Theme 14

Theme 14. Laboratory diagnosis of infection diseases caused by pathogenic cocci.

Theme 15. Causative agents of bacterial intestinal diseases: escherichiosis, typhoid fever, paratyphoids, salmonellosis. Microbiological diagnosis and prophylaxis of them.

 

Laboratory diagnosis of infection diseases caused by pathogenic cocci.

 

Gram-positive cocci

There are several types of symbiosis between cocci and the human body. Saprophytic and conditionally pathogenic types of staphylococci and streptococci live on the skin, mucous membranes, and in the respiratory tract. Meningococci may be harboured for long periods in the nasopharynx, and faecal streptococci (enterococci) in the intestine.

When body resistance is lowered or the skin and mucous membranes are injured, these bacteria penetrate the body tissues and cause infection. The various cocci possess different organotropic ability. This is distinctly manifest in meningococci, and gonococci but less so in staphylococci and streptococci. Cocci belong to the families Micrococcaceae, Streptococcaceae Peptococcaceae, Neisseriaceae.

Staphylococci. The staphylococcus, Staphylococcus aureus, was discovered by R. Koch (18,78), and later isolated from furuncle pus by L. Pasteur (1880). It has been described as the causative agent of numerous suppurative processes by A. Ogston (1881), and has been studied in detail by F. Rosenbach (1884).

Morphology. Staphylococci are spherical in shape, 0.8-1 mem in diameter, and form irregular clusters resembling bunches of grapes  (Fig. 1). In smears from cultures and pus the organisms occur in short chains, in pairs, or as single cocci. Large spherical (L-forms) or very small (G-forms) and even filterable forms may be seen in cultures which have been subjected to various physical, chemical, and biological (antibiotics) factors.

Figure 1. Staphylococci.

 

Staphylococci are Gram-positive organisms which possess no flagella and do not form spores. A macrocapsule can be seen on ultrathin sections of Staphylococci isolated from infected mice. The nucleoid occupies most of the cytoplasm and is filled with DNA fibrils. The G+C content in DNA ranges between 30.7 and 39.0 per cent.

 

Cultivation. Staphylococci are facultative-anaerobes. They grow well on ordinary nutrient media with a pH of 7.2-7.4 at a temperature of 37 °C but do not grow at temperatures below 10 °C and above 45 °C. At room temperature with adequate aeration and subdued light – the organisms produce golden, white, lemon-yellow, and other pigments known as lipochromes. These pigments do not dissolve in water but are soluble in ether, benzene, acetone, chloroform, and alcohol. They are most readily formed on milk agar and potatoes at a temperature of 20-25° C.

On meat peptone agar Staphylococci produce well defined colonies with smooth edges, measuring from 1-2 to 2.5 mm in diameter. Under the microscope the course granular nature of the colonies can be seen, the latter are opaque and have a dense centre. Their colour epends on the pigment produced by the organisms. Besides the typical colony types, Staphylococci produce R-, G-, and L-forms.  Growth of Staphylococci in meat-peptone broth produces diffuse opacity throughout the medium and, subsequently, a precipitate. In some cases when there is sufficient aeration, the organisms form a pellicle on the surface of the broth. Staphylococci grow well on potatoes and coagulated serum. After 24-48 hours of incubation there is usually abundant growth along the inoculation stab and liquefaction of gelatin media. On the fourth or fifth day the gelatin medium resembles a funnel filled with  fluid.

On blood agar pathogenic Staphylococci cause haemolysis of the erythrocytes. Rabbit and sheep erythrocytes are the most sensitive to the staphylococcal haemotoxin.

Fennentative properties. Staphylococci produce enzymes which cause the lysis of proteins and sugars (see Table 1). There is no indole production in young cultures. The organisms liquefy gelatin, coagulate milk and occasionally serum, reduce nitrates to nitrites, produce urease, catalase, phosphatase, ammonia, and hydrogen sulphide. They ferment glucose, levulose, maltose, lactose, saccharose, mannitol, and glycerin, with acid formation. A connection has been revealed between arginase activity and the level of g-toxin formation.

Toxin production. Staphylococci produce a-, b-, d– and g-haemolysins which are characterized by lethal, haemolytic, and necrotic activity. Filtrates of staphylococcal broth cultures contain an enterotoxin which causes food poisoning on entry into the gastro-intestinal tract. Staphylococci excrete exofoliatines which cause staphylococcal impetigo and pemphigus neonatomm in children.

 

Leucocidin, a substance which destroys leucocytes, haematoblasts of the bone marrow and nerve cells, is also produced by the Staphylococci. The organisms also coagulate blood plasma. Their ability to coagulate, plasma is a stable property and is used for differentiating various strains. Coagulase is thermoresistant. It can  be isolated from staphylococcal broth cultures.

Staphylococci produce fibrinolysin which when added to a blood clot dissolves the latter within 24-48 hours.

The Staphylococci produce hyaluronidase which breaks down hyaluronic acid, a component of connective tissue.

Coagulase, fibrinolysin, lecithinase, hyaluronidasa and phosphatasa all belong to the group of enzymes possessing destructive properties. Lecithinase destroys the lecithin protective membranes of the colloidal particles of a substance found in human, sheep, and rabbit erythrocytes. An anticoagulant which inhibits blood coagulation has also been derived from the staphylococcal cultures. This staphylococcal anticoagulant is produced in exudates of inflamed tissues, occurring during staphylococcal infections. Haemagglutmins which cause the agglutination of rabbit erythrocytes have also been found in staphylococcal culture filtrates. Virulent Staphylococci inhibit the phagocytic activity of leucocytes.

Many microbiologists believe that the Staphylococci isolated from patients produce alpha-haemolysins, while the organisms pathogenic for animals (e. g. itesponsible for mastitis in cows) more often produce beta-haemolysin.

Staphylococcal exotoxin, inactivated by treatment with 0.3-0.5 per cent formalin at 37 °C for 7-28 days, and injected parenterally to humans and animals, stimulates the production of a specific antitoxin capable of reacting with the toxin.

Antigenic structure. Polysaccharides A and B have been obtained from a staphylococcal suspension by treating the latter alternately with acid and alkali and removing the proteins with trichloracetic acid.

Polysaccharide A was extracted from pathogenic strains isolated from patients with septicaemia, furunculosis, osteomyelitis, and acute conjunctivitis, etc. Polysaccharide B is found in avirulent, non-pathogenic strains. Polysaccharides A and B differ not only in their serological reactions but also in their chemical structures.

Antigen C, containing a specific polysaccharide, has been recently isolated. Staphylococcal polysaccharides demonstrate a marked type specificity. Even in a 1 :1000 000 dilution they give a distinct precipitin reaction. The protein antigen is common to all species and types of  staphylococci.

Three types (I, II, III) of staphylococci have been revealed by the agglutination test and precipitin reaction. However, quite a number of cultures are unsuitable for serological typing. Recent studies have revealed fifteen type-specific staphylococcal antigens.

Classification. Staphylococci are included in the class Bacteria, family Micrococcaceae, genus Staphylococcus.

According to the contemporary classification, staphylococci are subdivided into three species: S. aureus, S. epidermidis, and S. saprophyticus.

Differentiation of Staphylococci

Note: S, sensitive; R, resistant, NT, not tested.

 

Resistance. Staphylococci are characterized by a relatively strong resistance to desiccation, freezing, sunlight, and chemical substances. After desiccation they can survive for more than 6 months. Repeated freezing .and thawing do not kill the organisms. They survive for many hours under direct sunlight. Staphylococci maintain their viability for more than 1 hour at 70 °C. At a temperature of 80 °C they are destroyed within 10-60 minutes and at boiling point, they instantly perish. A 5 per cent phenol solution kills the organisms in 15-30 minutes. Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant green which is used for treating pyogenic skin diseases caused by these organisms. Staphylococci possess high resistance to antibacterial agents, in 70-80 per cent of cases they are resistant simultaneously to 4-5 agents. Cross resistance to antibiotics of the macrolide group (erythromycin, oleandomycin, etc.) is encountered.

Pathogenicity for animals. Horses, cattle, sheep, goats, pigs, and, among laboratory animals, rabbits, white mice, and kittens are susceptible to pathogenic staphylococci.

An intracutaneous injection of a culture of pathogenic staphylococci produces inflammation and subsequent necrosis in the skin of the rabbit. An intravenous injection of a staphylococcal culture filtrate causes a condition similar to acute poisoning in rabbits, which is characterized by motonc excitation, respiratory disorders, convulsions, paralysis of the hind extremities, and sometimes, by diarrhoea and urine discharge. After complete fatigue the animal shortly dies.

Staphylococci or their toxin will cause vomiting, diarrhoea, and weakness in kittens if introduced per os or intraperitoneally. Functional disorders of the digestive tract arise owing to the effect of  tne enterotoxin which is distinguished from the other fractions of the staphylococcal toxin by its thermoresistance. It withstands a temperature of 100 °C for 30 minutes. The most reliable test for the presence of enterotoxin is an intravenous injection to adult cats.

Pathogenesis and diseases in man. Staphylococci enter the body through the skin and mucous membranes. When they overcome the lymphatic barrier and penetrate the blood, staphylococcal septicaemia sets in. Both the exotoxins and the bacterial cells play an important role in pathogenesis of diseases caused by these organisms. Consequently, staphylococcal diseases should be regarded as toxinfections.

The development of staphylococcal diseases is also influenced by the resulting allergy which in many cases is the cause of severe clinical forms of staphylococcal infections which do not succumb to treatment.

Staphylococci are responsible for a number of local lesions in humans: hidradenitis, abscess, paronychia, blepharitis, furuncle, carbuncle, periostitis, osteomyelitis, folliculitis, sycosis, dermatitis, eczema, chronic pyodermia, peritonitis, meningitis, appendicitis, and cholecystitis.

Diabetes mellitus, avitaminosis, alimentary dystrophy, excess perspiration, minor occupational skin abrasions, as well as skin irritation caused by chemical substances, are some examples of the conditions conducive to the formation of pyogenic lesions of the skin and furunculosis.

In some cases staphylococci may give rise to secondary infection in individuals suffering from smallpox, influenza, and wounds, as well as postoperative suppurations. Staphylococcal sepsis and staphylococcal pneumonia in children are particularly severe diseases. Ingestion of foodstuffs (cheese, curds, milk, rich cakes and pastry, ice cream, etc.) contaminated with pathogenic staphylococci may result in food poisoning.

Staphylococci play an essential part in mixed infections, and are found together with streptococci in cases of wound infections, diphtheria, tuberculosis, actinomycosis, and angina.

The wide use of antibacterial agents, antibiotics in particular, led to considerable changes in the severity and degree of the spread of staphylococcal lesions. Growth in the incidence of diseases and intrahospital infections m obstetrical, surgical and children’s in-patient institutions, intensive spread of the causative agent, and increase in the number of carriers among the medical staff and population have beeoted in all countries of the world. Intrauterme and extrauterine contamination of children with staphylococci has been registered, with the development of vesiculopustular staphyloderma, pemphigus, infiltrates, abscesses, conjunctivitis, nasopharyngitis, otitis, pneumonia, and other diseases.

It has been established that staphylococci become adapted rapidly to chemical agents and antibiotics due to the spread of R-plasmids among these bacteria. The high concentration of drugs  in the body of humans and in the biosphere has resulted in essential disturbance in the microflora and the extensive spread of resistant strains possessing more manifest virulence. The L-forms of staphylococci are especially marked by increased degree of resistance to antibiotics.

Immunity. The tendency to run a chronic flaccid course or relapse is regarded as a characteristic symptom of staphylococcal infections. This peculiarity gives a basis for concluding that postmfectional immunity following staphylococcal diseases is of low grade and short duration.

Immunity acquired after staphylococcal diseases is due to phagocytosis and the presence of antibodies (antitoxins, precipitins, opsonins, and agglutinins).

The inflammation restricts the staphylococci to the site of penetration and obstructs their spreading throughout the body. At the centre of inflammation the organisms undergo phagocytosis. Neutralization of the staphylococcal toxin by the antitoxin is an important stage of the immunity complex. As a result of capillary permeability, the antitoxin penetrates from the blood into the inflammation zone and renders harmless the toxin produced by staphylococci. Thus, the phagocytic and humoral factors act together and supplement each other.

The presence in the human organs and tissues of antigens which are also common in the staphylococci (mimicry antigens) is among the causes of low immunity. This causes a state of immunological tolerance to staphylococci and their toxins, which provides favourable conditions for uninhibited reproduction of the causative agent in the patient’s body. The wide use of antibacterial agents promotes intensive selection of staphylococcal strains resistant to the natural inhibitors of the microorganism.

Laboratory diagnosis. Test material may be obtained from pus, mucous membrane discharge, sputum, urine, blood, foodstuffs (cheese, curds, milk, pastry, cakes, cream, etc.), vomit, lavage fluids, and faeces.

 

The material is examined for the presence of pathogenic staphylococci. Special rules are observed when collecting the material since non-pathogenic strains are widespread iature.

Laboratory studies comprise the determination of the main properties of the isolated staphylococci (i. e their morphologic, cultural and biochemical characteristics), as well as their virulence. For this purpose the following procedures are carried out. Smears are made from pus and stained by the Gram method. Pus is inoculated onto blood agar and meat-peptone agar containing crystal violet. In cases of septicaemia blood is inoculated into glucose broth.The isolated pure culture is tested for its haemolytic (by inoculation onto blood agar plates), plasmacoagulative (by inoculation into citrated rabbit plasma), and hyaluronidase activities. Virulence is determined in rabbits by intracutaneous injection of 400 million microbial cells. Necrosis develops at the site of injection within 24-48 hours.

 

Pigment production of the isolated culture is also taken into account. For revealing sources of infection, particularly food poisoning and outbreaks of sepsis in maternity hospitals, serological typing and phage typing are carried out. To ensure effective therapy the isolated cultures are examined for sensitivity to antibiotics.

In cases of food poisoning presence of the enterotoxin in th isolated  staphylococcal culture is tested for by intravenous injection of the culture filtrate to adult cats. In cases when intoxication is due to ingestion of the milk of a cow suffering from mastitis, the culture grown on starch medium is tested directly for toxin production as a means of detecting staphylococci of animal origin. When the causative agent cannot be detected (osteomyelitis and other diseases), the patients’ serum is tested for agglutinins.

Treatment. Staphylococcal diseases are treated with antibiotics (penicillin, phenoxymethyl penicillin, tetracycline, gramicidin, etc.), sulphonamides (norsulphazol, sulphazol, etc.), and antistaphylococcal gamma-globulin.

When tr eating patients suffering from staphylococcal infections one should bear in mind that it is necessary to relieve intoxication and improve the immunological defence forces of the body (infusion of glucose, plasma, blood transfusion, injection of cardiac stimulants).

In cases of chronic staphylococcal lesions specific therapy is recommended: autovaccines, staphylococcal anatoxin, antitoxic serum, and diphage containing staphylococcal and streptococcal phages.

Staphylococd produce strains resistant to sulphonamides, antibiotics, and bacteriophage, which advances the wide distribution of staphylococcal infection. This variability is of particular importance in the therapy of staphylococcal pyogenic diseases. The medical services produce semisynthetic preparations of penicillin and tetracycline which are effectively used for the treatment of these diseases.

Prophylaxis. The general precautionary measures include: hygiene in working and everyday-life conditions, treatment of vitamin deficiency, prevention of traumatism and excess perspiration, observance  of rules of hygiene in maternity hospitals, surgical departments, children’s institutions, industrial plants and enterprises, particularly canneries, observance of personal hygiene and frequent washing of hands in warm water with soap.

Routine disinfection of hospital premises (surgical departments, maternity wards) and bacteriological examination of the personnel for carriers of pathogenic staphylococci resistant to antibiotics  are also necessary.

To prevent pyoderma protective ointments and pastes are used at industrial enterprises. For the treatment of microtraumas, besides iodine tincture and alcohol solutions of aniline dyes, solutions are widely applied which dry in one or two minutes and form an elastic film protecting the wound surface from dirt and infection. Patients with bums are kept in soft (plastic) isolating compartments which protect them against the entry of microflora from the external environment. In some cases specific prophylaxis by means of immunization with the staphylococcal anatoxin may be recommended for individuals subject to injury or infection with antibiotic-resistant staphylococci.

 

Streptococci. The streptococcus {Streptococcus pyogenes) was discovered by T. Billroth (1874) in tissues of patients with erysipelas and wound infections and by L. Pasteur and others (1880) in patients with sepsis. A. Ogston described the organisms in studies of suppurative lesions (1881). A pure culture of the organism was isolated by F. Fehleisen (1883) from a patient with erysipelas and by F. Rosenbach (1884) from pus. Streptococci belong to the family Streptococcaceae.

Morphology. Streptococci are spherical in shape, 0.6 to 1 mem in diameter, and form chains. They are non-motile (although motile forms are encountered), do not form spores and are Gram-positive. Some strains are capsulated. In smears from cultures grown on solid media the streptococci are usually present in pairs or in short chains, while in smears from broth cultures they form long chains or clusters.

 

Figure. Streptococci

 

 

The capsule is clearly demonstrated at the end of the phase of logarithmic growth. The microcapsule is seen on ultrathin sections, which forms in the phases of logarithmic and stationary growth. Under the microcapsule there is a three-layer cell wall 100-300 nm thick and then a three-layer cytoplasmic membrane which forms invaginations directed into the cytoplasm.

The cytoplasm is microgranular and contains ribosomes, some inclusions, and vacuoles with an electron-dense  material resembling volutin granules. The nucleoid occupies most of the cytoplasm. Division of the cell begins with protrusion of the cytoplasmic membrane after which a septum forms in the middle. New division of the cell begins before the previous one is completed, as the result of which dumbbell-like cells form. The G+C content in DNA ranges from 34.5 to 38.5 per cent.

Cultivation. Streptococci  are facultatively aerobic, and there are also  anaerobic species. The optimal temperature for growth is 37° C, and no growth occurs beyond the limits of 20-40° C  for enterococci the limits are 10-45 °C).

The organisms show poor growth on ordinary meat-peptone agar, and grow well on sugar, blood, serum and ascitic agar and broth, when the pH of the media is 7.2-7.6. On solid media they produce small (0.5-1.0 mm in diameter), translucent, grey or greyish-white, and granular colonies with poorly defined margins. Some streptococcal strains cause haemolysis on blood agar (Fig, 2, 2), others produce a green coloration surrounding the colony 1-2 mm in diameter as result a conversion of haemoglobin into methaemoglobin, while others do not cause any change in the erythrocytes. On sugar broth medium growth is in the form of fine-granular precipitates on the walls and at the bottom of the tube and only rarely does the broth become turbid.

Fermentative properties. Streptococci are non-proteolytic, do not liquefy gelatin, and do not reduce nitrates to nitrites. They coagulate milk, dissolve fibrin, ferment glucose, maltose, lactose, saccharose, mannitol (not always constantly), and break down salicin and trehalose, with acid formation.                                             

Toxin production. Streptococci produce exotoxins with various activities:

(1) haemolysin (haemotoxin, 0- and S-streptolysm) which loses its activity after 30 minutes at a temperature of 55 °C; disintegrates erythrocytes; produces haemoglobinaemia and haematuria in rabbits following intravenous injection;

 

 (2) leucocidin which is destructive to leucocytes; occurs in highly virulent strains and is rendered harmless by a temperature of 70 °C

 (3) lethal (dialysable) toxin which produces necrosis in rabbits when injected intracutaneously; it also causes necrosis in other tissues, particularly in the hepatic cells;

(4) erythrogenic toxin produces inflammation in humans who have no antitoxins in their blood;

(5) Streptococcus pneumoniae produces alpha-hae molysin secretedinto the culture fluid and  beta-haemolysin which is released after lysis of the streptococci.

Other substances produced by streptococci are harmful enzymes. They include hyaluronidase (which facilitates the spread of the organisms throughout the tissues and organs of the affected animal), fibrinolysin, desoxyribonuclease, ribqnuclease, proteinase, amylase, lipase, and diphosphopyridine nucleotidase. Streptococcal phages possess transduction properties and may be responsible for increased toxigenicity of C. diphtheriae and increased virulence of other bacteria occurring in association with streptococci.

Endotoxins, characterized by their thermoresistance and specific activity, are responsible for the pathogenic properties of streptococci together with the exotoxins and aggressive enzymes.

Antigenic structure. The study of the antigenic structure of streptococci is based on serologic examinations. F. Griffith used th e agglutination test, while R. Lancefield employed the precipitin reaction with an extract of a broth culture precipitate.

Four antigenic fractions were recovered from streptococci: the type-specific protein (M- and T-substances); group-specific  polysaccharide (C-substance), and nucleoprotein (P-substance). The M-substance is a protein which confers type specificity, virulence, and immunogenicity. The T-substance contains 0-, K-, and L-antigens. The C-substance is a polysaccharide common to the whole group of haemolytic streptococci. The P-substance belongs to the nucleoprotein fraction, being non-specific for haemolytic streptococci; it contains nucleoproteins common to other groups of streptococci, as well as staphylococci.

Group A and, partly, group C and G streptococci possess extracellular antigens: streptolysin 0, a protein which causes erythrocyte haemolysis, and streptolysin S, a lipoprotein complex possessing erythrocytolytic activity.

Classification. By means of the precipitation reaction founded on the detection of group specific carbohydrates, streptococci are subdivided into groups which are designated by capital letters from A to H and from K to T.

Five out of the known Streptococcal species cannot be related to any antigenic group. The haemolytic streptococci, rec overed from sick human beings, weresubdivided by F. Griffith into 51 serovars. He attributed 47 serovars to group A, serovars 7, 20, and 21 to group C, and serovar 16 to group G.

Streptococcus faecalis (enterococci) are pleomorphic oval cells which occur in pairs or in short chains. Some are oval or spear-shaped in form. The organisms are from 0.5 to 1 mem in diameter. Haemolytic types (Streptococcus faecalis) and organisms liquefying gelatin (S. faecalis, var. liquefaciens) are found. According to their antigenic structure, enterococci are divided into six 0-groups among which there are strains with K-antigens (capsular antigens). Some enterococcal and lactic streptococci possess identical antigens. On solid media enterococcal growths form a thin pellicle with smooth edges. On sugar broth they produce turbidity and precipitate. Certain enterococci are highly motile. Some of the strains produce a yellow pigment, and the pathogenic enterococci produce fibrinolysin. The organisms grow at tempera hires ranging from 10 to 45 °C. They are resistant to high temperature (e. g. withstand exposure to 60 °C for half an hour). Enterococci can be grown in broth containing 6.5 per cent common salt at pH 9.6 and on blood agar containing 40 per cent bile or an equivalent amount of bile salts. They ferment glucose, maltose, lactose, mannitol, trehalose, salicin, and inulin, with acid formation. They reduce and coagulate litmus milk in the presence of 0.1 per cent methylene blue. Enterococci differ from other streptococci in their ability to grow over a wide range of temperatures (10-45 °C) and in a medium of pH 9.6, in their resistance to high concentrations of salt and to penicillin (a number of strainsshow growth in media containing 0.5-1 U of antibiotic per 1 ml of  media). All enterococci decarboxylate tyrosine.

Enterococci inhabit the small and large intestine of man and warm-blooded animals. The organisms possess properties antagonistic to dysentery, enteric fever, and paratyphoid bacteria, and to the coli bacillus. In the child’s intestine the enterococci are more numerous than the E. coli. In lesions of the duodenum, gall bladder, and urinary tract enterococci are found as a result of dysbacteriosis. Isolation of enterococci serves as a criterion of contamination of water, sewage, and foodstuffs with faeces.

Streptococcus pneumonias (Diplococcus pneumoniae) belongs to the family Streptomycetaceae. For many years it was called pneumococcus. These are lanceolate or slightly elongated cocci measuring up to 0.5-1.25 mem in diameter and occurring in pairs, sometimes as single organisms or arranged in chains. In the body of humans and animals they have a capsule: they are Gram-positive, but young and old cultures are Gram-negative. The organisms are non-motile and  do not form spores. S. pneumoniae is a facultative anaerobe, the optimum temperature for growth is 37° C and the organism grows at 28-42 °C. The organisms are poorly cultivated on ordinary media but develop readily on serum or^ascitic agar at pH 7.2-7.6 as small colonies 1.0 mm in diameter. On blood agar they form small, rounded succulent colonies on a green medium. On sugar broth they produce turbidity and a precipitate. The organisms grow readily on broth to which 0.2 per cent of glucose is added. They usually do not form capsules on artificial  media, but the addition of animal protein to fluid medium promotes the formation of a capsule. There are 80 variants which are agglutinated ony by the corresponding type sera.

Resistance. Streptococci live for a long time at low temperatures, are resistant to desiccation, and survive for many months in pus and sputum. When exposed to a temperature of 70 °C, they are destroyed within one hour. A 3-5 per cent phenol solution kills the organisms within 15 minutes.

Pathogenicity for animals. Cattle, horses, and, among laboratory animals, rabbits and white mice are susceptible to the pathogenic streptococci.

The virulence of streptococci is tested on rabbits. The animals are infected by rubbing a culture suspension into a scratch made on the skin of the ear or on the back. This results in a local inflammation with the appearance of hyperaemia and swelling. An intravenous injection of pathogenic streptococci causes septicaemia or selectively affects the lungs, liver, kidneys, or joints.

Pathogenesis and diseases in man. The pathogenesis of streptococcal infections is brought about by the effect of the exotoxin and the-bacterial cells.The reactivity of the infected body and its previous resistance play an important part in the origin and development of streptococcal diseases. Such  diseases as endocarditis, polyarthritis, highmoritis, chronic tonsillitis, and erysipelas are associated with abnormal body reactivity, hyperergia. This condition may persist for a long period of time and serve as the main factor for the development of chronic streptococcal diseases.

With an exogenous mode of infection streptococci invade the human body from without (from sick people, and animals, various contaminated objects and foodstuffs). They gain access through injured skin and mucous membranes or enter the intestine with the food. Streptococci are mainly spread by the air droplet route. When the natural body resistance is weakened, conditionally pathogenic streptococci normally present in the human body become pathogenic. Penetrating deep into the tissues they produce local pyogenic inflammations, such as streptoderma, abscesses, phlegmons, lymphadenitis, lymphangitis, cystitis, pyelitis, cholecystitis, and peritonitis. Erysipelas (inflammation of the superficial lymphatic vessels) and tonsillitis (inflammation of the pharyngeal and tonsillar mucosa) are among the diseases caused by streptococci. Invading the blood, streptococci produce a serious septic condition. They are more commonly the cause of puerperal sepsis than other bacteria.

Streptococci may cause secondary infections in patients with diphtheria, smallpox, whooping cough, measles, and other diseases. Chronic tonsillitis is attributed to the viridans streptococci and adenoviruses. Contamination of wounds with streptococci during war results in wound suppurations, abscess formation, phlegmons, and traumatic sepsis.

Immunity. Immunity acquired  after streptococcal infections is ofa low grade and short duration. Relapses of erysipelas, fre quent tonsilitis, dermatitis, periostitis, and osteomyelitis occur as a result of sensitization of the body. This is attributed to low immunogenic activity and high allergen content of the streptococci, as well as to the presence of numerous types of the organisms against which no cross immunity is produced.

Immunity following streptococcal infections is of an anti-infectious nature. It is associated with antitoxic and antibacterial factors. The antitoxins neutralize the streptococcal toxin and together with the opsonins facilitate phagocytosis.

Laboratory diagnosis. Test material is obtained from the pus of wounds, inflammatory exudate. tonsillar swabs, blood, urine, and foodstuffs. Procedures are the same as for staphylococcal infections. Tests include microscopy of pus smears, inoculation of test material onto blood agar plates, isolation of the pure culture and its identification. Blood is sown on sugar broth if sepsis is suspected. Virulence is tested on rabbits by an intracutaneous injection of 200-400 million microbial cells. Toxicity is determined by injecting them intracutaneously with broth culture filtrate.

The group and type of the isolated streptococcus and its resistance to the medicaments used are also determined. In endocarditis there are very few organisms present in the blood in which they appear periodically. For this reason blood in large volumes (20-50 ml) is inoculated into vials containing sugar broth. If possible, the blood should be collected while the patient has a high temperature. In patients with chronic sepsis an examination of the centrifuged urine precipitate and isolation of the organism in pure culture are recommended.

Besides, the group and type of the isolated streptococcus are identified by means of fluorescent antibodies. Serological methods are also applied to determine the increase in the titre of antibodies, namely streptolysins O and antihyaluronidase.

Treatment. Usually  penicillin is used. For penicillin-resistant strains,and when penicillin is contraindicated, streptomycin, and erythromycin are required. Vaccine therapy (autovaccines and polyvalent  vaccines) and phage therapy are recommended in chronic conditions.

In some countries diseases caused by beta-haemolytic streptococci of groups A, C, G, and H and by alpha-streptococci (endocarditis) are treated with anti-infectious (antitoxic and antibacterial) streptococcal sera together with antibiotics and sulphonamides.

Prophylaxis. Streptococcal infections are prevented by the practice of general hygienic measures at factories, children’s institutions, maternity hospitals, and surgical departments, in food production, agricultural work, and everyday life.

Maintaining appropriate sanitary levels of living and working condi- tions, raising the cultural level of the population, and checking personal hygiene are of great importance.

Since streptococci and the macro-organism share antigenic structures in common and because streptococci are marked by weak immunogenic ability and there are a great number of types among them which do not possess the property of producing cross immunity, specific prophylaxis of streptococcal diseases has not been elaborated. Vaccines prepared from M-protein fractions of streptococci are being studied.

Role of Streptococcus in the Aetiology of Scarlet Fever

Scarlet fever has long been known as a widespread disease but at the present time its aetiology has not yet been ascertained. Four different theories were proposed: streptococcal, allergic, viral, and  combined (viral-streptococcal). Most scientists and medical practitioners favoured the streptococcal theory. G. Gabrichevsky in 1902 was the first to point out the aetiological role of the haemolytic streptococcus in scarlet fever. Usually he recovered the organisms from the pharynx of scarlet fever patients and from blood contained in the heart of those that had died of the disease. In 1907 he prepared vaccine from killed scarlet fever haemolytic streptococcal cultures. This vaccine was widely used for human vaccination.

In 1905 I. Savchenko, cultivating scarlet fever streptoco cci, obtained the toxin and used it for hyperimmunization of horses. The antitoxic antiscarlatinal serum was effectively used for treating people suffering from scarlet fever.

Data presented by Gabrichevsky and Savchenko concerning the streptococcal theory were confirmed by studies carried out in 1923-24 by G. Dick and G. Dick and by many other scientists.

The streptococcal aetiology o f scarlet fever is supported by the following arguments: (1) all people suffering from scarlet fever are found to harbour in their throats haemolytic streptococci which  are agglutinated by the sera of convalescents; (2) a subcutaneous injection of the scarlet fever toxin into susceptible people (volunteers) in some cases is followed by the appearance of a characteristic skin rash, vomiting, fever, tonsillitis, and other scarlatinal symptoms; (3) an intracutaneous injection of the toxin into susceptible children produces a local erythematous and oedematous reaction; the toxin produces no reaction in children who had previously suffered from scarlet fever and  were im-mune to the disease; (4) if 0.1 ml of antitoxic antistreptococcal serum or convalescent serum is introduced into the skin of a scarlet fever patient in the area of the rash, the latter turns pale (is ‘extinguished’); (5) hyper-immunization of animals with the scarlet fever toxin leads to the production of antitoxins, and a neutralization reaction takes place between  the toxin and antitoxins; (6) therapy with antitoxic sera and prophylaxis with combined vaccines consisting of the toxin and haemolytic streptococcal cells result in the appearance of less severe cases and decrease in morbidity and mortality.

At present many investigators accept the streptococcal theory in scarlet fever aetiology. In postwar years this theory has been confirmed by a number of investigations. Arguments against the streptococcal  theory are as follows: (1) people inoculated with scarlet fever streptococci or their toxins do not always display the characteristic symptoms of the disease, e. g. there is no peeling, only rarely are there instances of tonsillitis, and phlegmon, sepsis, and erysipelas occasionally develop; (2) in  severe hypertoxic forms the antitoxic ssswm has little effect, while the serum of convalescents gives better results; (3) the skin toxin test (Dick test) sometimes gives a negative reaction with susceptible  children and produces a positive reaction with those who are immune; (4) immunity acquired after scarlet fever is very stable and of long duration, while that acquired after other streptococcal diseases is unstable, of short duration, and is frequently accompanied by an increased susceptibility to streptococci.

It is assumed that scarlet fever is caused by group A beta-haemolytic streptococci which possess M-antigen and produce erythrogenic exotoxin. People become infected by the air droplet route. Sic k people, convalescents, and carriers of the causative agent of scarlet fever are all sources of infection. The disease is most commonly encountered in children from 1 to 8 years of age.

The causative agent sometimes enters the body through wounds on the skin and mucous membranes of the genitalia. This form of scarlet fever is known as extrabuccal or extrapharyngeal (traumatic, combustion, surgical, and puerperal). Certain objects (e. g. utensils, toys, books, etc.) as well as foodstuffs (e. g. milk), contaminated by adult carriers, may also be sources of infection. Of great importance in  the epidemiology of scarlet fever are the patients with atypical, unrecognizable forms of the disease. In its initial stage scarlet fever is chiefly characterized by intoxication, while in the second stage it is accompan ied by septic and allergic conditions.

Scarlet fever produces a relatively stable immunity. Reinfections are very rare. They have increased iumber in the last years as  a result of wide use of antibiotics which reduce the immunogenic activity of the pathogen and its toxin.

Data concerning  the correlation between a positive Dick test and susceptibility to scarlet fever provide evidence of the antitoxic nature of immunity acquired after scarlet fever. Children from 1 to 5 years are most susceptible.

Scarlet fever is  recognized mainly by its clinical course and on epidemiological grounds. Laboratory diagnosis for the detection of haemolytic streptococci and their typing is employed only in certain cases. This method is of no practical value since haemolytic streptococci are often isolated from people with various diseases and frequently from healthy individuals.

The phenomenon of rash ‘extinguishmenf is employed as an auxiliary diagnostic method. In the case of scarlet fever, the rash at the site of injection will disappear within 12-20 hours and the skin will turn pale.

Certain physicians apply the Dick test with the thermolabile fraction of the toxin. The diagnosis of scarlet fever is verified to a  certain extent if on a second injection of the toxin a positive Dick test reverts to a negative reaction.

Scarlet fever may also be diagnosed by detecting precipitins in the urine (urine precipitation test). A layer of type-specific  streptococcal sera or convalescent serum is transferred onto freshly filtered urine of patients in the first days of the disease. The appearance of a greyish-white ring at  the interface of the two fluids designates a positivereaction.

Scarlet fever patients are treated with  penicillin, tetracycline, sulphonamides (norsulphazol, etc.), and gamma-globulin  from human blood. The wide use of antibiotics has led to a significant decrease in the morbidity and mortality rate of scarlet fever and to a milder course of the disease. This fact also confirms the definite role played by  haemolytic streptococci in the aetiology and pathogenesis of scarlet fever, since it is known that these organisms are extremely sensitive to penicillin and other antibiotics. In the recent years, however, an increase in the incidence of scarlet fever and a more severe course of the disease are noted.

Prophylaxis consists of early diagnosis, isolation of patient s and hospitalization in the presence of epidemiological and clinical indications. Extremely hygienic cleaning and ventilation and observance  of correct hospital regime are also necessary. If cases of scarlet fever occur in children’s institutions, the children concerned must be isolated. Debilitated children who have been in contact with scarlet fever patients must be injected with 1.5-3.0 ml of human serum gammaglobulin.

Role of Streptococcus in the Aetiology of Rheumatic Fever

The majority of authors maintain that rheumatic fever develops as a result of the body becoming infected by group A beta-haemolytic streptococci. Acute or chronic tonsillitis and pharyngitis produce a change in the immunological reactivity of the body and  this gives rise to characteristic clinical symptoms and a pathological reaction. It should be noted, however, that recent research shows the leading role in rheumatism of virus agents with persisting properties. The active phase with an acute and subacute course is attended with virusaemia. Rheumatism is characterized by the virus remaining in the  body for a long period of time: the viral antigen penetrates the leucocytes and sensitizes them. Diminution of the specific and non-specific reactions to  the virus leads to tolerance of the body. Autoimmune reactions are encountered in rheumatism. Streptococci and other exogenous and endogenous factors contribute to exacerbation of rheumatism. Virusaemia is almost always revealed in the patients during exacerbation. Identical viruses are detected in the mother’s blood and in the blood of premature infants, stillborns, and in infants who die soon after birth.

Additional materials about laboratory diagnosis

STAPHYLOCOCCAL INFECTION. In diseases caused by pathogenic staphylococci (Staphylococcus aureus) the materials to be  examined include pus, mucosal secretions, blood, sputum, urine, and cerebrospinal fluid. In cases of food in­toxications, vomit, faeces and food remnants are also. studied.

In open suppurative lesions the material is taken with a cotton wool swab after removing the superficial layer of the pus, which may contaion-pathogenic staphylococci and other microorganisms usually present on the skin and in the air. When purulent foci are unruptured, they are punctured and the pus from the syringe is poured out into a sterile test tube. Mucosal secretion is obtained with a tampon. Urine and sputum are collected into sterile test tubes and jars. Blood withdrawn from a patient’s ulnar vein with a syringe as well as aseptically obtained cerebrospinal fluid are inoculated at the patient’s bedside into a vessel containing 100-200 ml of sugar broth (pH 7,2–7,4). Staphylococci propagate quite readily in simple media too but the use of sugar broth is preferable since septicaemia may be secondary not only to staphylococci but also to other microor­ganisms which are more demanding with regard to nutrient media.

Bacterioscopic examination. Pus, sputum, faucial secretion, and other biological samples to be studied, with the exception of blood. are examined microscopically. Place a specimen of high density pus into a drop of sterile water; make smears with the help of a swab or loop and stain them by Gram’s method. Staphylococci are Gram-positive and tend to be arranged in small clusters, in pairs, and in the form of short chains. Differentiation between staphy­lococci and streptococci by their distribution and tinctorial prop­erties proves rather difficult. Consequently, the examination is not confined to microscopy, but includes cultivation of the tested material.

Bacteriological examination. On the first day of investigation with the help of a loop or spatula, inoculate the specimen into dishes with 3-5 per cent of blood, milk-salt, and yolk-salt agar and place it into an incubator at 37 °C for 18-24 hrs.

To isolate staphylococci, it is advisable to use dry elective me­dium, representing a mixture of hydrolysin and aminopeptide (1:1), because this medium facilitates faster growth of staphylococci as compared with other nutrient media. It may be used as a base for preparing milk-yolk-salt agar for the  purpose of determining pig­ment formation and lecithinase activity.

On the second day the colonies are examined. On solid media staphylococci appear as convex, nontransparent medium-sized colo­nies of a homogeneous or fine-grain structure. Pathogenic strains on a blood agar with 0,25-0,5 per cent of glucose form a haemolytic zone around the colonies. On the yolk-salt agar most pathogenic staphylococci induce lecithovitellin (yolk) reaction manifested in the formation of a turbulent zone with an opalescent halo on the periphery around the colony.

Using dishes with milk-salt agar, pigment formation is deter­mined in strains displaying this ability. The pigment may be golden, white or of a lemon-yellow colour.

Microscopic examination reveals typical Gram-positive staphylo­cocci in such smears.

Further examination is aimed at isolating pure staphylococcal culture, for which purpose the colonies are transferred onto an agar slant.

Apart from haemolytic and lecithinase activity, pathogenetic staphylococci possess the ability to coagulate plasma, induce skiecrosis in rabbits, and to destroy DNA.

On the third day of bacteriological examination, the isolated cul­ture is introduced into a test tube with rabbit citrate plasma to identify plasmocoagulase. For this purpose 10 ml of blood obtained from the rabbit heart are poured into a test tube with 1 ml of 5 per cent solution of sodium citrate; the plasma separated by centrifugation or sedimentation is diluted with isotonic saline (1:4) and poured into sterile test tubes by 0.5-ml portions. At present, there is dry citrate plasma which is diluted with isotonic saline before use. The inoculated cultures are placed into an incubator at 37 °C to determine the time when plasma coagulation manifests itself. Milliard sus­pension is prepared from the same culture (1 milliard of microor­ganisms per ml) and 0.2 ml of this suspension is injected intra-cutaneously to a light-coated rabbit. One-two days later skie­crosis develops at the site of injection.

 

Some differential signs of staphylococci are present in table.

 

To perform the plasmoagglutination test, plasma is poured into a test tube and pure staphylococcal culture is ground with a loop at a distance of 0,5 cm from the plasma surface. Then, using a loop, the plasma is transferred to the culture. A positive result is recog­nized by the appearance of agglutinate flakes on the tube wall.

To demonstrate DNase activity, a 24-bour staphylococcal culture is inoculated in the form of small plaques into a Petri dish contain­ing Hottinger’s agar and DNA^[1][1][1].

Twenty-four hours later 5 ml of 1N HCl solution is introduced into the dish and zones of clearing indicating the presence of DNase are counted in 3-5 min.

Lysozyme activity of staphylococci is considered to be an addi­tional indicator of pathogenicity. To demonstrate it, solid nutrient medium with bacterial suspension of Micrococcus lysodeikticus is inoculated in a patch-like manner with 24-hour agar culture of staphylococcus. Zones of lysis form around the colonies when lysozyme is produced. Under anaerobic conditions pathogenic staphylococci break mannitol to acid.

 

Examination of cultural phagovar is of great epidemiological significance with regard to identifying the source of infection. The main international set of staphylococcal bacteriophages consists of 21 types divided into four groups, Staphylococcal bacteriophages in two working dilutions 1 TD (test-dilution), which should be no less than 10 ^­­­-3, and 100 TD, not less than 10^–1, are the  ones most com­monly used for typing. Phagotyping is started with 1 TD, then, if the result has beeegative, 100 TD is employed.

To diagnose staphylococcal diseases, one can use the  IHA test with an erythrocytic diagnosticum (red blood cells are sensitized with alpha-toxin of the staphylococcus), which allows the detection of antibodies.

Determination of sensitivity of the isolated staphylococci to antibiotics is essential for the purposeful and effective treatment of a given patient.

To isolate a haemoculture of staphylococci, sugar broth is inoculated with blood and incubated at 37 °C for 18-24 hrs. The  staphylococcus causes uniform cloudiness of the medium. On the second day of the study, prepare smears from the blood culture and streak the latter onto a meat-peptone agar slant and a blood agar plate to evaluate the haemolytic activity of staphylococci. On the third day, examine both the staphylococcal culture, which has grown on the agar slant, and the culture that has been isolated from the pus or other mate­rials.

In inoculation of exudate, pus from unruptured abscesses and phlegmons, meat-peptone agar or 5 per cent blood agar is employed. The inoculated cultures are placed into an incubator at 37 °C for 18-24 hrs, pure culture is isolated and then identified by the afore­mentioned methods.

In food intoxications the material to be studied is streaked onto plates with milk-salt and yolk-salt agar and also onto broth con­taining 1 per cent of glucose (for enrichment). Material contaminat­ed with extraneous microorganisms is inoculated onto blood agar containing 6-7 per cent of sodium chloride. Staphylococci demon­strate good growth and haemolytic action (they may propagate in the presence of 12 per cent of salt and 50 per cent of sugar) in this. medium; the development of enterobacteria and bacilli is inhibited, while Proteus is incapable of uninterrupted growth in the  form of a film.

The cultures are placed into an incubator at 37 °C. On the  next day, the colonies are examined, pure cultures are isolated, and staphylococci are identified. Pathogenic staphylococci responsible for food intoxications produce a golden, less commonly, white pigment, liquify gelatin, induce haemolysis, and coagulate plasma.

To establish the production of enterotoxin, the staphylococcal culture is streaked onto a special nutrient medium^[2][2][2]. The  inoculated cultures are placed into an exsiccator with 20 per cent of CO₂ and in­cubated at 37 °C for 3-4 days, and then filtered through Nos 3 and 4 membrane filters. The obtained filtrate (10-15 ml in total) is mixed with the equal amount of warm milk and fed to 1-2-month-old kit­tens or alternatively the filtrate is introduced into their stomach via a catheter. If enterotoxin is present, the kittens develop vomiting (the key sign of poisoning) in 30-60 min and diarrhoea in 2-3 hours, which persists for 2-3 days and may -result in death in grave cases. The filtrate may also be injected intraperitoneally, following its heating at 100 °C for 30 min to inactivate thermolabile fractions of a toxin. Currently, toxigenicity of isolated staphylococcal cultures is determined in vitro, making use of the  diffuse precipitation test in gel.

Pathogenic staphylococci may harbour in the air of operating-theatres, dressing rooms, post-delivery wards, and other hospital premises. To demonstrate them, air samples are inoculated on yolk-salt agar^[3][3][3].

Determination of the  staphylococcal antitoxin in the blood serum becomes of great importance in diagnosing chronic staphylococcal infections (osteomyelitis, septicopyaemia, etc.) when bacteriological studies conducted together with aggressive antibiotic therapy yield no results. The reaction is based on suppression of the haemolytic activity of the staphylococcal toxin by the patient’s serum antitoxin. For this purpose, add the patient’s serum diluted 1:5; 1:10; 1:15; 1:20, etc. to the staphylococcal toxin taken in a definite dose, mix thoroughly, and add 0.05 ml of rabbit erythrocytes to the resultant mixture. Incubate the test tubes for 1 h at 37 °C and for 1 h at room temperature, and then read the results.

The amount of serum with which the tested dose of toxin either retards or inhibits haemolysis is taken as the serum titre.

In clinically healthy children and individuals with a history of staphylococcal skin infection or staphylococcal infection that compli­cated surgical disease, the titre of the  staphylococcal antitoxin does not exceed 0,5-4 AU/ml. Patients with chronic staphylococcal infec­tions usually demonstrate higher titres.

 

STREPTOCOCCAL INFECTION. In diseases caused by pathogenic streptococci (Streptococcus pyogenes}, the material subjected to the study is pus, blood, mucosal secretions, urine, sputum, and cerebrospinal fluid. The procedure of obtaining the material to be examined is the same as in diseases caused by staphylococci.

Bacterioscopic examination. In Gram-stained smears from pus and mucosal secretions streptococci appear as short chains, less commonly as pairs or individual cocci. In the latter case streptococci cannot be distinguished from staphylococci, and they should be studied for other attributes.

Bacteriological study. Samples of pus, mucosal secretions, urine, sputum, and cerebrospinal fluid are inoculated onto Petri dishes with 5 per cent blood agar (defibrinated blood should be preferably used) and into a test tube with sugar broth. After an overnight incu­bation at 37 °C, the colonies and growth in the sugar broth are stud­ied. Streptococcus pyogenes develops with the formation of small flat rather dry granular colonies.

Streptococci producing beta-haemotoxin (streptolysin) form a zone of haemolysis around the colonies, while alpha-haemotoxin-producing streptococci are characterized by the appearance of green zones around the colonies as a result of methaemoglobin formation. There is no haemolysis in the absence of haemotoxin. In sugar broth strepto­coccal growth appears as flakes or granules on the bottom or walls whereas the medium remains transparent.

Smears from streptococcal colonies display no typical pattern of cocci in the form of chains. Cells are arranged singly, in pairs or in small aggregates. In liquid medium, a cultural smear exhibits typical chains of streptococci.

At further stages of examination the group and serovar of the streptococcus are determined.

Streptococcal groups (by Lancefield) are classified by the presence of a polysaccharide antigen which is revealed using the precipita­tion test with group sera (A, B, C, etc.).

Serovars (serotypes) of streptococci (by Griffith) are characterized by the presence of a specific protein antigen which is determined with the help of the slide agglutination test performed with typical agglutinating sera. For this purpose, a 24-hour broth culture of the isolated streptococcus is centrifuged and the sediment is diluted in 0,5-1 ml of isotonic sodium chloride solution. A drop of serum is mixed with a drop of the tested culture on a glass slide. The majority of pathogenic streptococci belong to group A.

To recover b-haemolytic streptococcus of group A, the immuno-fluorescence test may be employed. In this case a smear is prepared from the isolated streptococcal culture, fixed for 15 min with 95 per cent alcohol, and then stained with fluorescent serum for 15 min. After that, it is washed in running water, dried, and examined under the luminescent microscope.

Blood examination. The procedure of blood inoculation into sugar broth is the same as that employed in staphylococcal diseases. The presence of streptococci is indicated by the formation of floccular sediment on the bottom and haemolysis. Smears are characterized by long chains of streptococci. Microscopic examination allows a pre­liminary conclusion as to the presence or absence of streptococci. To detect haemotoxin, the culture is transferred onto a blood agar plate. Typical small colonies surrounded by a zone of haemolysis or a greenish halo develop in 24 hours (the third day of investigation). These findings permit the conclusion that the culture harbours S. pyogenes.

To isolate anaerobic streptococci (Peptestreptococcus anaerobius), which harbour the genital mucosa and may cause postpartal sepsis, blood is introduced into the Kitt-Tarozzi medium. Some strains of anaerobic streptococci form gas when they get into the liquid media.

Pathogenic streptococci show good growth in Garrod’s medium. It presents meat-peptone agar (pH 7,4) to which 5 per cent of blood and 0,1 per cent aqueous solution of gentian violet are added (0,2 ml per every 100 ml of meat-peptone agar). Growth of saprophytic air microflora and enterococci on Garrod’s medium is suppressed.

In order to determine pathogenic properties of streptococci, for­mation of toxins, in particular of fibrinolysin (streptokinase), is investigated. Human blood plasma is used for this purpose, which is obtained by adding 1 ml of 2 per cent citrate sodium solution to 10 ml of blood. Following sedimentation, the unstained plasma is separated and diluted in a 1 to 3 ratio. At the next stage, 0,5 ml of an 18–20-hour culture of the tested streptococcus and 0,5 ml of 0,25 per cent solution of calcium chloride are added. The test tubes are carefully shaken and placed in a water bath at 42 °C for 20–30 min.

A fibrin clot is formed during this period. The test tubes are left in the water bath for another 20 min. If the streptococcal culture pro­duces fibrinolysin, the clot is dissolved within 20 min.

As some strains of streptococci dissolve fibrin slowly, the test tubes are transferred from the water bath to an incubator two hours later, and the results are read on the following day.

Determination of the amount of superficial M-protein, which is observed only in pathogenic strains, may be considered as a method evaluating the virulence of streptococcal cultures. Young cultures are utilized to obtain hydrochloric extracts, and the content of M-protein is determined in the latter by the precipitation test.

No laboratory studies are usually used in the diagnosis of scarlet fever. Occasionally, secretion of the faucial mucosa is cultivated, and streptococci of various serogroups are isolated.

Haemolytic streptococci releasing a- and P-toxins may harbour the air of various hospital rooms. To demonstrate them, air samples are inoculated onto Garrod’s medium for the subsequent isolation and identification of pure culture.

In laboratory diagnosis, streptococci should be differentiated from enterococci (Streptococcus faecalis} which are characterized by the following specific features: ability to grow at temperatures vary­ing from 10° to 45 °C, resistance to high concentrations of sodium chloride, penicillin, and alkaline medium (pH 9.6). Enterococci occur in diseases affecting the duodenum, gallbladder, and urinary tract. Their presence in the environment serves as a criterion of faecal contamination of drinking water, sewage waters, and food-stuffs.

 

INFECTION CAUSED BY STREPTOCOCCI OF PNEUMONIA (PNEUMOCOCCI). Streptococci of pneumonia (pneumococci), Streptococcus pneumoniae, are the causative agents responsible for croupous pneumonia, focal pneumonia, other respiratory diseases, creeping cornea! ulcer, suppurative processes in the middle ear and maxillary sinus, and also for sepsis and meningitis.

Material to be studied includes sputum, pus, cerebrospinal fluid, blood, and post-mortem organs.

Bacterioscopic examination. Two smears are made of the tested material (with the exception of blood); one is stained by the Gram method, the other by the Burri or Kozlovsky technique. Microorgan­isms and Indian ink placed on a glass slide are mixed by circular movements to prepare the conventional smear. Indian ink is di­luted with isotonic saline in a ratio of’ 1 to 4. The smear is dried in the air, left unfixed and stained for 2–3 min with formol-gentian violet (10 ml of 40 per cent formalin and 1,5 g of gentian violet). Formol-gentian violet may be substituted with 0,33 per cent aqueous fuchsine solution and 3 per cent alkaline solution of methylene blue. Microscopic examination reveals unstained capsules which contain violet bacterial cells against a black-gray background. Pneumococci are arranged in pairs, their cells are elongated, resembling a candle flame, and are surrounded with a capsule. Hence, micro­scopic findings seem to suggest the presence of the causative agent. Yet, it is bacteriological investigation that provides most reliable data.

Bacteriological examination. To obtain pure culture, 5-10 ml of blood is inoculated into a serum broth (1 part of serum and 3 parts of meat-peptone broth, pH 7,2–7,4) or into sugar broth, or into a special medium which contains (in 100 ml): 1,8–2,0 ml of agar-agar, 70–75 ml of Hottinger’s hydrolysate or casein hydrolysate (1.8–2.0 g/l of amine nitrogen), 20-25 ml of bovine heart hydrolysate (1,40–1,60 g/I of amine nitrogen), 4-5 ml of defibrinated horse blood 0,5-0,7 ml of baking yeast extract. After 18–24-hour incubation in a heating block, the culture is transferred onto a plate with 10 per cent blood agar. Cerebrospinal fluid is centrifuged and the deposit is inoculated onto a blood agar. Colonies of the pneumococcus re­semble those of the streptococcus: they are small, almost flat, non transparent, with a halo of a green colour or, less commonly, of haemolysis. Another characteristic sign is an impression in the centre of the colony.

As a rule, direct inoculation of the material onto nutrient media (pus, sputum) does not give a positive result since saprophytes, es­pecially saprogenous microorganisms present in them, inhibit the growth of pneumococci. For this reason, pus and sputum are treated before cultivation: clumps are collected and comminuted in a por­celain mortar, then 1 ml of isotonic saline is added, and the resul­tant mixture is injected intraperitoneally to albino mice. Mice are very susceptible to the pneumococcus and die of pneumococcal sep­ticaemia in 18-72 hrs. The mouse’s carcass is dissected and the blood from the heart, pieces of the internal organs, and peritoneal fluid are inoculated into a blood agar plate and a test tube with serum broth.

Morphological and cultural characteristics do not allow any clear-cut differentiation between the pneumococcus and Streptococcus viridans. To achieve this, one can employ the reaction of pneumo­coccal lysis by bile: 1 ml of broth culture is introduced into a sterile test tube and then 0.5 ml of bovine bile is added. Ten-fifteen min­utes of incubation in a heating block is enough to bring about complete lysis of the pneumococci. A tube containing bile-free broth culture serves as a control. Streptococcus viridans whose colonies resemble those of the pneumococcus are not dissolved by bile. If lysis is present, the tested material is inoculated onto the Hiss me­dium. In contrast to the streptococcus, the pneumococcus splits inulin with the formation of oxygen and forms ammonia from arginine.

The conducted study allows the final identification of the isolated microorganism. The factors to be taken into consideration are: a lancet shape of diplococci, the presence of the capsule in the native material, high virulence for albino mice, dissolvement by bile, and inulin splitting.

 

Gram-negative cocci

Meningococci. The meningococcus (Neisseria meningitidis) was isolated from the cerebrospinal fluid of patients with meningitis and studied in detail in 1887 by A. Weichselbaum. At present the organism is classified in the genus Neisseria, family Neisseriaceae.

Morphology. The meningococcus is a coccus 0.6-1 mcm in diameter, resembling a coffee bean, and is found in pairs.

The organism is Gram-negative. As distinct from pneumococci, meningococci are joined longitudinally by their concave edges while their external sides are convex.Spores, capsules and flagella are not formed. In pure cultures meningococci occur as tetrads (in fours) and in pus they are usually found within and less frequently outside the leukocytes. The G+C content in DNA ranges from 50.5 to 51.3 per cent.

 

Meningococci in cerebrospinal fluid. The leukocytes have engulfed (phagocytized) large number of the diplococci

In culture smears, small or very large cocci are seen singly, in pairs, or in fours. Meningococci may vary not only in shape but also in their Gram reaction. Gram-positive diplococci appear among the Gram-negative cells in smears.

Cultivation. The meningococcus is an aerobe or facultative anaerobe and does not grow on common media. It grows readily at pH 7.2-7.4 on media to which serum or ascitic fluid has been added. Optimum temperature for growth is 36-37 °C and there is no growth at 22° C. On solid media the organisms form fine transparent colonies measuring 2-3 mm in diameter. In serum broth they produce turbidity and a precipitate at the bottom of the test tube, and after 3-4 day’s, a pellicle is formed on the surface of the medium.

Meningococci can be adapted to simple media by repeated subculture on media with a gradual change from the optimum protein concentration to media containing a minimal concentration of proteins.

Fermentative properties. Meningococci do not liquefy gelatin, cause no change in milk, and ferment glucose and maltose, with acid formation.

Toxin production. Meningococci produce toxic substances which possess properties of exo- and endotoxins. Disintegration of bacterial cells leads to the release of a highly toxic endotoxin. Meningococci readily undergo autolysis which is accompanied by accumulation of toxins in the medium. The meningococcal toxin is obtained by treating the bacterial cells with distilled water, or 10 N solution of soda, by heat autolysis, by exposure to ultraviolet rays.

Antigenic structure and classification. Meningococci were found to contain three fractions: carbohydrate (C) which is common to all meningococci, protein (P) which is found in gonococci and type III S. pneumoniae, and a third fraction with which the specificity of meningococci is associated. According to the International Classification, four groups of meningococci are distinguished, groups A, B, C, and D. Recently the number of types has increased to seven, but only the first two are dominant.

The organisms are characterized by intraspecies variability. A change of types takes place at certain times.

Resistance. The meningococcus is a microbe of low stability, and is destroyed by drying in a few hours. By heating to a temperature of 60° C it is killed in 10 minutes, and to 80 °C, in 2 minutes. When treated with 1 per cent phenol, the culture dies in 1 minute. The organism is very sensitive to low temperatures. Bearing this in mind, test material should be transported under conditions which protect the meningococcus against cooling.

Pathogenicity for animals. Animals are not susceptible to the meningococcus in natural conditions. The disease can be produced experimentally in monkeys and rabbits by subdural injections of meningococci. Intrapleural and intraperitoneal infection of guinea pigs and mice results in lethal intoxication. Septicaemia develops in experimental animals only when large doses are injected.

Pathogenesis and diseases in man. People suffering from meningococcal infection and carriers are sources of diseases. The infection is transmitted by the air-droplet route. The causative agent is localized primarily in the nasopharynx. From here it invades the lymph vessels and blood and causes the development of bacteriemia. Then as a result of metastasis the meningococci pass into the meninges and produce acute pyogenic inflammation in the membranes of the brain and spinal cord (nasopharyngitis, meningococcaemia, meningitis).

 

 

The disease usually arises suddenly with high temperature, vomiting, rigidity of the occipital muscles, severe headache, and increased skin sensitivity. Later paresis of the cranial nerves develops due to an increase in the intracranial pressure. Dilatation of the pupils, disturbances of accommodation, as well as other  symptoms appear. A large number of leukocytes are present in the cerebrospinal fluid, and the latter after puncture escapes with a spurt because of the high pressure.

In some cases meningococcal sepsis develops. In such conditions the organisms are found in the blood, joints, and lungs. The disease mainly attacks children from 1 to 5 years of age. Before the use of antibiotics and sulphonamides the death rate was very high (30-60 per cent).

The population density plays an important part in the spread of meningitis. During epidemic outbreaks there is a large number of carriers for every individual affected by the disease. Ion-epidemic periods the carrier rate increases in the spring and autumn. Body resistance and the amount and virulence of the causative agent are significant. Depending on these factors, the spread of infection is either sporadic or epidemic.

Meningitis can also be caused by other pathogenic microbes (streptococci, E. coli, staphylococci, bacteria of influenza, mycobacteria of tuberculosis, and certain viruses). These organisms, however, cause sporadic outbreaks of the disease, while meningococci may cause epidemic meningitis.

Immunity. There is a well-developed natural immunity in humans. Acquired immunity is obtained not only as a result of the disease but also as the result of natural immunity developed during the meningococcal carrier state. In the course of the disease agglutinins, precipitins, opsonins, and complement-fixing antibodies are produced. Recurring infections are rare.

Laboratory diagnosis. Specimens of cerebrospinal fluid, nasopharyngeal discharge, blood, and organs obtained at autopsy are used for examination.

 

The following methods of investigation are employed: (1) microscopic examination of cerebrospinal fluid precipitate; (2) inoculation of this precipitate, blood or nasopharyngeal discharge into ascitic broth, blood agar, or ascitic agar; identification of the isolated cultures by their fermentative and serologic properties; differentiation of meningococci from the catarrhal micrococcus (Branhamella catarrhalis) and saprophytes normally present in the throat. The meningococcus ferments glucose and maltose, whereas Branhamella catarrhalis does not ferment carbohydrates, and Neisseria sicca ferments glucose, levulose, and maltose; (3) performance of the precipitin reaction with the cerebrospinal fluid.

Treatment. Antibiotics (penicillin, oxytetracycline, etc.) and sulphonamides (streptocid, methylsulphazine) are prescribed.

Prophylaxis is ensured by general sanitary procedures and epidemic control measures (early diagnosis, transference of patients to hospital), appropriate sanitary measures in relation to carriers, quarantine in children’s institutions. Observance of hygiene in factories, institutions public premises, and lodgings, and prevention of crowded condition are also obligatory. An antimeningococcal vaccine derived from the C/B serogroup is now under test. It contains specific polysaccharides.

The incidence of meningitis has grown recently. The disease follows a severe course and sometimes terminates in death.

 

Gonococci. The causative agent of gonorrhoea and blennorrhoea (Neisseria gonorrhoeae) was discovered in 1879 by A. Neisser in suppurative discharges. In 1885 E. Bumm isolated a pure culture of the organism and studied it in detail. Gonococci belong to the genus Neisseria, familyNeisseriaceae.

Morphology. Gonococci are morphologically similar to meningococci. The organism is a paired, bean-shaped coccus, measuring 0.6-1 mcm in diameter. It is Gram-negative and occurs inside and outside of the cells. Neither spores nor flagella are formed. Under the electron microscope a cell wall, 0.3-0.4 mcm in thickness, surrounding the gonococci is visible. The G+C content in DNA is 49.5 to 49.6 per cent.

 

Drawing  of doughnut-shaped diplococci of Neisseria gonorrhoeae as they sometimes appear under the microscope.

 

Pleomorphism of the gonococci is a characteristic property. They readily change their form under the effect of medicines, losing their typical shape, and growing larger, sometimes turning Gram-positive, and are found outside the cells.

In chronic forms of the disease autolysis of the gonococci takes place with formation of variant types (Asch types). Usually gonococcal cells varying in size and shape are formed. The tendency toward morphological variability among the gonococci should be taken into account in laboratory diagnosis. L-forms occur under the effect of penicillin.

Cultivation. The gonococcus is an aerobe or facultative anaerobe which does not grow on ordinary media, but can be cultivated readily on media containing human proteins (blood, serum, ascitic fluid) when the pH of the media is in the range of 7.2-7.6. The optimum temperature for growth is 37° C, and the organism does not grow at 25 and 42° C. It also requires an adequate degree of humidity. Ascitic agar, ascitic broth, and egg-yolk medium are the most suitable media. On solid media gonococci produce transparent, circular colonies, 1-3 mm in diameter. Cultures of gonococci form a pellicle in ascitic broth, which in a few days settles at the bottom of the test tube.

Fermentative properties. The gonococcus possesses low biochemical activity and no proteolytic activity. It ferments only glucose, with acid formation.

Toxin production. The gonococci do not produce soluble toxin (exotoxin) An endotoxin is released as a result of disintegration of the bacterial cells. This endotoxin is also toxic  for experimental animals.

Antigenic structure and classification. The antigenic structure of gonococci is associated with the protein (O-antigen) and polysaccharide (K-antigen) fractions. No group specific or international types of gonococci have been revealed. Gonococci and meningococci share some antigens in common.

Resistance. Gonococci are very sensitive to cooling. They do not survive drying, although they may live as long as 24 hours in a thick layer of pus or on moist objects. They are killed in 5 minutes at a temperature of 56 °C, and in several minutes after treatment with a 1 : 1000 silver nitrate solution or 1 per cent phenol.

Pathogenicity for animals. Gonococcus is not pathogenic for animals. An intraperitoneal injection of the culture into white mice results in fatal intoxication but does not produce typical gonorrhoea.

Pathogenesis and diseases in man. Patients with gonorrhoea are sources of the infection. The disease is transmitted via the genital organs and by articles of domestic use (diapers, sponges, towels, etc). The causative agent enters the body via the urethral mucous membranes and, in women, via the urethra and cervix uteri. Gonorrhoea is accompanied by acute pyogenic inflammation of the urethra, cervix uteri, and glands in the lower genital tract. Often, however, the upper genito-urinary organs are also involved. Inflammations of the uterus, uterine tubes, and ovaries occur in women, vulvovaginitis occurs in girls, and inflammation of the seminal vesicles and prostata in men. The disease may assume a chronic course. From the cervix uteri the gonococci can penetrate into the rectum. Inefficient treatment leads to affections of the joints and endocardium, and to septicaemia. Gonococci and Trichomonas vaginalis are often found at the same time in sick females. The trichomonads contain (in the phagosomes) gonococci protected by membranes against the effect of therapeutic agents. Gonococcus is responsible for gonorrhoeal conjunctivitis and blennorrhea in adults and newborn infants.

Immunity. The disease does not produce insusceptibility and there is no congenital immunity. Antibodies (agglutinins, precipitins, opsonins, and complement-fixing bodies) are present in patients’ sera, but they do not protect the body from reinfection and recurrence of symptoms. Phagocytosis in gonorrhoea is incomplete. The phagocytic and humoral immunity produced in gonorrhoea is incapable of providing complete protection, so, in view of this fact, treatment includes measures which increase body reactivity. This is achieved by raising the patient’s temperature artificially.

 

Laboratory diagnosis.

Specimens for microscopic examination are obtained from the discharge of the urethra, vagina, vulva, cervix uteri, prostate, rectal mucous membrane, and conjunctiva. The sperm and urine precipitates and filaments are also studied microscopically, Smears are stained by Gram’s method  and with methylene blue by Loeffler’s method). Microscopy is quite frequently an unreliable diagnostic method since other Gram-negative bacteria, identical to the gonococci, may be present in the material under test. Most specific are the immuno-fluorescence methods (both direct and indirect). In the direct method the organisms under test are exposed to the action of fluorescent antibodies specific to gonococci. In the indirect method, the known organisms (gonococci) are treated with patient’s serum. The combination of the antibody with the antigen becomes visible when fluorescent antiserum is added.

 

If diagnosis cannot be made by microscopic examination, isolation of the culture is carried out. For this purpose the test material (pus, conjunctival discharge, urine precipitate, etc.) is inoculated onto media. The Bordeux-Gengou complement-fixation reaction and the allergic test are employed in chronic and complicated cases of gonorrhea.

Treatment. Patients with gonorrhoea are prescribed antibiotics (bicillin-6, ampicillin, monomycin, kanamycin) and sulphonamides of a prolonged action. Injections of polyvalent vaccine and autovaccine as well as pyrotherapy (introduction of heterologous proteins) are applied in complicated cases.

Improper treatment renders the gonococci drug-resistant, and this may lead to the development of complications and to a chronic course of the disease.

Prophylaxis includes systematic precautions for establishing normal conditions of everyday and family life, health education and improvement of the general cultural and hygienic standards of the population.

In the control of gonorrhoea great importance is assigned to early exposure of sources of infection and contacts and to successful treatment of patients.

The prevention of blennorrhea is effected by introducing one or two drops of a 2 per cent silver nitrate solution into the conjunctival sac of all newborn infants. In certain cases (in prematurely born infants) silver nitrate gives no positive result. Good results are obtained by introducing two drops of a 3 per cent penicillin solution in oil into the conjunctival sac. The gonococci are killed in 15-30 minutes.

In spite of the use of effective antibiotics the incidence of gonorrhoea tends to be on the increase in all countries (Africa, America, South-Eastern Asia, Europe, etc.). The number of complications has also increased: gonococcal ophthalmia of newborn infants (blennorrhea), vulvovaginitis in children, and inflammation of the pelvic organs (salpingitis) and sterility in  women. The rise in the incidence of gonorrhoea is caused by social habits (prostitution, homosexualism, etc.), inefficient registration of individuals harbouring the disease, deficient treatment, and the appearance of gonococci resistant to the drugs used.

The WHO expert committee has recommended listing the gonococcal infection among infectious diseases with compulsory registration and making a profound study of the cause of the epidemic character of gonococcal diseases in certain African countries. Stricter blennorrhea control measures, and elaboration of uniform criteria of clinical and laboratory diagnosis, and treatment of gonococcal infection and more efficient methods for determining the sensitivity of circulating gonococci to various drugs are also recommended by the committee.

 

Additional material about diagnosis of Neisseria diseases

MENINGOCOCCAL INFECTION. Meningococcal infection is caused by meningococci (Neisseria meningitidis). The material to be tested is secretions from the nasal portion of the throat, cerebrospinal fluid, blood, and scrapings from elements of the haemorrhagic rash on the skin.

Cerebrospinal fluid is collected into a sterile tube to be inoculated onto nutrient media or to be promptly sent (without allowing it to cool down) to the  laboratory. This requirement is necessitated by the fact that meningococci are very sensitive to temperature fluctua­tions.

Mucosal secretions in the nasal portion of the throat are collected with a special swab bent at a definite angle. The best results are obtained when the nasopharyngeal mucus is immediately streaked onto solid nutrient media. To achieve the maximal separation of bacterial cells, 2-3 plates with medium are utilized. If the material is to be studied 3-5 hrs after the collection, it is inoculated onto a liquid nutrient medium (casein hydrolysate of fermentative split­ting, which contains 1.5 g/1 of amine nitrogen and 250 U/ml of ristomycin) and then placed in a 37 °C water bath. Thereafter, it is streaked onto serum agar and placed into an incubator.

Bacterioscopic examination of cerebrospinal fluid and blood per­mits detection of the causative agent. If the cerebrospinal fluid looks like pus, smears are prepared without its preliminary treatment whereas in the presence of only mild turbidity the cerebrospinal fluid is centrifuged and the deposit is used to make smears. The latter are stained with aniline dyes (aqueous solution of basic fuchsine, methylene blue) since the Gram staining method is associated with alteration in the formed elements of the cerebro­spinal fluid and a large number of artefacts. Meningococci appear as bean-shaped diplococci situated within the leukocyte cytoplasm and touching each other with concave edges. A tender capsule is quite a frequent finding. In meningococcaemia meningococci may be demonstrated in blood smears. A thick-drop (film) preparation is made, stained for 2-3 min with aqueous solution of methylene blue without fixation, washed in tap water, and dried in the air. On a light blue background of the preparation one can see dark blue leukocytes with numerous small dark-blue cocci arranged in clusters, pairs, and singly in and around leukocytes.

Rapid diagnosis is performed by means of gel precipitation, counter-immunoelectrophoresis with group precipitating antisera or radioimmunoassay and based on the detection in the  patient’s cerebro­spinal fluid or blood of the specific meningococcal antigen.

Bacteriological examination. The cerebrospinal fluid or its sedi­ment is cultured simultaneously with conducting bacterioscopic study. The meningococcus grows on special nutrient media contain­ing native protein (serum broth and agar). One can also use Hottinger’s agar containing 0.15 per cent of insoluble starch, which does not change the cultural, fermentative, and agglutinating properties of the causative agent. It is preferable that the cerebrospinal fluid be cultured after centrifugation at 3500 X g for five minutes. Some 0.3-0.5 ml of the material is taken from the bottom and 2-3 drops are placed on the surface of heated nutrient medium. The  inoculated cul­ture is incubated at 37 °C and in conditions of elevated CO₂ contents. To do it, place onto the lid of a sterile Petri dish a sheet of filter paper soaked with 1.5-2.0 ml of 10 per cent pyrogallic acid and then cover it with a second sheet moistened with 1.5-2.0 ml of 20 per cent solution of sodium hydrocarbonate. The inoculated dish is covered with the lid containing the paper sheets and inverted (lid downward). The remainder of the cerebrospinal fluid is utilized for counter-immunoelectrophoresis.

On the second day of incubation at 37 °C, the growth is studied for its cultural properties. Meningococci form small, round, convex, and transparent colonies. Smears made of these colonies display polymorphic diplococci and tetracocci. The microscopic picture is so diverse that it creates the impression of unpure culture. The colonies are subcultured onto a serum agar slant.

On the third day of investigation, the isolated culture is agglu­tinated with meningococcal sera.

Prior to the use of sulpha nil amide drugs and antibiotics, it is nec­essary to determine the serovar of the meningococcus responsible for the disease since treatment is based on specific meningococcal sera. The agglutination test in Noble’s modification is currently employed for determining the meningococcal serovar with an epidemiological purpose. Three-drop portions of thick suspension of micro­organisms are poured into three test tubes, then three-drop aliquots of undiluted or diluted 1:10 meningococcal serum of A, B, and C serovars are added to them. The mixture is shaken for 2-4 min, then 10-20 drops of isotonic sodium chloride solution are added to each test tube, and the results are read.

To assay the fermentative activity of pure culture, it is transferred to media with lactose, glucose, maltose, sucrose, and fructose. Me­ningococci ferment glucose and maltose with the production of acid. The culture is also streaked onto a 5 per cent yolk agar and serum agar containing 5 per cent sugar. After a 48-hour incubation, 1 drop of Lugol’s solution is put on the surface of the grown colonies. The appearance of brownish staining indicates polysaccharide splitting. Neisseria are identified by the oxidase test which consists in the following. On the colony formed on the serum agar place a drop of the freshly-prepared 1 per cent solution of hydrochloric paradiethylphenylendiamine. As a result, colonies possessing oxidase activity turn pink and then black. Such colonies are transferred to a serum agar for further investigation.

To differentiate between the meningococcus and non-pathogenic Neisseria (Neisseria catarrhalis), the ability of the latter to grow on simple nutrient media and to form colonies at room temperature (22 °C) is utilized.

To demonstrate the meningococcus in the blood, introduce 5-10 ml of blood obtained from a vein under sterile conditions into vials with 50 ml of broth containing 0.1 per cent of agar-agar. Subculture onto a serum agar 24 hours later. The procedures of isolation and identification of the cultures are the same as in the examination of cerebrospinal fluid.

Indirect haemagglutination with erythrocytes sensitized with group-specific polysaccharides is employed for serological diagnosis.

GONOCOCCAL INFECTION. The causative agent of gonorrhoea is the gonococcus (Neisseria gonorrhoeae} which is morphologically similar to the meningococcus. Bacterioscopic, bacteriological, and serological techniques are em­ployed for the diagnosis of this disease.

Bacterioscopic examination is the main method for diagnosing acute gonorrhoea and blennorrhea. The  material for examination is taken from the urethra in the following manner: wipe the urethral opening with cotton wool moistened with sterile physiological salt solution, press with your finger onto the posterior wall of the urethra in the outward direction (in females the forefinger is inserted into the vagina for this purpose), and express a drop of pus. The secretion from the prostate is obtained by prostatic massage. The secretion of the cervical mucosa is collected with a swab, following intravaginal introduction of Cusco’s speculum. In patients with blennorrhea conjunctival secretion is removed with a loop and spread over a glass slide. The preparation is stained with alkaline solution of methylene blue and with the Gram stain (two smears). Upon microscopic examination gonococci appear as bean-shaped Gram-negative diplo­cocci positioned outside or inside the cells (neutrophilic granulocytes) similar to meningococci.

Gram’s staining allows differentiation of the gonococci from other bacteria. To ensure a more distinct outline of the gono­cocci, smears should be fixed by dimethylsulphoxide (dimexide). Pour dimexide on the smear until it is completely dry and then stain it.

Since the examined material may also contain other Gram-nega­tive bacteria resembling the gonococci, both direct and indirect immunofluorescence methods are employed. In the direct immunofluorescence test the smears are treated with fluorescent antibodies against gonococci, in the indirect one, gonococci and the patient’s serum are used. Conjugation between the antibody and the antigen be­comes evident when a fluorescent serum against human globulins is added.

Bacteriological examination is carried out when the study of smears reveals either no gonococci or only their atypical, altered forms. In view of extreme sensitivity-of the gonococcus to tempera­ture the material tested should not be transported. Moreover, the gonococcus is very sensitive to disinfectants, so it is advisable that 1 to 2 days before culturing the patients should temporarily discon­tinue the use of disinfectants and antibacterial drugs.

The material is inoculated immediately after its collection onto plates with a protein-containing meat-peptone agar. Ascitic-free media with casein digest, yeast autolysate, and native cattle serum are widely utilized for this purpose. Inclusion into the nutrient medium of ristomycin and poIymixin M (10 U/ml) significantly en­hances gonococcal growth. Prior to inoculation, the nutrient medium should be heated in an incubator. To facilitate better growth of the gonococci, the inoculated plates are placed into an exsiccator with a CO₂ concentration amounting to 10 per cent.

A 24-hour incubation at 37 °C brings about the formation of trans­parent, with smooth edges, convex, mucoid colonies of the gonococcus, which resemble drops of dew. Pure culture is isolated and identified. Biochemically, the gonococcus shows weak activity and breaks down only glucose with the formation of acid. To determine oxidase activity, the culture is introduced into yolk medium (to 100 ml of protein-containing meat-peptone agar add 1.5 g of glu­cose, 6 ml of phenol red solution, and 15 ml of egg yolk). Agglutina­tion with specific serum does not always yield positive results be­cause the gonococcus has many serovars and the serum may contain only low titres of the appropriate agglutinins.

Serological diagnosis is resorted to in chronic gonorrhoea when the patient has no discharge, and bacterioscopic and bacteriological examinations are impossible. In such cases the complement-fixation test with the patient’s blood serum or indirect immunofluorescence is used. A gonococcal vaccine or a special antigen prepared of killed (by variable methods, with antiformin being the most common one) gonococci is employed as the antigen.

 

 

Causetive agents of bacterial intestinal diseases: escherichiosis, typhoid fever, paratyphoids, salmonellosis

 

ENTEROBACTERIACEAE

The Enterobacteriaceae contain gram negative rodswhich, if motile, are peritrichously flagellated.

 

Because members of this family are morphologically and metabolically similar, much effort has been expended to develop techniques for their rapid identification. In general, biochemical properties are used to define a genus, and further subdivision frequently is based on sugar fermentation andantigenic differences. Yet, many paradoxes exist, for example, more than 2600 species of Salmonella have beennamed, whereas the equally complex species Escherichiacoli is divided into more than 1000 serotypes. Over the yearse, many taxonomists with different ideas have been involved in the classification of these bacteria, and disagreement still exists concerning family and genericnames. Table gives an outline of the taxonomic scheme proposed by Ewing and Martin for the Enterobacteriaceae, compared with that proposed in Bergey’s Manual of Systematic Bacteriology. As shown, Bergey’s has elimimated all tribes in the taxonomic division of this large family. Both schemes are used in various diagnostic laboratories, but this chapter adheres more closely to the Bergey classification.

Table

Classification of the Enterobacteriaceae

 

Biochemical Properties Used for Classification

Early taxonomic schemes relied heavily on the organism’s ability to ferment lactose, and numerous differential andselective media have been devised to allow one to recognize a lactose fermenting colony on a solid medium. The effectiveness of such differential media is based on the fact that organisms fermenting the lactose form acid, whereas nonlactose fermenters use the peptones present and donot form acids m these media. The incorporation of anacid base indicator into the agar medium thus causes acolor change around a lactose fermenting colony. Thus has been a valuable technique for selectingthe major nonlactose fermenting pathogens that causesalmonellosis or shigellosis, under special conditions, however, many lactose fermenters also cause a variety of infectious diseases.

Furthermore, many enterics ferment lactose onlyslowly, requiring several days before sufficient acid isformed to change the indicator. They all synthesize beta galactosidase, (the enzyme that splits lactose into glucoseand galactose) but lack the specific permease necessary for the transport of lactose into the cell One can easilydetermine whether an organism is a slow lactose or nonlactose fermenter by mixing a loopful of bacteria with orthonitrophenol beta galactoside (ONPG) dissolved ina detergent. The linkage of the galactose in ONPG is thesame as its linkage m lactose, inasmuch as the ONPG canenter the cell in the absence of a permease, an organism possessing beta galactosidase will hydrolyze ONPG to yield galactose and the bright yellow compound, orthonitrophenol. Thus, only the absence of a specific lactose permease differentiates the slow lactose fermenters fromthe normal lactose fermenters.

In addition, a number of selective media have been devised that contain bile salts, dyes such as brilliant greenand methylene blue, and chemicals such as selenite and bismuth. The incorporation of such compounds into thegrowth of medium has allowed for the selective growth of the enterics while inhibiting the growth of gram positive organisms.

Some other biochemical properties used to classify members of the Enterobacteriaceae include the ability to form H₂S; decarboxylate the ammo acids lysine, ornithine,or phenylalanine, hydrolyze urea into CO₂ and NH₃, form indole from tryptophan; grow with citrate as a sole source of carbon; liquefy gelatin; and ferment a large variety of sugars.

 

Serologic Properties Used for Classification

No other group of organisms has been so extensively classified on the basis of cell surface antigens as the Enterobacteriaceae. These antigens can be divided into threetypes, designated O, K, and H antigens.

O ANTIGENS. All gram-negative bacteria possess a lipopolysaccharide (LPS) as a component of their outer membrane. This toxic LPS (also called endotoxin) is composed of three regions, lipid A, core, and arepeating sequence of carbohydrates called the O antigen. Based on different sugars, alpha- or beta-glycosidic linkages, and the presence or absence of substituted acetyl groups, Escherichia coil can be shown to possess at least 173 different 0 antigens, and 64 have beendescribed in the genus Salmonella.

Sometimes, after continuous laboratory growth,strains will, through mutation, lose the ability to synthesize or attach this oligosaccharie O antigen to the coreregion of the LPS. This loss results in a change from a smooth colony to a rough colony type, and it is referred to as an S to R transformation Interestingly, the R mutants have lost the ability to produce disease.

K ANTIGENS. K antigens exist as capsule or envelope polysaccharidesand cover the O antigens when present, inhibiting agglutinarion by specific 0 antiserum. Most K antigens can be removed by boiling the organisms in water.

H ANTIGENS. Only organisms that are motile possess H antigens because these determinants are in the proteins that makeup the flagella. However, to complicate matters, members of the genus Salmonella alternate back and forth to formdifferent H antigens. The more specific antigens are called phase 1 antigens and are designated by lower-case letters (a, b, c, and so on), whereas the less-specific phase 2 H antigens  are giveumbers. The mechanism of this phase variation reveals an interesting way in which a cell canregulate the expression of its genes. In short, Salmonella possesses two genes. H1 encoding for phase 1 flagellar antigens, and H2 encoding for phase 2 flagellar antigens.The transcription of H2 results in the co-ordinate expression of gene rhl, which codes for a repressor that preventsthe expression of H 1. About every 10³ to 10^s generations, a 900-base-pair region, containing the promoter for the H2 gene, undergoes a site-specific inversion, stopping the transcription of both H2 and rhl. In the absence of the rhl gene product, the H1 gene is then transcribed until the 900-base pair region in the H2 promoter is again inverted, resulting in the expression ofH2 and rhl.

After obtaining the serologic data, an antigenic formula can be written, such as E.coli  O111:K-58:H6, meaning this E. coli possesses O antigen 111, K antigen 58, and H antigen 6. The formula Salmonella togo 4,12:1,w:1,6 indicates this serotype of Salmonella possesses O antigens 4 and 12, phase 1 H antigens  1 and w, and phase 2 H antigens  1 and 6.

 

Escherichia coli. The organism was isolated from faeces in 1885 by T. Escherich.  E. coli is a common inhabitant of the large intestine of humans and mammals. It is also found in the guts of birds, reptiles, amphibians, and insects. The bacteria are excreted in great numbers with the faeces and are always present in the external environment (soil, water, foodstuffs, and other objects).

Morphology. E coli are straight rods measuring 0.4-0.7 in breadth and 1-3 in length. They occur as individual organisms or in pairs and are marked by polymorphism. There are motile and non-motile types. The G+C content in DNA is 50-51 per cent. The cell surface has pili on which certain phages are adsorbed. The microcapsule is not always clearly defined.

Cultivation. E. coli is a facultative anaerobe. The optimum temperature for growth is 30-37 °C and the optimum pH value of medium up 7.2-7.5. The organism also grows readily on ordinary media at room temperature and at 10 and 45 °C, growth becomes visible in the first two days. E. coli from cold-blooded animals grows at 22-37° C but not at 42-43° C.

On meat-peptone agar E. coli produces slightly convex semitransparent, greyish colonies, and in meat broth it forms diffuse turbidity and a precipitate. The organism produces colonies which are red on Ploskirev’s medium, red with a metallic hue on Endo’s medium, and dark-blue on Levin’s medium.

Fermentative properties. E. coli does not liquefy gelatin. It produces indole and hydrogen sulphide, and reduces nitrates to nitrites; ferments glucose, levulose, lactose, maltose, mannitol, arabinose, galactose, xylose, rhamnose, and occasionally saccharose, raffinose, dulcitol, salycin, and glycerin, with acid and gas formation. It also coagulates milk. There are varieties of the bacteria which ferment saccharose, do not produce indole, have no flagella, and do not ferment lactose.

 

Endo’s medium

 

Toxin production. Certain strains of E. coli are conditionally pathogenic They contain a gluco-lipo-protein complex with which their toxic, antigenic, and immunogenic properties are associated. Some strains possess haemolytic properties (O124 and others) determined by plasmids. Pathogenic cultures possess endotoxins and thermolabile neurotropic exotoxins. The latter accumulate in broth cultures on the second-fourth day of cultivation, while the endotoxins appear only after the twentieth day. Haemotoxins and pyrogenic substances, proteinases, deoxyribonucleases, urease, phosphatase, hyaluronidase, amino acid decarboxylases have been obtained from pathogenic strains.

Antigenic structure. The antigenic structure of E. coli is characterized by variability and marked individuality. Along with the H- and O-antigens, the presence of other antigens has been shown m some strains, i.e. the surface somatic (membranous, capsular) K-antigens which contain the thermolabile L- and B-antigens and the thermostable A- and M-antigens.

Each antigen group in its turn is composed of a number of antigens designated by Arabic numbers, e.g. the O-group has 173 antigens, the K-subgroup 90, the H-subgroup 50, etc. On the basis of antigenic structure an antigenic formula is derived which fully reflects the antigenic properties of the strain For example, one of the most widely spread serotypes is designated 0111 : K58 : H2. Under the effect of transformation, lysogenic conversion, transduction, and conjugation E. coli may change its antigenic properties.

Numerous varieties of the organism are produced on cultivation under artificial conditions. Such varieties are not only of theoretical interest, but also of great practical importance in laboratory diagnosis of enteric infections.

Classification. Genus Escherichia includes one E. coli species consisting of several biotypes and serotypes. They are differentiated according to cultural, biochemical, and serological properties. The genus Escherichia includes E. coli, E. freundi, E. intermedia, and others. E. coli comprises several varieties which are differentiated by their cultural and biochemical properties. F. Kauffmann has detected 25 O-groups responsible for various diseases in humans.

About 50 phage variants have been revealed among E. coli organisms. They are used in laboratory diagnosis as confirmatory characteristics of the isolated serotypes.

Resistance. E. coli survives in the external environment for months. It is more resistant to physical and chemical factors of the external environment than the typhoid and dysentery bacteria. E. coli is killed comparatively rapidly by all methods and preparations used for disinfection. At 55° C the organism perishes in 1 hour, and at 60° C in 15 minutes. E. coli is sensitive to brilliant green.

E. coli is used as a test microbe in the assay of disinfectants and methods of disinfection and also in titration of certain antibiotics.

Pathogenicity for animals. The pathogenic serovars of E. coli cause severe infections in calf sucklings giving rise to an extremely high mortality. A parenteral injection of the culture into rabbits, guinea pigs, and white mice results in a fatal toxico-septical condition.

Pathogenesis and diseases in man. Definite E. coli serogroups are capable of causing various acute intestinal diseases in humans: (1) the causative agents of colienteritis in children are O-groups-25, -26, -44,   -55, -86, -91, -111, -114, -119, -125, -126, -127, -128, -141, -146, and others (they cause diseases in infants of the first months of life and in older infants); (2) the causative agents of dysentery-like diseases are E. coli of the O-groups-23, -32, -115, -124, -136, -143, -144, -151, and others; (3) the causative agents of cholera-like diarrhoea are the O-groups-6, -15, -78, -148, and others, they produce thermolabile and thermoresistant enterotoxins.

Colienteritis begins acutely with high temperature (38-39 °C), and frequently with severe meteorism, vomiting, diarrhoea, and general toxicosis. The disease usually occurs in infants of the first year of life.

The infection is acquired from sick children or carriers. Pathogenic E. coli serovars are found on various objects. It is assumed that colienteritis is transmitted not only by the normal route for enteric infections but also through the respiratory tract by the droplets and dust.

The pathogenesis of colienteritis depends entirely on the organism’s condition. In prematurely born infants and in infants during the first months of life the bactericidal activity of blood is considerably lower in respect to the pathogenic E. coli serovars in comparison to the nonpathogenic types. The reactivity of the child’s body at the time of infection plays an important role in the mechanism of resistance to the pathogenic strains. The pathological process develops mainly in the small intestine. Most probably, the mucous membrane of the small intestine in particular is exposed to the action of thermolabile toxic substances. Serovars O-124, O-151 and others cause diseases which are similar to dysentery.

E. coli may cause colibacillosis in adults (peritonitis, meningitis, enteritis, toxinfections, cystitis, pyelitis, pyelonephntis, angiocholitis, salpingooophontis, appendicitis, otitis, puerperal sepsis, etc.). Over-strain, exhaustion, and conditions following infectious diseases facilitate the onset of various E. coli infections. In a number of cases the organism is responsible for food poisoning.

Immunity. In individuals who had suffered from diseases caused by pathogenic E. coli serovars, cross immunity is not produced owing to which re-infection may occur. Over 85 per cent of E. coli strains contain inhibiting substances, colicins, marked by antagonistic properties in relation to pathogenic microbes of the enteric group, they are used as therapeutic and preventive agents, e.g. colibacterin (E. coli M 17, etc.).

Besides this, E colt as well as other common inhabitants of the intestine are capable of synthesizing various vitamins (K₂, E, and group B) which are indispensable to the human organism. The ability of various E. coli serovars to suppress the growth of Mycobacterium tuberculosis has also been observed. The suppression of E. coli and other members of the biocoenosis may result in a chronic disease known as dysbacteriosis.

Laboratory diagnosis. The patients’ faeces, throat and nasal discharges, material obtained at autopsy (blood, bile, liver, spleen, lungs, contents of the small and large intestine, pus), water, foodstuffs, and samples of washings from objects and hands of staff of maternity hospitals, hospitals, and dairy kitchens are all used for laboratory examination during colienteritis. If possible, faecal material should be seeded immediately after it has been collected. The throat and nasal discharges are collected with a sterile swab. Specimens of organs obtained at autopsy are placed in separate sterile jars.

The tested material is inoculated onto solid nutrient media (Endo’s, Levin’s) and, simultaneously, onto Ploskirev’s media and bismuth-sulphite agar for isolation of bacteria of the typhoid-paratyphoid and dysentery group. BIood is first inoculated into broth and then subcultured on solid media when development of a septic process is suspected. Pus is collected for examination in suppurative lesions. It is placed into a dry sterile vessel and then inoculated onto the differential media of Endo or Levin. The pure culture isolate is identified by its morphological, cultural, biochemical, serological, and biological properties.

T

he corresponding 0-group to which an enteropathogenic-serovars belong is determined by means of the agglutination reaction after the K-antigen of the culture that is being studied has been destroyed by boiling.

Besides, the immunofluorescence method employing type specific labelled sera is also used. It yields a preliminary answer in one to two hours.

In serological diagnosis of colienteritis beginning with the third to fifth day of the disease the indirect haemagglutination reaction is used which excels the agglutination reaction in sensitivity. It is positive when the antibody titre grows in the course of the diseased.

Treatment. Patients with colienteritis are prescribed antibiotics (tetracycline with vitamins C, B₁ and B₂) and biopreparations (coli autovaccine, coli bacteriophage, colicin, bacterin, lactobacterin, bificol, bifidumbacterin). Physiological solutions with glucose are injected for controlling toxicosis.

Prophylaxis. To prevent diseases caused by pathogenic serovars of E. coli, special attention is given to early identification of individuals suffering from colienteritis, and also to their hospitalization and effective treatment. Regular examination of personnel is necessary in children’s institutions as well as of mothers whose children are suffering from dyspepsia. Considerable importance is assigned to observation of sanitary regulations in children’s institutions, infant-feeding centres, maternity hospitals, and children’s nurseries. Protection of water and foodstuff’s from contamination with faeces, the control of flies, and gradual improvement of standards of hygiene of the population are also particularly important.

Sanitary significance of E. coli. This organism is widely spread iature. It occurs in soil, water, foodstuff’s, and on various objects. For this reason E. coli serves as an indicator of faecal contamination of the external environment.

Detection of E. coli is of great importance in estimating the sanitary index of faecal contamination of water, foodstuff’s, soil, beverages, objects, and hand-washings. The degree of contamination of water, soil and foodstuff’s is determined by the coli titre or coli index (these terms have been discussed in the chapter concerning the spread of microbes iature). Faecal contamination of articles of use is estimated by qualitative determination of the presence of E. coli.

Additional materials

Pathogenicity of Escherichia coli. Although E. coli is part of the normal flora of the intestinaltract, it is also the most common gram-negative pathogen responsible for nosocomially acquired septic shock, meningitis ieonates, cystitis and pyelonephritis in women, and for several distinct forms of diarrheal disease and dysentery affecting populations throughout the world. Strains of E coli capable of causing such diseases possess one or more virulence factors that are not found in E. coli strains comprising the normal flora. Such virulence factorscan be characterized as follows, the capacity to adhere to specific mammalian cells; the ability to invade and grow intracellularly in intestinal epithelial cells; the secretion of one or more enterotoxins that cause fluid loss, resulting mdiarrhea; the formation of a cytotoxin that blocks protein synthesis, causing a hemorrhagic colitis; and the possession of an antiphagocytic capsule that is responsible, at least in part, for the bacteremia and meningitis caused by E. coli. In addition, the ability to obtain iron from transferrin or lactoferrin by the synthesis of iron-binding siderophores markedly enhances the virulence of such strains through their ability to grow in host tissues. No one strain of E. coli possesses all of these properties but, as is discussedlater, all pathogenic strains must have one or more virulence factors to produce disease.

Diarrheal Diseases. It is estimated that during the American Revolutionary War there were more deaths from diarrhea than from English bullets, and during the American War between the states, over 25% of all deaths were because of diarrheaand dysentery. Diarrhea kills more people worldwide than AIDS and cancer, with about five million diarrheal deaths occurring annually primarily because of dehydrationMost of these occur ieonates and young children, anda large number are caused by pathogenic E. coli. Thedisease in adults, known by many names such as traveller’sdiarrhea or Montezuma’s revenge, may vary from a milddisease with several days of loose stools to a severe andfatal cholera-like disease. Such life-threatening E. coli infections occur throughout the world but are most com-mon in developing nations.

The virulence factors responsible for diarrheal diseaseare frequently encoded in plasmids, which may be spreadfrom one strain to another either through transduction^: or by recombination. As a result, various combinations of virulence factors have occurred, which has been used to place the diarrhea-producing strains of E. coli into variousgroups based on the mechanism of disease production

Enterotoxigenic Escherichia coli. Enterotoxin-producing E coli, called enterotoxigenic E.coli (ETEC), produce one or both of two different toxins – a heat labile toxin called LT and a heat-stable toxin called ST. The genetic ability to produce both LT and ST is controlled by DNA residing in transmissible plasmids called ent plasmids. Both genes have been cloned, and the ST gene has been shown to possess the characteristics of a transposon.

HEAT-LABILE TOXIN. The heat-labile toxin LT, which is destroyed by heating at 65 °C for 30 minutes, has been extensively purified, and its mode of action is identical to that described for cholera toxin (CT). LT has a molecula rweight of about 86,000 daltons and is composed of twosubunits, A and B Subunit A consists of one moleculeof A_i (24,000 daltons) and one molecule of A₂ (5000daltons) linked by a disulfide bridge. Each A unit is joinednoncovalently to five B subunits.

Like CT, LT causes diarrhea by stimulating the activity of a membrane-bound adenylate cyclase.This results in the conversion of ATP to cyclic AMP (cAMP):  ATP ® cAMP + PPi

Minute amounts of cAMIP induce the active secretion of Cl^– and inhibit the absorption of NaCI, creating an electrolyte imbalance across the intestinal mucosa, resulting in the loss of copious quantities of fluid and elec-trolytes from the intestine.

The mechanism by which LT stimulates the activityof the adenylate cyclase is as follows: (1) The B subunit of the toxin binds to a specific cell receptor, GM₁ ganglioside, (2) the A₁ subunit is released from the toxin and enters the cell; and (3) the A₁ subunit cleaves nicotinamide-adenic dinucleonde (NAD) into nicotinamide and ADP-ribose and, together with a cellular ADP-ribosylating factor, transfers the ADP-ribose to aGTP-binding protein. The ADP-ribosylation of the GTP-binding protein inhibits a GTPase activity of the binding protein, leading to increased stability of the catalytic cornplex responsible for adenylate cyclase activity. This results in an amplified activity of the cyclase and a corresponding increase in the amount of cAMP produced.

Two antigenically distinct heat labile toxins are produced by various strains of E. coli. LT-I is structurally andantigenically related to CT to an extent that anti-CT will neutralize LT I LT-II has, on rare occasions, beenisolated from the feces of humans with diarrhea, but it is most frequently isolated from feces of water buftalos and cows LT-II is biologically similar to LT-I, but it is notneutralized by either anti-LT-I or anti-CT.

LT will bind to many types of mammalian cells, and its ability to stimulate adenylate cyclase can be assayed incell cultures.

A report has also shown that CT stimulated an increase in prostaglandin E (PGE), and that PGE1 and PGE2 caused a marked fluid accumulation in the ligated lumen of rabbit intestinal segments. The mechanism whereby CT induces PGE release is unknown.

HEAT-STABLE TOXIN. The heat-stable toxin STa consists of afamily of small, heterogeneous polypeptides of 1500 to 2000 daltons that are not destroyed by heating at 100 °Cfor 30 minutes. STa has no effect on the concentrationof cAMP, but it does cause a marked increase m thecellular levels of cyclic GMP (cGMP). cGMP causes aninhibition of the cotransport of NaCI across the intestinal wall, suggesting that the action of STa may be primarily antiabsorptive compared with that of LT, which is both antiabsorptive and secretory.

STa stimulates guanylate cyclase only in intestinal cells, indicating that such cells possess a unique receptorfor Sta. The cell receptor for STa is known to be either tightly coupled to, or a part of, a particulate form of guanylate cyclase located in the brush border membranes of intestinal mucosal cells. Also, intimately associated with this complex is a cGMP-dependent protein kinase that phosphorylates a 25,000 dalton protein in the brush border. It has been proposed that this phosphorylated protein might be the actual mediator for the toxin-induced iontransport alterations that lead to fluid loss. The usual assay for STa is to inject the toxin intragastrically into a 1 – to 4-day old suckling mouse and measure intestinal fluid accumulation (as a ratio of intestinal/remaining body weight) after 4 hours. STa may also be assayed directly by measuring its effect on the increase in guanylate cyclasein homogenized intestinal epithelial cells.

A second heat stable toxin that is produced by somestrains of E. coli has been termed STb. This toxin is inactivein suckling mice but will produce diarrhea in weaned piglets. STb producers have not been isolated from humans. It does not seem to increase the level of adenylate or guanylate cyclase in intestinal mucosal cells, but maystimulate the synthesis of prostaglandin E2. The end resuit is to enhance net bicarbonate ion secretion.

Enterohemorrhagic Escherichia coli. The enterohemorrhagic E. coli (EHEC) were first described in 1982 when they were shown to be the etiologic agent of hemorrhagic colitis, a disease characterized bysevere abdominal cramps and a copious, bloody diarrhea. These organisms are also known to cause a condition termed hemolytic-uremic syndrome (HUS), which is manifested by a hemolytic anemia, thrombocytopenia (decrease in the number of blood platelets), and acuterenal failure. HUS occurs most frequently in children.

Although most initially recognized EHEC belong to serotype O157:H7, other EHEC serotypes such as O26, O111, O128, and O143 have been recognized. These organisms are not invasive, but they do possess a 60-megadalton plasmid that encodes for a fimbrial antigen that adheres to intestinal epithelium. In addition, the EHEC are lysogenic for one or more bacteriophages that encode for the production of one or both of two antigenically distinct toxins. These toxins are biologically identical and antigenically similar to the toxins formed by Shigella dysenteriae (Shiga’s bacillus), and are designated as Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT II). Because the Shiga-like toxins initially were characterized by their ability to kill Vero cells, a cell line developed from African green monkey kidney cells, they also arecalled Verotoxin I and Verotoxin II.

SLT I consists of an A subunit and five B subunits. The sequence of the B subunit from S. dysenteriae type 1 is identical to that of the B subunit of SLT I. The B subunit binds specifically to a glycolipid in microvillus membranes, and the released A subunit stops protein synthesis by inactivating the 60S ribosomal subunit. This inactivation results from the N-glycosidase activity of the toxin, which cleaves off an adenine molecule (A-4324) from the 28S ribosomal RNA, causing a structural modification of the 60S  subunit, resulting in a reduced affinity for EF-1 and, thus, an inhibition of aminoacyl- tRNA binding. The consequence of toxin action is a cessation of protein synthesis, the sloughing off of dead cells, anda bloody diarrhea. Notice that SLT 1 carries out the same reaction as the plant toxins ricin and abrin.

SLT II is biologically similar to SLT I, but because only a 50% to 60% homology exists between the two toxins, it is not surprising that they are antigenically distinct. Interestingly, both STL I and STL-II can be transferred to nontoxin producing strains of E. coli by transduction.

Outbreaks of hemorrhagic colitis have been traced to contaminated food as well as to person to person transmission iursing homes and day care centres. Contaminated, undercooked hamburger meat seems to be the most frequently implicated source of food borne illnesses followed by contaminated milk and water, indicating thatcattle are a common reservoir for EHEC. Of note is that E. coli  0157:H7 has been shown to survive up to 9 months at -20°C in ground beef.

Thus, the EHEC are able to cause hemorrhagic colitis as a result of their ability to adhere to the intestinal mucosa, and they presumably destroy the intestinal epithelial lining through their secretion of Shiga like toxins. The mechanism whereby the EHEC cause HUS is unclear but seems to follow bloodstream carriage of SLT II to the kidney. Experimental results have shown that humanrenal endothelial cells contain high levels of receptor for SLT-2. Moreover, in the presence of interleukin (IL)1/b, the amount of receptor increases, enhancing the internalization of the toxin and the death of the cell.

The section, “A Closer Look,” describes several epidemics of hemorrhagic colitis that have occurred in the United States and techniques that are used for the identification of this serotype

Enteroinvasive Escherichia coli

The disease produced by the enteroinvasive E. coli (EIEC) is indistinguishable from the dysentery produced by members of the genus Shigella, although the shigellae seem to be more virulent because considerably fewer shigellae are required than EIEC to cause diarrhea. The key virulence factor required by the EIEC is the ability to invade the epithelial cells.

EIEC INVASION. The specific property that provides these organisms with their invasive potential is far from understood. It is known, however, that this ability is encoded in a plasmid and that the loss of the plasmid results m aloss of invasive ability and a loss of virulence. Moreover, the shigellae seem to possess the same plasmid, because Western blots show that shigellae and EIEC plasmids express polypeptides that are similar in molecular weight and antigenicity.

EIEC TOXINS. Although the primary virulence factor of EIEC strains is the ability to invade intestinal epithelial cells, they also synthesize varying amounts of SIT I and SLT II. Based on the severity of the disease, however, it could assumed that the amount of toxin produced is considerably less than that formed by the highly virulent shigellae or the EHEC. Other enterotoxic products produced by the EIEC are under study.

EIEC can be distinguished from other E. coli by their ability to cause an inflammatory conjunctivitis in guinea pigs, an assay termed a Sereny test. A DNA probe thathybridizes with colony blots of EIEC and all species of Shigella also has been used to identify organisms producing Shiga-like toxins.

Enteropathogenic Escherichia coli. The enteropathogenic E. coli (EPEC) are diffusely adherent organisms that are particularly important in infantdiarrhea occurring in developing countries, where they may cause a mortality rate as high as a 50%. They comprise a mixture of organisms that seem to produce diarrhea by a two step process. The classic EPEC exist among a dozen or so different serotypes, all of which are characterizedby the possession of a 55 to 65-megadalton plasmid that encodes for an adhesin termed EPEC adherence factor (EAF). EAF causes a localized adherence of the bacteria to enterocytes of the small bowel, resulting m distinct microcolonies. This is followed by the formation ofunique pedestal-like structures bearing the adherent bacteria. These structures have been termed attaching and effacing lesions. The ability to form the effacing lesion resides in an attaching and effacing gene (eae). The lesions are characterized by a loss of microvilli and a rearrangement of the cytoskeleton, with a proliferation of filamentous actin beneath are as of bacterial attachment.

Thus, the ability of the EPEC to cause diarrhea involves two distinct genes, EAF and eae. The end result is anelevated intracellular Ca⁺² level in the intestinal epithelialcells and the initiation of signal transduction, leading to protein tyrosine phosphorylation of at least two eucaryotic proteins.

EPEC strains routinely have been considered noninvasive, but data have indicated that such strains can invadeepithelial cells in culture. However, EPEC strains do not typically cause a bloody diarrhea, and the significance of cell invasion during infection remains uncertain.

Other Diairhea-Producing Escherichia coli. All possible combinations, deletions, or additions of the various virulence factors responsible for intestinal fluid loss result in diarrhea producing strains that do not fitthe categories already described. Such has been found tobe the case.

The most recent of these has been termed the enteroaggregative E. coli. These strains seem to cause diarrhea through their ability to adhere to the intestinal mucosa and possibly by yet a new type of enterotoxin. It seems possible that the acquisition of other virulence factors may result in the discovery of additional pathogenic strains of E. coli.

E. coli Urinary Tract Infections. Escherichia coli is the most common cause of urinary tract infections of the bladder (cystitis) and, less frequently, of the kidney (pyelonephritis). In either case, infections usually are of an ascending type (enter the bladder fromthe urethra and enter the kidneys from the bladder). Many infections occur in young female patients, in persons with urinary tract obstructions, and in persons requiring urinary catheters, and they occur frequently in otherwise healthy women. Interestingly, good data support the postulation that certain serotypes of E. coli are more likely to cause pyelonephritis than others. Thus, the ability to produce P-fimbriae (so called because of their ability to bind to P blood group antigen) has been correlated withthe ability to produce urinary tract infections, seemingly by mediating the adherence of the organisms to human uroepithelial cells. Of note is that the rate of nosocomial urinary tract infection per person-day was significantly greater in patients with diarrhea, particularly in those with an indwelling urinary catheter.

In addition to fimbrial adhesins, a series of afimbrial adhesins has been reported. Their role in disease is not yet firmly established, but it has been demonstrated that at least one afimbrial adhesins mediated specific binding to uroepithelial cells.

Recurrent urinary tract infections in premenopausal, sexually active women frequently can be prevented by the postcoital administration of a single tablet of an antibacterial agent such as trimethoprim-sulfamethoxazole, cinoxacin, or cephalexin.

E. coli Systemic Infections. About 300,000 patients in United States hospitals develop gram-negative bacteremia annually, and about 100,000 of these persons the of septic shock. As might be guessed, E. coli is the most common organism involved in such infections. The ultimate cause of death in these cases is an endotoxin-induced synthesis and release of tumor necrosis factor-alpha and IL-1, resulting in irreversible shock.

The newborn is particularly susceptible to meningitis, especially during the first month of life. A survey of 132 cases of neonatal meningitis occurring in the Netherlands reported that 47% resulted from E. coli and 24% from group B streptococci. Notice that almost 90% of all cases of E. coli meningitis are caused by the K1 strain, which possesses a capsule identical to that occurring on group B meningococci.

Table  summarizes the virulence factors associated with pathogenic E. coli.

Table

Escherichia coif Virulence Factors

 

SALMONELLA

Enteric Fever and Paratyphoid Salmonellae. The causative agent of enteric (typhoid) fever, Salmonella typhi was discovered in 1880 by K. Eberth and isolated in pure culture in 1884 by G. Gaffky.

In 1896 the French scientists C. Archard and R. Bensaude isolated paratyphoid B bacteria from urine and pus collected from patients with clinical symptoms of typhoid fever. The bacterium responsible for  paratyphoid A (Salmonella paratyphi) was studied in detail m 1902 by the German bacteriologists A. Brion and H. Kayser, and the causative agent of paratyphoid B {Salmonella schottmuelleri) was studied in 1900 by H. Schottmueller.

Morphology. The morphology of the typhoid salmonella corresponds with the general characteristics of the Enterobacteriaceae family. Most of the strains are motile and possess flagella, from 8 to 20 iumber. It is possible that the flagella form various numbers of bunches.

The paratyphoid salmonellae do not differ from the typhoid organisms in shape, size, type of flagella, and staining properties.

The typhoid salmonellae possess individual and intraspecies variability. When subjected to disinfectants, irradiation, and to the effect of other factors of the external environment they change size and shape. They may become coccal, elongated (8-10 mcm), or even threadlike. The G+C content in DNA ranges between 45 and 49 per cent.

Cultivation. The typhoid and paratyphoid organisms are facultative anaerobes. The optimum temperature for growth is 37° C, but they also grow at temperatures between 15 and 41°C. They grow on ordinary media at pH 6.8-7.2. On meat-peptone agar S. typhi forms semitransparent fragile colonies which are half or one-third the size of E. coli colonies. On gelatin the colonies resemble a grape leaf in shape Cultures on agar slants form a moist transparent film of growth without a pigment and in meat broth they produce a uniform turbidity.

On Ploskirev’s and Endo’s media S. typhi and S. paratyphi form semitransparent, colourless or pale-pink coloured colonies. On Levin’s medium containing eosin and methylene blue the colonies are transparent and bluish in colour, on Drigalski’s medium with litmus they are semitransparent and light blue, and on bismuth-sulphite agar they are glistening and black. The colonies produced by S. paratyphi A outrient media (Ploskirev’s, Endo’s, etc.) are similar to those of S. typhi. 

Salmonella on Ploskirev’s mrdium

 

Colonies of S. schottmuelleri have a rougher appearance and after they have been incubated for 24 hours and then left at room temperature for several days, mucous swellings appear at their edges. This is a characteristic differential cultural property.

Salmonella on bismuth-sulphite agar

 

Fermentative properties. S. typhi does not liquefy gelatin, nor does it produce indole. It produces hydrogen sulphide, and reduces nitrates to nitrites.

 

Figure. Colonies of Salmonella paratyphi (1); colonies of Salmonella schottmuelleri (2); smear from Salmonella enteritidis culture (3)

 

The organisms do not coagulate milk, but they give rise to a slightly pink colouration in litmus milk and cause no changes in Rotberger’s medium. They ferment glucose,         mannitol, maltose, levulose, galactose, raffinose, dextrin, glycerin, sorbitol and, sometimes, xylose, with acid formation.

S. paratyphi ferments carbohydrates, with acid and gas formation, and is also distinguished by other properties. Two types of S. typhi occur iature: xylose-positive and xylose-negative. They possess lysin decarboxylase, ornithine decarboxylase and oxidase activity.

 

Differentiating Characteristics of Salmonella typhi, Salmonella paratyphi and Food-poisoning  Salmonella

 

In the process of dissociation S. typhi changes from the S-form to the R-form. This variation is associated with loss of the somatic 0-antigen (which is of most immunogenic value) and, quite frequently, with loss of the Vi-antigen.

Toxin production. S. typhi contains gluco-lipo-protein complexes. The endotoxin is obtained by extracting the bacterial emulsion with trichloracetic acid. This endotoxin is thermostable, surviving a temperature of 120° C for 30 minutes, and is characterized by a highly specific precipitin reaction and pronounced toxic and antigenic properties. Investigations have shown the presence of exotoxic substances in S. typhi which are inactivated by light, air, and heat (80° C), as well as enterotropic toxin phosphatase, and pyrogenic substances.

Note: A, acid formation;  AG, acid and gas formation; +, hydrogen sulfide formation;  —, absence of carbohydrate fermentation and hydrogen sulphide formation;   ±, arabinose fermentation and hydrogen sulfide formation do not always occur.

 

Antigenic structure. S. typhi possesses a flagellar H-antigen and thermostable somatic 0- and Vi-antigens. All three antigens give rise to the production of specific antibodies in the body, i. e. H-, 0-, and Vi-agglutinins. H-agglutinins bring about a large-flocculent agglutination, while 0- and Vi-agglutinins produce fine-granular agglutination.

The antigens differ in their sensitivity to chemical substances. The O-antigen is destroyed by formalin but is unaffected by exposure to weak phenol solutions. The H-antigen, on the contrary, withstands formalin but is destroyed by phenol.

S. typhi, grown on agar containing phenol in a ratio of 1:1000, loses the H-antigen after several subcultures. This antigen is also destroyed on exposure to alcohol. These methods are employed to obtain the 0-antigen in its pure form. The H-antigen is isolated by treating the bacterial emulsion with formalin or by using a broth culture which contains a large number of flagellar components. Immunization with H-and 0-antigens is employed for obtaining the corresponding agglutinating sera.

The discovery of the Vi-antigen isolated from virulent S. typhi is of great theoretical interest and practical importance.

Vi- and 0-antigens are located within the micro-organism, on the surface of the bacterial cell. It is assumed that the Vi-antigen occurs in isolated areas and is nearer to the surface than the 0-antigen. The presence of Vi-antigens hinders agglutination of salmonellae by 0-sera, and the loss of the Vi-antigen restores the 0-agglutinability. S. typhi, which contains Vi-antigens, is not agglutinated by 0-sera. Vi-agglutinating serum is obtained by saturation of S. typhi serum of animals inoculated with freshly isolated salmonellae, employing H- and O-antigens. The Vi-antigen is a labile substance. It disappears from the culture when phenol is added to the medium and also when the temperature is low (20 °C) or high (40 ° C). It is completely destroyed by boiling for 10 minutes and by exposure to phenol. Exposure to formalin and to temperature of 60° C for 30 minutes produces partial changes in the antigen.

Together with H-, O-, and Vi-antigens, other more deeply located antigens have been revealed. The latter are detected during the change transformation of the bacterial cell to the R-form when the superficial 0- and Vi-antigens are lost. The deeply located antigens are non-specific. Later, salmonellae were found to possess an M-mucous antigen (polysaccharide).

It has been ascertained that the Vi-antigen content of cultures varies, some serovars possessing a large quantity of this antigen, while others only a small quantity. F. Kauffmann subdivides all salmonellae containing Vi-antigens into three groups: (1) pure V-forms with a high Vi-antigen content; (2) pure W-forms which contaio Vi-antigens; (3) transitional V-W-forms which possess Vi-antigens and are agglutinated by O-serum. S. paratyphi have been found to have antigens in common with isoantigens of human erythrocytes.

Classification. The salmonellae of typhoid fever and paratyphoids together with the causative agents of toxinfections have been included in the genus Salmonella (named after the bacteriologist D. Salmon) on the basis of their antigenic structure and other properties. At present, about 2000 species and types of this genus are known.

F. Kauffmann and P. White classified the typhoid-paratyphoid salmonellae into a number of groups according to antigenic structure and determined 65 somatic 0-antigens. For instance, S. typhi (group D) contains three different 0-antigens — 9, 12, and Vi.

Serological Classification of Bacteria of the Genus Salmonella

 

S. paratyphi A alone constitutes  group A, and S. schottmuelleri belongs to group B. It has been proved by F. Andrewes that the flagellar H-antigen is not homogeneous but is composed of two phases: phase 1 is specific and agglutinable by specific serum, phase 2 is non-specific and agglutinable not only by specific, but also by group sera. Salmonellae, which possess two-phase H-antigens, are known as diphasic, while those which possess only the specific H-antigen are monophasic.

Resistance. Typhoid and paratyphoid A and B salmonellae survive in ice for several months, in soil contaminated with faeces and urine of patients and carriers for up to 3 months, in butter, cheese, meat and bread for 1-3 months, in soil, faecal masses, and water for several weeks, and in vegetables and fruits for 5-10 days. They remain unaffected by desiccation and live for a  long time in dry faeces. Salmonellae survive for only a short time (3-5 days) in polluted water owing to the presence of a large number of saprophytic microbes and substances harmful to pathogenic microorganisms.

S. typhi and S. paratyphi A are susceptible to heat and are destroyed at 56° C in 45-60 minutes, and when exposed to the usual disinfectant solutions of phenol, calcium chloride, and chloramine, perish in several minutes. The presence of active chlorine in water in a dose of 0.5-1 mg per litre provides reliable protection from S. typhi and S. paratyphi A.

Pathogenicity for animals. Animals do not naturally acquire typhoid fever and paratyphoids. Therefore, these diseases are anthroponoses. A parenteral injection of the Salmonellae organisms into animals results in septicaemia and intoxication, while peroral infection produces no disease. E. Metchnikoff and A. Bezredka produced a disease similar to human typhoid fever by enteral infection in apes (chimpanzee).

Pathogenesis and diseases in man. The causative agent is primarily located in the intestinal tract. Infection takes place through the mouth (digestive stage).

Cyclic recurrences and development of certain pathophysiological changes characterize the pathogenesis of typhoid fever and paratyphoids.

There is a certain time interval after the salmonellae penetrate into the intestine, during which inflammatory processes develop in the isolated follicles and Peyer’s patches of the lower region of the small intestine (invasive stage).

As a result of deterioration of the defence mechanism of the lymphatic apparatus in the small intestine the organisms enter the blood (bacteriemia stage). Here they are partially destroyed by the bactericidal substances contained in the blood, with endotoxin formation. During bacteraemia typhoid salmonellae invade the patient’s body, penetrating into the lymph nodes, spleen, bone marrow, liver, and other organs (parenchymal diffusion stage). This period coincides with the early symptoms of the disease and lasts for a week.

During the second week of the disease endotoxins accumulate in Peyer’s patches, are absorbed by the blood, and cause intoxication. The general clinical picture of the disease is characterized by status typhosus, disturbances of thermoregulation, activity of the central and vegetative nervous systems, cardiovascular activity, etc.

On the third week of the disease a large number of typhoid bacteria enter the intestine from the bile ducts and Lieberkuhn’s glands. Some of these bacteria are excreted in the faeces, while others reenter the Peyer’s patches and solitary follicles, which had been previously sensitized by the salmonellae in the initial stage. This results in the development of hyperergia and ulcerative processes. Lesions are most pronounced in Peyer’s patches and solitary follicles and may be followed by perforation of the intestine and peritonitis (excretory and allergic stage).

The typhoid-paratyphoid salmonellae together with products of their metabolism induce antibody production and promote phagocytosis. These processes reach their peak on the fifth-sixth week of the disease and eventually lead to recovery from the disease.

Clinical recovery (recovery stage) does not coincide with the elimination of the pathogenic bacteria from the body. The majority of convalescents become carriers during the first weeks following recovery, and 3-5 per cent of the cases continue to excrete the organisms for many months and years after the attack and, sometimes, for life. Inflammatory processes in the gall bladder (cholecystitis) and liver are the main causes of a carrier state since these organs serve as favourable media for the bacteria, where the latter multiply and live for long periods. Besides this, typhoid-paratyphoid salmonellae may affect the kidneys and urinary bladder, giving rise to pyelitis and cystitis. In such lesions the organisms are excreted in the urine.

In one, two, or three weeks following marked improvement in the patient’s condition, relapses may occur as a result of reduced immunobiological activity of the human body and hence a low-grade immunity is produced.

Due to the wide range in the severity of typhoid fever from gravely fatal cases to mild ambulant forms it cannot be differentiated from paratyphoids and other infections by clinical symptoms. Laboratory diagnosis of these diseases is of decisive importance. In recent years typhoid fever has changed from an epidemic to a sporadic infection, being milder iature and rarely producing complications. In the USSR typhoid fever mortality has diminished to one hundredth that in 1913. Diseases caused by S. paratyphi are similar to typhoid fever. The period of incubation and the duration of the disease are somewhat shorter in paratyphoid infections than in typhoid fever.

Immunity. Immunity acquired after typhoid fever and paratyphoids is relatively stable but relapses and reinfections sometimes occur. Antibiotics, used as therapeutic agents, inhibit the immunogenic activity of the pathogens, which change rapidly and lose their O- and Vi-antigens.

Laboratory diagnosis. The present laboratory diagnosis of typhoid fever and paratyphoids is based on the pathogenesis of these diseases.

1.     Isolation of haemoculture.

Blood collection

 

Bacteraemia appears during the first days of the infection. Thus, for culture isolation 10-15 ml of blood (15-20 ml during the second week of the disease and 30-40 ml during the third week) are inoculated into 100, 150 and 200 ml of 10 per cent bile broth, after which cultures are incubated at 37° C and on the second day subcultured onto one of the differential media (Ploskirev’s, Endo’s, Levin’s) or common  meat-peptone agar.

The isolated culture is identified by inoculation into a series of differential media and by the agglutination reaction. The latter is performed by the glass-slide method using monoreceptor sera or by the test-tube method using purified specific sera.

2. Serological method. Sufficient number of agglutinins accumulate in the blood on the second week of the disease, and they are detected by the Widal reaction. Diagnostic typhoid and paratyphoid A and B suspensions are employed in this reaction. The fact that individuals treated with antibiotics may yield a low titre reaction must be taken into consideration. The reaction is valued positive in patient’s serum in dilution 1 : 200 and higher.

The Widal reaction may be positive not only in patients but also in those who had suffered the disease in the past and in vaccinated individuals. For this reason diagnostic suspensions of O- and H-antigens are employed in this reaction. The sera of vaccinated people and convalescents contain H-agglutinins for a long time, while the sera of patients contain O-agglutinins at the height of the disease.

In typhoid fever and paratyphoids the agglutination reaction may sometimes be of a group character since the patient’s serum contains agglutinins not only to specific but also to group antigens which occur in other bacteria. In such cases the patient’s blood must be sampled again in 5-6 days and the Widal reaction repeated. Increase of the agglutinin titre makes laboratory diagnosis easier. In cases when the serum titre shows an equal rise with several antigens, 0-, H-, and Vi-agglutinins are detected separately.

The Vi-agglutination reaction is employed for identification of S. typhi carriers. This reaction is performed with sera (inactivated at 56° C for 30 minutes and diluted in the ratio of 1:10-1:80) and diagnostic Vi-suspensions. Individuals who give a positive Vi-agglutination reaction are subjected to microbiological examination for isolation of S. typhi from the bile, faeces, and urine. The best results are obtained when Vi-haemagglutination is employed.

For quick serological diagnosis of typhoid fever and paratyphoids Nobel’s agglutination method and agglutination on glass by the Minkevitch-Brumpt method are carried out. In the latter case the bacterial emulsion is agglutinated in a drop of undiluted blood placed on a slide.

3. A pure culture is isolated from faeces and urine during the first, second, and third weeks of the disease. The test material is inoculated into bile broth, Muller’s medium, Ploskirev’s medium, or bismuth sulphite agar.

Isolation and identification of the pure culture are performed in the same way as in blood examination.

Selective media are recommended for isolation of the typhoid-paratyphoid organisms from water, sewage, milk, and faeces of healthy individuals. These media slightly inhibit the growth of pathogenic strains of typhoid-paratyphoid organisms and greatly suppress the-growth of saprophytic microflora.

A reaction for the detection of a rise in the phage titre is employed in typhoid fever and paratyphoid diagnosis. This reaction is based on the fact that the specific (indicator) phage multiplies only when it is in contact with homologous salmonellae. An increase in the number of phage corpuscles in the test tube as compared to the control tube is indicative of the presence of organisms homologous to the phage used. This reaction is highly sensitive and specific and permits to reveal the presence of the salmonellae in various substrates in 11-22 hours without the necessity of isolating the organisms in a pure culture. The reaction is valued positive if the increase in the number of corpuscles in the tube containing the test specimen is not less than 5-10 times that in the control tube.

When unagglutinable cultures of the typhoid and paratyphoid organisms are isolated, the agglutination reaction is performed using Vi-sera. If the latter are not available, the tested culture is heated for 30 minutes at 60° C or for 5 minutes at 100° C. The agglutination reaction is carried out with a suspension of this heated culture.

In some cases a bacteriological examination of duodenal juice (in search for carriers), bone marrow, and material obtained from roseolas is conducted.

Phage typing of typho-paratyphoid organisms is sometimes employed. The isolated culture is identified by type-specific O- and Vi-phages. Sources of typhoid and paratyphoid infections are revealed by this method.

Water is examined for the presence of typho-paratyphoid bacteria by filtering large volumes (2-3 litres) through membrane filters and subsequent inoculation on plates containing bismuth sulphite agar. If the organisms are present, they produce black colonies in 24-48 hours. The reaction of increase in phage titre is carried out simultaneously.

Treatment. Patients with typhoid fever and paratyphoids are prescribed chloramphenicol, oxytetracycline, and nitrofuran preparations. These drugs markedly decrease the severity of the disease and diminish its duration. Great importance is assigned to general non-specific treatment (dietetic and symptomatic). Treatment must be applied until complete clinical recovery is achieved, and should never be discontinued as soon as the bacteria disappear from the blood, urine, and faeces since this may lead to a relapse. Mortality has now fallen to 0.2-0.5 per cent (in 1913 it was 25 per cent).

The eradication of the organisms from salmonellae carriers is a very difficult problem.

Prophylaxis. General measures amount to rendering harmless the sources of infection. This is achieved by timely diagnosis, hospitalization of patients, disinfection of the sources, and identification and treatment of carriers. Of great importance in prevention of typhoid fever and paratyphoids are such measures as disinfection of water, safeguarding water supplies from pollution, systematic and thorough cleaning of inhabited areas, fly control, and protection of foodstuff’s and water from flies. Washing of hands before meals and after using the toilet is necessary. Regular examination of personnel in food-processing factories for identification of carriers is also extremely important.

In the presence of epidemiological indications specific prophylaxis of typhoid infections is accomplished by vaccination. Several varieties of vaccines are prepared: typhoid vaccine (monovaccine), typhoid and paratyphoid B vaccine (divaccine).

Good effects are obtained also with a chemical associated adsorbed vaccine which contains 0- and Vi-antigens of typhoid, paratyphoid B, and a concentrated purified and sorbed tetanus anatoxin. All antigens included in the vaccine are adsorbed on aluminium hydroxide.

A new areactogenic vaccine consisting of the Vi-antigen of typhoid fever Salmonella organisms has been produced. It is marked by high efficacy and is used in immunization of adults and children under seven years of age. When there are epidemiological indications, all the above-mentioned vaccines are used according to instructions and special directions of the sanitary and epidemiological service.

 

ADDITIONAL MATERIALS FOR DIAGNOSIS

INFECTION CAUSED BY SALMONELLAE OF TYPHOID AND PARATYPHOID FEVERS

Typhoid fever and paratyphoid infections A and B are acute hu­man infectious diseases attended by bacteremia, intoxication, and characteristic ulcerous-necrotic damage to the lymphatic apparatus of the small intestine. They can be distinguished by laboratory methods only. The causative agent of abdominal typhoid is Salmo­nella typhi, of paratyphus A, Salmonella paratyphi A, of paratyphus B,  Salmonella schottmuelleri {paratyphi B).

Bacteriological examination is of the key significance in the diagnosis of typhoid-paratyphoid diseases since it allows both iso­lation and typing of the causal organism. The material to be studied for diagnostic purposes may include blood, faeces, urine, bile, secre­tions from scarified roseolas, and, occasionally, a puncture sample of bone marrow, cerebrospinal fluid, pus from septic foci, necrosis-affected tissues, etc.

The earliest and most reliable technique of bacterial diagnosis is the isolation of the causal organisms from the blood, the haemoculture method. Salmonellae of typhoid and paratyphoid fevers persist in the blood throughout the febrile period and even during the first days of temperature normalization (particularly in vaccinated sub­jects with typhoid fever). At an early stage of the disease, the in­tensity of bacteremia is higher than at the end of the pyrexial pe­riod. This explains why 10 ml of blood is sufficient to perform exami­nation at the onset of the disease, whereas at later stages 15-20 ml of blood is required. The sample of blood aseptically obtained at the patient’s bedside is inoculated into 10 per cent bile broth or Rapoport’s medium (10 per cent bile broth supplemented with 1 per cent of mannitol or 2 per cent of glucose, and 1 per cent of Andrade’s indicator; a float is placed into the vial with the medium to capture the gas formed), with the blood-medium ratio being 1:10.

The inoculated vials are incubated at 37 °C for 18-24 hrs. Prolif­eration of salmonellae as a result of mannitol or glucose splitting with the formation of acid is signalled by an alteration in the pH of the medium (it acquires red colour). Propagation of paratyphoid salmonellae is accompanied by the formation of gas and acid.

From enrichment media the material is transferred onto an agar slant or Olkenitsky’s medium and into a plate with Endo’s medium (the second day of examination). Inoculation into a plate with En­do’s medium allows the isolation of a pure culture in cases where the enrichment medium has been  contaminated by air flora (non-sterile syringe, inadequate disinfection of the skin prior to blood taking, etc.).

The inoculated cultures are examined after 18-24-hour incubation in a heating block . The growth of salmonellae on Endo’s medium is characterized by the appearance of colourless lactose-negative colonies.

On Olkenitsky’s medium the typhoid salmonellae ferment glucose with the formation of acid (yellowing of the agar column), do not split lactose (the colour of the slanted portion of the agar does not change), and produce hydrogen sulphide (blackening of the medium at the  borderline between the agar column and the slanted surface). The paratyphoid salmonellae ferment glucose with the formation of acid (yellowing) and gas (rupture of the agar column).

After the nature of the growth has been evaluated and the purity of the culture determined, it is time to identify the culture.

Inoculate the isolated culture (the third day of the investigation) onto Hiss’ media and perform the agglutination test with aggluti­nating adsorbed sera (abdominal typhoid, paratyphoid A. and pa­ratyphoid B), and make wet-mount or hanging drop preparations to estimate motility of the microorganisms.

The hemoculture of the salmonellae of typhoid and paratyphoid fevers may contain the Vi-antigen. If there is no agglutination with O-sera, introduce Vi-sera or destroy the Vi-antigen. For this purpose, heat the obtained culture at 60 °C for 30 min or at 100 °C for 5 min.

On the fourth day of the study, read changes in the Hiss’ media (Table 3) and the final results of the agglutination reaction, and make conclusive report.

The enrichment medium with a blood inoculum is left in the incu­bator for several days since in some cases propagation of the causa­tive agent in bile media may be slow. Subculturing to an agar slant, Olkenitsky’s and Endo’s media, is performed every 24-48 hrs over a period of 7 to 10 days.

Salmonellae may be isolated from the blood in chronic bacteria carriers when they present a dramatic change in immunoreactivity (helminthiasis, malignant tumours, etc.).

The faeces to be tested are collected in sterile test tubes or jars in an amount of 5-10 g. In taking the material, care should be exer­cised to exclude the action of disinfectants. The samples are inoculated into one plate with Ploskirev’s medium. Salmonellae in the faeces may be found by the direct and indirect immunofluorescence tests.

On the second day (following 18-24-hour incubation) in a heating block, inspect for colourless, lactose-negative colonies, study their morphology and structure, and subculture to Olkenitsky’s medium.

On the third day of investigation, pure culture of the isolated bacteria is transferred onto Hiss’ media, and presumptive and then standard agglutination tests are conducted.

On the fourth day of the investigation, consider changes in the Hiss’ media and the agglutination test results, and make the report. Thus, identification of pure culture isolated from faeces (coproculture) does not differ from identification of a hemoculture.

To detect possible bacteria carriers, examination is performed in individuals with a history of typhoid and paratyphoid fevers, as well as among the staff of child-caring institutions, food-catering and water supply services. Prior to collection of faeces, those examined are to drink on a fasting stomach 100ml of 30 per cent solution of magnesium or sodium sulphate that possess bile-expelling action. Faecal matter to be tested is collected 2-4 hours after ingestion of a purgative. Inoculate onto Ploskirev’s medium and simultaneously onto an enrichment medium (bile broth, selenite broth, Kauffmann’s medium-tetrathionate, etc.). After a 6-hour incubation, subculture the latter to Ploskirev’s medium. Further investigation is conducted in the aforementioned manner.

Following centrifugation, urine and its deposit are inoculated onto Ploskirev’s medium and into bile broth for enrichment. In the presence of the characteristic growth identification is performed, using the same procedure as in the case of a hemo- and coproculture.

Duodenal contents, scraping of roseolas, and section material are studied and identified in the like manner.

Typing of phages and colicins of Salmonellae isolated from patients and carriers is helpful in establishing the source of contamination.

In serological diagnosis Widal’s reaction is employed. Antibodies to the causative agents of typhoid, paratyphoid A and paratyphoid B fevers can be recovered in the patient’s blood serum beginning from the 8th-10th day of the disease. To perform the Widal test, draw 2-3 ml of blood from a vein or 1 ml of blood from  a finger or an ear lobe and obtain serum.

Schematic Description of the Widal Reaction

 

 

Successively dilute the serum in three parallel rows of test. tubes from 1:100 to 1:1600 and introduce 0-diagnosticums (usual or erythrocyte ones) of Salmonella typhi into test tubes of the first row, of Salmonella paratyphi A into test tubes of the second row, and of Salmonella paratyphi B into test tubes of the third row. The use of 0-diagnosticums makes it possible to reveal 0-antibodies which appear in the blood during the second week of the disease and disappear by the end of the illness. The diagnostic titre of antibodies in the Widal test ion-immunized subjects is 1:100 and higher.

Demonstration of the H-antibodies is of no diagnostic value since they are detected during convalescence, and also in vaccinated individuals and those with a history of the disease.

In some cases O-antibodies may be recovered in vaccinated persons. Hence, it is necessary that the Widal test be performed over time to look for an increase in its titre.

If the patient’s blood serum agglutinates two or three types of diagnosticums simultaneously, the titre of agglutination should be taken into account. Typically, the specific agglutination occurs at larger and the group one at lower serum dilutions.

Currently, the onset of antibiotic treatment at early stages of the disease poses difficulty in evaluating the results of the agglutina­tion reaction since the antibody titre in patients is small and cannot be considered diagnostically significant.

The indirect haemagglutination test with erythrocyte monoreceptor diagnosticums O₉, O₁₂, and Vi is a more sensitive test, yielding-positive results in a greater number of cases. Antibodies to the 0-an-tigens are detected beginning from the second week of the disease. Antibodies to the Vi-antigens are recovered at later stages. Vi-anti-bodies occur most commonly in carriers of Salmonella typhi. To identify bacteria carriers, indirect haemagglutination with demon­stration of antibodies belonging to Immunoglobulins G is employed (a signal method).

Examination, of water. The causal organisms of typhoid and para­typhoid fevers are contained in water in minute quantities. To obviate this problem, one utilizes techniques allowing for their concentration in water. The best of these methods is examination with the help of membrane filters.

Two litres of water or more is poured through No 2 or No 3 membrane fil­ters. If the water contains a large number of suspended particles which hinder filtration, it is first passed through a No 6 nitrocellulose filter which arrests gross particles. Filters with a deposit are immersed in a bile broth or placed on a bis­muth-sulphite agar. After a 6-10-hour incubation, subculture from the bile broth to Ploskirev’s medium. Further procedures are the same as in examination of faeces. After a 48-hour incubation in a heating block, harvest black colonies from the bismuth-sulphite agar and identify them in the way salmonellae are identified.

Examination of water for the presence of typhoid and paratyphoid phages is employed in cases where bacteria evade detection. The sewage is passed through a bacterial filter. Into a sterile Petri dish place 1-2 ml of the filtrate test­ed, pour in l5-20ml of meat-peptone agar cooled to 45 °C, and mix thoroughly. After the agar has solidified, streak (in sectors) cultures of salmonellae causing typhoid, paratyphoid A and paratyphoid B fevers. The appearance of negative colonies confirms the presence of the corresponding phages.

To examine drinking water, introduce it into a concentrated peptone solu­tion. To 100 ml of the water to be tested add 10 ml of peptone and 5 g of sodium chloride. Place the inoculated cultures in an incubator for 24 hours and then examine the filtrate for the presence of the phage.

Along with bacteriological and serological methods, an intra-cutaneous allergy test with the Vi-typhine of typhoid bacteria is used. This test becomes positive during recovery and may be utilized for retrospective diagnosis.

Salmonellae — Causative Agents of salmonellosis.  The genus Salmonella comprises many species and types of bacteria which possess properties similar to those of S. schottmuelleri. In 1885 in America D. Salmon isolated the bacterium S. cholerae-suis, which was long  considered the causative agent of plague in pigs. Later it was shown to be in association with the causative agent of this disease and the cause of human toxinfections. In 1888 during a large-scale outbreak of toxinfections in Saxony A. Gartner isolated S. enteritidis bacteria from the flesh of a cow which had to be killed, and also from the spleen of a dead person. The organisms proved to be pathogenic for mice, guinea pigs, rabbits, sheep, and goats. In 1896 in Breslau K. Kensche and in 1898 in Ertike G. Nobel discovered S. typhimurium (Bacillus Breslau) in cases of food poisoning and isolated a pure culture of the organism. It is now known that among the large number of organisms which comprise the salmonella group, about 440 species and types are pathogenic for humans and are the cause of food poisoning (toxinfections).

Morphology. Morphologically Salmonella organisms possess the general characteristics of the family Enterobacteriaceae. They are motile and peritrichous.

Cultivation. The organisms are facultative aerobes, the optimum temperature for growth being 37° C. They grow readily on ordinary nutrient media.

Fermentative properties. Salmonellae do not liquefy gelatine and do not produce indole. The majority of species produce hydrogen sulphide and ferment glucose, maltose, and mannitol, with acid and gas formation.

Toxin production. Salmonellae produce no exotoxin. Their ability to cause diseases in animals and humans is associated with an endotoxin which is a gluco-lipo-protein complex and is characterized by its high toxicity.

Antigenic structure. As was mentioned above, all salmonella® are divided into 65 groups according to their serological properties (see Table 4, Methodological Instructioo 35). Thus, according to the Kauffmann-White Scheme, S. enteritidis belongs to group D, S. typhimurium to group B, and S. cholerae-suis to group C.

Classification. The organisms are classified according to their antigenic, cultural, and biological properties (see Methodological Instructioo 35).

Virulence Factors of Salmonella Organisms. It is surprising that virulence factors for organisms that have caused so much disease still arc largely unknown. However, the ability to invade and grow inside of non-phagocytic cells undoubtedly comprises the major virulence determinant of the Salmonella because this intracellular location provides a compartment where they can replicate and avoid host defences. The mechanism whereby these bacteria accomplish this invasion is complex and only beginning to unfold.

Using various mutants of Salmonella typhimurium, John Pace and colleagues at the State University of New York determined that invasion of a host cell occurs in two separable steps: (1) adhesion to the host cell, and (2) invasion of the host cell. Furthermore, they found that invasion required that the organisms activate a growth factor receptor on the host cell known as epidermal growth factor receptor (EGFR). Mutants that could adhere, but not invade, were unable to activate EGFR. However, if EGF was added to the host cell-bacterium mixture, the EGFR was activated and the noninvasive mutant was internalized.

When EGFR is activated, a signal transduction process occurs, which results in at least two major events: (1) a rapid rise in the internal Ca²⁺ level occurs, and (2) enzymes are activated that lead to the synthesis of leukotriene D₄ (LTD₄). It is unclear how these events trigger the entry of Salmonella into the cell, but it is known that the Ca²⁺ level increase is essential because the addition of Ca²⁺ chelators blocked entry of the bacterium into the cell. It is also known that the addition of LTD₄ to cultured cells causes an increase in intracellular Ca²⁺ levels, permitting the internalization of an invasion-deficient mutant.

One can postulate, therefore, that the mediation of Ca²⁺ influx by LTD4 results in the opening of a Ca²⁺ channel, which, in turn, causes a reorganization of the host cell cytoskeleton, permitting entry of the bacterium.

It is also of note that the inflammatory diarrhea produced by the Salmonella may result from its ability to induce leukotriene synthesis because leukotrienes are well-known mediators of inflammation.

It is also known that a number of Salmonella, serotypes carry plasmids that greatly increase virulence in experimentally infected mice. Although many of these plasmids are distinct, all have a highly conserved 8-kb region that has beeamed the spv regulon. Interestingly, spv genes are not expressed during logarithmic growth in vitro but seem to enhance the growth of salmonellae within host cells. In experimentally infected mice, the expression of spv by intraccelular salmonellae in vivo has been postulated to lead to an increased rate of bacterial growth, resulting in early bacteremia and death before the infected mice can develop immunity.

The general types of infections that may be caused by the salmonellae usually are grouped into three categories: enterocolitis, enteric fevers, and septicemia.

Resistance. Salmonellae are relatively stable to high temperatures (60-75 °C), high salt concentrations, and to certain acids. They with stand 8-10 per cent solution of acetic acid for 18 hours, and survive for 75-80 days at room temperature. The endotoxins remain active within large pieces of meat for long periods (even after the meat has been cooked) as well as in inadequately fried rissoles and other foods.

A characteristic feature of foodstuffs contaminated by Salmonellae is that they show no changes which can be detected organoleptically.

Pathogenicity for animals. Salmonellae, the causative agents of toxinfections, are pathogenic micro-organisms which may give rise to paratyphoid in calves, typhoid and paratyphoid iewly-born pigs, typhoid in fowls and pullorum disease in chickens, typhoid in mice and rats, and enteritis in adult cattle.

Among laboratory animals, white mice are most susceptible to the organisms (S. typhimurium, S. enteritidis, S. cholerae-suis, etc.). Enteral and parenteral inoculations result in septicaemia in these animals.

Pathogenesis and diseases in man. Ingestion of food contaminated by salmonellae is the main cause of disease. Most frequently food poisoning is due to meat prepared from infected animals and waterfowls without observance of culinary regulations. Eggs of infected waterfowls are also sources of infection. Seabirds are frequent Salmonellae carriers. Meat may be infected while the animal is alive or after its death.

As distinct from typhoid fever and paratyphoids A and B, salmonellae toxinfections are anthropo-zoonotic diseases. S. typhimurium, S. cholerae-suis, S. Heidelberg, S. enteritidis, S. anatum, S. newport. S. derby, and others cause clinically manifest forms. Intoxication develops in a few hours following infection. Masses of microbes ingested with the food are destroyed in the gastro-intestinal tract and m me blood. This results in the production of large amounts of endotoxin which, together with the endotoxin entering the body with the ingested food, gives rise to intoxication. Salmonellae are known to be highly infestive. Bacteremia usually becomes manifest in the first hours after the onset of the disease.

The disease course is characterized by clinical manifestation of toxinfectional, gastroenteric, and typhoid- and cholera-like symptoms.

Along with typical zoonotic salmonella diseases, there are salmonelloses which occur as a result of infection from sick people and carriers. Such cases are predominant iewborn and prematurely born children, convalescents, and individuals with chronic diseases. In children’s institutions, maternity hospitals, somatic departments of pediatric clinics, and among children suffering from dysentery in departments for contagious diseases the main sources of infection are sick children and bacteria carriers. Children suffering from salmonelloses display symptoms of dyspepsia, colitis (enterocolitis), and typhoid fever, and often these conditions are accompanied by septicaemia and bacteremia. The diseases are of long duration or become chronic and are sometimes erroneously diagnosed as chronic dysentery.

Immunity acquired after salmonellosis is of low grade and short duration. Low titres of agglutinins (from 1:50 to 1 :400 and, rarely, up to 1:800) appear in the blood of convalescents during the second week.

Laboratory diagnosis. Specimens of food remains, washings from objects, stools, vomit, lavage water, blood, urine and organs obtained at autopsy are carefully collected and examined systematically. In the beginning, the specimens are inoculated into nutrient media employed for diagnosis of typhoid fever and paratyphoids A and B. Then the cultural, serological, and biological properties of the isolated cultures are examined (Table 3, Methodological Instructioo 35).

In some cases the biological test is performed not only with the cultures, but also with remains of the food which caused the poisoning.

For retrospective diagnosis blood of convalescents is examined for the presence of agglutinins on the eighth-tenth day after the onset of disease. This is performed by the Widal reaction with suspensions of the main diagnostic bacterial species which cause food toxinfections.

Table 4 (Methodological Instructioo 35) shows that differential laboratory diagnosis between S. typhimurium and S.  schottmuelleri is particularly difficult since they have group, somatic, and flagellar phase 2 antigens in common. Pathogenicity for white mice and appearance of mucous swellings and daughter colonies on agar serve as differential criteria.

Treatment. Therapeutic measures include antibiotics (chloramphenicol, oxytetracycline and tetracycline). Good effects are also obtained with stomach lavage, injections of glucose and physiological solution, and cardiac drugs.

Prophylaxis of salmonellae toxinfections is ensured by veterinary and sanitary control of cattle, slaughter-houses, meat factories and fish industries, laboratory control of meat intended for sale, and sterilization of meat which otherwise may not be sold. The medical hygiene service identifies carriers among people working in food factories, catering houses, and other food-processing establishments and controls the sanitary regulations at food enterprises, shops, store-houses, and in catering houses.

 

Diagnosis OF SALMONELLAL GASTROENTERITIS

(FOOD POISONING)

The primary reservoir for the salmonellae is the intestinal tracts ot many animals, including birds, farm animals, and reptiles. Humans become infected through the ingestionot contaminated water or food. Water, of course, becomes polluted by the introduction of feces from any animal excreting salmonellae. Infection by food usually results either from the ingestion of contaminated meat or by way of the hands, which act as intermediates in the transfer of salmonellae from an infected source. Thus, the handling of an infected – although apparently healthy – dog or cat can result in contamination with salmonellae. An-other major source of Salmonella infections has been pet turtles. In the early 1970s, almost 300,000 cases of turtle-associated salmonellosis were estimated to occur annually in the United States and, as a result, it is illegal to import turtles or turtle eggs or even to ship domestic turtles with shells less than 4 inches in diameter across state lines.

In the United States, poultry and eggs increasingly comprise the most common source of salmonellae for humans T his occurs because a large percentage of chickens routinely are infected with salmonellae. Thus, humans can acquire these organisms through direct contact with uncooked chicken or by the ingestion of undercooked chicken. And, because the organisms may occur both on the outer shell and in the yolk and egg white, consuming anything containing raw eggs (caesar salad, hollandaise sauce, mayonnaise, homemade ice cream) could result in a Salmonella infection. The CDC even cautions agains teating eggs sunny-side up and recommends that eggs be boiled for 6 to 7 minutes before being served.

On an industrial scale, slaughterhouse workers are faced with salmonellosis as an occupational hazard, primarily from poultry and pigs. Because humans can become asymptomatic carriers of Salmonella, infected food handlers also are responsible for the spread of these organisms.

Salmonella enterocolitis is one of the most frequent cause of food-borne outbreaks of gastroenteritis in the United States. It may be caused by any one of the hundreds of serotypes of Salmonella, and it is characterized by the fact that organisms do not cause an appreciable bacteremia. The hallmark of all Salmonella infections lies in the ability of the Salmonella to invade the intestinal epithelial cells, which are normally nonphagocytic. Those species involved in gastro-enteritis may reach the bloodstream early in the disease but arc rapidly taken up and killed by phagocytic cells. In general, bacteremia occurs only in persons having an impaired phagocyte system, AIDS, or chronic granulomatous disease. In the average case, symptoms of diarrhea may occur 10 to 28 hours after ingesting contaminated food, and the headache, abdominal pain, nausea, vomiting, and diarrhea may continue for 2 to7 days.

A search for salmonella toxins has not been as conclusive as one might wish, but there are multiple reports that many Salmonella species secrete a cholera-like enterotoxin that induces increased levels of cAMP, and that some strains produce a heat-stable enterotoxin. In addition, a cytotoxin that inhibits protein synthesis in intestinal epithelial cells has been described This toxin, characterized by its ability to kill Vero cells, is immunologically distinct from both Shiga toxin and the Shiga-like toxins produced by strains of E. coli and Shigella. The observation that those species of Salmonella causing the more severe enteric symptoms and inflammatory diarrhea also produce the highest levels of cytotoxin suggests that this toxin may be of paramount importance in the pathologic manifestations of gastrointestinal salmonellosis. Neither the molecular structure, its specific mechanisms of blockingprotein synthesis and causing cell death, nor the number of such Salmonella cytotoxins is known

Most cases of Salmonella enterocolitis formerly had not been treated with antibiotics because such treatment did not seem to shorten the duration of the infection. There have been reports, however, that the fluoroquinolones do decrease the period of illness but, interestingly, they do not eradicate the organisms from the intestinal tract.

At present, there are over 400 serovars of salmonellae known to be pathogenic for man and capable of inducing acute gastroenteritis. The most important of them are S. typhimurium, S. enteritidis, S. cholerae-suis, S. gallinarium, etc. (about 60 serovars). The material subject to laboratory examination includes vomited matter, waters from stomach lavage, bile, urine, cerebrospinal fluid, puncture sam­ple of the bone marrow, blood (in the first hours of the disease, for isolat­ing a haemoculture, and then in two weeks, usually after recovery, for demonstrating antibodies).

To identify carriers among the staff of food-catering or child-caring institutions, etc., samples of faeces are collected following the ad­ministration of a purgative. At autopsy one collects the contents of the stomach and intestines, heart blood, pieces of parenchymatous organs, and mesenteric lymph nodes.

In food toxinfections one should examine the remains of the food, foodstuffs from which it has been prepared, washings off from the tables, preparation boards, hands of the catering personnel, etc.

The material is collected in sterile jars and test tubes in the fol­lowing amounts: faeces and vomit, 50-100 ml; lavage waters, 100-200 ml; meat and meat products, several pieces weighing about 500 g; semi-solid and liquid foodstuffs (cream, milk, etc.), 100-200 ml.

Before being sent to the laboratory, the above materials are packed and sealed. Integrity of the packing or otherwise is noted in a special register. Before the examination, material that cannot be inoculated without preliminary treatment (e.g., solid faecal and vomited mat­ter, food remains, etc.) is ground in a porcelain mortar and suspend­ed in isotonic saline. The surface of the tested meat, sausage, and cheese is sterilized by applying to it a red-hot metallic spatula, and samples are cut from the depth, placed into a porcelain mortar with glass sand, and isotonic salt solution is added.

Bacteriological examination. To isolate a haemoculture of sal­monellae, the blood is introduced into a bile broth. The vomit, faeces, section material, pus, cerebrospinal fluid, foodstuffs, and washings off are inoculated into plates with Ploskirev’s medium and in enrichment media (bile broth and selenite medium) from which subinoculation is made into Ploskirev’s medium in 6-10 hrs. The inoculated cultures are incubated at 37 “C for 24 hrs. after which they are examined, colourless lactose-negative colonies are selected and transferred to OIkenitsky’s triple sugar medium or to an agar slant to enrich for pure culture. On the third day of the in­vestigation, the isolated pure cultures are identified: they are inocu­lated into Hiss’ cultures and the agglutination test with adsorbed group sera (A, B, C, D, E) is performed. If a positive result has been obtained with one of serum groups, one makes the agglutination test with the adsorbed 0-sera typical for the given group and then with monoreceptor H-sera (non-specific and specific phases) in order to determine the species and serovars of bacteria. For example, if the studied culture has agglutinated with a group B-serum, it is nec­essary to perform the agglutination test with sera against O, and OB antigens, which are typical of this group. If agglutination has been positive, the H-monoreceptor sera are utilized.

On the fourth day of the investigation, changes in Hiss’ media are assessed. The causative agents of salmonellal gastroenteritis, similar to the salmonellae responsible for paratyphus A and B, do not ferment lactose and sucrose, split glucose, mannitol, and maltose with the formation of acid and gas, do not form indol and, with minor excep­tions, release hydrogen sulphide.

Salmonella cultures can most frequently be isolated from patients’ faeces, somewhat less commonly, from vomit and stomach washings, and even less often from blood, urine, and bile. The results of bacte­riological examination of various biosubstrates are of varying diag­nostic significance. Isolation of salmonellae from the blood, bone mar­row, cerebrospinal fluid, vomit, and waters from the stomach lavage is a definite confirmation of the diagnosis. On the other hand, de­tection of salmonellae in the faeces, urine, and bile may be related to a bacteria carrier-state. The aetiological role of salmonellae in the development of gastroenteritis is corroborated by an increased titre of specific antibodies in an agglutination reaction with an autestrain.

Biological examination. Salmonellae of food poisoning, in con­trast to salmonellae of paratyphi A, are pathogenic for white mice. This property is used for the differentiation between the two types. On the first day of examination, along with inoculation of the patho­logical material and foodstuffs, white mice are infected per os. One-two days later the mice die of septicaemia. Post-mortem examination demonstrates a sharply enlarged spleen and, occasionally, liver, while inoculation of the blood from the heart and samples from the internal organs permits isolation of salmonella culture.

The agglutination reaction and indirect haemagglutination test are employed for serological diagnosis. These may be carried out from the first days of the disease and should be repeated in 7-10 days to determine whether the titre of specific antibodies tends to in­crease. In conducting these tests, salmonellal polyvalent and group (group A, B, C, D, E) diagnosticums (corpuscular and erythrocyte) are utilized.

A two-four-order elevation of the antibody titre is of diagnostic importance.

Salmonella Septicemia

Septicemia caused by Salmonella is a fulminating blood infection that does not involve the gastrointestinal tract. Most cases are caused by S. choleraesuis and are characterized by suppurative lesions throughout the body. Pneumonia, osteomyelitis, or meningitis may result from such an infection. Salmonella osteomyelitis is especially prevalent in persons who have sickle cell anemia, and focal infections, particularly on vascular prosthesis, also are common.

 

References: 

1.     Essential of Medical Microbiology /Wesley A. Wolk and al. / Lippincott-Raven Publishers, Philadelphia-Ney-York, 1995, 725 p.

2.     Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 88–96.

3.     Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002, P. 217-223, 225-228,