mYCOBACTERIA.

June 4, 2024
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mYCOBACTERIA.  Microbiological diagnosis of tuberculosis, leprosy and other mycobacteriosis.

 

 

 

Corynebacteria

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. 1) 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.

 

Описание: R_270_Cor_diphtheriae

Figure 1. 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.

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image003.gifAccording to cultural and biological properties, three varieties of C.diphtheriae can be distinguished, gravis, mitis, and intermedius, which differ in a number of properties (fig. 2).

 

Figure 2. Colonies of    Corynebacterium diphtheriae:

1 — gravis 2 — mitis

 

 

                1.                                2.

 

Corynebacteria of the gravis biovar produce large, rough (R-forms), rosette-like black or grey colonies (Fig. 2, 1) 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.

 

Описание: R_279_C_gravОписание: R_281_C_mitis Описание: R_283_C_intermedius

 

Growth of different biovars of C. diphtheriae

 

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.

Описание: DiphtoxinDiphtheria 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 3. 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. diphtheriae

+

+

+

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 beeoted 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 materials about laboratory 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.

Описание: R_289_Collection_materialОписание: R_272_Throat

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image015.gif 

 

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image016.gifОписание: R_275_diphther

 

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”.

 

Описание: R_277_Cor_diphther Описание: R_276_C_diphtheriae

 

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 2 ).

 

Table 2

Differential-Diagnostic Signs of Diphtheria and Non-Pathogenic

Type of corynebacteria

 

Fermentation

Toxigenicity

Additional signs

sucrouse

glucose

starch

cystinase test

urease test

agglutination with antiserum

Diphtheria corynebacteria

 

 

 

 

 

 

 

gravis

+

+

+

+

+

mitis

+

+

+

Diphtheroids

+

+

+

+

Pseudodiphtheria bacteria

+

 

Описание: R_284_Urease_Tube

 

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. 4)

 

Figure  4. Determination of the in vitro toxigenicity of Corynebacterium diphtheriae

 

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image026.gifОписание: R_286_toxin

 

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 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.

 

Описание: R_287_Bordetella_pertussis

 

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.

Описание: R_290_Bordet_Gengou_med Описание: R_291_B_per_chok_ag

                                                       Growth on:    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 3).

Table 3

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

 

 

Описание: R_288_B_bronchysepticaОписание: R_292_B_bronch_blood

 

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.

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image039.gifОписание: R_287A_pertussis Описание: R_287Б_whooping

 

 

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.

 

Описание: R_303_гемофилюс Описание: R_304_Haem_inf_liqour

 

H. influenzae                                                    H. influenzae in liquor

 

 

Cultivation. The organisms are facultative anaerobes. They do not grow on commoutrient 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 outrient 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.

Описание: R_307_Haemophilus_influenzae Описание: R_308_Haemophilus_influenzae_blood

 

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 commoame 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.

Описание: R_310_Haemophilus_influenzae

 

 

Описание: R_311_Haemophilus_parainfluenzae

 

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. 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.

 

 

Описание: R_312_H_ducr Описание: R_313_H_ducreyi Описание: R_316_Haemophilus_ducreyi

 

 

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.

 

Описание: R_315_sjanker

 

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.

Описание: R_326_listeria_monocytogenes

 

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.

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image065.gifОписание: R_329_listeriaoxford

 

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image068.gifОписание: R_330_listeria_agar

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.

 

Описание: R_332_listeria

 

 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 about 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 4).

Table 4

Differential-Diagnostic Criteria of Pathogenic Bordetella

Bordetella type

 

Growth on agar

 

Change in medium colour

Urea

breakdown

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 antigeeutralization 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.

 

There are such causative agents of tuberculosis:

Mycobacterium tuberculosis

Mycobacterium bovis

Mycobacterium africanum

 

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. 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.

 

Описание: R_333_Mycobacterium_tuberculosis Описание: R_334_Mycobacterium_bovis

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

Описание: R_335_M_africanum

 

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, betahydroxy 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.).

 

Описание: R_347_Mycobacterium_tuberculosis_cordes

 

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.

Описание: R_344_Lowenst_Jens

 

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.

Описание: R_345_MBTBC_colonies

 

Описание: R_346_MBTBC_agar

 

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 Table 5. There are also strains of M. tuberculosis which affect poikilotherms and acid-fast saprophytes.

Table 5

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 a sick person

M. africanum

+

+

+

Tuberculosis among inhabitants of tropical Africa (Senegal, and other countries)

M. bovis

+

+

Tuberculosis in calves, domestic and wild animals, humans and other primates

M. kansasii

+

+

+

Tuberculosis-like diseases 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 diarrhea 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

 

 

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 iatural 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.

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 ion-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.  

 

Описание: R_348_mycobact_tuberculosis_cord

 

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).

T

here 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.

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image089.gifОписание: R_349_tuberculin Описание: R_350_tuberculin Описание: R_351_Mantoux

 

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.

 

Описание: R_334_Mycobacterium_bovis

 

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.

 

Описание: R_338_Mycobacterium_ulcerans

 

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 6

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

 

 

Описание: R_334a_Mycobacterium_avium

 

M. avium

 

Описание: R_341_Mycobacterium_smegmatis

 

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.

 

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.

 

Описание: R_339_Mycobacterium_leprae

 

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 outrient 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 ºC), 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 outrient 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.

Описание: R_353_Lepra

 

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 iegative.

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.

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/med/lik/ptn/Microbiology,%20virology%20and%20immunology/3/16_Corynebacteria_Mycobacteria.files/image105.gif 

Описание: R_355_skin_biopsy_lepra

 

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 about 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 outrient 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.

 

 

References:

1.     Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 136-147.

2.     Essential of Medical Microbiology /Wesley A. Volk and al. / Lippincott– Raven Publishers, Philadelphia– Ney– York, 725 p.

3.     Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P. 112-114, 133-136, 142-148.

4.     Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P.188-192, 242-245, 275-284.

 

Internet address:

http://microbewiki.kenyon.edu/index.php/Corynebacterium

http://textbookofbacteriology.net/diphtheria.html

http://www.emedicine.com/med/byname/corynebacterium-infections.htm

http://www.ijmm.org/article.asp?issn=0255-0857;year=2002;volume=20;issue=1;spage=50;epage=52;aulast=Kanungo

http://www.cehs.siu.edu/fix/medmicro/coryn.htm

http://en.wikipedia.org/wiki/Bordetella_pertussis

http://textbookofbacteriology.net/pertussis.html

http://www.phac-aspc.gc.ca/msds-ftss/msds20e.html

http://en.wikipedia.org/wiki/Bordetella_bronchiseptica

http://www.ebi.ac.uk/2can/genomes/genomes.html?http://www.ebi.ac.uk/2can/genomes/bacteria/Bordetella_bronchiseptica.html

http://web.umr.edu/~microbio/BIO221_1999/B_bronchiseptica.html

http://iai.asm.org/cgi/content/abstract/66/12/5607

http://medic.med.uth.tmc.edu/path/00001492.htm

http://en.wikipedia.org/wiki/Mycobacteria

http://web.uct.ac.za/depts/mmi/lsteyn/lecture.html

http://www.ratsteachmicro.com/Mycobacteria_notes/HCOE_CAI_Review_Notes_Mycobacteria.htm

http://pathmicro.med.sc.edu/fox/mycobacteria.htm

http://healthlink.mcw.edu/article/954973743.html

http://www.erj.ersjournals.com/cgi/content/abstract/28/6/1204

http://en.wikipedia.org/wiki/Mycobacterium_leprae

http://www.bacteriamuseum.org/species/Mleprae.shtml

http://microbes.historique.net/leprae.html

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.section.1833

 

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