Salmonellae: Classification, Biochemical Reaction and Features

In this article we will discuss about Salmonellae:- 1. Classification of Salmonellae 2. Morphology and Staining of Salmonellae 3. Cultural Characteristics 4. Biochemical Reaction 5. Antigenic Structure 6. Pathogenesis and Clinical Features 7. Blood Culture 8. Relapse 9. Epidemiology 10. Salmonellae Food Poisoning.

Contents:

1. Classification of Salmonellae
2. Morphology and Staining of Salmonellae
3. Cultural Characteristics of Salmonellae
4. Biochemical Reaction of Salmonellae
5. Antigenic Structure of Salmonellae
6. Pathogenesis and Clinical Features of Salmonellae
7. Blood Culture of Salmonellae
8. Relapse
9. Epidemiology
10. Salmonellae Food Poisoning

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1. Classification of Salmonellae:

Practically, they may be divided into two groups on the basis of the common clinical symptoms:

2. Morphology and Staining of Salmonellae:

Salmonellae are Gram-negative non-sporing, non-capsulated bacilli, 2-4 µ 0.5 µ in size, fimbriate and actively motile with peritrichate flagella except S. pullorum causing “white diarrhoea” in chick and S. gallinarum fowl typhoid.

3. Cultural Characteristics of Salmonellae:

Aerobe and facultative anaerobe, optimum temperature 37°C, grow well on ordinary media, also on Salmonellae Shigella (SS) selective medium. Colonies on nutrient agar are moderately large, thick, greyish white, moist, circular disks, dome shaped and smooth; but on MacConkey agar and DC A medium, similar pale or colourless colonies are observed, since salmonellae do not ferment lactose.

On Wilson and Blair bismuth sulphite medium, S. typhi produces jet black colonies with a metallic sheen due to H₂ S production, whereas S. paratyphi A and other species that do not form H₂ S, produce green colonies.

Selenite F and tetrathionate broth are used as enrichment media. Triple Sugar iron (TSI) agar medium can be used for identification. On XLD, Salmonellae result in pink colonies with black centres produced by H₂S.

4. Biochemical Reaction of Salmonellae:

Glucose and mannitol are fermented without gas production- lactose and sucrose are not fermented nor is indole produced (Table 32.1)

5. Antigenic Structure of Salmonellae:

Salmonellae possess the following antigens based on which they are classified and identified:

(1) Somatic antigen O,

(2) Flagellar antigen H;

(3) Surface antigen Vi found in S. typhi and S. paratyphi C; but fimbrial antigen is not important for identification.

H. antigen is heat labile protein, destroyed by boiling or treatment with alcohol, but not formaldehyde; when mixed with specific antiserum. H suspensions agglutinate rapidly producing loose, large fluffy clumps. The H antigen is strongly immunogenic and induces antibody formation rapidly to a high titre following infection and immunization. It occurs in two phases—phase I and phase II.

O antigen is a phospholipid polysaccharide complex of cell wall, identical with endotoxin, can be extracted from the bacterial cell wall by trichloracetic acid treatment. When mixed with antisera, O antigen suspensions forms compact, chalky, granular clumps.

O agglutination takes place more slowly at higher temperature (50-55°C) than H agglutination (37°C).

O antigen is less immunogenic than H antigen;

O antigen titre is generally less than that of H antibody.

V. antigen interferes the O agglutination, is related to the virulence. The development of the carrier state is indicated by the persistence of V. antibody in the blood.

6. Pathogenesis and Clinical Features of Salmonellae:

Infection is by ingestion; from the small intestine, the organisms pass via the lymphatic’s to mesenteric glands, whence—after a period of multiplication—they invade the blood stream via the thoracic duct; the liver, gall bladder, spleen, kidney and bone marrow become infected during this bacteriaemic phase in the first seven to ten days of the disease-positive for blood culture.

From the gall bladder, further invasion of the intestine results—Peyer’s patches involved in an acute inflammatory reaction followed by necrosis, sloughing, characteristic typhoid ulcers and—less frequently—by intestinal perforation.

The incubation period of typhoid is usually 14 days. The onset is gradual with headache, malaise, anorexia, a coated tongue, abdominal discomfort with either constipation and diarrhoea. The typical feature is of a step-ladder pyrexia. “Rose spots” that fade on pressure appear on the skin during the second or third week. Some develop deafness and meningitis.

Laboratory diagnosis – Direct (blood, faeces, urine culture), Indirect (Widal test, demonstration of circulating antigen)

7. Blood Culture of Salmonellae:

About 5-10 ml of blood is collected by venepuncture and inoculated into a culture bottle containing 50- 100 ml. of 0.5% Bile broth or Hartley’s broth (Fig. 32.1). After overnight incubation at 37°C the Bile broth is sub-cultured on MacConkey’s medium. Pale colonies are picked up for biochemical and motility studies.

A loopful of the growth from the agar slant is emulsified in two drops of saline on a slide, then a loopful of O antiserum is added to a drop of bacterial emulsion; after rocking the slide gently there will be agglutination.

Clot culture is an alternative to blood culture. 5 ml of blood is allowed to clot in a sterile plain test tube, the serum is pipetted off for Widal test. The clot is broken with sterile glass rod and added to Bile broth medium containing Streptokinase (100 units per ml) which causes lysis of the clot which may yield higher rate of isolation then blood culture.

Faeces Culture:

Salmonellae can be isolated from the faeces Fig. 32.1: Blood culture bottle throughout the illness, most frequently during second and third weeks. Wilson and Blair medium is highly selective than MacConkey’s agar, SS and DCA media.

Urine Culture:

Clean mid-stream urine is collected and centrifuged. The deposit is inoculated into the enrichment media and then in selective media. Urine culture is less useful.

Culture of other materials (bile, rose spots, pus, CSF, sputum) can also be done.

Widal Test:

It is used to diagnose typhoid and paratyphoid by determining O and H agglutinin titres in patient’s sera. Two types of tubes (Felix tube with round bottom, Dreyer’s tube with narrow bottom) are used for O and H agglutination, respectively. The serially diluted serum (1: 10 to 1: 640) is mixed with equal quantity of H and O antigens.

After overnight incubation at 37°C, the agglutination was observed. O agglutination is caused by both S. typhi and S. paratyphi, whereas H agglutination is specific to each species of salmonellae.

8. Relapse:

It is mostly observed in typhoid fever and rarely in paratyphoid C due to presence of V.-antigen on Salmonella typhi. Some organisms escape the effects of chemotherapeutic agents and remain viable in gall bladder.

On withdrawal of drug, S. typhi reappears and in the blood causes relapse. In Relapse, V.-antigen masks “O” antigen of S. typhi and only V-antibody is produced in patient’s serum and “O” antibody production is inhibited. V-antigen is not routinely included Widal test. In relapse, diagnosis is made by detection of V-antibody by agglutination (1: 10 or more) test.

Interpretation:

If O and H agglutinations are positive with a significant titre, then it is paratyphoid due to S paratyphi A, B or C. Plate Widal test can also detect enteric fever but not the agglutinin titres.

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Demonstration of Circulating Antigen:

Typhoid bacillus antigen is consistently present in the blood in the early phase of the disease, and also in the urine. The sensitized staphylococcal co-agglutination test can detect this antigen; Staph, aureus (Cowan 1 strain) containing protein A is established with formaldehyde and coated with S. typhi antibody.

When a 1% suspension of this sensitized cells is mixed on a slide with serum from patients in the first week of typhoid, there will be visible agglutination within two minutes. The test is rapid, sensitive and specific.

Counter electro-immuno-electrophoresis and ELISA have also been used to detect typhoid antigen in blood and urine. The nested Polymerase Chain Reaction (PCR) technique is sensitive and specific to detect S. typhi in blood of polymerase typhoid patients.

Prophylaxis can be done by TAB vaccination. The vaccine is given in two doses of 0.5 ml subcutaneously at an interval of 4-6 weeks. Recent oral Ty 21 a typhoid vaccine can be used more than once in the expectation that they would stimulate an immune response.

9. Epidemiology of Salmonellae:

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 out-break may be water-borne or food borne:

1. Water-Borne:

Sewage contaminated by a carrier is responsible for drinking water pollution.

2. Food-Borne:

Polluted water or the hands of carriers may contaminate the food (e.g. “Typhoid Mary“, a cook in United States of America, was a first carrier in the world and was responsible for several outbreaks of Typhoid fever.

(a) Public Health:

Sanitary measures for clean water supply, proper disposal of sewage. Both S. typhi and S. paratyphi multiply very quickly in food. Tinned food may get contaminated during canning. Infected poultry, meat, contaminated tinned food should be properly cooked.

(b) Carriers:

They should not be allowed to prepare food and should follow strict personal hygiene.

(c) Immunization:

Vaccination is essential for those who live or travel in endemic areas of Typhoid fever.

(i) Killed whole Cell Vaccines:

The bacterial cultures of S. typhi—1,000 millions, S. paratyphi A and B, 750 millions each per ml, are killed by heating at 50-60°C and preserved in 0.5% phenol. This killed vaccine is injected subcutaneously in two doses of 0.5% ml each at an interval of 4-6 weeks followed by booster dose every 3-7 years.

In India a divalent vaccine containing S. typhi and S. paratyphi A are now in use. Local and general reactions are common after vaccination which vanish in 36 hours.

(ii) Live Oral (Ty 21 a) Typhoid Vaccine:

It is a recent vaccine prepared by using a live avirulent mutant strain of S. typhi (Ty 21 a), lacking UDP-galactose-4-epimerase (Gal E mutant). It is administrated orally and has given encouraging results in Egypt. The mutants intiate the infection in the intestine but “self-destructs” after four to five cell divisions and cannot produce any illness.

This oral vaccine is available in enteric coated capsule or in liquid form. It is safe and gives 65-96% protection for 3-5 years. Three doses of the vaccine are given to children on alternate days.

(iii) Purified Vi Polysaccharide Vaccine:

It contains purified Vi antigen and it is injected intramuscularly in a single dose of 25 µg. Its efficiency is about 75%.

Treatment:

Chloramphenicol is the drug of choice. Ampicillin, amoxycillin, furazolidone and cotrimoxazole are useful. Multi-drug resistant salmonellae or salmonellae resistant to chloramphenicol can respond to norfloxacin, ciprofloxacin therapy.

Drug Resistance:

Multiple drug resistance transmitted genetically by plasmids among the strains of S. typhi had been reported for the first time in 1972 from Mexico. The transmissible plasmids carry R. determinants to chloramphenicol, streptomycin, sulphonamide and tetracycline.

Multiple drug resistance has become a problem in India and South East Asia Chloramphenicol resistant typhoid fever had appeared first in epidemic form in Kerala (Calicut) India in 1972. The drug resistant strains of S. typhi that had been reported from India were originally confined to include phage D₁-N, but later to types C₅, A and O.

Phage types which are most prevalent throughout the world are E₁, A, B₂, C₁ D₁ and F₁ phage types prevalent in India are N, Dl-N and O.

10. Salmonellae Food Poisoning:

Human infection is due to the ingestion of food contaminated by salmonellae from poultry, meat, milk, cream and eggs. Clinically the food poisoning is characterised by diarrhoea, vomiting, abdominal pain and fever.

Laboratory diagnosis is made by isolating the salmonellae from the faeces, article of food. Treatment is symptomatic, antibiotics should not be used. Prevention of ingestion of contaminated food can be a measure to control the food poisoning.