Salmonella

SalmonellaGeneral Ch.Inhabitant of human and animal intestine.

Gram-negative bacilli, non capsulated, motile, non spore forming.

Non lactose fermenter (NLF).

Produce H2S from ‘thiosulphate’.

Pathogenicity1- 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 (1st week).

• Stool (2nd ,3rd week).

• Urine (3rd 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

O2facultative anaerobe

CO2

Temp37°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-endemicity2-antibiotics

3-vaccinations4-anamnestic reaction

5-time

Tube agglutination ‘Widal test’

O Ab H 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 present in a titre of more than 1:10 is

also suggestive of chronic typhoid carrier.

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