Salmonella

General Ch.  Inhabitant of human and animal intestine.  Gram-negative bacilli, non capsulated, motile, non spore forming.  Non lactose fermenter (NLF).  Produce H 2 S from ‘thiosulphate’.

Pathogenicity 1- Enteric fever (Typhoid fever, Paratyphoid fever) (S. typhi, S. paratyphi A,B,C). No animal reservoir. 2- Food poisoning (Gastroenteritis) (S. typhimurium, S. enteretidis). Zoonotic infection (animal). No systemic infection. 3- Septicemia S. cholerasuis.

Enteric fever

Salmonella : >1000 species  acc. to diff. Ag (Somatic, flagellar, virulence ) -O (Somatic or cell wall antigen). -H (flagellar antigen). -Vi (polysaccharide virulence).

Laboratory Diagnosis of Enteric Fever

I. Diagnosis of A case of Enteric Fever

A) Isolation of the organism 1- Sample: Blood (1 st week). Stool (2 nd,3 rd week). Urine (3 rd week)

a. From blood: Blood culture or clot culture

b. Culture of faeces: In at least 50% of the cases culture of faeces is positive in first week. The isolation of S. typhi greatly increases in the second or third week.

c. Urine culture: Urine culture is positive in one third of cases, and in great majority of cases, it will be free from organisms, before the faeces becomes negative.

2. Direct film stained by Gram: Gram-negative bacilli. motile. non capsulated. non sporulated.

a) Culture characters O2 facultative anaerobe CO2 Temp 37°C 3. Culture:

b) Media Selective Enrichment Indicator nutrient agar Selenite broth tetrathionate Mackonkey, DCA SS, HE, XLD

b) Culture media On nutrient agar: Grow as smooth colonies, 2-4 mm in diameter. Maximal recovery of salmonella is obtained by using enrichment broth medium as selenite or tetrathionate broth.

Selective media: for Salmonella are SS agar, Hekton enteric (HE) medium, and xylose-lysine-deoxycholate (XLD) agar, which contain selective ingredients. Indicator media: On MacConkey and DCA media: they produce pale colonies (NLF).

Salmonella- XLD media

Salmonella- SS media

4.Identification of growth Morphology BR Slide aggl Molecular

4. Identification of the obtained colonies by: 1. Film stained by Gram:

2. Biochemical reactions: Ferment glucose, mannitol, and maltose with acid (S. typhi) and A+gas (S. paratyphi). Lactose and sucrose is not fermented. H2S produced from thiosulfate.

3. Slide agglutination: using ‘O’ and ‘H’ antisera against salmonellae. 4. Molecular:

B)_ Serodiagnosis: Widal test ( tube agglutination test): Use: Widal test is an agglutination test for detection of antibodies against Salmonella typhi and Salmonella paratyphi, the common causal agents of enteric fevers.

Principle: When serum sample containing antibodies against S.typhi and S.paratyphi A, B or C are mixed with respective antigens, agglutination will take place. In S.typhi and S.paratyphi two types of antigens are recognized as diagnostically important: ‘O’ antigen “Somatic” antigen. ‘H’ antigen “Flagellar” antigen.

O antigens of various species have components in common and hence only one O antigen i.e. non species specific. H antigens of Salmonella spp. are species specific, and hence the H antigens of all S.typhi, S.paratyphi A and S.paratyphi B and S.paratyphi C are employed in the test. Serum antibodies against H and O antigens of salmonella usually appear by the 7th-10th day of infection and the titer reaches maximum during the 4th week.

Method Five sets of agglutination tubes are prepared. To each set, a different H suspension of the salmonella, i.e. H suspension of S. typhi, H suspension of S. paratyphi A, H of S. paratyphi B, H of S. paratyphi C and one set for O suspension of any species because it is a common antigen. Serial dilutions of patient’s serum (1/10, 1/20, 1/40, 1/80 …..) are made in each set of tubes.

Interpretation: The endemicity of the disease in the area. In Egypt, titers below 1/80 are of no significance. They are due to previous subclinical infection. Agglutination of O suspension indicates recent infection. The O suspension will not be agglutinated if vaccination was done a long time ago.

Recently vaccinated individuals possess agglutinins to S. typhi and S. paratyphi. Agglutination will occur with more than one suspension.

False –ve: If the test was done during the first week, it gives false negative results as the antibodies start to appear during the second week. If the patient received antibiotic treatment early in the disease, the antibody titer will be suppressed.

False +ve: Non-enteric infections may cause a non- specific rise on antibody titer; however, the titer is low and falls rapidly on recovery (anamnestic reaction). For proper interpretations of the Widal test, two serum samples separated by 10 days interval should be tested. The detection of a rising titer in the second serum sample indicates active enteric infection.

Percautions: 1-endemicity 2-antibiotics 3-vaccinations 4-anamnestic reaction 5-time

Tube agglutination ‘Widal test’

O AbH Ab ‘typhi’ H Ab ‘paratyphi A’ H Ab ‘paratyphi B’ H Ab ‘paratyphi C’ Result ++---S. Typhi inf S. P.typ. A inf S. P.typ. B inf S. P.typ. C inf Recent vaccine -++++Old vaccine

+ve widal S paratyphi B

+ve widal old vaccination

II. Diagnosis of Typhoid Carrier

-5% of cured - Asymptomatic  transmission. - Difficult treatment. In order to label a person as a typhoid carrier, the isolation of the organism should be done from urine or faeces. If these are repeatedly negative, bile or duodenal aspirate can be used. Vi antibodies Vi antibodies present in a titre of more than 1:10 is also suggestive of chronic typhoid carrier.

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