globulmaemia, or hypoagammaglobulinaemia.

Haifa century ago diphtheria was a menacing disease of children. In Russia every year more than 250 000 persons contracted the disease in 1886-1912. The death rate was very high (12 to 30 per cent).With the introduction of compulsory immunization against diphtheria great success has been gained in the control of this disease.

Other Corynebacteria. Many species of Corynebacterium exist in the soil; a few cause animal diseases, and a large number are plant pathogens. Such species, however, are only rare causes of human diseases. Interestingly, both Corynebacterium ulcerans and Corynebacterium pseudotuberculosis are known to cause occasional diphthena-like illnesses. Moreover, selected isolates of these species have been shown to produce a toxin that is indistinguishable from that of C. diphtheriae. The fact that human disease by these speciesis both rare and mild suggests that even though toxigemc, they may lack some virulence factor possessed by C. diphtheriae.

Additional materia for Diagnosis

Diphtheria is an acute infectious disease with the predominant localization of the causative organism in the mucosa of the fauces and upper respiratory pathways. The causative agent of the disease is Corynebacterium diphtheriae.

The material tested is diphtheritic films or secretions of the involved mucosal membrane of the fauces, nose, and occasionally of the external genitalia and conjunctiva. From carriers, secretions of the faucial and nasal mucosa are examined. At the requirement of the epidemiologist foodstuffs (milk, ice-cream) and washings from various objects (toys, etc.) are examined.

Secretions of the faucial mucosa should be taken on a fasting stomach or two hours after meal. It is recommended that no disinfectants or antibiotics be used before this procedure. Material from the fauces and nose is taken with two sterile tampons which are placed into test tubes and sent to the laboratory without delay. If transportation of specimens is to take over 3-4 hrs, use tampons soaked with a 5 per cent glycerol solution in isotonic saline.

Bacterioscopic examination of the material obtained from the patient is carried out only if the physician considers it advisable. In such cases the secretion of the mucosa or film is removed with two swabs: one of them is used for culturing. the other, for preparing smears. Smears may be stained with Gram's dye, acetic-acidic methyl violet, Loeffler's blue, toluidine blue, and Neisser's stain. In smears prepared from the film corynebacteria of diphtheria appear as single rods arranged at an angle to each other (V-like arrangement), less commonly they form clusters. They are Gram-positive; staining with acetic-acidic methyl violet or Loeffler's blue reveals intensely stained volutin granules. False diphtheria bacteria and diphlheroids are arranged in parallel ("a fence-like arrangement") and are ordinarily deprived of volutin granules. Volulin granules may be detected with the help of luminescent microscopy. For this purpose the preparation is stained with coryphosphine. Microscopic findings are yellow-green bodies of bacteria with orange-red volutin granules against a dark background. Upon the detection of typical corynehacteria. the laboratory immediately issues a preliminary result which reads "Diphtheritic corynebacteria have been detected, proceed with examination".

Bacteriological examination. The material is introduced onto one of the elective media: into test tubes with coagulated serum and in a Petri dish with telluric blood agar, cystine-tellurite-serum medium (Tinsdal-Sadykova), Buchin's quinosol medium, etc. It is recommended that one of the above media should be constantly used for the corynebacteria isolation as this practice makes it possible to obtain more clear-cut and comparable results.

Telluric blood agar. To 100 ml of melted 2-3 per cent agar cooled to 50 °C, add 5-10 per cent of defibrinated blood and 1 ml of a 2 per cent solution of potassium tellurite. Thoroughly mix the mixture and pour into sterile plates.

Cystine-tellurite-serum medium (Tinsdal-Sadykova medium). To 100 ml of melted meat-peptone agar cooled to 60 °C, add consecutively the following components: (1) 1 per cent solution of cystine in 0.1 N sodium hydroxide solution (12 ml); (2) 0.1 N solution of hydrochloric acid (12 ml); (3) 2 per cent solution of potassium tellurite (1.5-1.8 ml); (4) 2.5 per cent of sodium hyposulphite solution (1.8 ml); (5) normal horse or bovine serum (20 ml). The mixture is stirred and dispensed into sterile Petri dishes.

Buchin's quinosol medium is prepared from powder, according to the label instructions. It is boiled for 2-3 min and cooled to 50 °C after which 511 ml of defibrinated blood (rabbit or human) is added. The prepared medium is dark blue.

During inoculation the material is rubbed with a swab into the medium surface. The growth of diphtheria corynebacteria on coagulated serum is faster than that of other microorganisms, their colonies are small and separate. After 8-12 hrs of incubation smears are made; if the result is negative, microscopic examination is repeated in 18-24 hrs. If diphtheria corynebacteria cannot be found, the inoculated cultures are kept in the incubator for 48 hrs after which a negative result can be issued. If typical corynebacteria are demonstrated, a pure culture is isolated and identified by fermentative and toxigenic properties (Table ).

Table

Differential-Diagnostic Signs of Diphtheria and Non-Pathogenic

Type of corynebacteria	Fermenta-tion					Toxigenicity	Additional signs
	sucrouse	glucose		starch			cystinase test	urease test			Agglutination with antiserum
Diphtheria corynebacteria
gravis	–		+	+	+		+		–	+
mitis	–		+	–	–		+		–	+
Diphtheroids	+		+	–	–		+		+	–
Pseudodiphtheria bacteria	+		–	–	–		–		–	–

Urease production

Study the colonies in dishes in 24-48 hrs. On media with potassium tellurite diphtheria corynebacteria of the gravis type form relatively large, greyish-black, flat, rough colonies with radial lines and a wavy margin; colonies of the mitis type are small, protuberant, lustrous, black, with a smooth surface and an even margin. Diphtheroids grow in the form of protuberant moist colonies of a grey or brown colour. False diphtheria bacteria form dry. small mucoid colonies of a grey colour. On the Tinsdal-Sadykova medium colonies of diphtheria corynebacteria are surrounded with a dark brown halo, on Buchin's medium they are blue. while diphtheroids on the same medium form colourless colonies and false diphtheria bacteria form bluish colonies.

To obtain a pure culture and assess toxigenicity, suspicious colonies are examined microscopically, subcultured to a serum medium and onto a plate with a phosphate-peptone agar. Pure cultures are introduced into Hiss's media (glucose, sucrose, starch), cystine medium (cystinase test), and into a medium with urea (urease test).

Medium for cystinase determination. To 90 ml of melted 2 per cent meat-peptone agar (pH 7.6) add 2 ml of cystine solution (1 percent cystine solution with 0.1 M solution of sodium hydroxide), mix thoroughly, and add 2 ml of 0. 1 N solution of sulphuric acid. Sterilize the medium at 112 °C for 30 mill. To the melted medium cooled to 50 °C add 1 ml of 10 per cent solution of acetic-acidic lead (after its double sterilization with flowing steam), stir the mixture, and add 9 ml of normal horse serum. Decant the medium in 2-ml quantities into small test tubes under sterile conditions. When diphtheria corynebacteria are inoculated by injection, they induce blackening of the medium (combination of lead with hydrogen sulphide) along the course of the injection and around it in the form of a cloud.

Medium for demonstrating the urease enzyme. To 100 ml of a meat-peptone broth or Hottinger's broth (pH 7.0) add 1 g of urea and 0.2 ml of cresol red (1.6 per cent alcohol solution). Pour the resultant medium (in 2-3-ml aliquots) into sterile test tubes and sterilize with flowing steam for 10 min. Reddening of the medium observed 20-24 hrs after the inoculation of the diphtheroid culture into the urea broth witnesses the presence of the urease enzyme. Diphtheria corynebacteria do not alter the colour of the medium.

Simultaneously, the agglutination test is performed on a slide with monospecific diphtheria sera of the first-fourth serovars. Agglutinating sera are diluted 1:25 in advance. Using this reaction, 11 serological types or variants of the diphtheria causative organism have been established; in the USSR the second serovar is the most common one. In corynebacteria of diphtheria 12 phagovars have been identified, with the help of which the sources of the infection are established.

Upon the isolation of toxigenic strains of diphtheria corynebacteria the final answer may be issued in 48 hrs. It specifies a biological (gravis or mitis) and serological variants of the causative agent, a phagovar of the isolated microorganism, and its toxigenicity.

Determination of toxigenicity of cultures in vitro. For this purpose 12 ml of melted phosphate-peptone agar cooled to 50 °C are poured into a Petri dish.

Phosphate-peptone agar. 1. Preparation of marten peptone. Minced pieces of the pig stomach (250 g) are immersed with 1 l of 1 per cent aqueous solution of chemically pure hydrochloric acid and placed into a 37 °C incubator for 18-20 hrs. Following digestion, the peptone is heated at 80 °C for 10 min and allowed to settle down for 8-10 days in a cool place, then it is filtered, heated to 80 °C, alkalized with a 10 per cent solution of sodium hydroxide to pH 8-8.2, boiled for 10 min, filtered, dispensed into bottles, and, after being supplemented with 1 per cent. of chloroform, stored in a cool place.

2. Preparation of a phosphate agar. Per 11 of distilled water take 40 got agar-agar, 125 g of sodium hydrophosphate, and 3.75 g of potassium dihydrophosphate. Place the mixture into a sterilizer and allow it to stand there for 20 min at flowing steam and for 10 min at 115 °C. Leave the mixture in the sterilizer for 2 hrs to allow sedimentation to take place, then filter it and sterilize at 115 °C for 30 min.

To obtain a phosphate-peptone agar, mix 50 ml of heated peptone and 50 ml of a phosphate agar. Bring the pH to 7.8-8.0 by adding 0.5 per cent of sodium acetate and 0.3 per cent of maltose, dispense the mixture in 10-ml volumes and sterilize them with flowing steam for 3D min.

After the nutrient medium has solidified, on the middle of the plate place a strip of sterile filter paper (2.5 X 8 cm) soaked with an anti-toxic serum containing 500 AU per ml or with a specific gamma-globulin. The plate is dried for 15-20 min in an incubator, then the culture examined is streaked with strokes perpendicular to the filter paper or with patches 1 cm in diameter at a distance of 1 cm from the edge of the strip. From 3-4 to 10 cultures can be streaked onto one plate (one of the cultures, the control, is known to be toxigenic). Inoculated cultures are put into an incubator. The results are read in 24, 48, and 72 hrs. If the culture is toxigenic, lines of precipitation form at some distance from the paper strip, which coincide with the lines of the precipitate of the control culture. They are readily seen in transmitted light (Fig. )

Figure. Determination of the in vitro toxigenicity of Corynebacterium diphtheriae

Biological examination is conducted to determine the toxigenicity of isolated cultures in vivo.

Intracutaneous method. The day before the examination clip off hair from the sides of two guinea pigs (preferably with white hair). On the day of the examination prepare 100-200-million suspension of the culture to be studied and inject intracutaneously 0.2-ml portions of each suspension into two prepared guinea pigs. In 4 hrs administer intraperitoneally 100 IU of the antitoxic diphtheria serum to the control infected guinea pig. If the culture is toxigenic. the test guinea pig develops reddening, oedema, and then necrosis at the site of injection. The final assessment of the results is made in 72 hrs. Control animals present no alterations. The intracutaneous method of toxigenicity determination makes it possible to test 6 cultures in one guinea pig.

Guinea pigs weighing 240-300 g are used for the subcutaneous administration, of the material. One day before the test administer 1000 IU of the antitoxic serum to the control animal. On the examination day inject subcutaneously 0.5 ml of suspension of the culture tested (500 min and 1 mlrd of microorganisms per ml) to both test and control guinea pigs. If the examined strain of diphtheria is toxigenic, the test guinea pigs die on the 2nd-5th day. Post-mortem findings include oedema at the site of the culture administration, exudate in the peritoneal and thoracic cavity, hyperemia of the adrenal cortex. The control guinea pig remains alive.

Serological examination remains supplementary in the diagnosis of diphtheria. Sera of patients or convalescents are diluted with sodium chloride solution in ratios 1:-100, 1:200, 1:400, 1:800, 1:1600, etc. Add a specially prepared diagnosticum (diphtheria culture washed off with saline and killed with 0.2 per cent formalin solution) to the serum dilutions. The reaction is considered positive if the dilution of the serum is no less than 1:100. Agglutinins against diphtheria corynebacteria usually appear within the first days of the disease and disappear in 12-15 days. The usually employed test is IHA with an erythrocyte bacterial diagnosticum: a 1:8 or greater titre during the second week of the disease is considered diagnostically significant.

The current employment of Schick's test is limited. It is intended for detecting antitoxic immunity. For this purpose utilize diluted diphtherial toxin 0.2 ml of which contains 1/40 Dim for a guinea pig. The toxin is injected intracutaneously into a median internal surface of the upper arm. If 1 ml of the blood serum contains 1/30 IU of antitoxin or over, Schick's reaction is negative. If antitoxins are absent, redness and infiltrate develop at the site of toxin administration in 48-96 hrs.

Bordetella

Bordetella and Other Haemophilic Bacteria

Causative Agent of Whooping Cough. The causative agent of whooping cough (Bordetella pertussis) was discovered and isolated in pure culture from patients by J. Bordet and O.Gengou in 1906.

Morphology. The organisms are small oval-shaped non-motile rods, 0.2-0.3 mcm in breadth and up to 0.5-1.0 mcm in length. They are non-sporeforming and produce no capsules. The bacillus stains poorly with the usual aniline dyes, the ends staining more intensively. The organism is Gram-negative.

Cultivation. B. pertussis shows no growth on ordinary media but can be cultivated readily on glycerin-potato or blood agar media under aerobic conditions at pH 6.8-7.4 and at a temperature of 35-37°C. The organism does not grow at temperatures below 20° and above 38°C.The colonies are small, convex, and glistening, resembling globules of mercury, and may be granular or smooth. In blood broth the organisms produce turbidity and a small precipitate. At present, casein-charcoal medium is considered a very useful medium on which B. pertussis grows quite readily without the necessity of adding blood.

Borde-Gengou medium Chokolate agar

B. pertussis, grown on media which do not contain blood, dissociate into four different phases: the first and second phases are virulent cultures, while the third and fourth are avirulent.

Colonies of the first and second phases (S-forms) are small (1-2 mm),convex, and have smooth borders. Microscopic examination of smears reveals the presence of small ovoid-shaped organisms with rounded ends. Homologous sera to the titre readily agglutinate them. Colonies of the third and fourth phases (R-forms) are large (3-4 mm),flat, and glistening. The organisms from these colonies are not agglutinated by sera of the first and second phases but are agglutinated by homologous sera to the titre.

Fermentative properties. The bacteria do not ferment proteins, carbohydrates, or urea, but produce catalase.

Toxin production. B. pertussis produces a thermolabile exotoxin which causes haemorrhagic oedema, necrosis, and ulceration in rabbits and guinea pigs. It also produces histamin-sensitizing and lymphocytosis-stimulating factors.

A capsule, volutin inclusions, and vacuoles in the region of the nucleoid are demonstrated on ultrathin sections. The G + C content in DNA is 61 per cent.

B. pertussis coagulates human, calf, sheep, and rabbit blood plasma.

Antigenic structure. The causative agents of whooping cough share a common thermostable somatic O-antigen and superficial capsular antigens (a, e, f, h). Fourteen antigenic components (factors) have been identified in various Bordetella strains. Factor 7 is generic and common to all Bordetella organisms: factor 1 is characteristic of B. pertussis, factor 14 of B. parapertussis, and factor 12 of B. bronchiseptica. All the other factors are encountered in different combinations. Types 1, 2; 1,3; 1,2, 3 are most frequently found in B. pertussis, types 8,9, 10,11, and12 in B. parapertussis and types 8, 9, 10, 11, and 13 in B. bronchiseptica.

Classification. Besides the typical bacterium of whooping cough there are two other species (Bordetella parapertussis and Bordetella bronchiseptica) which also induce diseases in humans and animals (Table).

Table

Differentiation of B. pertussis, B. parapertussis and B. broncbiseptica

Differentiation signs	B pertussis	B parapertussis	B bronchiseptica
Reduction of nitrates to nitrites	No reduction	No reduction	Causes reduction
Change caused in litmus milk	Alkalizes on 12th-14th day	Alkalizes on 2nd-4th day
Assimilation of citrates as carbohydrate	–	+	+
Production of urease	–	+	+
Specific thermolabile antigen: factor 1
	+	–	–
factor 12	–	–	+
factor 14	–	+	–
G + C content, %	61	61	66

B. bronchyseptica B. bronchyseptica (blood agar)

Resistance. B. pertussis is very sensitive to environmental factors. It withstands exposure to direct sunlight for one hour and a temperature of 56° C for 10 to 15 minutes. It is relatively rapidly destroyed in 3 percent solutions of phenol and lysol.

Pathogenicity for animals. Animals are insusceptible to B. pertussis in nature. Whooping cough has been reproduced experimentally in monkeys and young dogs, the culture being isolated from the bronchi. The disease caused fever and catarrh. Laboratory animals (rabbits, guinea pigs, and white mice) infected with the cultures exhibit toxaemia and haemorrhagic foci in the internal organs.

Pathogenesis and diseases in man. Whooping cough is transmitted from the patient to .a healthy individual by the air-droplet route.

Patients are most contagious in the catarrhal stage. Various objects in the vicinity of the patient are insignificant in relation to the transmission of the infection as B. pertussis cannot withstand external environ-mental factors. Patients with atypical clinical forms of the disease and healthy individuals who have become temporary carriers of the organisms as a result of contact with patients are also sources of infection.

Whooping cough is a severe infectious disease of childhood. It is characterized by typical symptoms and a cyclic course (three stages):

(a) catarrhal stage, lasting about 2 weeks;

(b) paroxysmal (convulsive) stage, which is accompanied by a paroxysmal cough and lasts for another 4 or 6 weeks:

(c) final or convalescent stage, lasting for 2 or 3 weeks.

The organisms enter the body through the upper respiratory tract and multiply in its mucosa. The blood is not invaded. The organisms liberate toxins which cause inflammation of tracheal and bronchial mucosa. The toxins stimulate the receptors in the mucous membranes and give rise to a continuous flow of impulses to the central nervous system, thus forming a stable focus of excitation. It attracts stimulations from other parts of the nervous system, and, as a result, paroxysmal cough is produced not only by the effect of specific (toxins of pertussis bacilli) stimulations but also by non-specific stimulations (sound, injection, examination, etc.).

Immunity. The disease leaves a stable immunity of long duration, agglutinins, precipitins, and complement-fixing antibodies accumulating in the blood.

Laboratory diagnosis. Patient's sputum and discharge from the nasopharyngeal mucosa are examined. Specimens are collected with special swabs. Sputum is inoculated into Bordet-Gengou medium, milk-blood agar, casein-hydrolysate medium, casein-charcoal medium, etc., and antibiotics (penicillin, etc.) are added to inhibit the growth of other microflora. Favourable results are obtained by the cough-plate method. After 2-5-days' incubation on Bordet-Gengou medium the organism produces typical small colonies which are convex, glistening, and resemble mother-of-pearl or globules of mercury. The isolated pure culture is identified by its morphological, cultural, biochemical, antigenic, and biological properties (Table 1).

Agglutination and complement fixation reactions are employed beginning from the second week of the disease. These reactions are used for identifying both typical and atypical cases of the disease. The allergic test is also performed in which 0.1 ml of the antigen is injected intra-cutaneously, and an erythematous reaction measuring 2 cm in diameter and an infiltrate develop at the site of injection in 16-20 hours.

Treatment Patients are treated with antibiotics (streptomycin, chloramphenicol, and tetracyclines), human serum, gamma-globulin, and vitamins. Children undergoing treatment should have sufficient fresh air, and for this purpose the room must be frequently ventilated and the child taken for walks.

Prophylaxis is ensured by early recognition and isolation of children with whooping cough. Chemical disinfectants are not used due to the low resistance of the causative agent. The patient's room should be regularly ventilated. General measures are quite frequently of little effect since whooping cough is a very contagious disease.

At present a compound vaccine against whooping cough, diphtheria, and tetanus is employed.

Haemophilus Influenzae

M. Afanasyev in 1889 and R. Pfeiffer and S. Kitasato in 1892 encountered very small Gram- negative bacilli in the sputum of patients during an influenza pandemic. For forty years these organisms were mistakenly considered to be responsible for influenza. Later they were shown to be concomitants of influenzal infections and the causative agents of acute catarrhs and secondary infections.

Morphology. The influenza bacilli (H. influenzae) are very small organisms, measuring 0.5-2 mcm in length and 0.2-0.3 mcm in breadth. They appear as small rods with rounded ends. The organisms are non-motile, non-sporeforming, and Gram-negative. The virulent smooth strains are capsulated. The bacilli stain relatively well with dilute fuchsine, the ends staining more intensely than the central portion.

H. influenzae is pleomorphous, and sometimes grows in the form of long threads with round- or spindle-shaped swellings. The G+C con-tent in DNA ranges from 38 to 42 per cent.

H. influenzae H. influenzae in liquor

Cultivation. The organisms are facultative anaerobes. They do not grow on common nutrient media but multiply readily on blood agar at pH 7.3-7.5 and at a temperature of 37° C. The extremes of temperature for growth are 25° and 43° C. Small transparent colonies resembling drops of dew become visible on the medium after 24 hours .White flakes and slight turbidity are produced in blood broth.

On chocolate agar (heated blood agar) H. mfluenzae produces large transparent flat colonies. According to the form of their colonies, two types of bacilli are distinguished the smooth bacilli (typical) and the rough bacilli (atypical). H. mfluenzae grows on nutrient media only in the presence of two factors, the so-called X-factor which is thermostable and survives heating up to 120°C and the V-factor, which is thermolabile and occurs in blood, fresh potatoes, animal and vegetable tissues, and in a large number of bacteria.

Atypical forms often appear in cultures. M- and N-strains can be distinguished. The M-strains are more virulent and are isolated more frequently from meningitis patients, whereas the N-strains are less virulent and are usually found in the nasal mucus.

H. influenzae colonies

Fermentative properties. H. influenzae reduces nitrates to nitrites. The smooth typical strains produce indole and sometimes cause slow glucose fermentation, with acid formation.

Toxin production. The bacilli produce no exotoxin. Their pathogenicity is associated with an endotoxin which is liberated as a result of bacterial disintegration.

Antigenic structure and classification. The organisms are serologically heterogeneous. The smooth forms are characterized by type specificity due to the presence of polysaccharides. On the basis of their antigenic structure, the bacilli are differentiated into 6 (a, b, c, d, e, f) serological variants which are detected by the precipitin reaction between immune sera and capsular material. The rough atypical strains are heterogeneous, and their antigenic structure has not been sufficiently studied.

Resistance. H. influenzae are not very resistant organisms, and can survive only for a short period outside the body. The organisms are susceptible to physical and chemical factors and are easily killed by expo-sure to a temperature of 59° C, sun rays, desiccation, and disinfectants.

Pathogenicity for animals. Experimental animals (white mice) infected with H. influenzae cultures display symptoms of toxaemia. The bacteria do not normally invade the blood.

Pathogenesis and diseases in man. H. influenzae gives rise to acute catarrhs of the upper respiratory tract in combined action with other bacteria (staphylococci, streptococci, adenoviruses, etc.). Decrease in temperature facilitates the development of the infections, and for this reason they are known as colds and seasonal infections, and prevail during the cold months.

A sudden drop in temperature and exposure to the effect of influenza viruses weaken the general immunobiological defense mechanisms of the body, as a result of which certain bacteria which are present as commensals in the human throat become more active

In the human body H. influenzae localize in the mucous membranes of the respiratory tract and bronchi. They occur extra- and intracellularly and are sometimes found in the blood. The organisms are isolated quite frequently from acute catarrhal cases and are at times responsible for acute inflammatory conditions (laryngitis, tonsillitis, bronchitis, pneumonia, otitis, meningitis, etc.) They also give rise to various postinfectional complications, particularly in children

Immunity. Immunity acquired after H. mfluenzae infections has not been sufficiently studied. It is thought that acute catarrhal conditions produce no immunity. This is accounted for by the multibacterial aetiology of the disease. The commensals present in the upper respiratory tract and nose may cause various lesions in the weakened organism known under the common name of catarrhs.

Insusceptibility to acute catarrhs of the respiratory tract depends on the condition of the body's physiological defense mechanisms as well as on the ability of the body to endure changes in the temperature, humidity, and other factors of the environment.

Laboratory diagnosis. Specimens from sputum and nasal discharge serve as test material. Mucus from the tonsillar and nasopharyngeal mucosa is collected with a cotton-wool swab, and the following procedures are carried out:

(1) smears are prepared from sputum and stained with fuchsine for 5-10 minutes;

(2) purulent sputum clots washed in 0.85 per cent saline solution are inoculated into blood agar, chocolate agar, or Levithal's medium. The material may be plated by the cough-plate method when an open plate of medium is held at a distance of 5-8 cm in front of the patient's mouth when he coughs. The cultures are incubated at 37° C. From 15 to 25units of penicillin are added to the medium to inhibit the growth of coccal microflora. The isolated culture is differentiated from whooping cough bacilli by its biochemical and antigenic properties. Haemophilus parainfluenzae is present as a commensal in the respiratory tract mucosa of humans and cats. This organism is usually nonpathogenic.

Treatment. Patients are given streptomycin together with sulphonamides, and also chloramphenicol, oxytetracycline, polymyxin. Disinfectant gargles are also prescribed.

Prophylaxis includes prevention of cooling and body hardening by systematic physical exercises. Physical culture and sports, sufficient nourishment, with a full-value vitamin content in particular, and observance of rules of hygiene at work and in everyday life play an important part in the prophylaxis of catarrhs.

Conjunctivitis, caused by Haemophilus aegyptius, occurs in summer mainly in countries with a warm climate.

Causative Agent of Soft Chancre

The soft chancre bacillus (Haemophilus ducreyi) was discovered by P.Ferrari in 1885. Its aetiological role was shown in experiments in 1887 by O. Petersen, and described in detail by A. Ducrey in 1889, and studied by P. Unna in 1892.

Morphology. The organism is oval m shape and measures 1.5-2 mcm in length and 0.5 mcm in breadth. In smears from ulcers it occurs in-groups or long chains (Plate V). The organism forms neither spores, capsules, nor flagella. It is Gram-negative and exhibits bipolar staining. The G+C content in DNA is 38-42 per cent.

Cultivation. The causative agent of soft chancre is a facultative anaerobe. It does not grow on common media but grows on blood agar at37° C (35°-38°) and pH 7.2-7.8, on Martin's broth medium containing20 per cent defibrinated blood, and on medium consisting of one part of5 per cent glycerin agar and four parts of fluid egg medium. On blood agar the organisms are haemolytic and produce small, round, globe-shaped isolated colonies which measure 1-2 mm in diameter.

Fermentative properties. The organism is non-proteolytic. It ferments glucose, lactose, saccharose, and mannitol, with acid formation.

Toxin production. No soluble toxin is produced. All pathological changes are due to the effect of an endotoxin.

Antigenic structure and classification are still moot questions. The causative agent of soft chancre should be differentiated from Haemophilus vaginalis found in non-specific vaginitis and urethritis.

Haemophilus vaginalis is a Gram-variable facultative anaerobe. It does not grow on commonly used media but develops on a tellurate medium.

Resistance. The soft chancre bacillus is sensitive to various environ-mental factors. It withstands 55° C for 15 minutes and is destroyed in dilute disinfectant solutions.

Pathogenicity for animals. Monkeys are the only animals susceptible to H. ducreyi, and display a mild form of the disease. Guinea pigs and rabbits are insusceptible to inoculation.

Pathogenesis and disease in man. Soft chancre is a typical venereal disease and is transmitted via the genital organs. Individuals with an acute or chronic form of the disease are sources of infection.

The organism multiplies in the skin or mucous membranes of the genitalia. An inflammatory process develops at the site of penetration and is followed by ulceration. The ulcer is soft, with uneven edges and a purulent discharge, and is painful. Invasion of the adjacent parts of the body by the bacillus results in formation of a great number of painful ulcers and lesions of the lymphatic vessels with the development of lymphangitis and lymphadenitis. In the absence of ulcers the organism may localize in the mucous membranes of the vagina, cervix uteri, and the urethra.

Shancroid

Immunity. The disease leaves no immunity, although it gives rise to the production of complement-fixing antibodies and development of allergy.

Laboratory diagnosis comprises the following:

(1) microscopical examination of excretions obtained from deep ulcer layers, the smears being stained with methylene blue or with the Gram stain. In the microscope long chains of Gram-negative bacilli can easily be seen;

(2) inoculation into blood agar, isolation of the pure culture and its identification by the agglutination reaction with specific serum from the patients;

(3) employment of the allergic reaction (intracutaneous test) with an antigen derived from the soft chancre bacilli; a papule surrounded b\d zone of inflammation will appear at the site of injection of the antigen in 24-48 hours after inoculation.

Treatment. Sulphonamides and antibiotics (penicillin, streptomycin, tetracyclmes, and chloramphenicol) are prescribed.

Prophylaxis is ensured by social changes which have eliminated poverty, unemployment, and prostitution, improved cultural and hygienic standards of the population, established sound family relations, and bettered conditions of life.

Calymmatobacterium granulomatis, the causative agent of granuloma venereum, or Donovan granuloma, belongs to the genus Calymmatobacterium. It is a non-motile. Gram-negative, polymorphous rod, 1-2 mcm long and 0.5-0.7 mcm wide. In the body of sick individuals it forms a capsule. The genitals and the skin on the groin and perineum are mainly involved with the formation of persisting ulcers. The disease follows a chronic course and is encountered in tropical countries.

Listeria.

The causative agent of listeriosis (Listeria nionocvtogenes) was discovered in 1926 by E. Murray and named Listeria in honour of J. Listerin 1940 by J. Pirie.

Morphology. Listeria are small bacteria 0.5-2 mcm in length and0.4-0.5 mcm in breadth. They are motile, slightly curved, terminally flagellated and Gram-positive. The organisms occur singly or in pairs, and in smears from organs they are often seen arranged at an angle to each other in the form of the letter V or in chains. They do not form spores or capsules. The G+C content in DNA is 38 per cent.

Cultivation. Listeria are facultative anaerobes with simple growth requirements. They grow on all ordinary media at pH 7.0-7.2 and 37 C. No growth is shown below 2.5 and above 59 C. On solid nutrient media the organisms produce small, whitish, flat, smooth, and shiny colonies with a pearly hue. On liver agar the colonies are slimy. In broth listeria produce turbidity and a slimy deposit. On blood agar the colonies are surrounded by a narrow zone of haemolysis.

Listeria produce forms which are resistant to antibiotics as well as antibiotic-dependent varieties. The S-forms of the organisms are characterized by sensitivity to phagolysis while the R-forms are phage resistant. Eight phage types can be distinguished on the basis of phagolysis.

Fermentative properties. Litmus milk turns red but is not coagulated. Listeria produce no indole or hydrogen sulphide, do not reduce nitrates to nitrites, and do not liquefy gelatin. Glucose, levulose, and trehalose are fermented with acid but no gas formation. Fermentation of maltose, lactose, saccharose, dextrin, salicin, rhamnose, and soluble starch is variable and slow.

Toxin production. The organisms produce no soluble toxin (exotoxin).Listeria discharge a thermolabile haemolysin into the cultural fluid. This haemolysin is activated by cistein and causes haemolysis of pigeon, rabbit, guinea pig, and horse erythrocytes. The organisms also produce a lipolytic factor which causes cytolysis of a macrophage culture. An endotoxin is liberated when the bacterial cells disintegrate and is responsible for the characteristic manifestations of listeriosis in human beings and animals.

Antigenic structure and classification. There are two main serological variants of listeria: rodent and ruminant. The former variant was isolated from rodents, and is the most widespread. The latter variant was isolated from ruminants (bovine cattle). However, this classification is only relative since both serological variants have also been found in other animals, birds, and human beings. The main serovars possess somatic (O) and flagellar (H) antigens. The somatic 0-antigen contains four thermostable antigens (I, II, IV and V) and one variable antigen (III). The H-antigens contain antigens A, B, C, and D which are destroyed by exposure to formalin.

Resistance. Listeria are resistant to environmental factors. They maintain their pathogenic properties in the dried state for a period of 7 years, and withstand freezing. They survive at 55 C for one hour and 58 C for30 minutes. The organisms are killed in 3 minutes by boiling and in 20minutes by a temperature of 70 C. They are destroyed by exposure to1 and 0.5 per cent formalin solutions, 5 per cent phenol solution and to other disinfectants.

Pathogenicity for animals. Cattle, sheep and goats, horses, pigs, rabbits, chickens, and pigeons may naturally acquire the disease. The infection occurs among domestic and field mice and among wild rats which are probably the main reservoir of the causative agent in nature.

Rabbits, guinea pigs, and mice are most susceptible to the disease among the laboratory animals. Intracerebral inoculation results in sepsis which leads to death in 1 or 4 days. Protracted cases give rise to meningoencephalitis. The disease may also be brought about in laboratory animals by subcutaneous, intramuscular, and intranasal inoculation.

Pathogenesis and diseases in man. Listeriosis is a zoonotic infection. Human beings contract it from sick rodents, pigs, and horses. Meat products derived from pigs affected with listeriosis are most dangerous to man. Infection is possible through tick bites in enzootic listeriosis foci. The causative agent enters the body through injured skin, through the mucous membranes of the mouth, nasopharynx, and intestinal tract and through the eye conjunctiva. The diseases are characterized by sepsis (acute and chronic) and symptoms of meningoencephalitis which is fatal in most cases, particularly among newborn infants and people with cerebral injuries. Inflammatory processes develop in the pharynx, and a skin rash sometimes appears. Apart from cases with severe clinical manifestations, mild forms of the disease and carrier states' may occur.

Great significance in the pathogenesis of listeriosis is attributed to saturation of the whole body or of separate tissues and organs by endotoxin, the causative agent multiplying intensely in the body of infected man or animals.

The liver, spleen, lymph nodes, heart, central nervous system, meninges, uterus, and the internal organs of newborn infants are the most seriously involved.

There are two main forms of human listeriosis: anginose-septicaemic and nervous. Recovery from the former is normally possible, but death may sometimes occur with both forms. Septicogranulomatous (in foetus and newborn infants) and ocular-glandular forms occur in man besides the two above-mentioned main forms.

Immunity. Animals acquire immunity following listeriosis, regardless of the presence of a reservoir of the causative agent (infected rats and ticks). Immunity in man has not been studied sufficiently. Agglutinins, precipitins, and complement-fixing antibodies have been found to be present in patients' blood, but they do not show antibacterial effect in laboratory tests. Hyperimmune serum has no therapeutic properties. A rise in antibody titre is used in laboratory diagnosis.

Laboratory diagnosis is performed by isolating listeria cultures from the patients' blood. Specimens of brain tissue, pieces of liver and spleen are collected for examination at autopsy.

The best growth is obtained in glucose-serum broth. In addition, laboratory animals are infected.

Serological diagnosis comprises the agglutination reaction which is positive in patients' sera diluted in ratios ranging from 1 :250 to1 .5000 The precipitin reaction and the complement-fixation reaction are also employed.

When identifying listeria, it is necessary to differentiate them from the organisms responsible for swine erysipelas (Ehrysipelothrix rhusiopathiae). These organisms differ from listeria in that they are non-motile, incapable of fermenting salicin, and non-pathogenic for guinea pigs. The antigenic structure of both organisms is different and strictly specific.

Treatment and Prophylaxis. Treatment is accomplished by the use of antibacterial preparations of the tetracycline group, and sulphonamides. Prophylaxis is ensured by general sanitary measures carried out jointly with veterinary service. Laboratory control of meat which is to be marketed, routine control over domestic animals, timely recognition of rodent enzootics, and prevention of horses being infected by affected rodents and domestic animals are all necessary.

Additional material for diagnosis

INFECTION CAUSED BY BORDETELLA

The bacterial diagnosis of pertussis and parapertussis involves culturing of sputum by the "cough plates" method. For this purpose an open Petri dish with nutrient medium (Bordet's blood-potato-glycerol agar or casein-charcoal agar) is placed in front of the patient's mouth at a distance of 4-8 cm at the moment of coughing.

Preparation of Bordet's potato-glycerol agar: (a) boil in a sterilizer 100 g of finely cut potatoes with 200 ml of 4 per cent glycerol: (b) dissolve 5 g of agar in 150 ml of 0.6 per cent sodium chloride solution, add 50 ml of fluid A to the 150 ml of agar and sterilize. The day before the inoculation melt the medium and cool it to 50 X, then add 30-50 per cent of do fibrinated rabbit blood. To suppress the attendant flora, add 15-25 U of penicillin.

After several coughs, tile dish. is closed and put into an incubator. If the cough is absent, mucosal secretion from the posterior wall of the throat is collected with a tampon passed through the inferior nasal passages or the mouth and inoculated onto plates with the above-mentioned media. The investigation is conducted two times. The highest percentage of culturing is observed during the first and second week of the disease, after which tin* incidence of positive results tends to decrease.

On the 2nd-5th day after the inoculation of sputum, typical tiny colonies of B. pertussis appear on the agar. They are convex, moist, shiny, grey and resemble mercury drops. Colonies of B. parapertussis are somewhat larger. Smears are made from the colonies and stained by the Gram technique. The causative agent of pertussis appears as Gram-negative, small ovoid rods.

The employment of immunofluorescence is usually associated with good results. Two smears are made from the material taken with a throat tampon or colonies and treated with fluorescent, sera. The answer is obtained in 2-6 hrs. If the result is positive, use the remainder of the colonies for performing the slide agglutination reaction "with pertussis and parapertussis sera diluted 1:10. Then isolate a pure culture and identify it by a number of attributes (Table ).

Table

Differential-Diagnostic Criteria of Pathogenic Bordetella

Bordetella type	Growth on agar		Change in medium colour		Urea break-down
	MPA	with tyrosine	casein-charcoal agar	blood agar
B. pertussis	No growth	No growth	No change	No change	–
B. parapertussis	Growth with formation of brown colonies	Growth with formation of bright brown colonies	Growth with formation of brownish colonies	Darkening	+
B. bronchiseptica	Growth with formation of colourless colonies	Growth without any changes in colour of the medium	No change	No change	+

The indirect haemagglutination test with the use of red blood cells sensitized with immune pertussis serum is more sensitive than the agglutination test. Red blood cells are pretreated with an alizarine blue indicator.

For the purpose of serological diagnosis the agglutination and CF tests are employed. In view of widescale performance of inoculations, leading to the elaboration of specific antibodies in the blood of healthy persons, it is necessary that paired sera be utilized for serological diagnosis- Augmentation in the titre of antibodies in the dynamics of the disease confirms the diagnosis. A pertussis diagnosticum serves as an antigen in the serological reactions. It is recommended that pertussis and parapertussis immune sera he employed as an additional control in the serological tests.

Indirect haemagglutination is the most sensitive and convenient test for demonstrating antibodies in pertussis.

To evaluate the immunological alterations in children injected with a combined attenuated vaccine against pertussis, diphtheria. tetanus, the antigen neutralization test with a pertussis erythrocytic antibody diagnosticum is carried out. This reaction is more sensitive than the agglutination test.

INFECTION CAUSED BY HAEMOPHILUS INFUJENZAE

Haemophilus influenzae, which is present rather frequently on the mucosal membranes of the human upper respiratory pathways, may be responsible for the development of meningitis, bronchitis, pneumonia, empyema, conjunctivitis, otitis, and other diseases.

Bacteriological examination. The material tested (sputum, blood, cerebrospinal fluid, serous fluid) is inoculated onto nutrient media within 2-3 hrs after it has been collected. For a medium use a nutrient agar with 5-10 per cent of native blood or chocolate agar with heated or boiled blood since haemophilic bacteria do not grow on simple nutrient media.

Serous and cerebrospinal fluids are centrifuged and the sediment is transferred with a bacterial loop on blood-containing solid media.

For culture enrichment use Fildes' liquid nutrient medium (1 ml of the fluid tested per 5-10 ml of the medium).

Chocolate agar. Heat nutrient agar to 60 °C, add aseptically 5 per cent of whole or defibrinated human, horse, or rabbit blood, mix the medium obtained, and put it into a water bath for 2-3 min at 80 °C. After that add 5 per cent. of blood once again and replace the mixture into the water bath for the same time and at the same temperature. Store the medium for no longer than two weeks.

Fildes' medium. To obtain this medium, 150ml of 0.85 percent sodium chloride solution, 6 ml of chemically pure hydrochloric acid (with a relative density of 1.13), 50ml of defibrinated horse or sheep blood, and 1 g of pepsin are mixed until dissolution and allowed to stand for 24 hrs at 55 °C with occasional shaking (5-6 times). The liquid solidifies, appearing as a semi-solid agar in consistency. After digestion, add 12 ml of 20 per cent sodium hydroxide solution and the digest becomes liquid once more. Adjust the pH of the medium to 7.6 and then add by drops the concentrated hydrochloric acid until the pH becomes 7.2-7.0. After that, add 0.5 per cent of chloroform, let the medium stand for 2-3 days, while shaking it periodically, then pour it into ampoules and seal them. To prepare nutrient medium, add aseptically 5 percent of tildes' peptic digest to sterile broth or melted nutrient agar.

In cases of septicaemia 5-10 ml of the patient's blood is inoculated into 50-100 ml of nutrient medium and subcultured onto solid blood media 24 hrs later. Simultaneously, culturing is made onto simple nutrient agar (the absence of growth). In 24 hrs tiny transparent colonies appear on the solid blood media, while the colonies on the chocolate agar are larger and semi-transparent. From the colonies make smears and stain them by the Gram method. If tiny Gram-negative rods are detected, issue the first preliminary result. H. influenzae may appear in both capsular and non-capsular forms.

Following the identification of haemophilic bacteria, study their biochemical properties, namely, catalase, oxydase, urease, and P-galactosidase activity, nitrate reduction, carbohydrates fermentation, hydrogen sulphide and indol production, and haemolytic activity.

Haemophilic microorganisms have catalase activity, reduce nitrates, display P-galactosidase activity (apart from H. influenzae), and always split glucose arid lactose.

H. influenzae breaks down urea. form indol, and exhibit haemolytic activity, yet. they do not produce hydrogen sulphide and show no oxidase activity.

The serological identification of the cultures isolated is based on the capsular antigen, according to which all strains are divided into six groups (a, b, c, d, e, f). The agglutination reaction is made on a glass slide with type-specific poly- and mono-sera.

Catalase determination. On a glass slide place a drop of 10 per cent hydrogen peroxide, introduce the culture, and grind it with circular movements. A positive reaction is indicated by foam formation.

Oxidase determination. On the lid of a Petri dish put filter paper (5-7 cm ill diameter), place 2-3 drops of 1 percent tetramethylparaphenylendiamine solution, then introduce a loopful of the culture. In case of a positive reaction violet staining is observed at the site of culture inoculation in 10-15 s.

Determination of urease. Solution A; 2 ml of 95 per cent alcohol, 4 ml of distilled water, 2 g of urea. Solution B: 0.1 g of potassium dihydrophosphate, 0.1 g of potassium hydrophosphate, 0.5 g of sodium chloride, 1 ml of 0.2 percent phenol red solution, 100 ml of distilled water.

Sterilize the solutions for 30 min at 151.9 kPa (1.5 atm). Then add 19 parts of solution B to one part of solution A, dispense aseptically into test tubes (0.1 ml per tube), and introduce several loops of the culture studied into each test tube. The inoculated cultures are incubated for 30 min. If urease is present, the medium is stained crimson. In case of a negative reaction the inoculated cultures are observed for 24 hrs.

Mycobacteria

Causative Agent of Tuberculosis. The organism responsible for tuberculosis in man (Mycobacterium tuberculosis) was discovered in 1882 by R. Koch. He also studied problems concerning the pathogenesis of tuberculosis and immunity produced by the disease. A. Calmette's and Ch. Guerin's discovery in 1919 of the live vaccine against tuberculosis was very important since it permits widespread practice of specific preventive vaccination. The introduction of streptomycin, phthivazide, isoniazid, PAS, and other drugs has supplied modem medicine with powerful means of tuberculosis control.

Morphology. M. tuberculosis is a slender, straight or slightly curved rod, 1-4 mcm in length and 0.3-0.6 mcm in breadth (fig.1). It may have small terminal swellings. The organisms are non-motile, Gram-positive, pleomorphous, and do not form spores or capsules. They stain poorly by the ordinary methods but are stained well by the Ziehl-Neelsen method.

Rod-like, thread-like, branching, granular, coccoid, and filterable forms are encountered.

E. Metchnikoff and V. Kedrovsky observed certain forms in cultures, which were similar to actinomycetes. A. Fontes and others have put forward evidence of the existence of filterable forms of M. tuberculosis. On being injected into guinea pigs, they become acid-fast and may be seen under the light microscope. Occurrence of non-bacillary G-forms has also been ascertained, the majority of them-occurring under unfavourable conditions.

M. tuberculosis in smear. M. bovis in smear

Electron microscopy has revealed the presence of granules and vacuoles located terminally in the cells of mycobacteria. The cytoplasm of young cultures is homogeneous, while that of old cultures is granular. M tuberculosis is acid-fast due to the fact that it contains mycolic acid and lipids

M. africanum

The lipids of M. tuberculosis consist of three fractions: (1) phosphatide which is soluble in ether; (2) fat which is soluble in ether and ace-tone. (3) wax which is soluble in chloroform and ether.

Nonacid-fast granular forms, which readily stain violet by Gram's method and known as Much's granules, and acid-fast Slenger's fragments of M. tuberculosis also occur. The G +C content in DNA ranges from 62 to 70 per cent.

Chemical composition. The fact that as much as 40% of the dry weight of mycobacteria may consist of lipid undoubtedly accounts for many of their unusual growth and staining characteristics A comprehensive discussion of mycobacterial lipids is beyond the scope of this text, but one class of lipids, the mycosides, is unique to acid-fast organisms and is involved in some manner with the pathogenicity of the mycobacteria

Mycolic acid is a large alpha-branched, beta-hydroxy fatty acid that varies slightly in size from one species of Mycobacterium to another These acids occur free or bound to carbohydrates as glycolipids, which are referred to as mycosides. Free mycolic acid is, by itself, acid fast, but the observation that acid fastness is lost after the destruction of the cell integrity by sonication makes it unlikely that mycolic acid alone accounts for this property Cord factor is a mycoside that contains two molecules of mycolic acid esterified to the disaccharide trehalose. It is found in virulent mycobacteria, and its presence is responsible for a phenomenon in which the individual bacteria grow parallel to each other, forming large, serpentine cords (see Fig. ).

Figure. Young colony of virulent M. tuberculosis showing paralle growth.

Avirulent mycobacteria do not grow in such cords. Purified cord factor is lethal for mice, and it inhibits the migration of neutrophils and binds to mitochondrial membranes, causing functional damage to respiration and oxidative phosphorylation. Although its precise action is unknown, a report clearly shows that cord factor induces the synthesis of cachectin (also called tumor necrosis factor) in mice. When injected with cord factor, mice became severely wasted (cachexia) losing up to 25% of their weight within 48 hours. The observation that antibodies to recombinant cachectin would prevent this effect supports the conclusion that cachectin was responsible for the wasting induced by cord factor. It also strongly suggests that cord factor is responsible for the cachexia observed m tuberculosis patients as well as the fever and pulmonary necrosis that is characteristic of tuberculosis.

A group of glycolipids similar to cord factor are the sulfatides, which are multiacylated trehalose 2-sulfates. They have been shown to inhibit phagosome-lysosome fusion and, as such, seem to enhance survival of phagocytosed mycobacteria.

Wax D is another complicated mycoside in which 15 to 20 molecules of mycolic acid are esterified to a large polysaccharide composed of arabinose, galactose, mannose, glucosamine, and galactosamine—all of which seem to be linked to the peptidoglycan of the cell wall. When emulsified with water and oil, Wax D acts as an adjuvant to increase the antibody response to an antigen, and it is probably the active component in Freund's complete adjuvant, which employs intact tubercle bacilli emulsified with water, oil, and antigen.

Mycobacteria also possess some lipopolysaccharides, of which the best studied is a lipoarabinomannan (LAM). LAM appears to be an inducer of tumor necrosis factor-alpha synthesis by monocytes and macrophages.

M tuberculosis also possesses a number of protein antigens that by themselves do not seem to be toxic or involved in virulence. However, the host's cellular immune response to certain of these mycobacterial proteins apparently accounts for the acquired immunity and allergic response to the tubercle bacilli.

Cultivation. The organisms grow on selective media, e. g. coagulated serum, glycerin agar, glycerin potato, glycerin broth and egg media (Petroffs, Petragnani's, Dorset's, Loewenstein's, Lubenau's, Vinogradov's, etc.) They may be cultured on Soton's synthetic medium which contains asparagine, glycerin, iron citrate, potassium phosphate, and other substances.

Loewenstein's medium

Certain levels of vitamins (biotin, nicotinic acid, riboflavin. etc.) are necessary for the growth of M. tuberculosis. Scarcely visible growth appears 8-10 days after inoculation on glycerin (2-3 per cent) agar, but in 2-3 weeks a dry cream-coloured pellicle is produced. The best and quickest (on the sixth-eighth day) growth is obtained on Petroffs egg medium which consists of egg yolk, meat extract, agar, glycerin, and gentian violet.

On glycerin (4-5 per cent) meat-peptone broth the organisms produce a thin delicate film in 10-15 days, which thickens gradually, becomes brittle, wrinkled, and yellow; the broth remains clear. M. tuberculosis can be successfully cultivated by Pryce's microculture method or Shkolnikova's deep method in citrated rabbit or sheep blood. Growth becomes visible in 3-6 days. Synthetic and semisynthetic media are employed for cultivating M. tuberculosis in special laboratories. The organisms are aerobic, and their optimal growth temperature is 37 C. They do not grow below 24 and above 42 °C. The reaction of the medium is almost neutral (pH 6.4-7.0), but growth is possible at pH ranging from 6.0 to 8.0. M. tuberculosis dissociate from typical R-forms to the atypical S-forms. Some strains produce a yellow pigment in old cultures.

Fermentative properties. The organisms have been found to contain proteolytic enzymes which break down proteins in alkaline and acid medium. They also contain dehydrogenases which ferment ammo acids, alcohols, glycerin, and numerous carbohydrates. M. tuberculosis is cap-able of causing reduction (they reduce salts of telluric acid, potassium tellurite, and break down olive and castor oils, etc.). The organisms produce lecithinase, glycerophosphatase, and urease which ferment lecithin, phosphatides, and urea.

Toxin production. M. tuberculosis does not produce an exotoxin. It contains toxic substances which are liberated when the cell decomposes

In 1890 R. Koch isolated from the tubercle bacillus a substance known as tuberculin. There are several tuberculin preparations. The Old Koch's tuberculin is a 5-6-week-old glycerin broth culture sterilized for 30 minutes by a continuous current of steam (100 C), evaporated at 70 °C to one tenth of the initial volume, and filtered through a porcelain filter. The New Koch's tuberculin consists of desiccated M. tuberculosis which are triturated in 50 per cent glycerin to a homogeneous mass. A tuberculin has been derived from the bovine variety of M. tuberculosis, which contains protein substances, fatty acids, lipids, neutral fats, and crystalline alcohol. There is also a tuberculin free of waste sub-stances and designated PPD (purified protein derivative) or PT (purificatum tuberculinum).

Tuberculin is toxic for guinea pigs which are affected with tuberculosis (injection of 0.1 ml of the standard preparation is fatal for 50 percent of experimental animals). Small doses of tuberculin produce no changes in healthy guinea pigs.

The chemical composition of the toxic substances contained in M.tuberculosis has not yet been ascertained. It is known that the toxin of the tubercle mycobacteria is composed of proteins (albumins and nucleoproteins). Phosphatides have been isolated from the virulent types of the organism and are capable of producing characteristic lesions in rabbits. Phthioic acid is the most active.

Extremely toxic substances have been extracted from M. tuberculosis after boiling in vaseline oil. They are fatal to guinea pigs in doses of one-thousandth of a milligram.

Virulent mycobacteria differ from the non-virulent organisms in that they contain a great number of lipopolysaccharide components. The lipid fraction (cord factor) responsible for adhesion of mycobacteria and their growth in cords and strands is marked by high toxicity. The cord factor of M. tuberculosis destroys the mitochondria of the cells of the infected body and causes disorders in respiration and phosphorylation.

Antigenic structure. On the basis of agglutination and complement-fixation reaction a number of types of mycobacteria have been distinguished: mammalian (human, bovine, and rodent), avian, poikilotherm, and saprophytic. The human type does not differ serologically from the bovine or murine types. Mycobacterial antigens produce agglutinis, opsonins, precipitins, and complement-fixing antibodies in low titres. Tuberculin is considered to be a peculiar antigen (hapten).

A high molecular tuberculin may be considered to be a full-value antigen capable of stimulating the production of corresponding antibodies.

M. tuberculosis and tuberculin possess allergenic properties and produce local, focal, and generalized reactions in the body infected with tuberculosis.

According to data supplied by a number of investigators, the M tuberculosis antigen contains proteins, lipids, and particularly large amounts of phosphatides and lipopolysaccharides. Experiments on animals have proved that the lipopolysaccharide-protein complexes protect the body from infection with M. tuberculosis. Tuberculin is widely used for allergic tests, which are employed for determining infection with M. tuberculosis.

Classification of mycobacteria which are pathogenic for human beings, cattle, rodents, and birds is given in Table1. There are also strains of M. tuberculosis which affect poikilotherms and acid-fast saprophytes.

Resistance. Tubercle bacilli are more resistant to external effects as compared to other non-sporeforming bacteria as a result of their high lipid content (25-40 per cent).

The organisms survive in the flowing water for over a year, in soil and manure up to 6 months, on the pages of books over a period of3 months, in dried sputum for 2 months, in distilled water for several weeks, and in gastric juice for 6 hours. They are easily rendered harm-less at temperatures ranging from 100 to 120°C. The organisms are sensitive to exposure to sunlight.

Pathogenicity for animals. Tuberculosis is an infection which is wide-spread among cattle, chickens, turkeys, etc. Pigs, sheep and goats con-tract the disease less frequently.

Cattle, sheep and goats are quite resistant to the human type of tubercle mycobacteria. Guinea pigs are highly susceptible to the human type, and their infection results in a generalized pathological condition and death. Infection of rabbits produces chronic tuberculosis.

The bovine type of the organism is pathogenic for many species of domestic mammals (cows, sheep, goats, pigs, horses, cats, and dogs)and wild animals. Infected rabbits and guinea pigs contract acute tuberculosis, the condition always terminating in death.

Cattle and, less frequently, sheep and goats contract paratuberculosis (Johne's disease, a chronic specific hypertrophic enteritis) which is caused by Mycobacterium paratuberculosis.

The avian type of tubercle mycobacteria produces infection in chickens, turkeys, fowls, peacocks, pheasants, pigeons, and waterfowl in natural conditions. Domestic animals (horses, pigs, goats, and less frequently cattle) may naturally acquire the disease by infection with the avian type organisms. Man may also be infected in some cases.

Among laboratory animals rabbits are highly susceptible to the avian type of tuberculosis, small doses of the organism causing generalized tuberculosis. Guinea pigs are relatively resistant and subcutaneous injections of the culture affect the lymph nodes, which is accompanied with the development of caseous foci.

The murine type of M. tuberculosis is extremely pathogenic for field mice. Experimental inoculation of rabbits and guinea pigs with this type of mycobacteria produces chronic tuberculosis.

Table

Classification of Main Mycobacteriuni Species

Species	Formation of				Causes
	urease	nicotine amidase	paracin amidase	niacin
M. tuberculosis	+	+	+	+	Tuberculosis in humans and other primates, in dogs and other animals that were in contact with asick person
M. africanum	+	+	+	–	Tuberculosis among inhabitants of tropical Africa (Senegal, and other countries)
M. bo vis	+	–	+	–	Tuberculosis in calves, domestic and wild animals, humans and other primates
M. kansasii	+	+	+	–	Tuberculosis-like disaeses in humans, which is marked by weak activity
M. intracellulare	–	+	+	–	Severe forms of tuberculosis-like in humans, localized lesions in pigs
M. xenopi	–	+	+	–	Lesions of the lungs, urogenital system and granuloma of the skin in humans
M. ulcerans				+	Ulceration of the skin in persons dwelling in Mexico, New Guinea, Malaysia and Africa
M. paratuberculosis					Chronic diarrhoea in calves and sheep
M. microti	+	+	–	+	Generalized infection in field mice
M. avium	–	+	+	–	Tuberculosis in birds, some times in calves, pigs and other animals. Infection in humans is rare
M. leprae					Leprosy in humans
M. leprae-murium					Endemic affections of rats in different parts of the world

Pathogenesis and disease in man. It has been shown that tuberculosis in man is caused by several types of mycobacteria — the human type(M. tuberculosis), the bovine type (M. bovis), etc. The share of atypical mycobacteria which cause a variety of clinical forms of tuberculosis among humans has recently grown to 50 per cent.

Infection with tuberculosis takes place through the respiratory tract by the droplets and dust, and, sometimes, per os through contaminated foodstuffs, and through the skin and mucous membranes. Intrauterine infection via the placenta may also occur.

With air-borne infection, the primary infectious centre develops in the lungs, but if infection takes place through the alimentary tract, the primary focus is in the mesenteric lymgh nodes. When body resistance is low and conditions of work and life are unfavourable, the organisms may leave the site of primary localization and spread throughout the body, causing a generalized infection. At present, there is a point of view which maintains that localization of the infectious focus in the lungs is preceded by a lympho-haematogenic dispersion of M. tuberculosis throughout the body. The duration of the incubation period in tuberculosis is comparatively long, from several weeks to 40 years and more.

The development of the primary tuberculous foci takes a benign course if the conditions of life are favourable and there are no aggravating factors present. This stage usually terminates with resorption and healing of the caseous foci which become calcified and enclosed in a dense connective-tissue capsule. However, such result is not accompanied by the body becoming completely freed of the causative agents. About 70 per cent of people who are under 20 years of age are infected with M. tuberculosis but no disease is produced in them.

The organisms survive in the lymph nodes and other tissues and organs of the primary focus for many years and sometimes even for life. People infected in such a way acquire, on the one hand, relative immunity and, on the other hand, a potentially latent form of tuberculosis which may become active under the influence of a number of infectious diseases and psychic and physical traumas.

Under the effect of drugs and immunobiological factors of the macroorganism L-forms capable of reversion to typical mycobacteria form quite frequently.

In some cases primary tuberculosis can be quite severe in non-infected and non-immunized people, particularly if they were infected by massive doses as a result of contact with patients who discharge virulent mycobacteria.

Incidence of reinfection with tuberculosis increases 3-5 fold among individuals exposed to exogenous superinfection and the resulting condition is more severe than aggravation of primary tuberculosis. It involves the development of new foci in the lymphatic system, increased sensitization, and accumulation of irritations as a result of the body being affected by pathogenic mycobacteria which are extreme irritants.

Tuberculosis is characterized by a variety of clinical forms, anatomical changes, compensational processes, and results. The infection may become generalized and involve the urogenital organs, bones, joints, meninges, skin, and eyes.

Immunity. Man is naturally resistant to tuberculosis, this property being hereditary. On the basis of the allergic reaction. X-ray examination, and patho-anatomical changes it has been shown that in a great number of cases infection does not result in disease. There are approximately 80 per cent of adults over 20 years of age among infected persons and no more than 10 per cent of them become ill, and only 5 per cent immediately after infection.

There is a characteristic immunity produced by tuberculosis. Inoculation of M. tuberculosis into healthy guinea pigs causes no visible changes during the first days after infection. But a compact tubercle which undergoes ulceration is formed in 10-14 days. The lymph nodes become enlarged and hard, a generalized process develops, and the animal dies.

When tuberculous animals are inoculated with M. tuberculosis, an ulcer is formed at the site of injection. This ulcer shortly heals and no involvement of the lymph nodes or generalization of infection takes place. These facts were established by Koch and advanced the knowledge on a number of problems concerning pathogenesis and immunity in tuberculosis. Particular importance was attributed to non-sterile (infectious)immunity which has been widely reproduced artificially (by BCG vaccination). It is understood that immunity to tuberculosis is usually non-sterile. However, as in brucellosis, the phase of non-sterile immunity in tuberculosis is followed by the phase of sterile immunity.

Agglutinins, precipitins, opsonins, lysins, and complement-fixing antibodies are found to be present in the sera of tuberculosis patients. The presence of these substances, however, provides no evidence of the intensity of the immunity. Likewise, insusceptibility cannot be determined by the phagocytic reaction since phagocytosis in tuberculosis is frequently incomplete which is explained by lack of lymphokinins. Body reactivity and specific productive inflammation play the main role in production of immunity. This inflammation renders the M. tuberculosis harmless by formation of granulomas which consist of epithelioid cells surrounded by a zone of lymphoid and giant Langhans' cells.

Interference of M. tuberculosis with BCG strains and other non-virulent mycobacteria which are capable of blocking tissue and organ cells sensitive to virulent tuberculous mycobacteria plays a definite role in the complex of defence mechanisms of the body.

The genetic factor (which has been studied in detail in twins) plays an obvious role in immunity in tuberculosis. The concordance in affection with the disease is 67 per cent among monozygotic twins, 25.6 per cent among dizygotic twins, 25.6 per cent among brothers and sisters, and7 per cent in husband and wife.

A new component which affects M. tuberculosis has been found to be present in human blood. Individuals devoid of this component are more susceptible to tuberculosis.

Among the defence factors phages should be mentioned. They affect both virulent and avirulent M. tuberculosis strains. The discovery of phages is of certain practical importance. They may be used in diagnosis and, probably, in the treatment of tuberculosis.

Many tissues are capable of producing enzymes which break down mycobacteria. Such properties are characteristic of enzymes of the nuclease group.

The barrier function of tissues and organs which stops the organisms and prevents their dispersal throughout the body is of essential importance in body resistance to tuberculosis. Antituberculous antibacterial agents which have been found in the blood, muscles, skin, thyroid gland, pancreas, spleen, and kidneys are also of great significance. The role of tuberculous allergy in immunity has not been ascertained, although various points of view on this subject have been expressed (seethe section 'Relation of Allergy to Immunity'). The majority of phthisiotherapists hold that there is no correlation between allergy and immunity in tuberculosis.

Laboratory diagnosis. 1. Microscopy of smears from sputum, pus, spinal or pleural fluid, urine, faeces, lymph nodes, etc., stained by the Ziehl-Neelsen method.

For concentration of the organisms, the sputum is subjected to enrichment methods:

(a) homogenization (an equal volume of 1 per cent NaOH solution is added to the sputum, the flask is tightly stoppered and shaken for 5-15minutes until the sputum is dissolved completely; after centrifugation, the precipitate is neutralized by one or two drops of a 10 per cent hydrochloric acid solution and smears are prepared);

(b) flotation (the homogenized sputum is transferred into a flask which has a rubber stopper and heated in a water bath at 55°C for 30minutes, after which it is diluted with distilled water, and 1 or 2 ml of xylol, benzine or gasoline are added; the mixture is shaken for 10minutes and after it has been left to settle for 30 minutes, smears are made from the resulting cream-like layer).

There are other methods of sputum preparation which facilitate the demonstration of mycobacteria.

Good results are obtained by employing luminescent microscopy with auramine and examining the specimens under the phase-contrast microscope.

2. Isolation of the pure culture. The prepared sputum, pus, suspensions of parenchymatous tissues, and other material are inoculated into nutrient media.

Pryce's microculture method is the most effective. The material under test is spread thickly on a slide, dried, and treated with sulphuric acid which is then washed off with a sterile sodium chloride solution. The preparations are then put into flasks containing citrated blood and placed into a thermostat for a period of 2-3 days, or a maximum of 7-10days. The preparations may be stained after 48 hours' incubation. Virulent mycobacteria produce convoluted strands in the microcultures, while the non-virulent strains form amorphous clusters.

The virulent and non-virulent M. tuberculosis strains are differentiated by their growth on butyrate albumin agar (Middlebrook-Dubos test). The virulent strains grow in the form of plaits, and the non-virulent strains form irregular clusters. The above authors suggested the differentiation of the virulent and non-virulent strains by staining the smears with neutral red which has an affinity for virulent mycobacteria and stains them purple-pink (non-virulent strains are stained yellow).

3. Biological method. Inoculation of guinea pigs produces an infiltrate at the site of injection of the material, lymph node enlargement, and generalized tuberculosis. The animals die 1-1.5 months after inoculation. Post-mortem examination reveals the presence of numerous tubercles in the internal organs. Specimens are obtained from lymph nodes by puncture 5-10 days after inoculation and examined for the presence of tubercle bacilli. The tuberculin test is carried out 3-4 weeks after infection. The atypical strains and L-forms are non-pathogenic for guinea pigs.

4. Complement-fixation reaction (positive in 80 per cent of cases with chronic pulmonary tuberculosis, in 20-25 per cent of patients with skin tuberculosis, and in 5-10 per cent of healthy people).

5. Indirect haemagglutination reaction (Middlebrook-Dubos test).Sheep erythrocytes, on which polysaccharides of M. tuberculosis or tuberculin are adsorbed, are agglutinated in serum of tuberculosis patients.

6. Tuberculin (allergic) tests are used for detecting infection of children with M. tuberculosis and for diagnosis of tuberculosis.

Treatment is accomplished with antibacterial preparations. They include derivatives of isonicotinic acid hydrazide (tubazide, phthivazide, etc.), streptomycin, and PAS — preparations of the first series. Preparations of the second series (cycloserine, kanamycin, biomycin, etc) are used to enhance the therapeutic effect. The isolated M. tuberculosis are tested for sensitivity to drugs which are added to fluid or solid media indifferent concentrations. Surgical and climatic (health resort) treatment is also beneficial in certain cases. The complex of therapeutic measures for body desensitization includes the use of tuberculin. It restores body reactivity. Combined treatment with preparations of the first and second series is recommended in chronic forms of tuberculosis.

At present, in certain cases patients are given prednison together with chemotherapeutic agents and antibiotics. Tuberculin therapy is applied in incipient forms of primary tuberculosis.

Control. The incidence rate of tuberculosis in the United States declined an average of 5.6% a year between 1953 and 1985. However, since 1985, it has been increasing at 4% to 5% per year, largely because of the reactivation of latent tuberculosis in AIDS patients, as well as new infections in both AIDS patients and, to a lesser extent, in the homeless.

The control of tuberculosis in a population requires the location and treatment of infected persons who spread tubercle bacilli by way of pulmonary secretions. However, even though there arc annually over 26,000 new cases and 3000 deaths reported in the United States, tuberculosis is usually a slow, chronic disease, and it is exceedingly difficult to find infected persons until they have experienced months or years of active infection. For early detection, therefore, one must rely on the tuberculin skin test, and a positive reaction is interpreted as denoting an infected person, whether or not the disease is quiescent or active. For this reason, control relies heavily on preventive therapy, and the Tuberculosis Advisory Committee to the Centres for Disease Control has recommended that the following persons be considered potential candidates for active disease (in the order listed) and that they be treated with daily oral INH for 1 year:

1. Household members and other close associates of persons with recently diagnosed tuberculosis.

2. Positive tuberculin reactors with findings on a chest roentgenogram consistent with nonprogressive tuberculosis, even in the absence of bacteriologic findings.

3. All persons who have converted from a tuberculinnegative to a tuberculin-positive response within the last 2 years.

4. Positive tuberculin reactors undergoing prolonged therapy with adrenocorticoids, receiving immunosuppressive therapy, having leukaemia or Hodgkin's disease, having diabetes mellitus, having silicosis, or who have had a gastrectomy.

5. All persons younger than 35 years of age who are positive tuberculin skin reactors. INH therapy is not recommended for positive tuberculin reactors 35 years of age or older, because prolonged treatment with INH causes occasional progressive liver disease; although the risk is low for persons younger than 35 years of age, the incidence rate increases to 1.2% of persons between 35 and 49, and to 2.3% for those older than 50 years.

Some individuals older than 55 years of age may not respond to tuberculin even though they were at one time tuberculin positive. Such persons, however, may experience a booster effect from the initial testing and become tuberculin positive to a subsequent test given a year or more later, indicating a conversion resulting from an infection with M tuberculosis. Such an interpretation can be avoided if negative reactors are given a repeat test 1 week or 10 days after the first test. Positive reactions to the second test would then be attributed to a booster effect rather than to a new infection.

Prophylaxis is insured by early diagnosis, timely detection of patients with atypical forms of the disease, routine check up of patients and recovered patients, disinfection of milk and meat derived from sick animals, and other measures.

Active immunization of human beings is of great importance in the control of tuberculosis. It lowers significantly the incidence of the disease and the death rate, gives protection against the development of severe cases, and lowers the body sensitivity to the effect of tubercle mycobacteria and to the products of their disintegration. Active immunity makes the body capable of fixing and rendering harmless the causative agent, stimulates biochemical activity of tissues and intensifies the production of antibacterial substances. Immunization produces a certain type of infectious immunity.

Tuberculinum Mantoux test

For intracutaneous immunization and revaccination special dry BCG vaccine is produced. It is given in a single injection to newborn infants. Revaccination is carried out at the age of 7, 12, 17, 23, and 27-30 years.

Postvaccinal immunity is produced within 3 or 4 weeks and remains for 1-1.5 to 15 years.

To prevent tuberculosis among carriers or those who had recovered from the disease, preventive chemotherapy with Isoniazid (isonicotinicacid hydrazide) is applied.

Living conditions play an important part in the incidence of tuberculosis. Deterioration of the conditions increases the incidence of the disease and death rate (wars, famine, unemployment, economical crises, and other disasters).

According to WHO, a total of more than 15 million tuberculosis patients have been recorded in the world. The incidence of tuberculosis is very high in Latin America, India, and Africa.

Mycobacterium bovis. Mycobacterium bovis is closely related to M tuberculosis in growth characteristics, chemical composition, and potential for virulence. Because it is normally a pathogen of cattle, human infections ordinarily result from ingestion of contaminated milk. The organisms do not usually infect the lungs, but rather produce lesions primarily in the bone marrow of the hip, knee, and vertebrae, and in the cervical lymph nodes. However, if inhaled, M bovis produces a pulmonary disease indistinguishable from that of M tuberculosis.

Bovine tuberculosis has been essentially eradicated in many countries—including the United States—by a strict program for destruction of tuberculin-positive cattle and by the widespread use of pasteurized milk. However, it is still an occupational hazard for zoo workers, as evidenced by the observation that 7 of 24 such workers were infected while cleaning the cage of a white rhinoceros.

Mycobacterium ulcerans. An unusual organism, Mycobacterium ulcerans seems to be closely related to M. tuberculosis, but it is unable to grow above 33°C. As a result, it cannot cause a systemic infection, but it is the etiologic agent for a rare skin infection seen primarily in Australia, Africa, and Mexico.

The organism enters the skin through puncture wounds where it causes a necrotizing ulcer, sometimes referred to as a Buruli ulcer. Surprisingly, this infection induces neither fever nor a regional lymphadenopathy. Moreover, unlike other mycobacterial infections, M ulcerans is only rarely found inside macrophages. An explanation for these observations became available when it was shown that culture nitrates of M ulcerans suppressed T-cell proliferation and phagocytosis by murine macrophages. The mechanism of tissue destruction is unknown, but unlike other mycobacterial infections, it is not because of the host's immune response. Treatment frequently requires surgical intervention and skin grafting.

Atypical Mycobacteria

During the last several decades, it has become obvious that there is an extremely large group of mycobacteria that are apparently normal inhabitants of soil and water. In the United States, such organisms arc found predominantly in the South, where, as judged by specific tuberculin reactions (using tuberculin prepared from these organisms), between one third and one half of the population has been infected with them. The pulmonary disease in diagnosed cases usually is milder than that caused by M. tuberculosis and, strangely, does not seem to be communicable from person to person.

This overall group of organisms has had several names, such as the anonymous mycobacteria (because no one knew enough about them to name them) or the atypical mycobacteria (because, unlike M tuberculosis or M. bovis, they are completely avirulent for guinea pigs). The popular classification divides them into the following three groups: (1) photochromogens, which produce a yellow pigment only if grown in the light; (2) scotochromogens, which produce an orange pigment whether grown in the light or dark; and (3) nonchromogens, which do not produce pigment under any circumstances. All are acid-fast bacilli, but infection does not usually induce a strong skin reaction to the usual tuberculin prepared from M tuberculosis. However, tuberculin prepared from the atypical mycobacteria reacts intensely when injected into persons with the homologous infection. Such purified tuberculin is available and is designated as shown in Table 2. Infections caused by most of the atypical mycobacteria respond to treatment with rifampin in combination with streptomycin or cycloserine, although some skin infections may require years of therapy.

PHOTOCHROMOGENS. Mycobacterium kansasii is the most prevalent human pathogen in the photochromogen group. Antigenically, it is similar to M. tuberculosis, and PPD prepared from either organism shows considerable cross-reaction.

Table

Tuberculins Prepared From Various Species of Mycobacteria

Mycobacterium Species	Tuberculin Designation
M. avium	PPD A
M. intracellularis	PPD-B
M. fortuitum	PPD-F
M. scrofulaceum	PPD-G
M. kansasii	PPD-Y
M tuberculosis	PPD-S
M. marinum	PPD-platy
M. phlei	PPD-ph
M. smegmatis	PPD-sm

M. avium

Mycobacterium smegmatis

In the United States, infections by M kansasii occur most frequently in Texas and, to a lesser extent, in Chicago, California, Oklahoma, and North Carolina. The human disease is like that described for the tubercle bacillus and may occur as both pulmonary and extrapulmonary infections.

Mycobacterium marinum, another photochromogen, has been isolated from swimming pools and lakes. Infections occur at traumatized areas in the skin and are manifested by draining ulcers.

SCOTOCHROMOGENS. Mycobacterium scrofulaceum seems to be the most prevalent human pathogen of the scotochromogen group. The organism has been found worldwide, probably existing primarily as a soil saprophyte. Its most common clinical manifestation is a cervical adenitis. The fact that many such infections are asymptomatic or undiagnosed is confirmed by the observation that several large surveys show that about 50% of those tested gave a positive skin reaction to specific PPD prepared from M scrofulaceum (ie, PPD-G).

NONPHOTOCHROMOGENS-MAC COMPLEX. The organisms in the nonphotochrome group are heterogeneous, and their classification is still in a state of flux. The two major pathogens, M avium and Mycobacterium intracellularis, are so closely related that many refer to them as the M. avium–M. intracellularis complex (MAC).

The organisms are found worldwide and infect a variety of birds and animals. Both cause a pulmonary infection in humans similar to that caused by the tubercle bacillus, but such infections are seen most often in elderly persons with preexisting pulmonary disease.

The MAC has acquired a new significance in those individuals with AIDS in whom it is found to be the most common cause of a systemic bacterial infection. It usually is seen as a late opportunistic infection occurring after one or more episodes of Pneumocystis carinii infections. Such individuals often also experience an intestinal infection with these organisms.

Members of the MAC can be isolated from sputum, blood, and aspirates of bone marrow. Acid-fast stains of stools also may be valuable in making a diagnosis. Treatment is difficult because the MACs generally are resistant to the usual antituberculosis drugs. However, many physicians use a four- to six-drug regimen that includes INH, rifampin, ethambutol, and streptomycin. Experimentally, it has been reported that streptomycin that was encapsulated in liposomes was 50 to 100 times more effective in treating MAC infections in mice than was free streptomycin.

RAPIDLY GROWING MYCOBACTERIA. This group of mycobacteria has a generation time of less than 1 hour, and colonies become visible after 2 to 3 days of growth. The group includes nonpathogens such as Mycobacterium fhlei and Mycobacterium smegmatis, as well as several species that do cause human infections. Pathogens include Mycobacterium fortuitum, Mycobacterium chelonei, and Mycobacterium abscessus, but, because of uncertainty about their classification, these three organisms arc frequently grouped in an M. fortuitum complex.

Members of the M fortuitum complex are most frequently involved in wound infections, which may occur as skin abscesses or as deeper infections after surgery. One surprising postoperative wound infection caused by this group occurred when 24 patients became infected after open heart surgery. Cultures of equipment used in the operating room all gave negative results, and the source of these organisms remains unknown.

Leprosy

Causative Agent of Leprosy. The organism responsible for leprosy, Mycobacterium leprae, was discovered in 1874 by the Norwegian investigator G. Hansen. In 1901 V. Kedrowsky reported nonacid-fast forms of the organism and de-scribed their branching.

Morphology. M leprae have many properties in common with the tubercle bacilli. They are straight or slightly curved bacilli, and club-shaped swellings and granular forms sometimes occur. The organisms are 1-8 mcm in length and 0.3-0.5 mcm in breadth. They usually occur in groups resembling packets of cigars or clusters. They decolour more easily than M. tuberculosis. M. leprae is non-motile, produces neither spores nor capsules, and is Gram-positive.

The organisms are pleomorphous. Among the more typical forms long, short, and thin cells as well as larger cells which are swollen, curved, branched, segmented, or degenerate (splitting up into granules)may occur.

M. leprae are similar to M. tuberculosis in chemical composition. Their lipid content ranges from 9.7 to 18.6 per cent. Besides mycolic acid, they contain laeprosinic oxy acid, free fatty acids, wax (leprozine),alcohols, and polysaccharides.

Cultivation. Attempts to cultivate M. leprae on nutrient media employed for growth of M. tuberculosis have been unsuccessful. M. leprae found in the leprous tissues of humans are injected into the leg of mice where they reproduce in 20 to 30 days. In 1971 British scientists were successful in elaborating a quite satisfactory method for cultivating M. leprae in the body of armadillo. After infection with pathological material taken from humans suffering from leprosy, a copious number of typical granulomas develop in the animals. The body temperature of armadillos is rather low (30-35 C), at this temperature cell immunity against M. leprae is suppressed.

Experiments in which pieces of leproma enclosed in colloidal sacs were introduced into the peritoneal cavity of animals demonstrated the existence of a great variety of leprosy mycobacteria (nonacid-fast, capsulated, granular, coccal, spore-like, thread-like, L-forms and rod-like which resemble fungal mycelium.

Fermentative properties have been insufficiently studied. This research has been handicapped by failure to solve the problem of cultivation of M. leprae on nutrient media.

Toxin production. The organisms have not been shown to produce a toxin. They evidently produce allergic substances. It is difficult to study this problem because no experimental animal sensitive to M. leprae has been found over a period of more than 100 years.

Antigenic structure and classification have not been worked out.

Resistance. M. leprae are extremely resistant, and survive in human corpses for several years. Although the organisms retain their morphological and staining properties outside the human body for a long period of time, they quickly lose their viability.

Pathogenicity for animals. Leprosy-like diseases are known to occur among rats, buffaloes, and certain species of birds, but they differ essentially from human leprosy. M. leprae is pathogenic only for man. Leprosy in rats caused by M. lepraemurium has been studied quite thoroughly (V. Stefansky, 1903; Marcus and Sorel, 1912). The disease in rats takes a chronic course with involvement of the lymph nodes, skin, and internal organs, the formation of infiltrates and ulcerations, and loss of hair. Antituberculosis drugs proved to be most effective in the treatment of rat leprosy, on the basis of which it can be assumed that M. leprae is closer genetically to the causative agents of tuberculosis and paratuberculosis. The leprosy organisms are pathogenic for armadillos, in which typical granulomatous lesions are reproduced.

Pathogenesis and disease in man. Leprosy was well known in Egypt 3000-4000 years B.C. In the Middle Ages and during the Crusades, leprosy spread as epidemics. This period was characterized by continuous wars which caused bad sanitary conditions. There were 2000 lepra colonies in France in 1429.

Leprosy disappeared from Europe at the end of the 17th century. In France all lepra colonies were closed on August 24, 1693. A new in-crease in the disease incidence occurred from 1867, followed by a marked decline at the beginning of the 20th century. However, disease prevalence is still high.

The source of infection is a sick person. The causative agent is transmitted by the air-droplet route through the nasopharynx and injured skin. The infection may also be spread by various objects. However, intimate and prolonged contact between healthy individuals and leprosy patients is the main mode of infection.

After entering the body through the skin and mucous membranes, M. leprae organisms penetrate into the nerve endings, lymphatic and blood vessels, and disseminate gradually without causing any changes at the site of entry. In the presence of high body resistance, the majority of M. leprae perish. In some cases infection leads to the development of la-tent forms of leprosy. The duration of such latent forms depends on body resistance, and may persist for a lifetime and, as a rule, terminates in the death of the causative agent. The latent form may change to the active form with development of the disease, if living and working conditions become unfavourable. The incubation period may last for years, e.g. from a period of 3-5 to 20-35 years. The disease becomes chronic.

Three types of leprosy are distinguished on the basis of clinical manifestations: lepromatous, tuberculoid, and undifferentiated.

1. The lepromatous type is characterized by minimum body resistance to the presence, multiplication, and spread of the causative agent. M. leprae are constantly present at the sites of the lesions. The lepromin test in negative.

2. The tuberculoid type is distinguished by high body resistance to the multiplication and spread of M. leprae. Either no organisms are found at the site of the lesion, or only a small number of them may be present during the reactive state. The allergic test is usually positive.

3. The undifferentiated type (non-specific group) is characterized by varying body resistance, but tends to be resistant. Microscopic examination does not always reveal the presence of M. leprae. Allergic tests are negative or yield a slightly positive reaction.

Immunity. Little is known about immunity in connection with leprosy. Patients' blood contains complement-fixing substances. Phagocytosis does not play any significant role in leprosy. An allergic condition develops during the course of the disease. The mechanism of immunity in leprosy is similar to that in tuberculosis.

In individuals with high body resistance, the organisms are phagocytosed by histiocytes in which they are destroyed quite rapidly. In such cases leprosy assumes a benign tuberculoid type.

In individuals with low resistance, M. leprae multiply in great numbers even within the phagocytes (incomplete phagocytosis), and the organisms disseminate throughout the body. A severe lepromatous type of the disease develops in such individuals.

Resistance may vary from high to low in undifferentiated types of leprosy. Relatively benign lesions persist for years, but if body resistance lowers the disease assumes a lepromatous form with large numbers of mycobacteria present in the tissues and organs. The clinical picture changes to the tuberculoid type when immunity intensifies.

Immunity in leprosy is associated with the general condition of the host body. In the majority of cases the disease occurs among the poor who have a low standard of culture. Children are most susceptible to the disease. In 5 per cent of cases the disease is acquired through contact with sick parents.

Laboratory diagnosis. Specimens for examination are obtained from nasal mucosa scrapings (on both sides), skin lepromas, sputum, and ulcer excretions. Blood is examined during the fever period. Microscopic examination is the principal method of leprosy diagnosis. Smears are stained with the Ziehl-Neelsen stain.

Biopsy of leprotic lesions and puncture of lymph nodes are employed in some cases. M. leprae can be seen as clusters resembling packets of cigars; in preparations from nasal mucus they appear as red balls.

Skin biopsy

Leprosy is differentiated from tuberculosis by inoculating guinea pigs with a suspension of the pathological material in an 0.85 per cent solution of common salt. If tubercle bacilli are present, the animals contract the disease and die. Guinea pigs are unsusceptible to M. leprae.

The allergic Mitsuda test is considered positive when an erythema and a small papule (early reaction) are produced at the site of an 0.1 ml lepromin (a suspension prepared from a leproma after trituration and prolonged boiling) injection in 48-72 hours: this reaction either disappears completely at the end of the first week or changes to the late reaction. The latter is manifested by a nodule which appears at the site of injection in 10-14 days, and grows to a diameter of 1-2 cm with necrosis in the centre. This test is of no diagnostic value and is used to distinguish the clinical type of leprosy.

The complement-fixation reaction and the Middlebrook-Dubos haemagglutination test are employed for leprosy diagnosis.

Treatment. Leprosy is treated with sulphone drugs (dapson), diaminodiphenylsulphone and its derivatives (sulphetrone, promin, diazone, and promacetin). Carbonylid (Su 1906) is less toxic. In addition to this, conteben, desensitizing agents, and corticosteroid preparations (cortisone, prednisolone, etc.) are employed. Streptomycin and dehydrostreplomycin combined with PAS and isoniazid, and tybon, phthivazide, and biostimulators yield good effects. For a long period of time, leprosy patients were treated with chaulmoogra oil which was given per os. At present it is administered intramuscularly or intracutaneously. Chaulmoogra preparations promote the resolution of lesions and, sometimes, eliminate the visible leprosy manifestations. However, they give no protection from relapses and have no specific effect.

Prophylaxis. Leprosy patients which discharge the organisms are isolated in lepra colonies till clinical recovery. Patients who do not discharge leprosy organisms receive out-patient treatment. Routine epidemiologic control of endemic foci is carried out. If there is a leprosy patient in a family, all other members are subjected to a special medical examination at least once a year. Children born of mothers with leprosy should be taken away from them and fed artificially. Healthy children of leprosy parents are placed in children's homes or are looked after by relatives and are examined at least twice a year.

In the USSR, leprosy has become a sporadic disease. Only isolated cases are registered in some regions of the country.

According to WHO, over 10 million persons suffering from leprosy are registered throughout the world (6475000 in Asia, 3868000 in Africa, 385000 in America, 52000 in Europe, and 33000 in Oceania).The high prevalence of leprosy makes research into methods of its specific prophylaxis-necessary.

Additional material for diagnosis

TUBERCULOSIS. Causative organisms of tuberculosis in humans and animals are Mycobacterium tuberculosis, Mycobacterium bovis, and M. africanum.

Laboratory diagnosis of tuberculosis consists of bacterioscopic, bacteriological, biological, serological, and allergological examinations.

The material to be examined includes, depending on the localization of the process: sputum, pus, cerebrospinal fluid, faeces, and lavage waters from the stomach and bronchi. The obtained samples are collected in sterile vessels (sputum into jars, cerebrospinal fluid and other material into test tubes).

Bacterioscopic examination. Pour a sputum sample into a Petri dish, put it on the black surface of the table, pick up lumps of pus, place them onto a glass slide, and grind between two slides. A specimen of cerebrospinal fluid is kept in the cold. Examination of this specimen 18-24 hrs after the collection reveals a delicate film of fibrin, which contains M. tuberculosis and cell elements. Spread this film carefully on a glass slide. Centrifuge urine and make smears from the pellet.

Smears are stained with the Ziehl-Neelsen method. M. tuberculosis stained bright red (ruby) appear as either thin, long, slightly curved or short straight rods; occasionally, they may be characterized by granularity. M. tuberculosis are arranged singly or in irregular groups. In staining the urine sediment destaining should be , made not only with sulphuric acid but also with alcohol since the urine may harbour non-pathogenic acid-fast mycobacteria of smegma (Mycobacterium smegmatis) which, unlike M. tuberculosis, are de-stained by alcohol. If mycobacteria evade detection because of their small numbers present in ordinary smears, this difficulty is obviated with the employment of such enrichment methods as homogenization and floatation.

Homogenization technique. Pour a 24-hour sample of sputum into a vessel or a jar, add an equal volume of 1 per cent water solution of sodium hydroxide, stopper the vessel tightly with a rubber plug, and shake vigorously until the mixture is completely homogenized (for 10-15 min). Centrifuge sputum specimens which have lost their viscosity, pour out the liquid, and neutralize the residue by adding 2-3 drops of 10 per cent hydrochloric or 30 per cent acetic acid. Make smears from the sediment and stain them with the Ziehl-Neelsen method.

Floatation method. Using the above mentioned procedure, homogenize a 24- or 48-hour sample of sputum. To eliminate any possibility of mucous lumps remaining in the material, the jar with the homogenized sputum should be placed into a water bath at 55 °C for 30 min. Then, add 1-2 ml of xylol (benzene, petrol, petroleum ether, etc.), shake for 10 min, and let the mixture settle for 20 min at room temperature. Xylol droplets with adsorbed microorganisms float up, forming a cream-like layer which is pipetted onto a glass slide that is mounted on a glass plate heated to 60 °C in a water bath.

A dried smear is covered with a new portion of the cream-like layer. the procedure being repeated until the entire floatation layer is transferred onto the glass slide. The preparation is fixed and stained by the Ziehl-Neelsen technique.

Lavage waters from the stomach are also studied by the floatation technique. In the morning make a fasting patient drink 200 ml of distilled water and immediately withdraw it from the stomach by means of a thick probe into a sterile glass, pour the obtained material into a 250-300-ml flask, and add 2-3 ml of 0.5 per cent solution of sodium hydroxide. Then, shake the mixture for 5 min, add 1-2 ml of xylol or petrol, and shake the mixture once again for 5-10 min. After that allow the flask to stand at room temperature for 30 min. A cream-like layer formed in the shape of a ring at the neck of the flask is removed and treated in a manner similar to that employed in sputum examination. The result is considered positive if microscopy reveals even individual mycobacteria. Positive results obtained in repeated examinations are more reliable.

Lavage waters from the bronchi are studied for M. tuberculosis in patients producing no sputum. In the morning spray 1 per cent solution of tetracaine hydrochloride (2 ml) onto the tongue, palatal arches, and throat of a fasting patient. In 2-3 min infuse into the larynx 1-2 ml of 2 per cent solution of tetracaine hydrochloride with the aid of a laryngeal syringe. In another 2-3 min place the patient on the side corresponding to the examined lung and, using a laryngeal syringe, slowly pour onto the middle of the tongue root 10-20 ml of isotonic sodium chloride solution heated to 37 °C. The fluid runs along the lateral wall of the pharynx into the larynx and then into the main bronchus. The entry of the solution into the bronchus is manifested by characteristic rales. Make the patient cough up the infused solution and mucus from the deep portions of the respiratory tract into a sterile glass. Thereafter, examination is performed in the same manner which is used in investigating lavage waters from the stomach.

In the rapid diagnosis of tuberculosis luminescent microscopy is utilized. The preparation is stained with auramine in a 1:1000 dilution and then destained with hydrochloric alcohol and counter-stained with acid fuchsine which "extinguishes" fluorescence of elements of tissues and mucus. M. tuberculosis fluoresce with a bright golden-green light against a dark background.

Bacteriological examination is more effective than bacterioscopic one and makes it possible to reveal in the examined material 20-100 and over mycobacteria per ml and also to determine their resistance to drugs, their virulence, type, etc.

Add a double volume of 6 per cent sulphuric acid killing acid-sensitive microorganisms to the examined material in a sterile test tube and shake the tube for 10 min. Then, centrifuge the resultant mixture, pour off the fluid, neutralize the pellet by adding 1-2 drops of 3 per cent sodium hydroxide or by washing it off several times with isotonic sodium chloride solution, and streak on the appropriate medium. Faeces are treated with 4 per cent solution of sodium hydroxide, the mixture is placed in an incubator for 3 hrs, centrifuged, and the residue is neutralized by 8 per cent hydrochloric acid, after which inoculation on special media is carried out.

International Loewenstein-Jensen medium is recommended by WHO as the-standard medium for the primary growth of M. tuberculosis and for determining their resistance to antibacterial drugs. Dissolve 3.6 g of asparagin, 2.4 g of potassium hydrophosphate, 0.24 g of magnesium sulphate, O.B g of magnesium citrate, and 0.4 g of potato starch and malachite green in 600 ml of distilled water containing 12 ml of glycerol. Sterilize the mixture obtained for 15 min at 120 °C. Then pour it into 100 ml of homogeneous suspension from fresh eggs, mix thoroughly, filter through a cotton-gauze filter, decant into test tubes, and obtain a slant medium by coagulation at 85 °C for 45 min.

Petragnani's medium. To 150 ml of whole milk, add (with constant stirring) 6 g of potato starch, 1 g of peptone and one finely-chopped egg-sized potato. Heat tile mixture until paste is formed, cool to 50 "C, and add four chicken eggs. and one yolk. Mix all the components, pour in 12 ml of glycerol, and 10 ml of 2 per cent of malachite green solution, filter the mixture through a gauze filter dispense into test tubes, and coagulate in a slant position at 85 °C for 2.5 hrs.

Glycerol potato as proposed by Pavlovsky. Peel a potato and immerse it in 1 percent solution of mercuric chloride for 30 min, wash for 12 hrs in running water, and cut out cylinders by making diagonal cuts. Slanted potato is placed into a Roux test tube- Pour 1 ml of 5 per cent glycerol solution onto the bottom and sterilize the test tube.

Sauton's synthetic medium. In 200 ml of distilled water dissolve (while constantly heating) 4 g of asparagin, 2 g of citric acid, 0.5 g of potassium dihydrophosphate, and 60 g of glycerol. Filter the obtained mixture, supplement it with 800 ml of distilled water, add ammonium to bring pH to 7.2, decant into flasks, and sterilize for 20 min at 115 °C. To protect the mixture from drying, the plugs. of test tubes with nutrient media are sealed with paraffin.

The composition of the Finn-2 medium is similar to that of Loewenstein-Jensen's medium, but asparagin is replaced in it with sodium glutamate.

Samples of the cerebrospinal fluid, exudate, pus, and blood are pipetted onto a nutrient medium without any preliminary treatment and thoroughly rubbed into it with the aid of a loop, spreading them over the entire surface of the-medium. Cotton plugs are sealed with paraffin (to prevent drying), the inoculated cultures are placed into a 37 °C incubator, and kept there for 6-8 weeks. An intensive growth of M. tuberculosis is observed on the 15th-25th day on the Loewenstein-Jensen medium and on the 21th-35th day on Petragnani's medium. Colonies of M. tuberculosis are wrinkled, dry, irregular, and protrude above the surface. If no growth is observed within 6 weeks, make a scraping from the medium surface and examine it microscopically for the presence of acid-fast bacteria.

To improve growth of M. tuberculosis, it is recommended that the-material examined be treated with detergents possessing a bactericidal action (sodium laurilsulphate, rodolan, teapol, laurosept, cetavlon, etc.) or their combination with sodium hydroxide. These-methods make it possible to achieve a better homogenization of the material, to reduce the time during which colonies form on nutrient media, and to do away with the stages of centrifugation, resuspension, and neutralization.

If the results are negative, the study is repeated several times (at least 5), and the period of culture inoculation is lengthened.

Rapid methods of the bacterial diagnosis of tuberculosis. The method of microcultures (Price's method). Samples of sputum, pus. urine residue, and lavage waters are spread in a thick layer on several sterile glass slides. Take a dried preparation with a sterile forceps and immerse it for 5 min in 6 per cent sulphuric acid, and then in a sterile isotonic sodium chloride solution to remove acid. After that place the preparations into vials with citrate blood (add 2 ml of 5 per cent sodium citrate to 10 ml of rabbit or sheep blood, dilute the contents in a 1:4 ratio "with distilled water, and pour the mixture into test tubes). Put the inoculated cultures into an incubator. In 48-72 hrs the preparation is retrieved, fixed, and then stained with the ZiehI-Neelsen method. Microcolonies in the preparation appear as plaits which form under the impact of the lipid fraction of mycobacteria (the cord factor); the maximal growth is observed on the 7th-10th day.

In-depth growth in haemolysed blood (Shkolnikova's method). Into tubes with citrate blood introduce material treated with sulphuric acid and washed with isotonic sodium chloride solution. After 6-8 days of incubation, centrifuge the medium and make smears from the pellet.

Resistance of the M. tuberculosis to drugs is determined by a serial dilution technique. For inoculation, one may use both initial material containing no less than 5 mycobacteria per a microscopic field (direct method) and the culture isolated from it (indirect method). WHO recommends that the resistance of mycobacteria on Loewenstein-Jensen's medium should be determined by adding into it, prior to coagulation, various doses of drugs.

Resistance of mycobacteria can also be determined in liquid media (with an addition of drugs in corresponding concentrations) in which M. tuberculosis grow in a way similar to that described by Price and Shkolnikova. At the present time the biological examination fails to find wide employment in laboratory diagnosis since experimental animals are insensitive to the strains of mycobacteria resistant to tubazid, phthivazid, isoniazid, and other anti-tuberculosis drugs.

Biological tests are utilized for determining the virulence of isolated M. tuberculosis which are inoculated subcutaneously into guinea pigs with negative Mantoux's test. Two-three weeks after inoculation one should weigh the infected guinea pig, measure its regional lymph nodes, and make Mantoux's test which is then repeated in 6 weeks. If the results are negative, sacrifice the animal

4 months after inoculation, examine histologically the internal organs (liver, spleen, lungs, lymph nodes), and inoculate nutrient media. The virulence of the strain is determined by the number of specific changes in organs (development of tubercles), changes in the expected life span of the animal, weight loss, etc.

The allergy cutaneous test (Mantoux's intracutaneous test with tuberculin) is largely employed for the determination of contamination of individuals with M. tuberculosis. The results are read in 24-48-72 hrs.

If the diameter of the infiltrate at the site of tuberculin administration does not exceed 1 mm, the test is considered negative. If the diameter of the infiltrate is 2-4 mm, the test is doubtful, if over

5 mm, it is positive. The tuberculin reaction may be attended with the development of lymphangitis, regional lymphadenitis, and the appearance of vesicles or necrosis. A positive allergic response to tuberculin administration indicates the presence of M. tuberculosis in the body. A negative reaction in adults points to the absence of immunity to tuberculosis. As a diagnostic test, this technique is helpful in recognizing tuberculosis in children, identifying populations requiring revaccination against tuberculosis, and assessing the prevalence of tuberculosis as an epidemiological indicator. Pirquet's cutaneous test that was extensively used in the past has become outdated and is no longer utilized.

Serological diagnosis. The complement-fixation reaction is rarely employed in the diagnosis of tuberculosis. The IHA reaction, as proposed by Middlebrook and Dubos is used more extensively. Sensitized red blood cells (tannin-treated sheep or human 0-group erythrocytes) are utilized as an antigen. They are mixed with an extract of M. tuberculosis or purified tuberculin (0.5 ml of erythrocyte sediment and 10 ml of the extract), incubated for 2 hrs at 37 °C, and washed off with centrifugation to remove excessive antigen. To run the test, the patient's serum is depleted by a suspension of erythrocytes that have not been treated with the antigen, which excludes the possibility of a non-specific reaction. The serum to be assayed is diluted, beginning with 1:2, 1:4, 1:8, etc. A positive reaction in a 1:8 dilution is definitely diagnostic. Positive results are recorded in 70-90 per cent of tuberculosis patients.

To reveal antibodies, the agglutination reaction may be performed. The patient's blood serum is diluted with isotonic sodium chloride solution in dilutions varying from 1:40 to 1:640. As an antigen, use non-acid fast cultures of M. tuberculosis obtained as a result of penicillin action and serologically similar to native M. tuberculosis. This reaction is extremely sensitive. It should be remembered that even when the results of bacterioscopic and bacteriological studies are negative, the diagnosis of tuberculosis may be based on clinical and X-ray findings.

BURULI’S ULCER. The causative agent of the disease is Mycobacterium ulcerans. The material to be investigated includes pus or granulation tissue from the bottom of an ulcer or from cavities formed by the overhanging edges of the ulcer. The material should be taken with a curette rather than a tampon. Examination of biopsy tissue samples yields good results.

Bacterioscopic examination consists of preparation of smears from the obtained material and staining them by the Ziehl-Neelsen or Gram techniques. The Ziehl-Neelsen staining reveals red rods which are arranged singly, in pairs (parallel to each other), or in the form of beads. M. ulcerans are Gram-positive.

Bacteriological examination. To obtain a pure culture, the material to be studied is streaked on the Petragnani and Loewenstain-Jensen media and cultivated at 33 °C. Seven weeks later one can observe tiny, light-pink, flat or protruding colonies. A pure culture is identified by morphological, tinctorial, and cultural properties, as well as by the fermentative activity and antigenic structure.

No methods of the serological diagnosis of this illness have been developed.

Apart from clinical considerations, one should also inquire whether the patient has been to endemic areas {Uganda, Nigeria, Zaire, and other countries with a hot climate), which may warrant the necessity of conducting special examinations.

LEPROSY. The causative agent of leprosy (a chronic generalized infections disease characterized by involvement of the skin, mucosa. peripheral nervous system, and internal organs) is Mycobacterium leprae.

Bacterioscopic examination is the main method to diagnose leprosy. When the skin is affected, study a scraping from its indurated portions (after you have cut off the epidermis with a razor blade): when the lungs are affected, sputum is examined; in any other form study a scraping from the nasal mucosa. For this purpose introduce deep into the nose a metallic spoon and scrape the mucosa until drops of blood make their appearance. Smears are stained by the Ziehl-Neelsen method, yet, in view of low acid resistance of the leprosy causative agent, it is decolourized with 0.5 per cent solution of sulphuric acid. Semenovich-Martsinovsky staining is also used.

M. leprae are arranged inside the cells filling them. The cytoplasm and nucleus of these cells are pushed to the periphery. Involved tissues also contain a large number of mycobacteria located extra-cellularly. They are clustered as cigar packs, which allow? their differentiation from M. tuberculosis, the latter being similar to the causative agents of leprosy both morphologically and tinctorially. Streaking of leprosy material onto the nutrient media used for cultivating M. tuberculosis induces no growth.

Guinea pigs are resistant to the leprosy causative agents. An experimental leprosy infection with the formation of typical multiple nodules (lepromas) in tissues and organs has been successfully induced in armadillos. Functional tests with various pharmacological drugs make it possible to reveal an early involvement of the peripheral nervous system characteristic of leprosy.

Most commonly used for this purpose is the test with histamine (1:10000), morphine (1 per cent), and ethylmorphine hydrochloride (2 per cent). Place a drop of one of these solutions onto damaged and intact portions of the skin. With a sharp needle make a puncture to such a depth that the point of the needle reaches the live part of the epidermis (no blood should appear). The solution is removed with cotton wool. In 0.5-1 min an erythema develops on the intact skin, which transforms within 1-2 min into a blister or an oedematous papule whose development is attended by itching. These changes are either absent or less pronounced on the affected skin.

The "inflammation" test consists of intravenous administration of nicotinic acid (3-7 ml of 1 per cent solution). This is followed by the formation of blisters and pronounced hyperaemia at the site of leprosy spots. A great diagnostic importance is ascribed to Minor's test: apply 2-5 per cent alcohol solution of iodine to the suspicious site of skin and after it has dried up powder this area with a thin layer of starch;

then make the patient perspire profusely by using a dry air bath, profuse hot drinking, etc. There is no perspiration at the damaged sites and hence no blue staining as a result of iodine-starch interaction occurs in such spots.

The allergy test with lepromin (Mitsudas reaction) is employed for determining the patient's reactivity. A suspension (0.1 ml) of M. leprae taken from a leproma and killed by boiling is injected intracutanecusly into the forearm. Three weeks after the inoculation both healthy subjects and patients with tuberculoid leprosy will develop an inflammatory infiltrate at this spot, which may turn ulcerous.

Clostridia Responsible for Anaerobic Infections. Anaerobic infections (gas gangrene) are polybacterial. They are caused by several species of clostridia in association with various aerobic micro-organisms (pathogenic staphylococci and streptococci).

The organisms responsible for anaerobic infections are: (1) Cl. perfringens, (2) Cl. novyi, (3) Cl. septicum, (4) C. histolyticum, and (5) Cl. sordellii. Cl. chauvoei, Cl. fallax, and Cl. sporogenes are pathogenic for animals. Cl. aerofoetidum and Cl. tertium are non-pathogenic organisms which have significance in the pathogenesis of anaerobic infections only in association with pathogenic bacteria.

Cl. Perfringens Cl. novyi

Cl. Septicum C. histolyticum,

Anaerobic infections may be caused by any one of the first four species mentioned above but usually several members of a parasitocoenosis acting in a particular combination are responsible for them. The less pathogenic and non-pathogenic species cannot be responsible for anaerobic infections by themselves, but they cause tissue destruction, lower the oxidation-reduction potential, and thus create favourable conditions for the growth of pathogenic species.

Clostridium perfringens. The causative agent was discovered in 1892 by W. Welch and G. Nut-tall. This organism occurs as a commensal in the intestine of man and animals. Outside of the host's body it survives for years in the form of spores. It is almost always found in the soil. The organism was isolated from 70-80 per cent of anaerobic infection cases during World War I, and from 91-100 per cent of cases during World War II.

Morphology. Cl. perfringens is a thick pleomorphous non-motile rod with rounded ends 3-9 mcm in length and 0.9-1.3 mcm in breadth (Fig. ). In the body of man and animals it is capsulated, and in nature it produces an oval, central or subterminal spore which is wider than the vegetative cell. Cl. perfringens stains readily with all aniline dyes and is Gram-positive but in old cultures it is usually Gram-negative.

Figure. Pure culture and colonies of Clostridium perfringens

Electron microscopy demonstrates a homogeneous cell wall with no clearly demarcated layers. The cytoplasmatic membrane consists of one layer, the cytoplasm is granular and contains ribosomes and polyribosomes. The nucleoid is in the centre of the cell. Spore formation begin safter 3 to 3.5 hours of growth, the spores are enclosed by sporangia. The G+C content in DNA ranges from 24 to 27 per cent.

Cultivation. Cl. perfringens is less anaerobic than the other causative agents of anaerobic infections. It grows on all nutrient media which are used for cultivation of anaerobes. The optimum temperature for growth is 35-37 °0 (it does not grow below 16 and above 50°C), and optimal pH of medium is 6.0-8.0. A uniform turbidity and large volumes of gas are produced in cultures grown on Kitt-Tarozzi medium.

Brain medium is not blackened (Tabl. ). The colonies resemble discs or lentils deep in agar stabcultures (see Fig. 1). On blood agar containing glucose smooth disc-like grey colonies are formed, with smooth edges and a raised centre.

Many strains of Cl. perfringens lose their anaerobic properties on exposure to antibiotics, bacteriophage, and X-rays and may be cultivated under aerobic conditions. Catalase and peroxidase, enzymes typically present in aerobic organisms, were revealed in the variants thus obtained. The aerobic variants are non-toxic and non-pathogenic for laboratory animals.

Fermentative properties. Cl. perfringens slowly liquefies gelatin, coagulated blood serum and egg albumen (Tabl. 1). The organism reduces nitrates to nitrites and normally no indole or only traces are produced. Volatile amines, aldehydes, ketones, and acetyl methyl carbinol, are produced. Milk is vigorously coagulated and a sponge-like clot is formed. In meat medium the organism yields butyric and acetic acids and large quantities of gases (CO₂ H₂, H₂S, NH₃). It ferments glucose, levulose, galactose, maltose, saccharose, lactose, starch, and glycogen with acid and gas formation. Mannitol is not fermented.

Toxin production. The organism produces a toxin which has a complex chemical structure (lethal toxin, haemotoxin, neurotoxin, and necrotic toxin). The toxins and enzymes produced by the various species of the gas gangrene group are similar from one species to another. Actually, many of them have not been purified or characterized, and are grouped together under the general name lethal toxins. The products produced by C perfringens have received the most study: at least 12 different toxins and enzymes have been described and labeled with Greek letters (Table 2), but not all serologic strains of C perfringens produce all 12 products or even similar quantities of certain toxins and enzymes.

The most extensively studied toxin is the alpha-toxin, a phospholipase-C (lecithinase) that hydrolyzes the phospholipid, lecithin, to a diglyceride and a phosphorylcholine. Because lecithin is a component of cell membranes, its hydrolysis can result in cell destruction throughout the body. Lecithinase C acts as digestant enzyme in human intestine.Another toxin produced by this group is the m, toxin, a lethal hemolytic product characterized by its effect on the heart—more precisely, its cardiotoxic properties. C. perfringens type E is the only one of this group to produce the iota (i) toxin, which is believed to be responsible for an acute enterotoxemia in both domestic animals and humans. iToxin is a binary product in which two nonlinked proteins are required for activity. One molecule binds to a cell (iota–b), functioning as a receptor to transport the active toxin molecule (iota–a)across the membrane. Like botulism C2 toxin, i toxin will ADP–ribosylate poly L–arginine and skeletal muscle and nonmuscle actin, but its true substrate within the cell is unknown. Other toxic enzymes produced by the gas gangrene group include a collagenase that hydrolyzes the body's collagen; a hyaluronidase; a fibrinolysin, which breaks down blood clots; a DNase; and a neuraminidase, which can remove the neuraminic acid from a large number of glycoproteins. With such an array of toxic sub–stances, it is no wonder that gas gangrene was one of the major causes of death in the American Civil War, and, undoubtedly, in many other wars.

Lecithinase production

Due to such a complex of toxic substances and enzymes Cl. perfringens is capable of causing rapid and complete necrosis of muscular tissue. This process is the result of a combined effect of lecithinase, collagenase, and hyaluronidase on the muscles. Collagenase and hyaluronidase destroy the connective tissue of the muscles, and lecithinase C splits lecithin, a component in the muscle fibre membranes. Haemolysis in anaerobic infection is due to the effect of lecithinase on lecithin of the erythrocyte stroma. The animal dies from rapidly developing asphyxia which is the result of intensive erythrocyte destruction and disturbance of the nerve centres.

Table

Toxins and Toxigenic Types of Clostridium perfringens

Toxins		Bacterial Types
		A	B	C	D	E
a	Lecithinase	+++	+++	+++	+++	+++
b	Lethal, necrotizing		+++	+++	–	–
g	Lethal	–	++	++	–	–
d	Lethal, hemolytic	–	+	++	–	–
e	Lethal, necrotizing	–	+++	–	+++	–
h	Lethal	+	?	?	?	?
q	Lethal, hemolytic	+	++	+++	+++	+++
i	Lethal, necrotizing	–	–	–	–	+++
k	Collagenase	+	+	+++	++	+++
l	Proteinase	–	+	–	++	+++
m	Hyaluronidase	++	+	+	++	+
n	Deoxyribonuclease	++	+	++	++	++

Note: “+++” – most strains, “++” – some strains, “+” – a few strains, “–“ – not produced

In addition to battlefield casualties, automobile and farm equipment accidents also may cause traumatic wounds resulting in gas gangrene. Also, because C. perfringens can be part of the normal flora of the female genital tract, induced abortions may result in uterine gas gangrene.

Clostridia may also cause a diffuse spreading cellulitis accompanied by an overwhelming toxemia. Such infections probably originate from the large intestine, either from a bowel perforation or from a contaminated injection site. Gas may be produced, but the cellulitis differs from the classic gas gangrene in that muscle necrosis is not involved.

Antigenic structure and classification. Six variants of Cl. perfringens are distinguished: A, B, C, D, E, and F. These variants are differentiated by their serological properties and specific toxins.

Variant A is commonly found as a commensal in the human intestine, but it produces anaerobic infections when it penetrates into the body by the parenteral route. Variant B is responsible for dysentery in lambs and other animals. Variant C causes hemorrhagic enterotoxaemia in sheep, goats, sucking pigs, and calves. Variant D is the cause of infectious enterotoxaemia in man and animals, and variant E causes enterotoxaemia in lambs and calves. Variant F is responsible for human necrotic enteritis.

Resistance. The spores withstand boiling for period of 8 to 90minutes. The vegetative forms are most susceptible to hydrogen peroxide, silver ammonia, and phenol in concentrations commonly employed for disinfection.

Pathogenicity for animals. Among laboratory animals, guinea pigs, rabbits, pigeons, and mice are most susceptible to infection. Postmortem examination of infected animals reveals oedema and tissue necrosis with gas accumulation at the site of penetration of the organism. Most frequently clostridia are found in the blood.

Clostridium novyi. The organism was discovered by F. Novy in 1894. Its role in the aetiology of anaerobic infections was shown in 1915 by M. Weinbergand P. Seguin. It ranks second among the causative agents of anaerobic infections. Soil examination reveals the presence of the organism in 64per cent of the cases.

Morphology. Cl. novyi is a large pleomorphous rod with rounded ends, 4.7-22.5 mcm in length and 1.4-2.5 mcm in width, and occurs often in short chains (Fig.). The organism is motile, peritrichous, and may possess as many as 20 flagella. It forms oval, normally subterminal spores in the external environment. In the body of man and animals it is non-capsulated. The organism is Gram-positive. The G+C content in DNA amounts to 23 per cent.

Figure. Pure culture and deep colonies of Clostridium novyi

Cultivation. C/. novyi is the strictest of the anaerobes. Its optimal growth temperature is 37-45 C (growth temperature ranges from 16 to 50 C), and optimal pH of medium is 7.8. Growth on Kitt-Tarozzi medium is accompanied by gas accumulation, precipitation, and clearance of the medium. On sugar-blood agar the colonies are rough, raised in the centre, and have fringed edges surrounded by zones of haemolysis. In agar stab cultures the organisms produce flocculent colonies with a dense centre from which thin filaments grow outwards.

Fermentative properties. The organisms slowly liquefy and blacken gelatin. They coagulate milk, producing small flakes. Glucose, maltose, and glycerin are fermented with acid and gas formation. Acetic, butyric, and lactic acids as well as aldehydes and alcohols are evolved as a result of the breakdown of carbohydrates.

Toxin production. Cl. novyi A produces alpha, gamma, delta, and epsilon toxins; Cl. novyi B produces alpha, beta, zeta, and eta toxins. Cl.novyi C is marked by low toxigenicity. In cultures Cl. novyi liberates active haemolysin which possesses the properties of lecithinase.

Antigenic structure and classification. Cl. novyi is differentiated into four variants A, B, C and D. Variant A is responsible for anaerobic infections in man, and type B causes infectious hepatitis, known as the black disease of sheep. Variant C produces bacillary osteomyelitis in buffaloes, and variant D is responsible for haemoglobinuria in calves.

Resistance. Spores survive in nature for a period of 20-25 years with-out losing their virulence. Direct sunlight kills them in 24 hours, boiling destroys them in 10-15 minutes. Spores withstand exposure to a 3 percent formalin solution for 10 minutes. Coal-tar is an extremely active disinfectant.

Pathogenicity for animals. Cl. novyi causes necrotic hepatitis (black disease) in sheep. In association with non-pathogenic clostridia it produces bradsot (acute hemorrhagic inflammation of the mucous membranes of the true stomach and duodenum, attended with formation of gases in the alimentary canal and necrotic lesions in the liver) and haemoglobinuria in calves.

A subcutaneous injection of the culture into rabbits, white mice, guinea pigs, and pigeons results in a jelly-like oedema usually without the formation of gas bubbles. Postmortem examination displays slight changes in the muscles; the oedematous tissues are pallid or slightly hyperaemic.

Clostridium septicum. The organism was isolated from the blood of a cow in 1877 by L. Pasteur and J. Joubert. In 1881 R. Koch proved the organism to be responsible for malignant oedema. It is found in 8 per cent of examined soil specimens.

Morphology. The clostridia are pleomorphous and may be from3.1-14.1 mcm long and from 1.1-1.6 mcm thick; filamentous forms, measuring up to 50 mcm in length, also occur. The organisms are motile, peritrichous, and produce no capsules in the animal body. The spores are central or subterminal. The clostridia are Gram-positive but Gram-negative organisms occur in old cultures.

Cultivation. Cl. septicum are strict anaerobes. Their optimal growth temperature is 37-45° C, and they do not grow below 16° C. The pH of medium is 7.6. The organisms grow readily in meat-peptone broth and meat-peptone agar to which 5 per cent glucose has been added. On glucose-blood agar they produce a continuous thin film of intricately interwoven filaments lying against a background of haemolysed medium. In agar stab cultures the colonies resemble balls of wool. In broth a uniform turbidity is produced, and an abundant loose, whitish, and mucilaginous precipitate later develops.

Fermentative properties. Cl. septicum liquefies gelatin slowly, produces no indole, reduces nitrates to nitrites, and decomposes proteins, with hydrogen sulphide and ammonia formation. Force-meat is reddened but not digested; the culture evolving a rancid odour. Levulose, glucose, galactose, maltose, lactose, and salicin are fermented with acid and gas formation. Milk is coagulated- slowly.

Toxin production. Cl. septicum produces a lethal exotoxin, necrotic toxin, haemotoxin, hyaluronidase, deoxyribonuclease, and collagenase. The organism haemolyses human, horse, sheep, rabbit, and guinea pig erythrocytes.

Antigenic structure and classification. On the basis of the agglutination reaction, serovars of Cl. septicum can be distinguished, which produce identical toxins, the differential properties being associated with the structure of the H-antigen Cl. septicum possesses antigens common to Cl. chauvoei which is responsible for anaerobic infections in animals.

Resistance is similar to that of Cl novyi.

Pathogenicity for animals. Among domestic animals horses, sheep, pigs, and cattle may contract the disease. Infected guinea pigs die in18-48 hours. Postmortem examination reveals crepitant haemorrhagic oedema and congested internal organs. The affected muscles have a moist appearance and are light brown in colour. Long curved filaments which consist of clostridia are found in impression smears of microscopical sections of the liver.

Clostridium histolyticum. The organism was isolated in 1916 by M. Wemberg and P. Segum. It produces fibrinolysin, a proteolytic enzyme, which causes lysis of the tissues in the infected body. An intravenous injection of the exotoxin into an animal is followed shortly by death. The fact that the organisms are pathogenic for man has not met with general acceptance in the recent years The organism's responsibility for anaerobic infections during World War II was insignificant.

Pathogenesis and diseases in man. Anaerobic infections are characterized by a varied clinical picture, depending on a number of factors. These include the number of pathogenic anaerobic species and their concomitant microflora, i. e. non-pathogenic or conditionally pathogenic anaerobes and aerobes which occur in particular association reflecting the complex process of parasitocoenosis. The type of wound and the immunobiological condition of the body are also among the factors.

The causative agents of anaerobic infections require certain conditions for their development after they have gained entrance into the body, i. e. favourable medium (the presence of dead or injured tissues)and a low oxidation-reduction potential (state of anaerobiosis) which arises due to the presence of necrotized cells of the affected tissues and aerobic microflora. Later the pathogenic anaerobes cause the necrosis of the healthy tissues themselves.

This process develops particularly intensively in the muscles owing to the fact that they contain large amounts of glycogen which serves as a favourable medium for pathogenic anaerobes responsible for anaerobic infections. Oedema is produced during the first phase of the infection, and gangrene of the muscles and connective tissue, during the second phase.

The exotoxins which are produced by clostridia anaerobic infections exert not only a local effect, causing destruction of muscular and connective tissues, but affect the entire body. This results in severe toxaemia. The body is attacked also by toxic substances produced by the decaying tissues. Investigations have shown that exotoxins produced by the causative agents of anaerobic infections possess potentiation activity. Simultaneous injections of one-fourth of a lethal dose of both Cl. perfringens and Cl. novyi toxins produce a reaction which is more marked than that produced by separate injections of the toxins into different parts of the body.

As a result of the vasoconstrictive effect of the toxins, development of oedema, and gas formation, the skin becomes pale and glistening at first and bronze-coloured later. The temperature of the affected tissues is always lower than that of the healthy areas. Deep changes occur in the subcutaneous cellular, muscle, and connective tissues, and degenerative changes take place in the internal organs.

The organisms themselves play an essential part in the pathogenesis of anaerobic infections owing to their high invasive activity. An extremely important role in the development of the disease is attributed to the reactivity state of the macro-organism (trauma, concomitant diseases, etc.).

Ingestion of food (sheep's milk cheese, milk, curds, sausages, cod, etc.) contaminated abundantly with C/. perfringens results in toxinfections and intoxications. These conditions are characterized by a short incubation period (from 2 to 6 hours), vomiting, diarrhoea, headache, chills, heart failure, and cramps in the gastrocnemius muscle; the body temperature may either be normal, or elevated to 38 °C.

Immunity. The immunity produced by anaerobic infections is associated mainly with the presence of antitoxins which act against the most commonly occurring causative agents of the wound infection. For example, Cl. perfringens loses its lecithinase activity completely in the presence of a sufficient amount of antitoxin against its alpha-toxin.

The toxin-antitoxin reaction depends to a great extent on the presence of lecithin which acts as substratum for toxin activity. The antitoxin cannot neutralize lecithinase if the former is added at certain periods of time after the toxin had been in the presence of lecithin, the reaction being simply somewhat delayed in such cases. A definite role is played by the antibacterial factor, since the existence of bacteraemia in the pathogenesis of anaerobic infections has been shown.

Laboratory diagnosis. Material selected for examination include spieces of affected and necrotic tissues, oedematous fluid, dressings, surgical silk, catgut, clothes, soil, etc. The specimens are examined in stages:

(1) microscopic examination of the wound discharge for the presence of C/. perfringens;

(2) isolation of the pure culture and its identification according to the morphological characteristics of clostridia, capsule production, motility, milk coagulation, growth on iron-sulphite agar, gelatin liquefaction, and fermentation of carbohydrates (see Table 1);

(3) inoculation of white mice with broth culture filtrates or patient's blood for toxin detection;

(4) performance of the antitoxin-toxin neutralization reaction on white mice (a rapid diagnostic method).

C. perfringens is found in 70% to 80% of all cases of gas gangrene, and of the five serologic types of this organism, type A is the most prevalent. Any exudate is cultivated on thioglycolate broth and on blood–agar plates that are incubated both aerobically and anaerobically. The presence of large gram–positive rods that grow only anaerobically is strong evidence for clostridia C. perfringens is characterized by a stormy fermentation in milk, in which the coagulated milk is blown apart by gas formed during the fermentation of the lactose in milk. Organisms producing an a toxin hydrolyze the lecithin in an egg yolk medium, breaking down the lipid emulsion and, in turn, causing an opaque area to appear around the colony. Individual clostridial species are identified by a series of biochemical tests.

Treatment and prophylaxis comprise the following procedures:

– surgical treatment of wounds (surgical cleansing of wounds to eliminate extraneous material or necrotic tissue is, undoubtedly, the most important control mechanism for gas gangrene);

– early prophylactic injection of a polyvalent purified and concentrated antitoxin “Diaferm 3” in a dose of 10000 units against Cl. perfringens, Cl. novyi, Cl. septicum. For treatment the doses of antitoxin are increased five-fold;

– use of antibiotics (streptomycin, penicillin, chlortetracycline, and gramicidin), sulphonamides, anaerobic bacteriophages, diphage, antistaphylococcal plasma and antistaphylococcal gamma globulin. In a number of cases treatment with antitoxin alone does not give the desired effect, while the combined use of antitoxin and antibiotics significantly lowers the mortality rate.

Transfusion of blood, oxygen therapy, administration of inhibitors of proteolytic enzymes and biologically active preparations which normalize metabolism are auxiliary therapeutic measures. Hyperbaric oxygen chambers, in which an infected area is placed in a chamber containing pure oxygen under pressure, have been used with some success to stop the growth of these obligate anaerobes.

CLOSTRIDIUM PERFRINGENS AND FOOD POISONING

In addition to being the major etiologic agent in wound infections, C perfringens also is an important cause of food poisoning. Most outbreaks follow the ingestion of meat or gravy dishes that are heavily contaminated with vegetative cells of C perfringens. Interestingly, C perfringens type A strains produce a heat–labile enterotoxin only when the vegetative cells form spores in the small intestine, releasing the newly synthesized enterotoxin. Symptoms of acute abdominal pain and diarrhea begin 8 to 24 hours after ingestion of the contaminated food and usually subside within 24 hours. The toxin appears to bind to specific receptors on the surface of intestinal epithelial cells in the ileum and jejunum. The entire molecule then is inserted into the cell, membrane, but does not enter the cell. This induces a change in ion fluxes, affecting cellular metabolism and macromolecular synthesis. As the intracellular Ca²⁺ levels increase, cellular damage and altered membrane permeability occurs, resulting in the loss of cellular fluid and ions.

Rare, but severe, cases of food poisoning, characterized by hemorrhagic enteritis and a high mortality rate, usually are caused by C perfringens type C. Such cases have been reported primarily from Germany and New Guinea. Those in New Guinea (known as pig–bel) have been associated with the eating of pork or other high–protein foods. Type C organisms produce a sporulation enterotoxin indistinguishable from that produced by C. perfringens type A, but they also produce large amounts of a toxin and the lethal necrotizing b toxin. It seems that the severe hemorrhagic enteritis is primarily a result of the action of the b toxin.

C perfringens type C has been reported to occur in the feces of over 70 % of the villagers in Papua, New Guinea. Because of a diet low in protein, the organisms in the gut do not ordinarily grow and produce sufficient toxin to cause any pathologic manifestations. Meat and other high–protein foods (which are seldom eaten), however, stimulate growth and toxin production by the clostridia. The disease occurs primarily in young children because of their poor immunity to b toxin. Also, because of a diet low in proteins, such children have an abnormally low level of intestinal proteases that could destroy the toxin before intestinal damage occurs. Furthermore, even this low level of protease activity is inhibited by protease inhibitors present in sweet potatoes that are consumed in large quantities at New Guinea feasts.

Because of the severity and high incidence of this disease, a program of active immunization with C perfringens b toxoid was initiated in 1980. Data indicate that the use of this vaccine has resulted in a dramatic decrease in the incidence of pig–bel in the New Guinea highlands.

C perfringens also has been reported to cause an infectious diarrhea, in which the organisms seem to be spread from person to person. Such infections are characterized by large numbers of C perfringens and high titers of enterotoxin in stool specimens, as well as a considerably longer duration of illness.

CLOSTRIDIUM DIFFICILE. Pseudomembranous colitis, a severe, necrotizing process that may occur in the large intestine after antibiotic therapy and produces severe diarrhea, has been associated with a number of antimicrobial agents, but the antibiotics clindamycin, ampicillin, amoxicillin, and the cephalosporins have been incriminated most often. One mechanism of this diarrhea was elucidated in 1978, when it was observed that the use of these antibiotics resulted in an over growth of an organism in the intestine identified as Clostridium difficile. C difficile can cause a spectrum of symptoms, ranging from asymptomatic carriage, mild to severe cholera–like diarrhea with 20 or more watery stools per day, and, in its most serious form, pseudomembranous colitis. Evidence indicates that C difficile is responsible for virtually all cases of pseudomembranous colitis and for up to 20% of cases of antibiotic–associated diarrhea without colitis. C difficile seems to be part of the normal intestinal flora of about 7% to 10% of adults; but only when antibiotic–sensitive organisms are eliminated from the intestine is it able to grow to sufficient numbers to produce disease. Interestingly, as many as 50% to 75% of neonates may become colonized with C difficile acquired as a nosocomial infection. Fortunately, most infants re–main asymptomatic, but they do serve as a reservoir for the spread of toxigenic C difficile to others both in the hospital and at home.

To demonstrate the nosocomial acquisition of this organism in adult patients, the University of Washington (Seattle) carried out a study in which 428 consecutive patients were cultured for C difficile over an 11–monthperiod. They reported that 7% had positive results on admission, but of the patients with negative culture re–sults, 21% acquired the organism during their hospital stay. Of these, 37% had diarrhea. Moreover, of the hospi–tal personnel carrying for the patients, 59% were positive for C difficile.

C difficile produces disease by the elaboration of two distinct exotoxins, which have been designated as A and B. Toxin A is an enterotoxin that is primarily responsible for the diarrhea associated with this disease. Its mechanism of action seems to result from tissue damage after an inflammatory process induced by the toxin. Toxin A acts as a strong chemoattractant for neutrophils, and it is thought that the release of inflammatory cytokines from these cells results in altered membrane permeability, fluid secretion, and hemorrhagic necrosis. Toxin B is a cytotoxin that demonstrates a lethal effect on cultured tissue cells. Its cytotoxic action is thought to involve depolymerization of filamentous actin, resulting in a change in the cell cytoskeleton and a rounding of the cell.

In addition, an enzyme with ADP–ribosylating activity has been described in one strain of C difficile. This toxin has been shown to modify cell actin in a manner similar to that of Clostridium botulinum C^ and C per–fringens t toxin.

The diagnosis of C difficile diarrhea usually is based on the demonstration of the presence of toxin A, toxin B, or both. Toxin B can be detected by its effect on cell cultures, but this requires 18 to 24 hours. Latex beads coated with antibody to toxin A also are commercially available, as is an enzyme–linked immunosorbent assay kit, for detecting both toxins A and B.

The primary treatment is to discontinue the implicated antibiotic. Most patients then recover spontaneously. An agent can be substituted that is unlikely to cause an antibiotic–associated diarrhea such as a quinoline, sulfonamide, parenteral aminoglycoside, metronidazole, or trimethoprim–sulfomethoxazole.

Clostridium sordellii occasionally is one of the etiologic agents of clostridial myonecrosis. It is mentioned here because pathogenic strains of C sordellii produce two toxins that share biologic and immunologic properties with toxins A and B of C difficile, and it may be responsible for some cases of antibiotic–associated diarrhea.

Additional materials for diagnosis

GAS ANAEROBIC INFECTION. Gas anaerobic infection is a disease developing in the wake of extensive deeply penetrating wounds of muscles and other tissues provided they are contaminated with anaerobes from the environment, particularly from soil. The pathogens responsible for this disease are Clostridium perfringens, Cl. septicum, Cl. sordellii, Cl. novyi, etc.

The material to be studied is damaged and necrotic tissues taken at the borderline between pathologically-altered and healthy tissues, exudate, pus, secretions from wounds, and blood. Post-mortem material examined includes secretions from wounds, pieces of altered muscles, blood from the heart, and pieces of the spleen and liver. In food poisoning vomits, waters of stomach lavage, faeces, blood, and food remains are examined.

Bacteriological and biological examination. The material is stained by the Gram technique, examined microscopically, paying attention to the presence of gross Gram-positive spore rods or individual spores, and then introduced into casein or meat liquid and solid media (blood agar, Wilson-Blair medium).

The inoculated cultures are cultivated in an anaerobic jar, while columns with medium are placed into a 37 °C incubator.

Make preparations from the inoculated cultures, stain them by Gram's method, note the nature of the growth on liquid nutrient media, and subculture the material onto solid media.

Filtrates of the cultures or centrifugates are examined for the presence of toxin in experiments on mice or guinea pigs and utilized for conducting the neutralization reaction with diagnostic sera of Cl. perfringens, Cl. septicum, Cl. sordellii, Cl. oedematiens of A and B types.

The nature of growth on solid nutrient media is determined on the third day. Using a needle, pick up colonies and inoculate, with the help of column technique, into a semi-solid agar containing 0.5 per cent of glucose. Assay the morphology of the bacteria isolated, their motility, capacity to ferment carbohydrates, change the colour of litmus milk, liquefy gelatin, and coagulated serum or yolk. For this purpose emulsify the colony on a glass slide in a drop of acridine orange, cover it with a cover slip, and examine under the immersion objective of a luminescent microscope. Detection of only green rods is indicative of toxigenic species.

The presence of red rods or those of a green colour with red fragments points to weak or no toxigenicity of bacteria.

For rapid diagnosis the material tested is centrifuged and the pellet is used to make the in vitro neutralization test with specific antitoxic sera. Other rapid methods of the diagnosis include demonstration of lecithinase in filtrates and its neutralization with type-specific sera.

The material is centrifuged, diluted with isotonic sodium chloride solution 1:2, 1:4 . . ., an activator (0.005 M CaCl₂) is added, and 1 ml of each dilution is mixed with 0.1 ml of type-specific serum. The mixture is incubated at 20 °C for 40 min, and then 0.2 ml of lecithovitellin is added, and the mixture is reincubated at 37 °C for 2 hrs. The same mixture, only without serum, serves as the control. In the control a filtrate of the lecithinase-positive microorganism Cl. perfringens forms turbidity and opacification, with no such changes being observed in test tubes with the serum.

FOOD POISONING CAUSED BY CLOSTRIDIUM PERFRINGENS

Food poisoning in man is most often caused by Clostridium perfringens of types A and C.

The material used for examination is food remains, such clinical specimens as vomited matter, faeces, and blood in anaerobic sepsis, and such autopsy samples as blood and pieces of the internal organs.

Bacteriological examination is conducted for the isolation and identification of the causative agent, determination of the degree of colonization of the material examined by this microorganism and the type of the toxin produced by the latter.

Day 1. The material to be examined is diluted ten-fold with peptone water to 10^–10 and 1-ml portions from the respective dilutions are transferred into the melted Wilson-Blair medium which has been cooled to 45 °G. In some cases the material is introduced into blood or yolk agar which is then decanted into plates. After the agar has solidified, the inoculated culture is immersed with a 2 per cent meat-peptone agar and incubated for 6-8 hrs at 45-46 "C or for 20 hrs at 37 °C.

In addition, homogenates of the materials examined are streaked onto liquid nutrient media (Kitt-Tarozzi's medium). The inoculated cultures are incubated at 37 °C. Growth of Clostridia may he noted in 6-8 hrs. If this is the case, further examinations are carried out within 24 hours.

Day 2. Count black colonies in the Wilson-Blair medium, select the specimen where some 10-30 colonies have formed (20-100 per plate) and recalculate the number per ml (taking into account the dilution and the dose of the inoculum).

To obtain a pure culture after microscopy, subculture 3-5 colonies into the Kitt-Tarozzi medium and 2-3 colonies onto litmus milk. The inoculated cultures are incubated at 37 °C for 24 hrs.

Day 3. Study the nature of growth in the Kitt-Tarozzi medium. Cl. perfringens grow with intense gas formation. On litmus milk one can observe characteristic fermentation with lightening of the serum and formation of a sponge clot of brick colour.

To detect exotoxin and determine its type, the neutralization reaction is performed with a filtrate of the broth culture. The test is performed and the results are read as it is done in botulism.

The diagnosis is considered confirmed if the food products responsible for the disease contain large numbers of Clostridium (10⁶ and more per g), if the cultures of the material examined show Cl. perfringens of types A and C. if the Clostridia isolated produce exotoxins and strains of Cl. perfringens of any type (A, B, C, D, E) are found in the patient's blood.

To speed up the diagnosis, examination is carried out according to the following scheme.

1. The material is heated for 15 min at 80 °C, introduced into defatted milk containing 0.5 per cent of glucose, and cultivated at 37 °C.

If the material harbours Cl. perfringens, milk peptonization is seen in several hours.

2. After a clot has formed, the serum is centrifuged and 0.5-1.0 ml administered intraperitoneally to white mice.

If a toxin is demonstrated, the neutralization test with serum against Cl. perfringens of type A only is performed. The toxin formed in the serum treated with trypsin (proteolytic activation of toxin) is also determined.

Tetanus Clostridia

A. Nicolaier discovered the causative agent of tetanus in 1884, and S.Kitasato isolated the pure culture in 1889.

Morphology. The causative agent of tetanus (Clostridium tetani) is a thin motile rod, 2.4-5 mcm in length and 0.5-1.1 mcm in breadth. It has pentrichous flagellation and contains granular inclusions which occur centrally and at the ends of the cell. The organism produces round terminal spores which give it the appearance of a drumstick (Fig. ). Cl. tetani is Gram-positive.

Figure. Clostridium tetani with terminal spores

Electron microscopy shows that the cell wall is composed of five layers and the cytoplasmatic membrane of three layers; the cytoplasm is dense, granular and contains ribosomes and polysomes. During maxi-mum liberation of the exotoxin, the cytoplasmatic membrane draws away from the cell wall and the main bulk of the cell is lysed. The nucleoid is compact and occupies a small part of the cell. The spores are enclosed by a sporangium. The G+C content in DNA is 25 per cent.

Cultivation. The organisms are obligate anaerobes. They grow on sugar and blood agar at pH 7.0-7.9 and at a temperature of 38 °C (no growth occurs below 14 and above 45 °C) and produce a pellicle with a compact center and thread-like outgrowths at the periphery. Some-times a zone of haemolysis is produced around the colonies. Brain medium and bismuth-sulphite agar are blackened by Cl. tetani. Agar stab cultures resemble a fir-tree or a small brush and produce fragile colonies which have the appearance of tufts of cotton wool or clouds (Fig.). A uniform turbidity is produced on Kitt-Tarozzi medium with liberation of gas and a peculiar odour as a result of proteolysis.

Figure. Clostridium tetani. Colonies in stab agar culture.

Fermentative properties. Cl. tetani causes slow gelatin liquefaction and produces no indole. Nitrates are rapidly reduced to nitrites. The organisms coagulate milk slowly, forming small flakes. No carbohydrates are usually fermented (see Table 1, Mettodological instructions).

Toxin production. Cl tetani produces an extremely potent exotoxin which consists of two fractions, tetanospasmin, which causes muscle contraction, and tetanolysin, which haemolyses erythrocytes.

A 0.0000005 ml dose of toxin obtained from a broth culture filtrate kills a white mouse which weighs 20 g; and 0.000000005 g of dry toxin obtained by ammonium sulphate precipitation is fatal to the mouse. Several million lethal mouse doses are contained in 1 mg of crystalline toxin.

The mode of action of the tetanus toxin is similar to that of enzymes which catalyse chemical reactions in the bodies of affected animals.

Tetanus toxin (also termed tetanospasmin) is synthesized in the bacterium as a single polypeptide chain, but after its release by lysis of the organism, a bacterial protease cleaves one peptide bond to yield two chains that remain linked together through a disulfide bond. The larger chain (H chain) has a molecular weight of 100,000 daltons, and it possesses the specific receptors that bind the toxin to the neuronal gangliosides. The smaller peptide (L chain) has a molecular weight of 50,000 daltons and is thought to exert the biologic effect of the toxin.

The mechanism of action of the toxin is not fully understood, but it is known that the toxin is first bound to neuronal cells at the neuromuscular junction. The complete toxin then crosses the nerve cell membrane and is transported retrogradely to the inhibitory interneurons. There, by an as yet unknown mechanism, the toxin enters the interneurons and blocks the exocytosis of inhibitory transmitters, namely, glycine and gamma-aminobutyric acid. In an analogous situation, tetanus toxin has been reported to inhibit the secretion of lysosomal contents from stimulated human macrophages. The final effect is a spastic paralysis characterized by the convulsive contractions of voluntary muscles. Because the spasms frequently involve the neck and jaws, the disease had been referred to as lockjaw. Death ordinarily results from muscular spasms affecting the mechanics of respiration.

Interestingly, all toxin-producing strains of C tetani possess a large plasmid, which encodes for the synthesis of the toxin. Loss of the plasmid converts the cell to an avirulent, non-toxin-producing organism.

A second toxin produced by C tetani is called tetanolysin. This toxin is related functionally and serologically to streptolysin O and belongs to a large group of oxygensensitive hemolysins from a variety of bacteria. In addition to erythrocytes, tetanolysin lyses a variety of cells such as polymorphonuclear neutrophils, macrophages, fibroblasts, ascites tumor cells, and platelets. It is unknown, however, whether it plays any significant role in infections by C tetani.

Antigenic structure and classification. Cl. tetani is not serologically homogeneous and 10 serological variants have been recognized. All 10variants produce the same exotoxin. The I, III, VI, and VII types exhibit a manifest specificity. The motile strains contain the H-antigen, and the non-motile strains contain only the O-antigen. Variant specificity is associated with the H-antigen and group specificity with the O-antigen.

Resistance. Vegetative cells of the tetanus organism withstand a temperature of 60-70° C for 30 minutes and are destroyed quite rapidly by all commonly used disinfectants. The spores are very resistant, and survive in soil and on various objects over a long period of time and with-stand boiling for 10-90 minutes or even, as with spores of certain strains, for 1-3 hours. The spores are killed by exposure to a 5 per cent phenol solution for 8-10 hours, and by a 1 per cent formalin solution, for 6 hours. Direct sunlight destroys them in 3-5 days.

Pathogenicity for animals. Horses and small cattle acquire the disease naturally, and many animals may act as carriers of Cl. tetani.

Among experimental animals, white mice, guinea pigs, rats, rabbits, and hamsters are susceptible to tetanus.

The disease in animals is manifested by tonic contractions of the striated muscles and lesions in the pyramid cells of the anterior cornua of the spinal cord. The extremities are the first to be involved in the process, the trunk being affected later (ascending tetanus).

Pathogenesis and disease in man. Healthy people and animals, who discharge the organisms in their faeces into the soil, are sources of the infection. Spores of Cl. tetani can be demonstrated in 50-80 per cent of examined soil specimens, and some soils contain the spores in all test samples (manured soil is particularly rich in spores). The spores may be spread in dust, carried into houses, and fall on clothes, underwear, foot-wear, and other objects.

The majority of tetanus cases in adults occur among farm workers, and more than 33 per cent of the total incidence of the disease is associated with children from 1 to 15 years old. In more than 50 per cent of cases tetanus is acquired as the result of wounds of the lower extremities inflicted by spades, nails, and stubbles during work in the orchard or in the field.

Cl. tetani may gain entrance into the body of a newborn infant through the umbilical cord and into a woman during childbirth, through the injured uterine mucosa.

The organisms produce exotoxins (tetanospasmin and tetanolysin) at the site of entry. In some cases tetanus is accompanied by bacteraemia.

Microbes and spores, washed-off from the toxin, normally produce no disease and are rapidly destroyed by phagocytes.

The tetanus toxin reaches the motor centres of the spinal cord via the peripheral nerves (it moves along the axial nerve cylinders or along the ecto- and endoneural lymphatics).

According to the school of thought of A. Speransky, the specificity of the tetanus toxin is manifest only at the onset of the disease. In its further stages the infection is governed by other phenomena, primarily by the neurodystrophic factors. Sites of high and increasing excitation develop under the influence of irritation stimuli.

The toxin enters the blood and is thus distributed throughout the whole body, causing subsequent excitation of the peripheral nerve branches and the cells of the anterior cornua of the spinal cord.

Receptors situated in the neuromuscular apparatus play a significant role in the development of tetanus. Impulses sent out from these receptors give rise to a dominant excitation focus in the central nervous sys-tem The effect of the toxin produces an increased reflex excitation of the motor centres, and this, in its turn, leads to the development of attacks of reflex tonic muscular spasms which may occur often in response to any stimuli coming from the external environment (light, sound, etc.).

The onset of the disease is characterized by persistent tonic muscular spasms at the site of penetration of the causative agent. This is followed by tonic spasms of the jaw muscles (trismus), face muscles (risus sardonicus), and occipital muscles. After this the muscles of the back (opisthotonus) and extremities are affected. Such is the development of the clinical picture of descending tetanus. The patient lies in bed, resting on his head and hips with his body bent forward like an arc. The death rate varies from 35 to 70 per cent, being 40 per cent on the average and 65 per cent in the USA. More than 50000 people die every year from tetanus in the world. According to incomplete WHO data, more than one million people contracted the disease within a period of 10 years (1951-1960) and about 500000 of them died.

Immunity following tetanus is mainly antitoxic in character, and of low grade. Reinfections may occur.

Laboratory diagnosis is usually not carried out because clinical symptoms of the disease are self-evident. Objects of epidemiological significance (soil, dust, dressings, preparations used for parenteral injections)are examined systematically.

Wounds, dressings, and medicaments used for parenteral injections are examined for the presence of Cl. tetani and their spores by the following procedures. Specimens are inoculated into flasks or test tubes. The sowings are kept at a temperature of 80° C for 20 minutes to sup-press the growth of any non-sporeforming microflora which may be present. After 2-10 days' incubation at 35° C, the culture is studied microscopically and tested for the presence of toxin by injection into mice. If Cl. tetani is present, tetanus of the tail develops during 24-48 hours, followed by tetanus of the body and death. The disease does not occur in mice which have been inoculated with antitetanus serum.

If no tetanus toxin is detected in the first inoculation but microscopic examination reveals the presence of organisms morphologically identical with Cl. tetani, the initial culture is inoculated into a condensated water of coagulated serum. A thin film will appear over the entire surface of the medium after 24 hours' growth in anaerobic conditions. Experimental animals are infected with a culture grown on liquid nutrient medium and kept for 4-5 days at 35° C.

A biological test is employed for detecting the exotoxin in the test material extract. Two white mice are given intramuscular injections of0.5-1.0 ml of a centrifuged precipitate or filtrate of the extract. An equal amount of the filtrate is mixed with antitoxic serum, left to stand for 40 minutes at room temperature, and then injected into another two mice in a dose of 0.75 or 1.5 ml per mouse. If the toxin is present in the filtrate, the First two mice will die in 2-4 days while the other two (control mice) will survive.

Treatment. Intramuscular injections of large doses of antitoxic antitetanus serum are employed. The best result is produced by gamma-globulin obtained from the blood of humans immunized against tetanus. Anticonvulsant therapy includes intramuscular injections of 25 per cent solutions of magnesium sulphate, administration of diplacine, condelphine, aminazine, pipolphen or andaxine and chloral hydrate introduced in enemas. To reproduce active immunity, 2 ml of toxoid is administered two hours before injecting the serum; the same dose of toxoid is repeated within 5-6 days. Uninoculated persons are subjected to active and passive immunization. This is achieved by injecting 0.5 ml of toxoid and 3.000 units of antitoxic serum and then 5 days later, another 0.5 ml of toxoid. The tetanus antitoxin is also introduced into previously inoculated individuals suffering from a severe wound. Injection of the total dose of antitoxin is preceded by an intracutaneous test for body sensitivity to horse protein. This is carried out by introducing 0.1 ml of antitoxin, previously diluted 1 :100, into the front part of the forearm. If the intracutaneous test proves negative, 0.1 ml of whole antitoxin is injected subcutaneously and if no reaction is produced in 30 minutes, the total immunization dose is introduced.

The complex of prophylactic measures includes adequate surgical treatment of wounds. The organisms are sensitive to penicillin, but the antibiotic has no effect on the neutralization of the toxin. However, after surgical cleansing of the wound, antibiotic therapy can be helpful in preventing any additional growth of the organisms.

Prophylaxis is ensured by prevention of occupational injuries and traumas in everyday life. Active immunization is achieved with tetanus toxoid. It is injected together with a tetravalent or polyvalent vaccine or maybe a component of an associated adsorbed vaccine. The pertussis-diphtheria-tetanus vaccine and associated diphtheria-tetanus toxoid are employed for specific tetanus prophylaxis in children. Immunization is carried out among all children from 5-6 months to 12 years of age, individuals living in certain rural regions (in the presence of epidemiological indications), construction workers, persons working at timber, water-supply, cleansing and sanitation, and peat enterprises, and railway transport workers.

Immunization with tetanus toxoid stimulates the production of sufficient amounts of antitoxin. Immunity lasts for a period of 2 or 3 years.

The effectiveness of the immunization has made tetanus a relatively rare disease in the developed countries(36 cases in the United States during 1994). In Third World countries, however, most persons are not immunized, and infections of the umbilical cord give rise to large numbers of neonatal tetanus. For example, special surveillance programs indicate death rates from neonatal tetanus to be 2% of all live births in Bangladesh, 6% in Papua, New Guinea, and 14.5% in Haiti. One study relating the proportion of pregnant females with tetanus antitoxin titres adequate to provide protection for the newborn found 96% protected in New Haven, Connecticut, and only 19% in Santiago, Chile.

Oral immunization may become possible using a live attenuated strain of Salmonella typhimurium that was transfected with a plasmid engineered to express a 50–kdfragment of the tetanus toxin. Given orally, this strain provided protective immunity in mice.

After an injury, human tetanus immune globulins should be administered to those who have never been immunized with tetanus toxoid or to those who did not receive the full three doses of toxoid. Booster injections of toxoid also are given if the immune status of the patient is unknown, or if it has been over 5 years since the last dose of toxoid.

Clostridia Responsible for Botulism

The causative agent of botulism (L. botulus sausage, botulism poisoning by sausage toxin), Closlridium botulinum, was discovered in Hollandin 1896 by E. van Ermengem. The organism was isolated from ham which had been the source of infection of 34 people and from the intestine and spleen on post-mortem examination. In Western Europe botulism was due to ingestion of sausages, while in America it was caused by canned vegetables, and in Russia, by ingestion of red fish. In the recent 50 years 5635 persons contracted botulism, 1714 of them died.

Morphology. Cl. botulinum is a large pleomorphous rod with rounded ends, 4.4-8.6 mcm in length and 0.3-1.3 mcm in breadth. The organism sometimes occurs in short forms or long threads. Cl. botulinum is slightly motile and produces from 4 to 30 flagella per cell. In the external environment Cl. botulinum produces oval terminal or subterminal spores which give them the appearance of tennis rackets (Fig.). The organisms are Gram-positive.

Figure. Pure culture and deep colonies of Closlridium botulinum

On ultrathin sections the cell wall in A, B, and E types consists of five layers, the cytoplasmatic membrane of three layers. By the time of maxi-mum exotoxin liberation (on the 5th-7th day) cell lysis with the discharge of crystalline structure occurs. The cytoplasm is granular and contains inclusions of various size. The nucleoid is compact and occupies a small part of the cytoplasm. Spore formation takes place on the3rd or 4th day of cultivation 1 he G +C content in DNA ranges between 26 and 28 per cent.

Cultivation. Cl. botulinum are strict anaerobes. The optimal growth temperature for serovars A, B, C, and D is 30-40 C, for serovar E 25-37 C, for serovar G 30-37 C They grow on all ordinary media at pH 7.3-7.6 Cultivation is best on minced meat or brain which the organisms turn darker. The cultures have an odour of rancid butter.

On Zeissler's sugar-blood agar irregular colonies are produced which possess filaments or thin thread-like outgrowths. The colonies are surrounded by a zone of haemolysis.

In agar stab cultures the colonies resemble balls of cotton wool or compact clusters with thread-like filaments (Fig. 3).

On gelatin the organisms form round translucent colonies surrounded by small areas of liquefaction. Later the colonies turn turbid, brownish, and produce thorn-like filaments.

In liver broth (Kitt-Tarozzi medium) turbidity is produced at first, but a compact precipitate forms later, and the fluid clears.

Fermentative properties. Cl. botulinum (serovars A and B) are proteolytic organisms, and decompose pieces of tissues and egg albumin in fluid medium. The organisms liquefy gelatin, produce hydrogen sul-phide, ammonia, volatile amines, ketones, alcohols, and acetic, butyric, and lactic acids. Milk is peptonized with gas formation. Glucose, levulose, maltose, and glycerin are fermented, with acid and gas formation (see Table 1, Mettodological instructions no 43).

Toxin production. Cl. botulinum produces an extremely potent exotoxin. The toxin is produced in cultures and foodstuffs (meat, fish, and vegetables) under favourable conditions in the body of man and animals. Multiplication of the organism and toxin accumulation are inhibited in the presence of a 6-8 per cent concentration of common salt or in media with an acid reaction. Heating at 90 C for 40 minutes or boiling for 10 minutes destroys the toxin.

The toxin produced by Cl. botulinum, as distinct from the tetanus and diphtheria toxins, withstands exposure to gastric juice and is absorbed intact. The toxin produced by serovar A Cl. botulinum can kill 60000million mice having a total weight of 1 200 000 tons. The toxin has been obtained in crystalline form and is the most potent of all toxins known to date. Curiously, the toxins seem to be secreted as progenitor toxins which, even though some have been crystallized, are composed of two polypcptide subunits linkedby disulfide bonds. Also, the toxicity of those toxins thathave been extensively studied can be increased from four–fold to 250–fold by treatment with trypsin. This phenomenon is not understood at the molecular level.

The botulinum toxin is a globulin and does not change on recrystallization. Its activity is similar to that of enzymes which catalyse chemical processes in the body of man and animals with formation of large amounts of toxic substances. These substances produce the clinical manifestations of poisoning.

The toxin acts primarily as a neurotoxin, inducing paralysis in three basic steps (1) binding of the toxin to a receptor on the nerve synapse, (2) entrance of the toxin(or possibly one polypeptide subunit) into the nerve cell, and (3) blocking of the release of acetylcholine from the cell, resulting m a flaccid muscle paralysis.

C botulinum type C produces two distinct toxins that have been designated Cl and C2 The Cl toxin functions like other botulism toxins to block the release of acetylcholine at the myoneural junction C2 toxin, however, is a binary complex consisting of two unlinked components designated as I and II Component II recognizes the cell receptor and thus facilitates the entrance of component I into the cytoplasm The C2 toxin causes a necrotic enteritis, which seems to result in an increase in vascular leakage of the intestinal mucosa. Its mechanism of action is unclear, but it has been shown to ADP-ribosylate G-actin as well as the synthetic substrate, homo-poly l arginine

C botulinum organisms, types C and D, also produce an additional toxin which has been termed exoenzymeC3 The DNA encoding C3 is located on both phage C and phage D, the phages that also encode for botulism toxins C and D, respectively Its function is to ADP-ribosylates Rho protein, a eucaryotic member of the ras superfamily of proteins Because the ras superfamily of proteins are GTP-binding proteins involved in enzyme regulation, this exoenzyme could function as a virulence factor, but the exact consequence of the C3 ADP-ribosylation is unknown.

Antigenic structure and classification. Six serovars of Cl. botulinum are known: A, B, C, D, E, and F, serovars A, B, and F being the most toxic. Each of the serovars is characterized by specific immunogenicity associated with the H-antigen and is neutralized by the corresponding antitoxin. Variants C and D are responsible for neuroparalytic lesions in animals. As has been proved recently, serovar C may produce diseases also in man. The 0-antigen is common to all variants.

Resistance. The vegetative forms of the organisms are killed in 30minutes at 80 C, while the spores withstand boiling for periods from 90minutes to 6 hours-and survive 115" C for 5-40 minutes and 120° C, for3-22 minutes. Spores remain viable in large pieces of meat and in large cans even after autoclaving for 15 minutes at 120° C. In 5 per cent phenol solutions they survive for up to 24 hours and in cultures they may live for a year.

Pathogenicity for animals. Horses, cattle, minks, birds, and among the laboratory animals, guinea pigs, white mice, cats, rabbits, and dogs are susceptible to the botulinum toxin.

Paralysis of the deglutitive, mastication, and motor muscles is usually produced in horses 3 days after infection. The mortality rate reaches 100per cent. Botulism in bovine cattle is accompanied with bulbar paralysis, and in birds it causes limbemeck and paresis of the legs.

Infection of guinea pigs results in muscular weakness which appears in 24 hours, followed by death in 3-4 days. Autopsy displays hyperaemia of the intestine, gastric flatulence, and a urinary bladder filled beyond capacity. White mice die on the second day after infection manifesting relaxed abdomen muscles and paresis of the hind limbs. Paralysis of the eye muscles, disturbances of accommodation, aphonia, pendulous and protruding tongue, and diarrhoea are caused in cats.

Pathogenesis and disease in man. Botulism is contracted by ingesting meat products, canned vegetables, sausages, ham, salted and smoked fish (red fish more frequently), canned fish, chicken and duck flesh, and other products contaminated with Cl. botulinum. The organisms enter the soil in the faeces of animals (horses, cattle, minks, and domes-tic and wild birds) and fish and survive there as spores.

Natural nidality of botulism among ducks and other wild birds has been ascertained. Extremely widespread epizootics occur in the western regions of Canada, in Uruguay, and in the USA. Natural foci develop in territories where there are stagnant reservoirs rich in vegetable debris which provides favourable conditions for anaerobiosis in warm weather. Intensive processes of decay are accompanied with oxygen uptake which contributes to the growth of serovar C botulism clostridia and production of high concentrations of the exotoxin in the water and alkaline mud in swamps. Besides birds, muskrats and frogs may also contract botulism. Migrating birds spread the causative agent of botulism from the natural foci during their flights.

Cl. botulinum spores occur both in cultivated and virgin soil. They were isolated from 70 per cent of examined soil samples in California, and from 40 percent of samples in the Northern Caucasus. Spores have been isolated from littoral soil at the Sea of Azov, sea water, and silt. They have also been found on the surface of vegetables and fruit, in the intestine of healthy animals, in 5.4 per cent of cases in the guts of fresh red fish (sturgeon, beluga, etc.), and in 15-20 per cent of cases in the guts and in 20 per cent of cases in the tissues of dead fish.

The infectious condition is caused by the exotoxin which is absorbed in the intestine, from where it invades the blood, and affects the medulla oblongata nuclei, cardiovascular system, and muscles. It has been ascertained that Cl. bolulinum may enter the body through wounds. Usually, the wounds themselves were not serious, but wound botulism should be suspected in any persons with even minor wounds who present the typical symptoms of botulism: blurred vision, weakness, and difficulty in swallowing. In the past botulism was considered to be only of a toxic nature. Recent investigations have proved the Cl. botulinum to be present in various organs of individuals who have died from botulism. Therefore, this disease is a toxinfection. The incubation period in botulism varies from 2 hours to10 days, its usual duration is 18 to 24 hours.

Botulism symptoms include dizziness, headache, and, sometimes, vomiting. Paralysis of the eye muscles, accommodation disturbances, dilatation of the pupils, and double vision occur. Difficulty in swallowing, aphonia, and deafness also arise. The death rate is very high (40-60 per cent).

Botulism in child

Immunity. The disease does not leave a stable anti-infectious immunity (antitoxic and antibacterial).

Laboratory diagnosis. Remains of food which caused poisoning, blood, urine, vomit, faeces, and lavage waters are examined. Post-mortem examination of stomach contents, portions of the small and large intestine, lymph nodes, and the brain and spinal cord is carried out.

The test specimens are inoculated into Kitt-Tarozzi medium which has previously been held at 100 C for 10-20 minutes. To free the cultures from foreign non-sporeforming microflora, 50 per cent of the test tubes containing the inoculated medium is heated at 80 C for 20minutes and then incubated in anaerobic conditions. The isolated pure culture is identified by its cultural, biochemical, and toxigenic properties.

For toxin detection a broth culture filtrate, patient's blood or urine, or extracts of food remains, are injected subcutaneously or intraperitoneally into guinea pigs, white mice, or cats. One of the control animals is infected with unheated material, while the other animal is injected with the heated specimen. In addition, 3 laboratory animals are given injections .of the filtrate together with serovar A antitoxin, with serovar B antitoxin, and with serovar E antitoxin.

The indirect haemagglutination reaction and determination of the phagocytic index are also performed. This index is significantly lowered in the presence of the toxin.

A rapid method of detection of serovar A, B, C, D, and E toxins in water has been developed in which the toxin is absorbed by talc and a suspension of the talc and toxin is injected into the animals.

Treatment. The stomach is lavaged with potassium permanganate or soda solutions Polyvalent botulinum antitoxin is injected intramuscularly (intravenously or into the spinal canal) m doses of 10000 IU (serovars A, C, and E) and 5000 IU (serovar B). If there is no improvement, the injection is repeated at the same dosage within 5-10 hours. All individuals who had used food which caused even a single case of food poisoning are given 1000-2000 IU of antitoxin as a preventive measure. Simultaneously with the antitoxin, 0 5 ml of each serovar of botulinum toxoid is injected three times at intervals of 3-5 days, for production of active immunity. Penicillin and tetracycline are recommended

General measures include subcutaneous injections of saline and glucose solutions Camphor, caffeine, vitamin C, and thiamine are prescribed if necessary. Strychnine is given 2-3 times a day as a stimulant.

Prophylaxis. Proper organization of food processing technology at food factories, meat, fish, and vegetable canning in particular, and preparation of smoked and salted fish and sausages is essential for the prevention of botulism. Home-preserved fish products (smoked and salted)as well as canned mushrooms and canned vegetables of a low acid con-tent (cucumbers, peppers, eggplant), stewed apricots, etc. are very dangerous since they are usually prepared without observance of sanitary rules.

Fish should be gutted after being caught, and placed in the refrigerator. The established temperature regimen must be observed during transportation, and the fish must be protected from pollution with soil and bowel contents. Vegetables must be washed thoroughly. The cooking of meat and fish in small pieces is recommended. Foodstuffs (ham, fish) should not be stored in large hunks and in many layers. The weight of a canned product should not exceed 0.5 kg. Cl botulinum which have with stood sterilization cause swelling of the can lids. The contents of such cans have an odour of rancid butter Such canned goods must not be put on the market and must be withdrawn and thoroughly examined. Fish must be salted in strong salt solutions (brine) with a minimal concentration of 10 per cent. Canned goods must be stored in a cool place.

Active immunization of man, horses, and cows with the toxoid is recommended by many authors in view of Cl botulinum being wide-spread in nature

Botulism occurrence in the USSR is extremely rare as a result of continuous improvement of the standard of living of the population, and of technological methods in food processing and canning industries, observance of the rules of hygiene and strict state and sanitary control of the production, storage, and sale of foodstuffs.

Infant Botulism

A new variety of botulism was recognized during 1976with the report of five cases of infant botulism. These cases occurred in babies as young as 5 weeks, some of whom were breast fed, although all had had some exposure to other foods. Since then, hundreds of additional cases of infant botulism have been diagnosed, and it has become a significant paediatric clinical entity.

Epidemiology and pathogenesis of infant botulism. Infant botulism has been diagnosed in infants ranging from 3to 35 weeks of age. It is well-established that the disease is acquired by the ingestion of C botulinum spores that subsequently germinate in the intestine and produce botulism toxin. Such spores are ubiquitous and, in fact, soil and dust samples from many homes have been shown to contain such spores. Thus, even breast-fed infants are susceptible through contaminated dust. Honey also has been shown to contain spores of C botulinum, and a number of cases of infant botulism have followed the ingestion of honey.

The major initial symptom of infant botulism is 2to 3 days of constipation followed by flaccid paralysis, resulting in difficulty in nursing and a generalized weakness that has been described as "overtly floppy."

The mortality rate of infants admitted to the hospital has been about 3%, and some patients have required mechanical respirators because of respiratory distress. Death, however, may occur more frequently in undiagnosed cases, and considerable data link infant botulism to at least some cases of the sudden infant death syndrome.

Because, by definition, infant botulism is the result of toxin production by organisms that have colonized the gut, it is not surprising that there have been a few cases of adult-infant botulism that occurred after antibiotic therapy or gastric surgery. Even in cases of food-borne botulism, it is usual for the gut to be colonized with C. botulinum, providing a continuing source of toxin.

Diagnosis and treatment of infant botulism. A tentative clinical diagnosis of botulism can be made for an infant with several days of constipation, an unexplained weakness, difficulty in swallowing, or respiratory distress. A laboratory diagnosis, however, requires the demonstration of botulism toxin in the feces, which is determined by the injection of fecal extracts intraperitoneally into a mouse. Death of the mouse within 96 hours (which did not occur in controls in which the fecal extracts were first neutralized with botulism antitoxin) is taken as positive evidence for the presence of the toxin.

Infants are not usually treated with antitoxin, primarily because it is a horse product and may induce lifelong hypersensitivity. Attempts to eradicate the bacteria are not recommended because of the fear that the organisms might lyse in the intestine, releasing large amounts of toxin. Treatment thus far has been mostly symptomatic, requiring an average of 1 month of hospitalization.

Additional materials for diagnosis

TETANUS

Tetanus is an acute infectious disease caused by Clostridium tetani and attended by tonic and clonic muscular contractions. The clinical picture is so typical that, as a rule, the bacteriological examination for diagnosis is unnecessary. To detect the causative agent, surgical dressings and various preparations intended for parenteral administration are usually checked.

In cases of an obscure course of the disease examine pus, blood, pieces of tissue cut from the wound, as well as post-mortem specimens of organs, tissues, and blood. From tissues and thick pus prepare suspensions in isotonic sodium chloride solution. Cotton wool and gauze are cut with scissors and placed into nutrient media.

Bacterioscopic examination. Detection of thin long Gram-positive rods with round terminal spores in smears from the material obtained from the patient or corpse suggests the presence of Cl. tetani.-. Yet, one cannot derive the conclusion as to the presence of Clostridia of tetani on the basis of bacterioscopic findings alone since the material tested may contain other morphologically similar microorganisms, e.g., Cl. tetanomorphum, Cl. paratetanomorphum, etc.

Bacteriological examination. The material to be examined is streaked on the Kitt-Tarozzi enrichment medium and placed in the incubator for 3-4 days after which it is subcultured to solid media to obtain separate colonies. Following incubation in a microanaerostatic jar, Cl. tetani colonies on a blood sugar agar appear as small spiders or dew-drops, whereas in the column of sugar agar they resemble balls of wool or cotton.

The isolated pure culture is identified and examined for toxigenicity. Cl. tetani form a toxin on the 4th-5th day of cultivation. The culture formed on the Kitt-Tarozzi medium is centrifuged and 0.3-0.4 ml of the supernatant is injected intramuscularly (at the root of the tail) to two white mice. Two control mice receive the same amount of the tested liquid which is mixed with an antitoxic anti-tetanus serum, following the incubation for 1 h at 37 °C. The mice are observed for 4-5 days. In 2-4 days infected animals present signs of tetanus (rigidity of the tail and muscles at the site of the toxin administration) and soon die, while the control mice survive unaffected.

If white mice are injected the material tested (together with the inoculated culture), they present the same clinical picture of tetanus that is seen after administration of the toxin.

Isolation of a pure culture of Cl. tetani by the Fildes technique. For this purpose a 3-4-day culture in the enrichment medium is heated for 1.5 hrs at 60 °C, after which several drops are inoculated into condensation water of the coagulated serum. The inoculated culture is kept under strictly anaerobic conditions. In 1-2 days, a "creeping" growth of Cl. tetani is observed. From the upper part of the inoculated culture it is again subcultured with a loop into condensation water of the coagulated serum. Such passages are reiterated until a pure culture is isolated.

BOTULISM

Botulism is acute food poisoning characterized by the predominant damage to the central and vegetative nervous system. The causal organism of this disease is Clostridium botulinum.

To carry out the examination, one usually collects at least 10-12 ml of blood, which is supplemented with sodium citrate in a 3:1 ratio, 100-200 ml of vomited matter, lavage waters of the stomach, faeces, and urine, as well as 200-300 g of remains of the food-stuffs that are suspected to be the cause of the disease. All materials should be taken prior to the administration of a therapeutic serum. At necropsy blood, pieces of the internal organs, lymph nodes, contents of the stomach and intestines, brain, and spinal cord are examined.

Examination has a double purpose: detection in the material tested of the botulin toxin (two-thirds of the specimen) and isolation from the material of the causative agent (one-third of the specimen).

Demonstration of the botulin toxin and identification of its type with the help of the neutralization reaction are very important with regard to the prescription of a specific therapy.

Preparation of material. Lavage waters of the stomach (25-30 ml) containing food lumps are ground in a sterile mortar; two-thirds of the sample are kept at room temperature for 1 h for extracting and then filtered through a cotton-gauze filter or centrifuged at 3000 X g for 15-20 min.

Citrate blood or serum obtained from the patient should not be diluted before the examination; it is administered to mice only in the form of intraperitoneal injection.

Patients' faeces (20-25 g) are ground in a sterile mortar with a double volume of isotonic sodium chloride solution and kept at room temperature for 1-1.5 hrs, and then filtered through a water-gauze filter. Extracts from post-mortem material are prepared in a similar manner.

Procedure of the test. To perform the neutralization test, use dry diagnostic antitoxic sera of A, B, C, and E types, which are diluted with isotonic sodium chloride solution to 100-200 lU/ml, which ensures neutralization of the homologous toxin in the specimen tested.

The neutralization reaction is carried out with either a mixture of sera (a preliminary reaction) or with monovalent sera (for detection of a specific type of toxin).

The prepared material to hp studied (in the form of a filtrate or pullet) or blood is dispensed in 1-mt volumes into live test tubes; into each of the first four tubes 1 ml of anti-botulinal serum of types A, B, C, and E is added respectively, into the last one, 1 ml of the normal serum is introduced. The tubes are incubated for 30 min after which 1-ml amounts of the mixture from each test tube are introduced to five pairs of white mice weighing 1(3-18 g (blood is injected intraperitoneally; other biomaterials, subcutaneously. The animals are observed for four days.

If the material studied contains the botulinal toxin, only one pair of mice survives due to the neutralization of the toxin by the antitoxic serum of the corresponding type (a positive reaction). If all mice die, the neutralization test should be repeated after diluting the biomaterial by 5-, 10-, 20- and even 100fold. If the material tested contains the botulinal toxin, mice develop paresis of the limbs. Autopsy findings include hyperaemia of the internal organs, pneumonic foci in the lungs, overfilling of the stomach, bladder, and gallbladder.

The laboratory conclusion about the presence in the material examined of the botulinal toxin should refer to its particular type.

Bacteriological examination. Prior to inoculation, the material to be tested is ground in a porcelain mortar. Some 10-12 ml of the material are introduced into the Kitt-Tarozzi medium, casein-acid, or casein-mycotic medium. One specimen is inoculated into four vials, two of which are heated: one at 60 °C for 15 min (for the selection of E type Cl. botulinum), the other at 80 °C for 20 min. Enrichment for cultures of Clostridia of types E and F occurs in a 28 °C incubator. To grow Clostridia of types A, B, and C, the cultures are cultivated at 35 °C for 48 hrs.

To activate toxin E from the protoxin, add trypsin to the nutrient medium to achieve the final concentration (0.1 per cent). The remains of samples of the material studied are stored in a refrigerator till the end of the analysis.

After 24-48 hrs of incubation, the enrichment medium becomes turbid and gas formation is observed. From the medium presenting growth prepare smears and stain them by the Gram technique. Upon detection of typical Clostridia with spores subculture them to solid nutrient media for obtaining separate colonies. Isolation of a pure culture presents some difficulty as Cl. botulinum often form associations with some aerobic bacteria. Sometimes, only multiple passages make it possible to obtain a pure culture. On a sugar blood agar Cl. botulinum form irregularly-shaped colonies with a smooth or rough surface surrounded by a zone of haemolysis. Deep in the sugar agar column these colonies appear as fluffs or lentils.

To identify the obtained pure culture, it is inoculated into Hiss's media. Cl. botulinum displays proteolytic properties: it liquefies gelatin and serum and splits yolk and pieces of meat. Most strains ferment glucose, mannitol, maltose, and other carbohydrates with acid and gas formation. Antigenic attributes are studied with the help of the agglutination test, using type specific sera.

Simultaneously with the investigation of the fermentative properties the botulinal toxin is demonstrated in the filtrate of a broth culture, and its type is identified.

Detection of the botulinal toxin with the help of the phagocytic parameter. The botulinal toxin inhibits the phagocytic activity of leucocytes, whereas specific sera eliminate this action by neutralizing the toxin. The employment of this method is particularly advisable for detecting the toxin in blood.

The rapid method of detecting the botulinal toxin in drinking water is based on the adsorption of toxin from water with the help of talcum powder and the subsequent administration to mice of the talc suspension obtained.

Epidemiological data and characteristic clinical manifestations (the paralytic syndrome) play an important role in the diagnosis of botulism. Negative results of laboratory studies do not exclude the presence of botulism.

Peptostreptococcus. Clinically significant anaerobic cocci include peptostreptococci, Veillonella species, and microaerophilic streptococci. The genus Peptostreptococcus contains very small bacteria that grow in chains. Peptostreptococcus is a genus of anaerobic, Gram-positive, non-spore forming bacteria. The cells are small, spherical, and can occur in short chains, pairs or individually. Peptostreptococcus are slow-growing bacteria with increasing resistance to antimicrobial drugs.These anaerobic counterparts of Streptococcus are usually not harmfull. They are known to be normal flora of the skin, urethra, and the urogenital tract. If given an opportunity, however, they can cause infections of bones, joints and soft tissue. Their increasing resistance to such antibiotics as penicillin G and clindamycin makes them especially important to clinical work. P. magnus is the species that is most often isolated from infected sites.

Peptostreptococcus infections can occur in all body sites, including the CNS, head, neck, chest, abdomen, pelvis, skin, bone, joint, and soft tissues. Inadequate therapy against these anaerobic bacteria may lead to clinical failures. Because of their fastidiousness, peptostreptococci are difficult to isolate and are often overlooked. Isolating them requires appropriate methods of specimen collection, transportation, and cultivation. Their slow growth and increasing resistance to antimicrobials, in addition to the polymicrobial nature of the infection, complicate treatment.

Peptostreptococcus is the only genus among anaerobic gram-positive cocci encountered in clinical infections. This group also includes species within the genus formerly known as Peptococcus, with the exception of Peptococcus niger. This change in taxonomy was based on the results of a guanine-plus-cytosine content analysis. Additionally, Gaffkya anaerobia was renamed Peptostreptococcus tetradius. The species of anaerobic gram-positive cocci isolated most commonly are Peptostreptococcus magnus, Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus prevotii, and Peptostreptococcus micros.

Anaerobic gram-positive cocci that produce large amounts of lactic acid during the process of carbohydrate fermentation were reclassified as Streptococcus parvulus and Streptococcus morbillorum from Peptococcus or Peptostreptococcus. Most of these organisms are anaerobic, but some are microaerophilic. Based on DNA homology and whole-cell polypeptide-pattern study findings supported by phenotypic characteristics, the DNA homology group of microaerobic streptococci that was formerly known as Streptococcus anginosus or Streptococcus milleri is now composed of 3 distinct species: S anginosus, Streptococcus constellatus, and Streptococcus intermedius. The microaerobic species S morbillorum was transferred into the genus Gemella. A new species within the genus Peptostreptococcus is Peptostreptococcus hydrogenalis; it contains the indole-positive, saccharolytic strains of the genus.

Pathophysiology: Peptostreptococcus organisms are part of the normal florae of human mucocutaneous surfaces, including the mouth, intestinal tract, vagina, urethra, and skin. They are isolated with high frequency from all specimen sources. Anaerobic gram-positive cocci are the second most frequently recovered anaerobes and account for approximately one quarter of anaerobic isolates. Anaerobic gram-positive cocci are usually recovered mixed with other anaerobic or aerobic bacteria from infections at different sites of the body.

Many of these infections are synergistic. Bacterial synergy, the presence of which is determined by mutual induction of sepsis enhancement, increased mortality, increased ability to induce abscesses, and enhancement of the growth of the bacterial components in mixed infections, is found between anaerobic gram-positive cocci and their aerobic and anaerobic counterparts. The ability of anaerobic gram-positive cocci and microaerophilic streptococci to produce capsular material is an important virulence mechanism, but other factors also may influence the interaction of these organisms in mixed infections.

Frequency: In the US: The exact frequency of Peptostreptococcus infections is difficult to calculate because of inappropriate methods of collection, transportation, and cultivation of specimens. These infections are found more commonly in patients with chronic infections. Recovery rates in blood cultures are 2-5% and are higher in patients who have predisposing conditions. In 1974, Martin reported that anaerobic cocci were isolated in 8.5-31% of clinical specimens that yielded any anaerobic bacteria at the Mayo Clinic.

In 2 studies published in 1988 and 1989, Brook reported that anaerobic gram-positive cocci accounted for 26% of all anaerobic bacteria recovered at Bethesda Navy Hospital and Walter Reed Army Hospital from 1973-1985. The infected sites where the organisms predominated were ears (53% of all anaerobic isolates), cysts (40%), bones (39%), and obstetrical and gynecological sites (35%). They were occasionally found in the CNS, abdomen, lymph nodes, bile, and eyes. Most isolates were found in abscesses, wounds, and obstetrical and gynecological infections.

The recovery rates differed for the different anaerobic gram-positive cocci. In descending order of frequency, the most common anaerobic gram-positive cocci were P magnus (18% of all anaerobic gram-positive cocci and microaerophilic streptococci), P asaccharolyticus (17%), P anaerobius (16%), P prevotii (13%), P micros (4%), Peptostreptococcus saccharolyticus (3%), and Peptostreptococcus intermedius (2%).

The highest recovery rates of P magnus were in bone and chest infections. The highest recovery rate of P asaccharolyticus and P anaerobius were with obstetrical/gynecological and respiratory tract infections and wounds. Isolates of each of the most frequently recovered anaerobic gram-positive cocci were recovered from abscesses, wounds, and obstetrical and gynecological infections.

Although most of the infections were polymicrobial when anaerobic and facultative cocci were recovered, these organisms were isolated in pure culture in 45 (8%) of 559 children who had infections involving anaerobic gram-positive cocci, in 12 (10%) of 121 children who had infections due to microaerophilic streptococci, and in 15 (9%) of 176 patients who had P magnus infection. The most frequent types of infections from which anaerobic gram-positive cocci were isolated in pure culture were soft tissue infections, osteomyelitis, arthritis (especially in the presence of a prosthetic implant), and bacteremia. Most patients from whom microaerophilic streptococci were recovered in pure culture had abscesses (eg, dental, intracranial, pulmonary), bacteremia, meningitis, or conjunctivitis.

P magnus is the most commonly isolated anaerobic cocci. It is most often recovered in pure culture. The most common peptostreptococci in the different infectious sites are P anaerobius in oral infections; P magnus and P micros in respiratory tract infections; P magnus, P micros, P asaccharolyticus, Peptostreptococcus vaginalis, and P anaerobius in skin and soft tissue infections; P magnus and P micros in deep organ abscesses; P magnus, P micros, and P anaerobius in gastrointestinal tract–associated infections; P magnus, P micros, P asaccharolyticus, P vaginalis, P tetradius, and P anaerobius in female genitourinary infections; and P magnus, P asaccharolyticus, P vaginalis, and P anaerobius in bone and joint infections and leg and foot ulcers.

Internationally: The frequency of these infections appears to be higher in developing countries, where therapy is often inadequate or delayed.

Mortality/Morbidity: Mortality has decreased over the past 3 decades.

Age: Peptostreptococcus infections can occur in patients of all ages; however, head and neck infections occur more frequently in children than in adults.

Physical: Although anaerobic cocci can be isolated from infections at all body sites, a predisposition for certain sites has been observed. In general, Peptostreptococcus species, particularly P magnus, have been recovered more often from subcutaneous and soft tissue abscesses and diabetes-related foot ulcers than from intra-abdominal infections. Peptostreptococcus infections occur more often in chronic infections and in association with the predisposing conditions below.

CNS infections

Anaerobic gram-positive cocci and microaerophilic streptococci can be isolated from subdural empyema and from brain abscesses that develop as sequelae of chronic infections of the ears, mastoid, sinuses, and teeth.

Anaerobic gram-positive cocci and microaerophilic streptococci have been isolated from 18 (46%) of 39 brain abscesses.

Upper respiratory tract and dental infections

The high rate of anaerobic cocci colonization of the oropharynx accounts for the organisms' significance in these infections. Anaerobic gram-positive cocci and microaerophilic streptococci are often recovered from acute and chronic upper respiratory tract infections. These organisms have been recovered in 15% of patients with chronic mastoiditis, 30% of patients with chronic sinusitis, 33% of patients with peritonsillar and retropharyngeal abscesses, and 50% of patients with purulent parotitis. They have also accounted for two thirds of isolates from periodontal abscesses.

In more than 90% of cases, other organisms also present in the oral florae have been found mixed with anaerobic gram-positive cocci and microaerophilic streptococci. These include Staphylococcus aureus, Streptococcus species, Fusobacterium species, and pigmented Prevotella and Porphyromonas species.

Anaerobic pleuropulmonary infections

Anaerobic gram-positive cocci and microaerophilic streptococci account for 10-20% of anaerobic isolates recovered from properly obtained specimens of pulmonary infections. The pulmonary infections in which these organisms have been found most frequently include aspiration pneumonia, empyema associated with aspiration pneumonia, lung abscesses, and mediastinitis.

Obtaining appropriate culture specimens of these organisms requires the use of transtracheal aspiration, aspiration through double-lumen catheterization, or direct lung puncture.

Intra-abdominal infections

Because anaerobic gram-positive cocci are part of the normal gastrointestinal florae, they can be isolated in approximately 20% of specimens from intra-abdominal infections, such as peritonitis and abscesses of the liver, spleen, and abdomen.

Anaerobic gram-positive cocci are generally recovered mixed with other organisms of intestinal origin that include Escherichia coli, Bacteroides fragilis group, and Clostridium species.

Female pelvic infections

Anaerobic gram-positive cocci and microaerophilic streptococci can be isolated in 25-50% of patients with endometritis, pyoderma, pelvic abscess, Bartholin gland abscess, postsurgical pelvic infections, or pelvic inflammatory disease. The origin of these organisms is probably the vaginal and cervical florae.

The predominant anaerobic gram-positive cocci are P asaccharolyticus, P anaerobius, and P prevotii.

Bacteremias with anaerobic gram-positive cocci and microaerophilic streptococci are often associated with septic abortion.

Anaerobic gram-positive cocci are generally found mixed with Prevotella bivia and Prevotella disiens.

Osteomyelitis and arthritis

Anaerobic gram-positive cocci are frequently isolated from anaerobically infected bones and joints. In studies, they accounted for 40% of anaerobic isolates of osteomyelitis caused by anaerobic bacteria and 20% of anaerobic isolates of arthritis caused by anaerobic bacteria.

P magnus and P prevotii are the predominant bone and joint isolates. In a 1980 study by Bourgault and colleagues, most patients with infections involving these organisms underwent orthopedic surgery and had foreign prosthetic material in place at the time of infection. Management of these infections requires prolonged courses of antimicrobials and is enhanced by removal of the foreign material.

Skin and soft tissue infections

Anaerobic gram-positive cocci and microaerophilic streptococci are often recovered in polymicrobial skin and soft tissue infections (eg, necrotizing synergistic gangrene; necrotizing fasciitis; decubitus ulcers; diabetes-related foot infections; paronychia; burns; human or animal bites; infected cysts; abscesses of the breast, rectum, and anus). Anaerobic gram-positive cocci and microaerophilic streptococci are generally found mixed with other aerobic and anaerobic florae that originate from the mucosal surface adjacent to the infected site or that have been inoculated into the infected site.

Gastrointestinal florae can cause infections such as gluteal decubitus ulcers, diabetes-related foot infections, and rectal abscesses.

Vaginal and cervical florae can cause scalp wound infections in newborns after fetal monitoring.

Because anaerobic gram-positive cocci and microaerophilic streptococci are part of the normal skin florae, care must be used when obtaining specimens to avoid contamination by these florae.

Bacteremia and endocarditis

Anaerobic gram-positive cocci and microaerophilic streptococci may be responsible for 4-15% of anaerobic bacteria isolated from blood cultures of patients with clinically significant anaerobic bacteremia. They are often recovered in persons with puerperal sepsis.

Peptostreptococci can cause fatal endocarditis, paravalvular abscess, and pericarditis.

The most frequent source of bacteremia due to Peptostreptococcus is infections of the oropharynx, lower respiratory tract, female genital tract, abdomen, skin, and soft tissues.

Predisposing factors for bacteremia due to Peptostreptococcus include malignancy; recent gastrointestinal, obstetrical, or gynecological surgery; immunosuppression; dental procedures; and oropharyngeal, female genital tract, abdominal, and soft tissue infections.

Microaerophilic streptococci typically account for 5-10% of cases of endocarditis; however, peptostreptococci have only rarely been isolated.

Causes:

The following are the major predisposing conditions to infection with anaerobic gram-positive cocci and microaerophilic streptococci:

Previous surgery

Immunodeficiency

Malignancy

Trauma

Diabetes

Steroid therapy

Presence of a foreign body

Sickle cell anemia

Reduced blood supply

Vascular disease

Infection with aerobic bacteria can make the local tissue conditions more favorable for the growth of anaerobes, including anaerobic cocci. Anaerobic conditions and anaerobic bacteria can impair host defenses. Anaerobic infection often manifests as suppuration, thrombophlebitis, abscess formation, and gangrenous destruction of tissue associated with gas. Anaerobes, including peptostreptococci, are common in chronic infections. Therapy with antimicrobials (eg, aminoglycosides, trimethoprim-sulfamethazine, older quinolones) often does not eradicate anaerobes.

Lab Studies:

Microbiology

Anaerobic, microaerophilic, and facultative gram-positive cocci have minor morphological differences. P magnus has a larger diameter than other anaerobic gram-positive cocci. P micros has a smaller diameter than other anaerobic gram-positive cocci and usually forms short chains. P anaerobius and Peptostreptococcus productus are elongated and often appear in pairs or chains.

LABORATORY INDICATIONS:

Esculin hydrolysis –

Hydrogen sulfide –

Catalase –

Lactose –

Gas-liquid chromatography and biochemical tests are required for genus-level identification and separation of most anaerobic gram-positive cocci. These organisms are fastidious, and their complete identification is often difficult. Because of ill-defined differences in the pathogenic potential for the different species, the need for exact specification is controversial.

Anaerobic cocci show slow but adequate growth on all nonselective anaerobic growth media. Vancomycin-containing selective media inhibit their growth.

Recovery in clinical specimens

Anaerobic and facultative gram-positive cocci are often isolated from clinical specimens mixed with other anaerobic or aerobic bacteria and, on rare occasions, are isolated as the sole pathogen. As a group, these organisms are the most frequently recovered anaerobes in cutaneous, oral, respiratory tract, and female genital tract infections.

Collecting anaerobic bacteria specimens is important because documentation of an anaerobic infection is through culture of organisms from the infected site. Documentation requires proper collection of appropriate specimens, expeditious transportation, and careful laboratory processing.

Obtain uncontaminated specimens. Inadequate culture techniques or media can lead to faulty results and the incorrect conclusion that only aerobic organisms are present in a mixed infection. Specimens must be obtained free of contamination. Inadequate techniques or media can lead to missing the presence of anaerobic bacteria or the assumption that only aerobic organisms are present in a mixed infection.

Because anaerobes are present on mucous membranes and skin, even minimal contamination with normal florae can be misleading.

Unacceptable or inappropriate specimens can also yield normal florae and therefore have no diagnostic value. Obtain appropriate specimens using techniques that bypass the normal florae.

Direct-needle aspiration is the best method of obtaining a culture. Direct-needle aspiration is probably the best method of obtaining a culture, and the use of swabs is much less desirable.

Specimens obtained from normally sterile sites, such as blood, spinal, joint, or peritoneal fluids, are collected after thorough skin decontamination.

Two approaches are used to culture the maxillary sinus following sterilization of the canine fossa or the nasal vestibule, either via the canine fossa or via the inferior meatus.

Urine collected is collected by percutaneous suprapubic bladder aspiration.

Other specimens can be collected from abscess contents, from deep aspirates of wounds, and by special techniques, such as transtracheal aspirates or direct lung puncture.

Specimens of the lower respiratory tract are difficult to obtain without contamination with indigenous florae. Double-lumen catheter bronchial brushing and bronchoalveolar lavage, cultured quantitatively, can be useful.

Culdocentesis fluid obtained after decontamination of the vagina is acceptable.

Transportation of specimens should be expeditious. Place specimens into an anaerobic transporter as soon as possible. These devices generally contain oxygen-free environments provided by a mixture of carbon dioxide, hydrogen, and nitrogen plus an aerobic condition indicator.

Liquid or tissue specimens are always preferred to swabs.

Inoculate liquid specimens into an anaerobic transport vial or a syringe. All air bubbles are expelled from the syringe. Insertion of the needle tip into a sterile rubber stopper is no longer recommended. Because air gradually diffuses through the plastic syringe wall, specimens should be processed in less than 30 minutes.

Transport tissue specimens in an anaerobic jar or a sealed plastic bag rendered anaerobic.

If swabs are used, place them in sterilized tubes containing carbon dioxide or prereduced, anaerobically sterile Carey and Blair semisolid media.

Gram stain of a smear of the specimen provides important preliminary information regarding types of organisms present, suggests appropriate initial therapy, and serves as a quality control. Immediately place cultures under anaerobic conditions and incubate for 48 hours or longer. An additional 36-48 hours is usually required for species- or genus-level identification using biochemical tests; kits containing these tests are commercially available.

A rapid enzymatic test enables identification after only 4 hours of aerobic incubation. Gas-liquid chromatography of metabolites is often used. Nucleic acid probers and polymerase chain reaction methods are also being developed for rapid identification. Detailed procedures of laboratory methods can be found in microbiology manuals.

Antimicrobial susceptibility test results of peptostreptococci have become less predictable because of the increasing resistance of peptostreptococci to several antimicrobials. Routine susceptibility testing is time consuming and often unnecessary; however, it is important to test the susceptibility of isolates recovered from sterile body sites, those that are clinically important and have variable susceptibilities, and especially those isolated in pure cultures from properly collected specimens. These include isolates associated with bacteremia; endocarditis; and bone, joint, or skull infections.

Perform testing with antibiotics. Recommended methods include agar microbroth and macrobroth dilution. Newer methods include the E-test and the spiral gradient end point system. Agents that should be tested include penicillin, broad-spectrum penicillin, penicillin plus a beta-lactamase inhibitor, clindamycin, chloramphenicol, second-generation cephalosporins (eg, cefoxitin), newer quinolones, metronidazole, and carbapenems.

Imaging Studies:

Radiological or imaging studies are helpful. The presence of gas in the infected site is a strong indication of anaerobic infection.

Medical Care: A patient's recovery from anaerobic infection depends on prompt and proper treatment according to the following principles: (1) neutralizing toxins produced by anaerobes, (2) preventing local bacterial proliferation by changing the environment, and (3) limiting the spread of bacteria.

Control the environment by debriding necrotic tissue, draining pus, improving circulation, alleviating obstruction, and increasing tissue oxygenation. Certain types of adjunctive therapy, such as hyperbaric oxygen therapy, may be useful but remain unproven.

In many cases, antimicrobial therapy is the only form of therapy required, but it can also be an adjunct to a surgical approach. Because anaerobic bacteria, including peptostreptococci, are generally recovered mixed with aerobic organisms, choose antimicrobial agents that treat both types of pathogens, taking into consideration their aerobic and anaerobic antibacterial spectrum and their availability in oral or parenteral form.

Penicillin G is most effective for treating anaerobic gram-positive cocci and microaerophilic streptococci.

Other effective agents include other penicillins, cephalosporins, chloramphenicol, clindamycin, vancomycin, telithromycin, linezolid, quinupristin/dalfopristin, and carbapenems.

The efficacy of macrolides (eg, erythromycin) and imidazoles (eg, metronidazole) is variable and unpredictable. Imidazoles are ineffective against some anaerobic gram-positive cocci and all aerotolerant strains.

The newer quinolones are effective against more than 90% of anaerobic cocci; ciprofloxacin is less effective.

Occasionally, certain strains are resistant to antimicrobials, especially after administration of these agents.

When mixed with other beta-lactamase–producing bacteria, anaerobic gram-positive cocci and microaerophilic streptococci may survive penicillin or cephalosporin therapy because of the protection provided by the free enzyme. In such instances, antimicrobials with wider spectrums of activity may be more effective.

Surgical Care: In most cases, surgical therapy is critically important. Surgical therapy includes (1) draining abscesses, (2) debriding necrotic tissues, (3) decompressing closed-space infections, and (4) relieving obstructions. If surgical drainage is not used, the infection may persist and serious complications may develop.

BACTEROIDES

Bacteroides are not E. coli! They are not even that closely related to eachother. However they can both be found in the same place: the intestine. Each and every one of us contain many billions of these bugs inside their gut. Bacteroides are specialists in this environment as they are adapted to grow where there is no oxygen. E. coli can grow both with and without oxygen and is consequently a generalist and not as good at growing in either condition as a true anaerobe (B. fragilis) or a true aerobe (Bacillus subtillus). In fact Bacteroides are one of the most numerous of the intestinal bugs and we get to see a great many everyday as about 30 % of what comes out of the intestine is bacteria! Most of the time we get on perfectly well with Bacteroides, in fact they assist in breaking down food products and supply some vitamins and other nutrients that we cannot make ourselves. The problem with Bacteroides is when they get out of the intestine and into our bodies. One of the most common results of this is an abscess, which is a big ball of puss comprised mostly of bacteria (especially B. fragilis). If the ball breaks then billions of bacteria wreak havok in the body often resulting in death. Luckily this dosn't happen too often as bacteria are susceptable to antibiotics. Unfortunately the Bacteroides are very good at finding ways to become resistant to all of the antibiotics that we use so developing new ways to fight the bugs is a great importance.

Phylogeny

Anaerobes comprise the majority of bacteria in the human colon; the most numerically predominant of these are members of the genus Bacteroides. Originally described in 1898, for many years the Bacteroides were a vague conglomeration of host-associated, obligately anaerobic, gram-negative, pleomorphic rods that could not be convincingly assigned to any other genera. Physiological analysis of this genus revealed considerable heterogeneity with regard to their biochemical properties, indicating these bacteria did not represent a true phylogenetic grouping. With the advent of phylogenetic analysis techniques, several investigators have tried to redefine this group of bacteria using physiological characteristics, serotyping, bacteriophage typing, lipid analysis, oligonucleotide cataloging, and 5S - 16S rRNA sequence comparisons. Based on this information, the original Bacteroides members have been partitioned into three genera: Bacteroides, Prevotella, and Porphyromonas. The Bacteroides are found predominantly in the colon of mammals, while the Prevotella and Porphyromonads generally are associated with the oral cavity and the rumen. The current definition of Bacteroides species is as follows: a) obligately anaerobic, Gram-negative, b) saccharolytic, producing acetate and succinate as the major metabolic end products, c) contain enzymes of the hexose monophosphate shunt-pentose phosphate pathway, d) have a DNA-base composition in the range 40-48 mol% GC, e) membranes contain sphingolipids, and contain a mixture of long-chain fatty acids, mainly straight chain saturated, anteiso-methyl, and iso-methyl branched acids, f) possess menaquiones with MK-10 and MK-11 as the major components, and g) contain meso-diaminopimelic acid in their peptidoglycan. This definition restricts the Bacteroides to ten species: B. fragilis, B. thetaiotaomicron, B. vulgatus, B. ovatus, B. distasonis, B. uniformis, B. stercoris, B. eggerthii, B. merdae, and B. caccae, with B. fragilis as the type strain. The Bacteroides, along with Prevotella and Porphyromonas, form one major subgroup in the bacterial phylum Cytophaga-Flavobacter-Bacteroides. This phylum diverged quite early in the evolutionary lineage of bacteria, and thus the Bacteroides, although gram-negative organisms, are not closely related to the enteric gram-negatives such as Escherichia coli.

Bacteroides as Commensal Organisms

  The Bacteroides inhabit the human colon, which contains the largest, most complex bacterial population of any colonized area of the human body. The colonic contents contain in excess of 1011 organisms per gram of wet weight, representing over 400 species.

The Bacteroides are the most numerous members of the normal flora, representing nearly 1011 organisms per gram of feces (dry weight). Gut organisms are involved in numerous metabolic activities in the colon, including fermentation of carbohydrates, utilization of nitrogenous substances, and biotransformation of bile acids and other steroids. In order to maintain their high numbers, the Bacteroides are evidently able to compete with other members of the flora, as well as transient organisms, for utilization of these resources. While the role of the microflora in the physiology of the human intestine is not well studied, it is clear that the anaerobic members of this ecosystem play a fundamental role in the processing of complex molecules into simpler compounds, and through their metabolic activities the human microflora participate in the complex physiology of the host.

Most intestinal bacteria are saccharolytic, obtaining carbon and energy by hydrolysis of host and dietary carbohydrate molecules. Simple sugars are rarely encountered in the colon as most are absorbed in the small intestine, however it is estimated that approximately 2% of simple sugars can pass through the upper gastrointestinal tract when large amounts of starch and complex carbohydrates are also present during digestion. Bacteroides species are able to utilize simple sugars when present, but due to their limited availability, simple sugars are probably not the main source of energy for the Bacteroides. Much more prevalent in the colon are polysaccharides, from dietary sources and host cells. Polysaccharides from plant fibers, such as cellulose, xylan, arabinogalactan, and pectin, and vegetable starches such as amylose and amylopectin contain Bacteroides have been shown to have a variety of glucosidase activities, including a beta-1,3-glucosidase activity responsible for laminarin degradation, and a variety of a and b-1, 4 and -1, 6 xylosidase and glucosidase activities induced by the presence of hemicellulose. Originally it was believed that these enzymatic activities were extracellular, and the short oligosaccharides and monosaccharides produced by hydrolysis were taken up into the cell for fermentation. Analysis of the B. thetaiotaomicron starch utilization system (sus), has revealed the polysaccharides to be bound to an outer membrane receptor system, and pulled into the periplasm for degradation into monosaccharides. The Bacteroides use a similar approach for uptake and degradation of chondroitin sulfate, indicating this technique may provide a competitive advantage in the human gut, as polysaccharides sequestered in the periplasm are less likely to be "stolen" by other intestinal organisms or lost by diffusion.

Interestingly, utilization of chondroitin sulfate by Bacteroides thetaiotaomicron is repressed in the presence of glucose, while utilization of other sugars in B. thetaiotaomicron is tightly regulated in the presence of mannose. This implies the Bacteroides may have a catabolite repression mechanism to allow for the utilization of select carbon sources in preference to others. If so, this system is probably not similar to the catabolite repression systems of enteric bacteria, as the Bacteroides do not possess cyclic AMP. It is likely that most Bacteroides polysaccharide utilization systems are controlled by repressor/inducer mechanisms, as B. ovatus and B. thetaiotaomicron are able to utilize several sugars simultaneously, and several polysaccharide utilization genes have been shown to be activated in the presence of their substrate. Carbohydrate fermentation by the Bacteroides and other intestinal bacteria results in the production of a pool of volatile fatty acids, predominately acetate, propionate (from succinate), and butyrate. These short chain fatty acids are reabsorbed through the large intestine, and utilized by the host as an energy source. It has been estimated that absorption of the short chain fatty acids could provide up to 540 kcal/d, a significant proportion of the host's daily energy requirement.

The utilization of nitrogen sources by the intestinal Bacteroides is not well understood, as most work in the area of nitrogen uptake has been done with rumen organisms. However, several parallels may be drawn between intestinal and rumen bacteria, providing a paradigm of nitrogen utilization in the human gut. There are three major sources of nitrogen in the mammalian intestine: dietary protein, epithelial cell and mucin glycoproteins, and ammonia. Most dietary protein is degraded and absorbed before reaching the large intestine, but once in the colon, these peptides and amino acids are not able to be absorbed by the host. Instead, a two step degradation process occurs, during which peptides are proteolysed to amino acids, which are subsequently deaminated to form ammonia, CO2, volatile fatty acids, and branched chain fatty acids. The ammonia is utilized by the intestinal bacteria as a nitrogen source. Bacteroides fragilis has been shown to produce three major proteases, with activity against a variety of proteins, including casein, trypsin, and chymotrypsin, but not collagen, elastin, or gelatin. The Bacteroides also encode glutamine synthetase and glutamate dehydrogenase, which are important for ammonia assimilation but the regulation of these activities is not yet understood.

The Bacteroides play a key role in the enterohepatic circulation of bile acids. Cholic acid and chenodeoxycholic acid are the two main bile acids synthesized in the human liver, where they are conjugated to taurine or glycine polar side groups before secretion in bile. Once bile enters the gut, the conjugated bile acids assist in the absorption of dietary fats. If the bile acids are not reabsorbed in association with fat in the upper intestine, they are deconjugated by bacteria to secondary bile acids, primarily deoxycholic and lithocholic acid, although the microflora can generate 15-20 other secondary bile acids from these same precursors. Deconjugation allows the bile acids to reenter the enterohepatic circulation via the portal system, where they are returned to the liver and reconjugated for further use. The secondary bile acids deoxycholic and lithocholic acid are produced by 7 alpha-dehydrogenation of the primary bile acids; once these secondary bile acids are produced, a variety of other bacterial reactions can occur, including oxidation-reduction, desulphation, and dehydrogenation, producing a variety of isomers of secondary bile acids. The Bacteroides have been found to play a major role in the biotransformation of bile acids, and contain many enzymes required for these reactions, including a hydrolase, dehydrogenase, and dehydroxylase. The direct benefit to the host is obvious, as deconjugation of the primary bile acids allow them to be reabsorbed in the large intestine instead of lost in the feces. The benefit to the Bacteroides and other intestinal bacteria is not clear, but may contribute to energy metabolism.

Aside from their metabolic activities, the Bacteroides and other anaerobes provide an additional benefit to their host in excluding pathogenic organisms from colonizing the intestine. Colonization resistance mediated by anaerobes is thought to occur by four mechanisms: competition for nutrients, competition for intestinal wall attachment sites, production of volatile fatty acids, and release of free bile acids. The intestinal microflora adhere to the surface of epithelial cells and mucin associated with the intestinal wall, with Bacteroides being the most common anaerobic colonizer. By coating the walls of the intestine, it is believed that the microflora prevent transient bacteria from obtaining a binding site on the intestinal surface, and the transients are subsequently lost with the luminal contents during peristalsis. The volatile fatty acids produced as metabolic end products by the Bacteroides are also believed to play a role in colonization resistance, by lowering the pH and oxidation-reduction potential of the intestinal milieu, resulting in unfavorable growth conditions for transient bacteria. The most notable pathogens inhibited under these conditions are Salmonella enteritidis, and Shigella flexineri. Production of free bile acids also plays a role in inhibition of pathogens, as bile salts are toxic to many organisms, including Clostridium botulinum.

Pathogenicity and Virulence

  While the Bacteroides occupy a significant position in the normal flora, they also are opportunistic pathogens, primarily in infections of the peritoneal cavity. B. fragilis is the most notable pathogen; although it makes up only 1-2% of the normal flora, it is the Bacteroides species isolated from 81% of anaerobic clinical infections. B. fragilis is not overtly invasive, but is capable of participating in intraabdominal infections in the event the mucosal wall of the intestine is disrupted. Incidences during which Bacteroides infections may be initiated include gastrointestinal surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma, and inflammatory bowel disease.

The current model for development of abdominal infections is based on the concept of synergism, during which cooperation between different species of bacteria aids in the establishment of persistent infection. Synergism has been most clearly established in infections involving both E. coli and B. fragilis, although other combinations of aerobes and anaerobes also are synergistic. After disruption of the intestinal wall, members of the normal flora infiltrate the normally sterile peritoneal cavity, and during the early, acute stage of infection (approximately 20 hours), the aerobes, such as E. coli, are the most active members of infection, establishing preliminary tissue destruction and reducing the oxidation-reduction potential of the oxygenated tissue. Once sufficient oxygen has been removed to allow the anaerobic Bacteroides to replicate, these bacteria begin to predominate during the second, chronic stage of infection.

The Bacteroides contribute to development of a synergistic infection in three ways: stimulation of abscess formation, reduced phagocytosis by polymorphonuclear leukocytes (PMN's), and inactivation of antibiotics by b lactamase production. Abscess formation is a major complication of intestinal infections, and results in the formation of a fibrous membrane surrounding a mass of cellular debris, dead PMN's, and a mixed population of bacteria. If not removed, the abscess will expand, possibly causing intestinal obstruction, erosion of resident blood vessels, and ultimately fistula formation. Abscesses may also metastasize, resulting in bacteremia and disseminated infection. Formation of the abscess is a pathological response of the immune system to the presence of the Bacteroides capsular polysaccharide. B. fragilis is the only bacterium that has been shown to induce abscess formation as the sole infecting organism. Purified capsule can stimulate formation of a histologically identical abscess, indicating that it is this component of the bacterium which stimulates the host immune system to deposit fibrin, forming the outer membrane of the abscess. The Bacteroides capsule has been shown to have an unusual structure, composed of repeating units of two distinct polysaccharides, each of which contains exposed positively and negatively charged side-chains. Most bacterial polysaccharides stimulate an antibody-mediated immune response, but the B. fragilis capsule stimulates a T cell-mediated response. Presumably, the intention of the cell-mediated immune response is to wall off the infection and protect the host from dissemination, but in fact, formation of an abscess protects the Bacteroides and neighboring bacteria from exposure to high concentrations of antibiotics and further attack from the immune system.

Another important synergistic virulence factor of B. fragilis is the ability to inhibit phagocytosis. Once the Bacteroides actively begin to replicate, they are able to interfere with attack by the immune system in two ways. First, production of the capsule itself is able to reduce the ability of the PMN's to phagocytose the bacterial cells. Secondly, the Bacteroides are able to secrete an as yet uncharacterized factor which degrades complement proteins, and thus inhibits both chemotaxis of PMN's and opsonization of itself and neighboring bacteria.
A final contribution of the Bacteroides to a successful synergistic infection is the production of b-lactamase. Most Bacteroides strains express constitutive b-lactamase activity; the enzyme is extra-cellular, and thus is capable of diffusing within an abscess or other site of infection. Production of extra-cellular b-lactamases has been shown to protect other organisms in the vicinity during a mixed infection. These bacteria have several other features that contribute to their pathogenicity. The Bacteroides are among the most aerotolerant of anaerobes, able to tolerate atmospheric concentrations of oxygen for up to three days. During initiation of an intraabdominal infection, oxygen tolerance is believed to allow the bacteria to survive in the oxygenated tissue of the abdominal cavity until E. coli and other synergistic organisms are able to reduce the redox potential at the site of infection. Additionally, this oxygen tolerance may help in surviving free radical production by the immune system PMNs. Bacteroides have been found to encode two major oxidative stress response genes, catalase and superoxide dismutase, as well as approximately 28 other oxygen-induced proteins.

Although a commensal organism, Bacteroides can occasionally cause diarrhea. Strains of Bacteroides isolated from some patients with undiagnosed diarrhea were found to be enterotoxigenic, and in patients less than three years age they were associated with intestinal cramping, vomiting, and bloody stools. The purified toxin, fragilysin, was found to be a metalloprotease capable of hydrolysing gelatin, actin, tropomyosin, and fibrinogen. In a study comparing the frequency of B. fragilis enterotoxigenic and non-enterotoxigenic bacteria involved in various infection sites, the enterotoxic strains were found in higher frequencies in bacteremias. It is possible that fragilysin is involved in releasing the organism from an abscess or other site of infection and allowing it to enter the blood stream, thus disseminating infection throughout the body.

The Bacteroides genus of anaerobic bacteria comprise the majority of microorganisms that inhabit the digestive tract. 50% of most fecal matter is actually Bacteroides fragilis cells! Bacteroides organisms are the anaerobic counterpart of E. coli except they are somewhat smaller. They grow well on blood agar, and under the microscope, they may contain large vacuoles that are similar in appearance to spores. Members of Bacteroides species are not spore-forming, but they do produce a very large capsule. Their pathogenicity is limited, however, because they possess no endotoxin in their cell membrane. Infection only occurs after severe trauma to the abdominal region. Infection could lead to abscess formation and possibly fever. Antibiotic treatment usually consists of metronidazole or clindamycin.

Antibiotic Resistance

B. fragilis is the most common anaerobic organism isolated from clinical infections, and untreated has a mortality rate of 60%. This mortality rate can be greatly improved, however, with use of appropriate antimicrobial therapy. The Bacteroides are potentially resistant to a broad range of antibiotics, and resistance to a given antimicrobial can vary greatly between institutions. Resistance to any antimicrobial agent may occur by three mechanisms: altered target binding affinity, decreased permeability for the antibiotic, or the presence of an inactivating enzyme. The Bacteroides are adept at antimicrobial evasion, and may use any or all of the above mechanisms to thwart effective clinical therapy. Antimicrobial agents may target several areas of bacterial physiology: protein translation, nucleic acid synthesis, folic acid metabolism, or cell wall synthesis. Protein synthesis inhibitors bind either the 30s subunit of the ribosome (aminoglycosides, tetracycline), or the 50s subunit (macrolides, lincosamides, chloramphenicol). Bacteroides are inherently resistant to aminoglycosides, as uptake of this drug is energy dependent, and requires an oxygen or nitrate dependent electron transport chain which is lacking in these anaerobes. The Bacteroides have acquired resistances to the other protein synthesis inhibitors; resistance to clindamycin/erythromycin (macrolide-lincosamide antibiotics), and tetracycline will be discussed as pertinent examples.

Laboratory Identification:

Collection of specimens of anaerobic bacteria is important because documentation of an anaerobic infection is through culture of organisms from the infected site. Appropriate documentation of anaerobic infection requires proper collection of appropriate specimens, expeditious transportation, and careful laboratory processing.

Specimens must be obtained free of contamination. Inadequate techniques or media can lead to missing the presence of anaerobic bacteria or the assumption that only aerobic organisms are present in a mixed infection.

Because anaerobes are present on skin and mucous membranes, even minimal contamination with normal florae can be misleading.

Unacceptable or inappropriate specimens can yield normal florae and, therefore, have no or little diagnostic value.

Appropriate materials should be obtained by using techniques that bypass the normal florae.

Direct-needle aspiration is the best method of obtaining a culture; the use of swabs is much less desirable.

Specimens obtained from normally sterile sites, such as blood or spinal, joint, or peritoneal fluids, are collected after thorough skin decontamination.

Two approaches are used to culture the maxillary sinus following sterilization of the canine fossa or the nasal vestibule, via either the canine fossa or the inferior meatus.

Urine is collected by percutaneous suprapubic bladder aspiration.

Other specimens can be collected from abscess contents, from deep aspirates of wounds, and by special techniques, such as transtracheal aspirates or direct lung puncture.

Specimens of the lower respiratory tract are difficult to obtain without contamination with indigenous florae. Double-lumen catheter bronchial brushing and bronchoalveolar lavage, cultured quantitatively, can be useful.

Culdocentesis fluid obtained after decontamination of the vagina is acceptable.

Transportation of specimens should be prompt unless transport devices are available. Transport devices generally contain oxygen-free environments provided by a mixture of carbon dioxide, hydrogen, and nitrogen, plus an aerobic condition indicator. Specimens should be placed into an anaerobic transporter as soon as possible.

Liquid or tissue specimens are always preferred to swabs.

Liquid specimens are inoculated into an anaerobic transport vial or a syringe and a needle.

All air bubbles are expelled from the syringe. Insertion of the needle tip into a sterile rubber stopper is no longer recommended. Because air gradually diffuses through the plastic syringe wall, specimens should be processed in less than 30 minutes.

Swabs are placed in sterilized tubes containing carbon dioxide or prereduced anaerobically sterile Carey and Blair semisolid media.

Tissue specimens can be transported in an anaerobic jar or in a sealed plastic bag rendered anaerobic.

Gram stain of a smear of the specimen provides important preliminary information regarding the types of organisms present, suggests appropriate initial therapy, and serves as a quality control.

Cultures should be immediately placed under anaerobic conditions and should be incubated for 48 hours or longer. An additional 36-48 hours is generally required for species- or genus-level identification by using biochemical tests. Kits containing these tests are commercially available.

A rapid enzymatic test enables identification after only 4 hours of aerobic incubation.

Gas-liquid chromatography of metabolites is often used.

Nucleic acid probers and polymerase chain reaction methods are also being developed for rapid identification.

Detailed procedures of laboratory methods can be found in microbiology manuals.

Antimicrobial susceptibility testing of AGNB has become less predictable because their resistance to several antimicrobials has increased. Screening of AGNB isolates for beta-lactamase activity may be helpful. However, occasional strains may resist beta-lactam antibiotics through other mechanisms.

Routine susceptibility testing is time-consuming and often unnecessary. However, testing the susceptibility of isolates recovered from sterile body sites and/or those that are clinically important (ie, blood cultures, bone, CNS, serious infections) and have variable susceptibilities, especially those isolated in pure culture from properly collected specimens, is important.

Antibiotics that should be tested include penicillin, a broad-spectrum penicillin, a penicillin plus a beta-lactamase inhibitor, clindamycin, chloramphenicol, a second-generation cephalosporin (eg, cefoxitin), newer quinolones, metronidazole, and a carbapenem.

The recommended methods include agar microbroth and macrobroth dilution.

Newer methods include the E-test and the spiral gradient end point system.

  Specimen collection to avoid contamination with normal flora

  Oxygen-free transport medium system

  Avoid drying

Bacteroides spp. grow rapidly (within two days) but most other anaerobes are slow growers on selective media

B. fragilis are resistant to kanamycin, vancomycin and colistin

B. fragilis growth is stimulated in the presence of 20% bile

LABORATORY INDICATIONS (B. fragilis):

Indole -

Catalase +

Esculin hydrolysis +

Glucose fermentation

Lactose +

Treatment, Prevention & Control:

  Surgical drainage of abscess(es) and removal of necrotic tissue(s)

  Long-term course of antibiotics

  Prophylatic use of antibiotics

  Prior to invasive surgical procedures that disrupt mucosal barriers

  Immediately following trauma that disrupts mucosal barriers

PREVOTELLA

P. albensis; P. baroniae; P. bergensis; P. bivia; P. brevis; P. bryantii; P. buccae; P. buccalis; P. corporis; P. dentalis; P. denticola; P. disiens; P. enoeca; P. genomosp. C1; P. intermedia; P. loescheii; P. marshii; P. melaninogenica; P. multiformis; P. multisaccharivorax; P. nigrescens; P. oralis; P. oris; P.oulorum; P. pallens; P. ruminicola; P.ruminicola 23; P. aff. ruminicola Tc2-24; P. salivae; P. shahii; P. tannerae; P. veroralis; P. sp

Prevotella sp. are among the most numerous microbes culturable from the rumen and hind gut of cattle and sheep, where they help the breakdown of protein and carbohydrate foods. They are also present in humans, where they can be opportunistic pathogens. Prevotella, credited interchangably with Bacteroides melaninogenicus, has been a problem for dentists for years. As a human pathogen known for creating periodontal and tooth problems, Prevotella has long been studied in order to counteract its pathogenesis (AAP).

Prevotella strains are Gram-negative, non-motile, rod-shaped, singular cells that thrive in anaerobic growth conditions. They are known for being host-associated, colonizing the human mouth. Prevotella bacteria colonize by binding or attaching to other bacteria in addition to epithelial cells, creating a larger infection in previously infected areas. Another survival mechanism is Prevotella cells' natural antibiotic resistant genes, which prevent extermination.

Pathology

About twenty identified species of Prevotella are known to cause infection, including Prevotella dentalis, which was previously known as Mitsuokella dentalis. Prevotella species cause infections such as abscesses, bacteraemia, wound infection, bite infections, genital tract infections, and periodontitis (Pavillion). Specific infections caused by Prevotella include the disease of tissues surrounding an individuals teeth (see photo at right) and of the supporting tooth and gingivitis (TIGR). Symptoms of Prevotella infections can include pain, swelling, and in some cases a "wet" canal (Gomes).

Disease shown in Xray cause by Prevotella oralis.

Antibiotics for treating Prevotella include metronidazole, amoxycillin/clavulanate, ureidopenicilins, carbapenems, cephalosporins, clindamycin, and chloramphenicol (Pavillion).

Prevotella is also well-known as a preventative agent for the bovine disease of rumen acidosis. Rumen acidosis greatly affects milk production of cattle by disrupting the typical digestive processes of the stomach. This leads to an increased susceptibility to other pathogenic forces which also affect the health of food provided from the cattle. With an estimated twenty percent of all American cattle suffering from some form of acidosis, it has been calculated that the bovine market loses one billion dollars annually (ARS).

CLINICAL MANIFESTATIONS: Bacteroides and Prevotella species from the oral cavity can cause chronic sinusitis, chronic otitis media, dental infection, peritonsillar abscess, cervical adenitis, retropharyngeal space infection, aspiration pneumonia, lung abscess, empyema, or necrotizing pneumonia. Species from the gastrointestinal tract are recovered in patients with peritonitis, intra-abdominal abscess, pelvic inflammatory disease, postoperative wound infection, or vulvovaginal and perianal infections. Soft tissue infections include synergistic bacterial gangrene and necrotizing fasciitis. Invasion of the bloodstream from the oral cavity or intestinal tract can lead to brain abscess, meningitis, endocarditis, arthritis, or osteomyelitis. Skin involvement includes omphalitis in newborn infants, cellulitis at the site of fetal monitors, human bite wounds, infection of burns adjacent to the mouth or rectum, and decubitus ulcers. Neonatal infections, such as conjunctivitis, pneumonia, bacteremia, or meningitis, occur rarely. Most Bacteroides infections are polymicrobial.

ETIOLOGY: Most Bacteroides and Prevotella organisms associated with human disease are pleomorphic, nonspore-forming, facultatively anaerobic, gram-negative bacilli. Bacteroides and Prevotella species produce enzymes that play a role in the pathogenesis of disease.

EPIDEMIOLOGY: Bacteroides and Prevotella species are part of the normal flora of the mouth, gastrointestinal tract, or female genital tract. Members of the Bacteroides fragilis group predominate in the gastrointestinal tract flora; members of the Prevotella melaninogenica (formerly Bacteroides melaninogenicus) and Prevotella oralis (formerly Bacteroides oralis) groups are more common in the oral cavity. These species cause infection as opportunists, usually after an alteration of the body’s physical barrier, and in conjunction with other endogenous species. Endogenous transmission results from aspiration, spillage from the bowel, or damage to mucosal surfaces from trauma, surgery, or chemotherapy. Mucosal injury or granulocytopenia predispose to infection. Except in infections resulting from human bites, no evidence for person-to-person transmission exists.

The incubation period is variable and depends on the inoculum and the site of involvement but usually is 1 to 5 days.

DIAGNOSTIC TESTS: Anaerobic culture media are necessary for recovery of Bacteroides or Prevotella species. Because infections usually are polymicrobial, aerobic cultures also should be obtained. A putrid odor suggests anaerobic infection. Use of an anaerobic transport tube or a sealed syringe is recommended for collection of clinical specimens.

TREATMENT: Abscesses should be drained when feasible; abscesses involving the brain or liver may resolve with effective antimicrobial therapy. Necrotizing lesions should be débrided surgically.

The choice of antimicrobial agent(s) is based on anticipated or known in vitro susceptibility testing. Bacteroides infections of the mouth and respiratory tract generally are susceptible to penicillin G, ampicillin sodium, and broad-spectrum penicillins, such as ticarcillin disodium or piperacillin sodium. Clindamycin is active against virtually all mouth and respiratory tract Bacteroides and Prevotella isolates and is recommended by some experts as the drug of choice for anaerobic infections of the oral cavity and lungs. Some species of Bacteroides and Prevotella produce ß-lactamase. A ß-lactam penicillin active against Bacteroides combined with a ß-lactamase inhibitor can be useful to treat these infections (ampicillin-sulbactam sodium, amoxicillin-clavulanate potassium, ticarcillin-clavulanate, or piperacillin-tazobactam sodium). Bacteroides species of the gastrointestinal tract usually are resistant to penicillin G but are predictably susceptible to metronidazole, chloramphenicol, and usually, clindamycin. More than 80% of isolates are susceptible to cefoxitin sodium, ceftizoxime sodium, and imipenem. Cefuroxime, cefotaxime sodium, and ceftriaxone sodium are not reliably effective.

ISOLATION OF THE HOSPITALIZED PATIENT: Standard precautions are recommended.

CONTROL MEASURES: None.

Porphyromonas

Porphyromonas, which are commonly found in the human body and especially in the oral cavity, were originally classified in the Bacteroides genus. Porphyromonas gingivalis are an oral anaerobe associated with periodontal lesions, infections, and adult periodontal disease. Approximately 70-90% of people pubescent and older have gingivitis, an oral inflammatory process and a possible precursor to adult periodontal disease, which is associated with Porphyromonas gingivalis. Gingivitis allows Porphyromonas gingivalis to further infect the areas near the root of the teeth causing tooth decay and infection.

Porphyromonas gingivalis possesses an armamentarium of cell-surface associated and extracellular activities, which are studied intensively for their virulence potential. Several are putative adhesins which interact with other bacteria, epithelial cells, and extracellular matrix proteins. Secreted or cell-bound enzymes, toxins, and hemolysins may play a significant role in the spread of the organism through tissue, in tissue destruction, and in evasion of host defenses.

Three groups reported oral epithelial cell invasion by laboratory and clinical isolates of P. gingivalis. Recently it was reported that P. gingivalis invasion was accompanied by intracellular calcium-fluxes and inhibited by cytochalasin D and nocodazole, indicating that rearrangements in the cytoskeleton of the epithelial cell are necessary for internalization. The authors also report that protein kinase signaling pathways within epithelial cells are associated with P. gingivalis invasion as in other systems. The genes associated with invasion are being investigated.

Biochemical, immunological and genetic evidence indicates that P. gingivalis fimbriae are involved in adhesion to both saliva-coated hydroxy-apatite and to human oral epithelial cells. Animal studies suggest fimbriae play a role in host colonization.

Porphyromonas gingivalis possesses three major proteolytic activities with a) trypsin-like, b) collagenolytic, and c) glycylprolyl peptidase activities. Numerous proteins with thiol-dependent trypsin-like cleavage specificity can be isolated from cells and culture supernatants, but biochemical and genetic analyses indicate that many of these are derived by proteolytic processing of a larger, cell-associated, primary gene product. Structurally, the proteases contain a propeptide sequence, an N-terminal active site and a C-terminal putative adhesin domain with repeat regions. Recent studies indicate that P. gingivalis possesses a family of genes which contains part of the protease sequences. These proteases also possess hemagglutinating activity and share extensive DNA sequence homology with hemagglutinin genes hagA and D.

The black pigmentation of P. gingivalis is due to the accumulated hemin used as an iron source for growth. The organism appears to lack known siderophore activities and must use alternate mechanisms to sequester and transport exogenous iron. The expression of several outer membrane proteins is induced or repressed by heme, and a heme-repressible outer surface protein, which is translocated to the outer surface under heme-limiting conditions, is able to bind hemin. It was showed that hemin binding was induced by growth in hemin, a discrepancy which is attributed to differences in bacterial growth and assay conditions. P. gingivalis hemolytic activity is associated with the cell surface and outer membrane vesicles and two hemolysin genes have been cloned. Hoover and Yoshimura reported the isolation of pigment-deficient (non-heme accumulating) transposon mutants also defective in trypsin-like protease activity. These data suggest that these virulence functions may be genetically linked. Using a similar screen, Genco et al. observed that non-pigmented mutants had increased proteolytic activity.

Cell Structure and Metabolism

Porphyromonas gingivalis.

Porphyromonas are Gram-negative, nonsporeforming, anaerobic, rod-shaped bacteria that produce porphyrin pigments (dark brown/black pigments). Like Bacteroides, Porphyromonas are more closely related to Gram-positive bacteria than other Gram-negative bacteria. Also like Bacteroides, Porphyromonas have an outer membrane, a peptidoglycan layer, and a cytoplasmic membrane.

The black pigmentation of P. gingivalis is from the accumulation of hemin used as an iron source for bacterial growth. This may be a reason that people with higher metal intakes, such as iron, have more of a risk for getting gingivitis and periodontitis. Also, cell surface adhesion molecules on the surface of Porphyromonas, which interact with other bacteria, epithelial cells, and extracellular matrix proteins, assist the bacteria in living in their human host. The mouth generally has a consistant flow sugars and other simple carbohydrates, so it is likely that P. gingivalis living in peridontal tissue receive their energy from these materials. However, another common idea is that the main source of P. gingivalis energy and cell materials come mainly from peptides instead of single amino acids. However, due to the complicated amino acid composition of peptides or proteins, the amino acid metabolic pathway of P. gingivalis has been difficult to determine. Also, some enzymes involved in amino acid metabolism in these bacteria are known to be oxygen labile (changes in the presence of oxygen), which further complicates the detection and analysis of P. gingivalis metabolic enzymes.

Ecology

P. gingivalis may be one of the natural bacterial flora in the oral cavity that is comprised of over 400 different species of microorganisms. However, isolating it in a health oral cavity has proven difficult. It constitutes approximately 5% of the bacterial flora in an oral cavity with gingivitis and more than 5% in a mouth with advanced periodontitis. P. endodontalis also does not appear in a healthy mouth but can be detected in a diseased mouth. Porphyromonas also favor a slightly alkaline environmental pH. Like other bacteria that live in the human mouth, Porphyromonas favor an average temperature of around 95 degrees and a 100% humidity. It has been reported that anywhere from 1,000 to 1 billion bacteria can live on each tooth surface. P. asaccharolytica has been isolated from many nonoral sites such as the cervix, ear, intestine, genitalia, and from many infections throughout the body (only limited reports of P. asaccharolytica in the mouth). Other strains have been found in samples of blood, amniotic fluid, umbilical cord, empyema, peritoneal and pelvic abscesses, endometritis, and infections.

Pathology

Cell surface adhesion molecules on the surface of Porphyromonas interact with other bacteria, epithelial cells, and extracellular matrix proteins; they are currently being studied for their pathogenic potential. P. gingivalis is thought to spread through tissue, destroy tissue, and evade host defenses by the use of secreted cell-bound proteases, immunoactive cellular compounds, and toxins. P. gingivalis cytotoxic metabolic end products, which include butyrate, propionate, have low molecular weights which allows them to easily penetrate periodontal tissue and disrupt the host cell activity.

In the past, more research papers have been devoted to P. gingivalis that to any other dental pathogens. This is due to the high frequency in which P. gingivalis is associated with peridontal lesions, infections, and periodontitis. Projects such as The Forsyth Institute and TIGR's Porphyromonas gingivalis genome project hope to switch the method of treating periodontal diseases by surgery and tooth scaling to antibiotic or vaccine therapies.

Diagram of gingivitis.

Gingivitis, which is inflamation of the gums that causes bleeding and exposes the base of the teeth, allows Porphyromonas gingivalis to infect the areas near the root of the teeth causing tooth decay and infection. the most common type of gingivitis is brought on by the accumlation of microbial plaques in people who do not take proper care of their mouth. Pockets form around the teeth, lesions can form, bacterial infections occur, and, eventually, peridontal ligaments break down and destruction of the local aveolar bone occurs. The teeth then loosen and fall out or can be broken off. Once bacterial infections occur, the gingivitis takes on a new, infectous form called acute necrotizing ulcerative gingivitis (ANUG). ANUG can cause an accelerated destruction of affected tissues as well as local or systemic spread of infection. When ANUG spread beyond the gingiva (gums) and invades the local tissues of the mouth and face, the syndrome is called noma (cancrum oris).

fusobacteria

Fusobacterium necrophorum is part of anaerobic normal throat flora has a predisposition to abscess formation (termed 'necrobacillus' - this is very rare - affecting one per million of population) platelet aggregation and virulent toxin production results in internal jugular venous thrombosis (Lemierre's syndrome) cavitating pulmonary lesions and haemoptysis occur as a result of septic embolisation other possible features include empyema, septic arthritis, and abscesses in the liver, spleen and muscles if fusibacteria isolated on a throat swab consult local microbiologist for guidance re: treatment some strains are beta-lactimase producers so there may be advantages of prescribing a beta-lactimase inhibitor such as co-amoxiclav .

The bacterial species Fusobacterium nucleatum is one of the most frequently detected cultivable organisms in subgingival dental plaque from both inactive and active gi n givitis and periodontitis sites. F. nucleatum is the most frequent cause of gingival inflammation that initiates periodontal disease and that it is the most common predominant pathogen in subsequent periodontal destruction.4 Moreover, it is found in a number of extra-oral sites where, together with other organisms, it causes polymicrobial infections.

Despite some confusion surrounding their validity, the heterogeneous collection of bacteria characterized as F. nucleatum has been divided into a number of subspecies; namely, subspecies n u c l e at u m , vincentii, polymorphum, fusiforme and animalis, the first two of which are believed to be associated with sites of periodontal disease. In an attempt to explain differences in pathogenicity, studies in this laboratory have accordingly focused on various aspects of the physiology and metabolism of the Type strain and a clinical isolate from within each of the putative sub-species.

The growth and metabolism of Fusobacterium nucleatum

The growth and nutritional aspects of the metabolism of the various strains of F. Nucleatum were studied by growing them under continuous culture conditions in a chemostat using methods described previously. Briefly, the growth medium was a filter-sterilized chemically-defined medium (CDM). It contained a range of amino acids, a

number of vitamins, nucleotides, salts, trace elements and a fermentable carbohydrate such as glucose or fructose.10 Tween 80 was added to aid cell dispersion, as was thioglycollic acid to maintain a low redox potential. The growth temperature was 37°C, the pH controlled by the automatic addition of either 2 mol/L KOH or 2 mol/L HCl and anaerobic conditions maintained by gassing both the culture vessel and medium reservoir with a N₂/CO₂ (90:10)

mixture. Under varying conditions of growth rate and pH, growth parameters such as biomass – as measured by cell dry mass and protein content – and metabolic end-products were determined . All strains showed similar physiological and metabolic properties. For example, they grew well in various CDMs, with or without added carbohydrate, over a pH range of about 6 to 8, the optimum being between 7.0 and 8.0. In the absence of fermentable carbohydrate – most likely to occur in the subgingival environment – energy and carbon were obtained

from the fermentation of the amino acids glutamate (Glu), histidine (His), lysine (Lys) and serine (Ser). Most strains appeared to be auxotrophic for His and some for Lys. The end-products of fermentation were acetate:butyrate: formate (3:2:1), irrespective of growth rate. It is worth noting here that butyrate was shown to be a potent in vitro inhibitor of gingival fibroblast proliferation and could, therefore, be a virulence factor. In terms of interbacterial nutritional co-operation – a key to explaining the coexistence of dental plaque bacteria – the formate produced by F. nucleatum could act as an electron donor for Wolinella recta with acetate performing the same function for Eubacterium species.

The breakdown and utilization of peptides. The low levels of free amino acids usually present in the oral environment would probably be insufficient to sustain the growth of Gram-negative anaerobes, including F. nucleatum, that obtain energy from the fermentation of amino acids. Since it lacks endopeptidase activities, F. nucleatum will not grow on proteins such as casein or albumin, but organisms such as Porphyromonas gingivalis, which is often found together with F. nucleatum in active disease sites, does possess such activities. Provided that they contained the appropriate residues, resultant peptides would thus be potential energy sources for

F. nucleatum. Accordingly, the ability of resting cells of F. nucleatum to attack unsubstituted peptides containing the appropriate residues – present C- or N - terminally or buried in the peptides – was investigated. The ability of growing cells to utilizean essentially amino acid-free peptide fraction prepared from a commercial peptone was also studied.

The pathogenic potential of Fusobacterium nucleatum and its significance in development of periodontal diseases, as well as in infections in other organs, have gained new interest for several reasons. First, this bacterium has the potential to be pathogenic because of its number and frequency in periodontal lesions, its production of tissue irritants, its synergism with other bacteria in mixed infections, and its ability to form aggregates with other suspected pathogens in periodontal disease and thus act as a bridge between early and late colonizers on the tooth surface. Second, of the microbial species that are statistically associated with periodontal disease, F. nucleatum is the most common in clinical infections of other body sites. Third, during the past few years, cloning and sequencing and the application of new techniques such as PCR have made it possible to obtain more information about F. nucleatum on the level, thereby also gaining better knowledge of the structure and functions of the outer membrane proteins (OMPs). OMPs are of great interest with respect to coaggregation, cell nutrition, and antibiotic susceptibility. Several studies have shown that OMPs are involved in the pathogenicity of gram-negative bacteria.

F. nucleatum is the type species of the genus Fusobacterium, which belongs to the family Bacteroidaceae. The name Fusobacterium has its origin in fusus, a spindle; and bacterion, a small rod: thus, a small spindle-shaped rod. The term nucleatum originates from the nucleated appearance frequently seen in light and electron microscope preparations owing to the presence of intracellular granules F. nucleatum is nonsporeforming, nonmotile, and gram negative, with a G1C content of 27 to 28 mol% and a genome size of about 2.4 3 106 bp (34). Most cells are 5 to 10 mm long and have rather sharply pointed ends. Colony morphology is not a consistent parameter of the fusobacteria and is not sufficient for species identification. The bacterium is anaerobic but grows in the presence of up to 6% oxygen (204). The production of butyric acid as a major product of the fermentation of glucose and peptone, together with characteristic lipid constituents, differentiates Fusobacterium species from other anaerobic, gram-negative, nonsporeforming rods. F. nucleatum has no sialidase activity.

The species F. nucleatum is considered to be rather heterogeneous. On the bases of electrophoretic patterns of whole-cell proteins and DNA homology, there were proposed dividing F. nucleatum into three (or four) different subspecies: subspecies nucleatum, polymorphum, and vincentii.

Occurrence and Role in Periodontal Diseases. F. nucleatum is one of the most common species in human

infections and can be found in body cavities of humans and other animals.

Of the periodontal species that are statistically associated with periodontal disease, it is the most common in clinical infections of other body sites. It has been isolated from several parts of the body and from infections such as tropical skin ulcers, peritonsillar abscesses, pyomyositis and septic arthritis, bacteremia and liver abscesses, intrauterine infections, bacterial vaginosis, urinary tract infections, pericarditis and endocarditis, and lung and pleuropulmonary infections. The origin of F. nucleatum in infection has been dental in several cases. Fusobacteria, including F. nucleatum, are recovered from a variety of infections in children. Studies of the predominant cultivable oral microflora reveal that only a small number of the over 300 species found in human subgingival plaque are associated with periodontal disease. Collective microbiological studies implicate the gram-negative species Porphyromonas gingivalis,

Prevotella intermedia, Bacteroides forsythus, F. nucleatum, Capnocytophaga rectus, Eikenella corrodens, Capnocytophaga spp., certain spirochetes, and the gram-positive Eubacterium spp. in adult periodontitis. Actinobacillus actinomycetemcomitans seems to be the prime candidate in the etiology of juvenile periodontitis.

The role of F. nucleatum in the development of periodontal diseases has lately attracted new interest. Of over

51,000 isolates examined by Moore and Moore (208), F. Nucleatum and Actinomyces naeslundii were the most commonly occurring species in the human gingival crevice. From the early to the late stages of plaque formation, there is a shift from a gram-positive to a gram-negative microflora in which, among others, F. nucleatum increases in proportion as plaque forms. From studies on the bacteriology of experimental gingivitis in children (4 to 6 years) and young adults (22 to 31 years), F. nucleatum appeared to be one of the nonspirochetal organisms most closely correlated with gingivitis, and it appeared to be more common in young adults. This also seems to be the case in naturally occurring gingivitis. F. nucleatum has been detected less frequently in the first 6 months of life compared with older age groups, ranging from 25% of children below 6 months to 67% of children by 2 years, but of total anaerobic CFU, the proportion of F. Nucleatum was generally low (85, 173). In children 5 to 7 years of age, F. nucleatum is found commonly in plaque, being isolated from 60 to 70% of children examined (86). Even in juvenile periodontitis lesions, F. nucleatum has been reported in large amounts at active sites of inflammation. F. nucleatum is detected more commonly in dental plaque than on the tongue or in saliva, but these sites are a more common habitat of the organism than are the tonsils.

It has been suggested that certain combinations of bacterial species (clusters) present at the same time in the periodontal pocket are more prone to elicit periodontitis than other bacterial clusters. In experimentally induced infections in mice, strains of F. nucleatum were pathogenic when administered in pure culture; however, a mixed culture of F. nucleatum with either P. gingivalis or Prevotella intermedia was significantly more pathogenic than F. Nucleatum in pure culture. Positive correlations for disease production between F. nucleatum, C. rectus, Prevotella intermedia, and Peptostreptococcus micros have been found in periodontal as well as endodontal lesions. Recently, it was demonstrated positive associations between F. nucleatum, P. gingivalis, Prevotella intermedia, and B. Forsythus in subgingival plaque samples from untreated Sudanese patients with periodontitis. The most important finding was the effect exerted by F. nucleatum on the colonization of Prevotella intermedia; Prevotella intermedia was never detected in a site unless F. nucleatum also was present. Combinations of F. nucleatum, B. forsythus, and C. rectus or of P. gingivalis, Prevotella intermedia, and Streptococcus intermedius in sites that had the most attachment loss and the deepest pockets have been reported. F. nucleatum was also present in the majority of instances when B. forsythus was detected

However, F. nucleatum is rather widespread in periodontal pockets in general, and F. nucleatum and C. rectus were the most frequently recovered species in an analysis of the subgingival flora of randomly selected subjects; 80 to 81% of the subjects were found positive for these microorganisms. F. nucleatum has been isolated from both active and inactive sites of disease, and it has been suggested that different subgroups may vary in pathogenesis and be related to different levels of disease activity. The most common subspecies in the gingival crevice is F. nucleatum subsp. vincentii (this is also the case for other body sites), with F. nucleatum subsp. nucleatum and F. nucleatum subsp. Polymorphum following in a ratio of 7:3:2.

Growth and Metabolism

Fusobacteria require rich media for growth and usually grow well in media containing Trypticase, peptone, or yeast extract. Much attention has been paid to the utilization of amino acids and peptides by F. Nucleatum. F. nucleatum seems to be one of the few nonsporulating anaerobic species that uses amino acid catabolism to provide energy, and some strains of F. nucleatum utilize and apparently need peptides for growth. ATCC 10953 did not use any peptides to a noticeable extent (16), whereas all other strains examined utilized peptides containing glutamate and aspartate. All strains used amino acids, and glutamate, histidine, and aspartate utilization was common to all strains. The glutamate and histidine pools were characteristically depleted before the other amino acids were attacked, and at that time all strains except ATCC 10953 started to utilize peptides at a noticeable rate.

The utilization of peptides by these species is in accordance with available substrates in the environmental niches that these bacteria colonize. In the gingival crevice, the saccharolytic bacteria utilize the available carbohydrates. Peptides are generated by the hydrolytic activity of P. gingivalis, and therefore the levels of protein and ammonium ions are high and probably available to F. nucleatum. Carbohydrate metabolism and uptake by F. nucleatum have

been the focus of interest for several studies.

F. nucleatum utilizes glucose to a low extent compared with other species, and F. nucleatum does not grow with sugars as the main energy source. Available data on fusobacterial species indicate that glucose is used for the biosynthesis of intracellular molecules and not energy metabolism. The ability of F. nucleatum to metabolize its storage glycopolymers before utilizing amino acids has recently been demonstrated (254). F. nucleatum possesses an amino aciddependent (only glutamine, lysine, and histidine are effective) carbohydrate transport system for glucose, galactose, and fructose that operates exclusively under anaerobic conditions and results in the production of polysaccharides inside the cell. Catabolism of these polysaccharides is controlled by the same amino acids, and the polymer can be degraded to yield butyric, lactic, formic, and acetic acids. Addition of glutamine, lysine, or histidine to the anaerobic cell suspension inhibits polymer degradation. Polymer catabolism is resumed when specific enzymes required for amino acid fermentation are inactivated by exposure of the cells to air. The energy necessary for active transport of the sugars (acetylphosphate and ATP) is derived from the anaerobic fermentation of glutamine, lysine, and histidine, and these compounds must provide the energy for glucose and galactose accumulation by a three-stage process involving membrane translocation, intracellular phosphorylation, and polymer synthesis. The capacity of F. nucleatum to form intracellular polymers from glucose, galactose, and fructose under conditions of amino acid excess and to ferment this sugar reserve under conditions of aminoacid deprivation may contribute to the survival of F. nucleatum in the environment of the oral cavity and to the persistence of this organism in periodontal disease. Certain strains of F. nucleatum can catabolize dextrans, and the dextran hydrolase is found to be cell associated. Since dental plaque bacteria can synthesize and partly utilize dextran, it is suggested that this polysaccharide can act as a carbohydrate storage compound.

The major product from metabolism of peptone or carbohydrate by fusobacteria is butyrate without any iso-acids but often with acetate and lactate and lesser amounts of propionate, succinate, formate, and short-chained alcohols. F. Nucleatum produces propionate from threonine but not from lactate; it does not hydrolyze esculin, but it produces indole. Butyrate, propionate, and ammonium ions inhibit proliferation of human gingival fibroblasts (21), may have the ability to penetrate the gingival epithelium (, and are present in elevated levels in plaque associated with periodontitis. Because of this, they may have an etiological role in periodontal disease.

Although the effect of the metabolites is not sufficient to cause cell death, inhibition of fibroblast proliferation is serious because the potential for rapid wound healing is compromised. Proteases from pathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes, the bacterial proteases may participate in periodontal destruction. F. nucleatum is capable of desulfuration of cysteine and methionine, resulting in the formation of ammonia, hydrogen sulfide, butyric acid, and methyl mercaptan. Hydrogen sulfide and methyl mercaptan account for 90% of the total content of volatile sulfur compounds in mouth air. A biotin-dependent sodium ion pump from F. nucleatum, glutaconyl-coenzyme A decarboxylase, has been characterized.

From a nutritional point of view, the organization of different bacterial species, for example, saccharolytic and asaccharolytic species, aerobic and anaerobic species, and clusters of bacteria in the tooth environment, is fascinating and logical. There exists a symbiotic life in the periodontal pocket that apparently several species make use of. This is best illustrated by the coexistence of different bacterial species in clusters and by coaggregation of F. nucleatum and P. gingivalis in intimate contact, which probably supplies each with essential metabolites. The saccharolytic aerobic bacteria found mostly in supragingival plaque convert carbohydrates into short-chain organic acids, lowering the pH in the local environment. The asaccharolytic bacteria are nearly always anaerobic and generally found subgingivally, where they utilize nitrogenous substances for energy, are usually weakly fermentative, and tend to raise the local pH. More than 90% of the carbohydrates utilized by bacteria in dental plaque are used for energy production, but carbohydrates are also utilized by asaccharolytic species like F. nucleatum in which, e.g., glucose is used for biosynthesis of intracellular macromolecules and not energy metabolism (. Most of the carbohydrate utilized by the subgingival microflora is probably derived from the carbohydrate side chains of glycoproteins. Removal of the carbohydrate residues leaves the protein core available for further hydrolysis by the asaccharolytic species.

The fusobacteria are susceptible to many of the most commonly used antibiotics, but they have reduced susceptibility or may be resistant to vancomycin, neomycin, erythromycin, amoxicillin, ampicillin, and phenoxymethylpenicillin. Penicillinase-producing strains of F.nucleatum have been isolated, and isolation of beta-lactamase-producing strains of fusobacteria is increasing. As beta-lactamase production and beta-lactam resistance have been increasingly found in gram-negative bacteria, including F. nucleatum, the susceptibility of different bacteria to new agents has been tested. Biapenem, imipenem, the penem WY-49605, and trospectomycin were active against F. Nucleatum in vitro, as were the commonly used agents chloramphenicol and metronidazole.

Antimicrobial agents have been used in periodontal treatment either alone or preferentially in combination with conventional treatment to eliminate putative periodontal pathogens. The most extensively used antimicrobial agents as an adjunct in the treatment of periodontal disease have been the broad-spectrum bacteriostatic tetracyclines, which inhibit protein synthesis in the bacterial cells. Tetracycline, doxycycline, and minocycline concentrate in gingival crevicular fluid at concentrations up to five times those found in serum. As many as 75% of the bacteria in the subgingival pocket may be resistant to tetracycline after long-term, low-dose treatment. Besides systemic administration, antibiotics can be delivered locally to the periodontal pocket. Examples of antibiotics and antibiotic vehicles used for sustained release subgingivally are tetracycline-impregnated fibers and metronidazole gel. Tetracycline-resistant F. nucleatum strains have been found in subgingival plaque samples from patients with periodontal disease.

Eikenella corrodens

Eikenella corrodens was first described in 1948 as a slow-growing, anaerobic, Gram-negative rod.  A distinguishing feature of this organism is the ability to pit or corrode the agar in plated culture.  The colonies grow in the little grooves and for this reason it was called a corroding bacillus.  It was classified as Bacteroides corrodens.  Further studies proved that the classification had been applied to two organisms.  The major difference between the two being that one was a facultative anaerobe and the other was an obligate anaerobe.  The facultative anaerobe was renamed Eikenella corrodens.

E. corrodens inhabits the mucous membrane surfaces of humans, most commonly the respiratory tract.  E. corrodens can cause infections in humans when their immune system is weak.  Once an infection has occurred it can travel to other parts of the body.  E. corrodens is usually found with other bacteria in infections, commonly streptococci. E. corrodens is also responsible for about a quarter of human hand-bite wound infections and clenched-fist injuries.  It is also a putative periodontal pathogen, found at high levels in humans with periodontitis. E. corrodens infections can be treated with antibiotics such as penicillin, ampicillin and tetracycline.

E. corrodens does not grow on selective media.  When it is incubated aerobically it requires hemin.  However, when it is incubated anaerobically it does not require hemin.  Plate growth may be stimulated in a 3-10% CO₂ enviroment, even though CO₂ is not required.  E. corrodens grows so slowly that sometimes it is hidden by other faster-growing bacteria.  However, adding 5 ug/ml of clindamycin increases recovery.

E. corrodens must be incubated for 2-3 days before the colonies grow to a size sufficient for counting.  When plated the organism is dry, flat and has a yellow-pigmented colony.  The colony growth has three zones.  There is a clear and moist center, a highly visible ring that appears like droplets, and an outer growth ring.  The organism can produce either a musty smell or a bleach smell.

E. corrodens is small, straight, nonsporeforming, nonencapsulated and nonmotile.  It is biochemically inactive for most biochemical tests.  It does not produce catalase, urease, indole or H₂S₂.  They are oxidase-positive, catalase-negative, urease-negative, indole-negative and reduce nitrate to nitrite.

Medical importance

E. corrodens is a commensal of the human mouth and upper respiratory tract. It is an unusual cause of infection and when it is cultured, it is most usually found mixed with other organisms. Infections most commonly occur in patients with cancers of the head and neck, but it is also the common in human bite infections, especially "reverse bite" or "clenched fist injuries". It has also causes infections in insulin-dependent diabetics and intravenous drug users who lick their needles. It is one of the HACEK group of infections which are a cause of culture-negative endocarditis.

E. corrodens infections are typically indolent (the infection does not become clinically evident until a week or more after the injury). They also mimic anaerobic infection in being extremely foul-smelling.

Treatment

E. corrodens can be treated with penicillins, cephalosporins or tetracyclines. It is innately resistant to macrolides (e.g., erythromycin), clindamycin and metronidazole. It is susceptible to fluoroquinolones (e.g., ciprofloxacin) in vitro but there is no clinical evidence available to advocate its use in these infections.

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