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SALMONELLA

Eberth (1880) first observed the typhoid bacillus in mesenteric lymph nodes and spleen in fatal cases of typhoid fever and Gafllcey (1884) successfully isolated.Sahnonellae produce 3 main types of disease in man but mixed forms are frequently observed. 1. Enteric fevers : Salmonella typhi causes typhoid fever and S. paratyphi A, B and C cause paratyphoid fever. Some strains of S. paratyphi B occasionally found in animals (cattle, pigs, poultry). 2. Food poisoning (gastroenteritis) :The food poisoning salmonellae are primarily pathogenic for animals from whom sporadic human infections occur. These are S. typhimurium, S. enteritidis, S. thompson, S. newport and S. dublin. 3. Septicaemia :This is commonly associated with S. choleraesuis but other salmonellae may also cause septicaemia.

Morphology These are Gram-negative, motile, nonsporing, noncapsulated bacilli measuring 2-4 X 0.6 m. Most strains are motile due to presence of peritrichous flagella except S. gallinarum and S. pullorum which are non-motile.

Cultural character The salmonellae grow on ordinary culture media, and in MacConkey’s agar and DCA media, and produce small, circular, translucent, colorless non-lactose fermenting colonies. In Wilson and Blair bismuth sulphite medium (selective medium for salmonellae), the colonies are jet black with metallic sheen due to formation of hydrogen sulphite. Selenite F or tetrathionate broth are commonly used as enrichment media, both of these media inhibit the growth of normal intestinal bacteria and selectively permit the growth of salmonellae.

Biochemical reaction

The salmonellae do not ferment lactose or sucrose and do not produce indole or ferment gelatin. They ferment glucose, mannitol, maltose and dextrin with production of acid and gas except S. typhi which produces only acid and no gas. Most strains produce H2S in triple sugar iron agar (except S. paratyphoid A and

S. choleraesuis), utilize citrate and are M.R. positive.

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Antigenic structure :Salmonellae possess three main types of antigens on the basis of which they are serologically classified. These are (i) flaellar antigen ‘H’, (ii) somatic antigen ‘0’ and (iii) a surface antigen ‘Vi’ present in some species. Several strains possess fimbrial antigens that are not important in their identification.

1.0-antigen It is a lipopolysaccharide complex. It is heat stable and can withstand boiling for several hours, present both in motile and non motile bacilli. 0 antigens are also resistant to alcohol and dilute acids. For serological tests, 0 suspensions is prepared either from non-motile strains or by heat or alcohol treatment of motile strains which destroy H antigens.

The 0 antigen is not a single factor but a mosaic of two or more antigenic factors. 0 polysaccharide possess a core structure that is common to all enterobacteria, but the side chains of sugar attached to the core determine 0 specificity. Salmonella 0 antigen is detected by agglutination of heated suspensions of organisms by rabbit antisera raised against boiled organisms.

2. H antigen The H antigens of Salmonella are found in this genus only and-not shared by other enterobacteria. It is a heat labile protein and gets destroyed after boiling for a few minutes and also by treatment with alcohol and acids but not by formalin. The H suspension for Widal .test is prepared by addition of formalin to young motile broth culture. H agglutination occurs rapidly forming loose and large floccules (fluffy clumps). H antigens are strongly immunogenic and corresponding antibodies are mainly IgG. Within a single serotype, flagellar antigens are present in either or both of two forms called phase 1 and phase 2.

3. Vi antigen

some strains of S. typhi possess surface (K) antigens external to the cell-wall, referred to as Vi, which interferes with agglutination of freshly isolated organisms by 0 antigen. Vi antigen is a polysaccharide and heat labile.

Antigenic variation

The antigens of salmonellae undergo several types of phenotypic and genotypic variations.

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1. OH → 0 variations: Motile strains lose their flagella and become non motile in H-O variation. The change may be temporary (reversible) or stable (irreversible). Some strains of Salmonella, like S. gallinarum and S. pullorum, are permanently non flagellated.

2. Phase variation: The flagellar antigens of most salmonellae exist in one of two phases, phase 1 and phase 2. Phase I antigen is more specific and is shared by a few species only and designated as a, b, c, d etc. up to z, and after z as z1, z2, z3up to z14. Phase 2 antigen is nonspecific or group phase and is shared by several unrelated species of salmonellae.

The identification of serotypes of salmonellae is made mainly on the basis of H antigens on the specific phase (phase 1). Many salrnonellae strains are diphasic. Diphasic bacteria existing in one phase tends to mutate spontaneously and reversibly to the other phase variation.

3. V →W variation: Fresh isolates of S. typhi with Vi antigen are generally inagglutinable by 0-antiserum because Vi antigen completely masks the 0 antigen. These are called V forms.

The V forms are only agglutinable by Vi antiserum. Organisms lose their Vi antigen either partially or completely after repeated subculture in the laboratory. With partial loss of Vi antigen, S. typhi are agglutinable by both 0 and Vi antisera and those intermediate forms are called Vw forms. When there is complete loss of Vi antigen, the strain is agglutinable only by 0-antiserum. These are called W-form.

4. S→R variation: The rough variants are organisms with defective capacity to synthesize the polysaccharide of somatic antigen and the loss of 0-antigen may be partial or total. The smooth to rough variation occurs due to mutation and is associated with (a) change of colonial morphology from smooth to rough, (b) loss of 0 antigens and virulence of the strain. S-R variation is not common under natural conditions but is frequent-when strains are maintained for long periods in laboratory medium with high content of carbohydrates.1tcan be avoided by maintaining laboratory cultures in Doreset’s egg medium or by lyophilisation.

5. Variation in 0 antigens: A change in structural formulae of 0 antigens may be brought about by some phages through lysogenisation resulting in the alteration of the bacterial serotype.

Classification The genus Salmonella is broadly divided into four sub-groups on the basis of biochemical reactions.

Kauffmann White scheme: The Kauffmann-White diagnostic scheme, simplified by Edwards and Kauffmann (1952) forms the basis of serotyping of salmonellae based

on the identification of 0 and H antigens. This is done by agglutination tests with absorbed sera. In Kauffmann-

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White scheme each serotype of Salmonella is given a species status. There are about 2,000 serotypes of salmonellae and only strains belonging to groups A to E are of medical importance.

Toxin Like all other enteric Gram-negative bacilli the cell walls of salmonellae contain lipopolysaccharide which, upon lysis of bacteria, act as endtoxin.

Pathogenesis S.typhi and perhaps, S. paratyphi A and S. paratyphi B primarily cause infections in human which remain confined to man. The vast majority of salmonellae are primarily infective for animals and human beings are secondarily infected from the animals, e.g. poultry, pigs, rodents, cattle, turtles, parrots etc. Infection occurs almost always via oral route, usually with water or food contaminated by sewage or via the hands of a carrier.

I. Enteric Fever This clinical entity includes typhoid (S. typhi) and paratyphoid (S.paratyphi A.B .C; S. enteritidis) fevers. Infection due to S. typhi and paratyphi A is prevalent in India and other Asian countries. Although enteric fever is most usually caused by S. typhi, S. paratyphi, A, B or C, but it can be caused by any serotype. Clinical features due to S. typhi infection tend to be more severe. The incubation period ranges from 10 to 14 days.

A. Typhoid fever

a) Incubation stage Infection is transmitted by the faecal oral route through contaminated food and water. The ingested organisms are mostly destroyed in the stomach. Sufficient number of bacilli passes through the gastric acid barrier and reaches the duodenum, where they multiply in alkaline medium. In the small intestine, the bacilli attach themselves on the surface of epithelial cells of the villi and pass through them to the sub mucous coat, where they are phagocytosed by neutrophils and macrophages (monocytes). The virulent bacilli resist intracellular killing and multiply within these cells. These cells enter the mesenteric lymph nodes, where after a period of multiplications, the bacilli invade the blood stream via thoracic duct and a transient bacteraemia follows (primary bacteraemia). Blood stream is rapidly cleared of by cells of the mononuclear phagocytic system (MPS) in the liver, bone marrow, spleen, lung and lymph node. Thus, the internal organs are infected during primary bacteraemia in first 7-10 day.

b) Septicemia stage During primary bacteraemia the bacilli are able to live and multiply in cells of MPS and by about 10th day the parasitized cells undergo necrosis and the bacilli pass into blood leading to a secondary and heavier bacteraemia, which corresponds with the onset of clinical illness at about 14th day after

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ingestion (infection). During this period some organisms undergo lysis liberating endotoxin in the circulation. The bacteraemia and toxaemia cause pyrexia and other signs of clinical illness.

c) Stage of localization

From the blood stream some organisms localize in organs. E.g. gall bladder, liver, spleen, bone etc. Some bacilli are discharged from the gall bladder into the intestine which causes inflammation of Payer’s patches of intestine and lymphoid follicles producing necrosis and sloughing of the affected follicles with resultant typhoid ulcers

which may lead to hemorrhage and perforation.

The febrile illness often causes severe mental clouding of consciousness (typhus means cloud) and includes headache, anorexia and congestions of mucous membrane which result from toxic effects of endotoxin liberated by the organisms. Characteristically there is splenohepatomegaly, bradycardia, step-ladder pyrexia and leucopenia. Skin rashes known as rose spots (1-5 mm red macules that fade on pressure) may appear on chest and abdomen in 2nd and 3rd week. The rose spots contain the infecting organism. The disease lasts for 4 to 5 weeks. The organisms appear in stool during second to third week and in urine during third to fourth week. Although most of the organisms are sensitive to chioramphenicol but relapses after treatment occurs in about 5 to 10 per cent cases, about a week after the primary illness.

Lesions in internal organs like spleen and liver are other complications of infection.

Laboratory Diagnosis (Enteric fever) 1. Isolation of the bacteria from the patient 2. Demonstration of antibodies in serum 3. Demonstration of circulating antigen and 4. General blood picture.

A. Bacteriological investigations Specimens: Blood, faeces, aspirated duodenal fluid; other materials like bone marrow and rose spots may be examined. 1. Blood culture Sample of blood should be collected before starting treatment.

B. 2. Stool and Urine Culture Cultures of faeces and urine are always positive during 3rd and 4th week of illness. Stool culture may give positive result in 2nd week. In S. paratyphi B infection, bacilli appear in stool at the end of 1st week of illness. Repeated cultures are required for positive result.

C. 3. Duodenal juice or bile culture Bile is cultured to detect chronic carriers in whom the organisms are present in the biliary tract.

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B. Serological test Widal Test: It is an agglutination test which detects presence of serum agglutins (H and 0) in patient’s serum with typhoid and paratyphoid fever. Salmonella antibody starts appearing in serum at the end of first week and rises sharply during the 3rd week of enteric fever. It is preferable to test two specimens of sera at an interval of 7 to 10 days to demonstrate a rising antibody titre.

Procedure: In Widal test, two types of tubes were originally used: (1) Dreyer’s tube (narrow tube with conical bottom) for H agglutination and (ii) Felix tube (short round-bottomed tube) for 0 agglutination. Nowadays 3 x 0.5 ml Kahn tubes is used. A serial two-fold dilution of patient’s serum in normal saline (1:20, 1:40 and so on up to 1280 or more) is prepared in 5 small (3 x 0.5 ml) test tubes for each series; 4 for serum dilutions and 5th for a non-serum control. To the diluted serum and control saline equal volume (0.4 cc) of antigen suspensions (TH, TO, AH and BH) are added and mixed thoroughly by shaking the rack and then the mixtures are incubated at 37°C for 4 hours and read after overnight refrigeration at 4°C. Observation: Loose and cotton-wooly clumps are formed in H agglutination and a disc-like granular deposit in 0 agglutination at the bottom of tube. Control tube shows a compact deposit. The maximum dilution of serum at which agglutination occurs indicates the titre of antibodies. The routinely used antigens are H and 0 of S.typhi, H of S. paratyphi A and B. As paratyphoid 0 antigens cross reacts with typhoid 0 antigens due to the sharing of factor 12 by them, paratyphoid 0 antigens are not used.

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Result: The highest dilution (titre) of patient’s serum in respect of each salmonella antigen is read i.e. if this dilution is 1 in 160, the titre is 160. Interpretation of Widal test 1. Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week and the titre remains steady till 4th week after which it declines. 2. Rising titre: Demonstration of rising titre of four-fold or greater of both H and 0 agglutinins at an interval of 4 to 7 days is the most important diagnostic criterion. A rising titre of H or 0 antibodies between tests made in the first and third weeks is highly significant. 3. In a single test, 1:160 titre of 0 or more and 1:200 titre of H signifies presence of active infection, but that has to be interpreted taking into consideration the following factors: (i) Local titre: Due to sub-clinical infectiotion of salmonellosis in endemic area, low titre of agglutinins is present in the serum of normal individuals, which may cause positive reaction. This is known as local titre. It differs slightly from place to place. (ii) Immunization: In immunization with TAB vaccine, vaccinated individuals may show high titers of antibody (H antibody titre 1:160 or more) to each of the salmonellae, and only when a marked rise of titre to one serotype is observed, the result can be regarded as diagnostically significant. H agglutinins tend to persist in titres of 1:50

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to 1:800 for many months and even years after TAB inoculation, but 0 agglutinins do not reach a high titre (up to 1:100) and disappears sooner, e.g. within 6 months. (iii) H-agglutination: In a non vaccinated individual, presence of H agglutinin in serum indicates enteric fever or a latent infection. H agglutinin is more reliable than 0 agglutinin as different salmonellae have same 0 antigens in common. Moreover, 0 antigens are also widely distributed among other enterobacteria. (iv) Anamnestic reaction: Persons who had past entenc infection or who have been vaccinated may develop anamnestic reaction during unrelated fever like malaria, influenzae etc. In such cases, there will be a transient rise in H antibody, whereas the rise is sustained in enteric fever. (v) Nonspecific antigens: Test suspensions of bacteria may contain nonspecific antigens (e.g. fimbrial antigen) which may produce false positive result by reacting with serum of some uninfected persons. (vi) Effect of antibiotic, treatment: Early chloramphenicol therapy exerts a profound effect on antibody response. When treatment is started before the appearance of agglutinins, they are unlikely to appear subsequently; if the antibody is already present, no further rise in titre is expected. (vii) Carriers: Widal tests may be positive in many healthy carriers. Other serological tests ELISA is a sensitive method of measuring antibody against the lipopolysaccharide of, salmonellae, titre of 1gM antibody corresponds fairly well with the Widal 0 titre.

Detection of carriers Chronic carriers of typhoid and paratyphoid fever are important sources of infection and their identification is necessary for epidemiological and public health purposes.

Treatment Enteric Fever 1. Chloramphenicol is the antibiotic of choice for enteric fever, which became available only in 1948. it does not prevent relapse or carriage. 2. Antimicrobial therapy with ampicihin or trimethoprim-sulfamethoxazole gives encouraging result. Ciprofhoxacin is also effective against salmonellae. Carriers 1. Ampicilhin is the most successful drug but the treatment involves prolonged treatment with large doses, 6 gm orally daily for 3 months. Ciprofloxacm, a newly introduced drug, in early trials have shown promising results. 2. Cholecystectomy is indicated in those permanent carriers who do not show any response to antibacterials. It cures carriage but the operation is risky as it carries a small mortality rate.

Epidemiology The faeces of convalescent and healthy carriers are a more important source of contamination of food and drink than the frank clinical cases. Several typhoid outbreak have occurred involving very large number of people, which

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may be water-borne or food-borne. 1. Water-borne: Sewage contaminated by a carrier is responsible for polluting drinking water.

2. Food-borne: Foodstuff gets contaminated via polluted water or via the hands of carriers. “Typhoid Mary”, a cook in USA was a famous carrier, and caused several outbreaks in the beginning of this century in U.S.A. Both typhoid and paratyphoid bacilli multiply rapidly in food. Tinned food may also be contaminated during canning.

Control 1. Public Health: Sanitary measures for clean water supply, disposal of sewage and supervision of food processing and handling must be taken. Infected poultry, meats and eggs should be thoroughly cooked. 2. Carriers: Carriers must not be engaged in food preparation and should observe strict personal hygiene. 3. Immunization: Vaccine is indicated for those persons who travel or live in areas where typhoid fever is endemic.

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