Salmonella

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1. Salmonella

2. Family: Enterobacteriaceae
Gram-negative rods
Motile except Salmonella Gallinarum and S. Pullorum
Aerobic and facultatively anaerobic
Catalase positive; oxidase negative
Attack sugars by fermentation and produces gas
Citrate utilization usually positive except S. Typhi and S.
Paratyphi A
Lysine decarboxylase usually positive except S.
Paratyphi A
G+C content 50-53 mol%

3. Morphology
Gram-negative rods
Motile
Nonsporing, noncapsulated meas. 2-4 x0.6
micron

4. Cultural character
Grow on ordinary culture media
In MacConkey agar and DCA: Small, circular,
translucent, nonlactose fermenting colonies.
In Wilson and Blair Bismuth sulfite medium: Black
colonies with metallic sheen due to production of
H
2
S
Selenite F and tetrathionate broth (enrichment
media for stool specimen culture)

5. Biochemical reaction
Do not ferment lactose or sucrose
Do not produce indole
Ferment glucose, mannitol, maltose with
production of acid and gas except S. Typhi
(produces acid only)
Most strain produces H
2
S in TSI agar except S.
Paratyphi A and S. Choleraesuis
Methyl red positive

6. Classification
Based on DNA-DNA hybridization: 2 species
a.Salmonella enterica and
b.Salmonella bongori
Salmonella enterica comprises 6 sub species
S. enterica subspecies enterica
subspecies salamae
subspecies arizonae
subspecies diarizonae
subspecies houtanae
subspecies indica

7. Popoff, et. al.
2541 serotypes
Sub spp enterica 1504
Sub spp salamae 502
Sub spp arizonae 95
Sub spp diarizonae 333
Sub spp houtanae 72
Sub spp indica 13
S. bongori 22

8. Biochemical reaction patterns of S.
Typhi and S. Paratyphi (d=delayed)
S.
Typhi
S. Paratyphi
A
S. Paratyphi
B
S. Paratyphi C
GlucoseA AG AG AG
Xylosed - AG AG
D-
tartrate
A - - AG
Mucated - AG -

9. Biochemical reactions of some Salmonella serotypes of
subsp. enterica (subsp,I)
Reaction Most serotypes Typhi Paratyphi-A Choleraesuis Gallinarum Pullorum
Gas from sugar + - + + - +
Citrate utilization + - - d + -
H
2
S + w - - - -
Lysine decarboxylase + + - + + +
Ornithine decarboxylase+ - + + - +
Motility + + + + - -

10. Antigenic structure
 3 types
a.O antigen (Somatic)
b.H antigen (Flagellar): present in either or both of two forms- phase 1 and
phase 2.
c.Vi antigen (capsular) includes
F antigen (fimbrial)
M antigen
R antigen
Vi antigen is also found in other bacteria than S. Typhi eg
S. Paratyphi C, S. Dublin
Some strains of E. coli and Citrobacter

11. Antigenic variation
a.H O variation:
Lose flagella and becomes non-motile.
When cultivated in hard agar (phenol 1:800)
b. Phase variation
Occurs in one of two phase i.e. phase 1 or phase 2.
Phase 1 is more specific and is shared by few species.
Phase 2 is non-specific or group phase.
Phase 2 is shared by several unrelated species of
Salmonellae.

12. Antigenic variation contd……
c. S R variation: Smooth to rough variation
It is due to
Change of colonial morphology from smooth to rough
Loss of O antigen and
Loss of virulence
It can be avoided by
Maintaining culture in Dorset’s egg medium or by lyophilization
d. V W variation:
Vi antigen completely mask the O antigen and render O antisera
inagglutinable.
These are agglutinable with Vi antisera
Can be removed by boiling or
By repeated subcultivation in the laboratory media

13. Kauffman and White scheme of classification
Antigenic notation: consists of 3 parts
a.O antigen: In arabic numerals
b.Phase-1 H antigen: a to z and then z1 to z83
c.Phase-2 H antigen: arabic numerals 1-12
O-ag serogroup Serotype O antigens H antigen

Phase-1 Phase- 2
2 A S. Paratyphi A 1,2,12 a [1,5]
4 B S. Paratyphi B 1,4,[5],12 b 1,2
7 C1 S. Paratyphi C 6,7[vi] c 1,5
9 D S. Typhi 9,12[vi] d -

14. Virulence factors
Endotoxin- LPS of cell wall
Invasins - mediates adherence to and penetration of
intestinal epithelial cells.
Resistance to phagocytosis
Vi antigen – antiphagocytic property
Resistance to acid pH – acid tolerance response gene
(ATR gene)
Quorum sensing

15. Cardinal feature of Salmonella
Ability to withstand phagocytosis
(intracellular multiplication)
Produces endotoxin
Resistance to bile

16. Pathogenesis

17. Salmonellae causes the following clinical
syndrome in human beings
1.Enteric fever
2.Septicaemia with or without local
suppurative lesion
3.Gastroenteritis or food poisoning

18. Enteric fever
Typhoid fever caused by S. Typhi and paratyphoid fever
caused by S. Paratyphi A,B and C.
The name typhoid was given by Louis (1829) who
distinguish it from typhus fever.
In 1869, based on anatomical site of infection, the term
enteric fever was proposed.
It is systemic disease characterized by fever and abdominal
pain.

19. Enteric fever contd…,
Epidemiology
Disease of underdeveloped and developing countries (global health
problem)
13-17 million case/year
600,000 deaths/year
Transmission: close contact with acutely infected individuals or chronic
carriers
Faeco-oral rare
Most cases via contaminated food and water

20. Epidemiology contd….
Antibiotic resistant among salmonellae is a rising concern
and has been linked to antibiotic use in live stock.
Ciprofloxacin resistance either plasmid or chromosomally
mediated has been observed.
ESBL producing strains have been reported from different
part of world including Nepal.
Food handlers and cooks who become carriers are
particularly dangerous
Mary Mallon (‘Typhoid Mary’) - a New York cook - over a 15
years- at least 7 outbreaks affecting over 200 individuals.

21. Clinical course
Incubation period: 3-21 days
Fever: > 75% and abdominal pain: 20-40% at presentation.
Most prominent symptom: prolonged fever(101.8
o
c-104.9
o
c)
Chills, headache, weakness, dizziness and muscle pain.
GI symptoms are quite variable (Diarrhea or constipation).
Early physical findings: rose spots in the trunk and chest
region, hepatosplenomegaly and relative bradycardia.
Late complication (untreated adults): Intestinal perforation
and/or gastrointestinal hemorrhage
Rare complication: pancreatitis, hepatic and splenic
abscess, endocarditis, pericarditis, orchitis, hepatitis,
meningitis, nephritis, myocarditis, pneumonia, arthritis,
osteomylitis, and parotitis.
Approx 1-5% of the patients become asymptomatic

22. Laboratory diagnosis
Four principles
1.Isolation of bacteria from blood
2.Demonstration of antibody
3.Demonstration of circulating antigen
4.General blood picture
The choice of specimen depends upon stage
of the disease

23. Lab diagnosis contd…
Bacteriological investigation
Blood culture
Clot culture
Bone marrow culture
Bile culture
Urine culture
Stool culture
Rose spot biopsy
culture

24. Lab.diagnosis contd…
Media for Blood culture
0.5% bile broth (WHO recommended)
0.5% glucose broth
Brain heart infusion broth
Nutrient broth
Trypticase soy broth
Thioglycollate broth
Castaneda’s culture (Biphasic medium)
Liquiod broth (0.025% SPS)
Agar Slant
Broth

25. Lab.diagnosis contd…
Blood culture
Blood: Broth ratio- 1:10
Larger volume of media helps to dilute the
antibacterial substance present in the blood.
Incubation up to 7 days at 37
0
c.
Sub culture 1
st
after 24 hours and then after
every 48 hours or if culture appears turbid.

26. Bone marrow culture
More sensitive(abt.90%) than blood
culture.
Even after starting antibacterial
therapy(<5days) it remains positive.

27. Urine/Stool culture
Positive during 3
rd
and 4
th
week of illness.
If blood, bone marrow and intestinal
secretions are all cultured, the yield of
a positive culture is >90%.

28. Bactec system
Monitors bacterial growth by detecting
14
C-
labelled CO
2
produced by bacterial
metabolism of
14
C-labelled substrate in the
liquid growth medium.

29. Widal test
Detects O and H agglutinins for typhoid and
paratyphoid bacilli.
Two types of tubes are used
Dreyer’s tube for H agglutination: narrow tube with
conical bottom
Felix tube for O agglutination: short round bottomed
tube
H agglutination: loose, cottony agglutinates
O agglutination: compact granular agglutinates.

30. Antigen preparation for widal test
H-antigen
Organisms are cultured in liquid media
(Hazana broth)- overnight
Preserved by adding 0.1% formalin

31. Demonstration of circulating antigen
Coagglutination
Latex agglutination

32. General blood picture
In 15-25% of cases, leukopenia and
neutropenia.
In majority of cases WBC normal despite
high fever
Leukocytosis in children during the 1
st
10
days or in the complicated case like
intestinal perforation.

33. Carrier detection
This is important for epidemiological and
public health purpose
For detection
Bile or duodenal aspiration culture
Stool and urine culture
Vi antigen detection

34. Serotyping
Growth on agar slope is used for
agglutination
a.Polyvalent O ( Groups A-G)
b.Group specific sera
c.H-antisera
d.Polyvalent-H, specific and non-specific
e.Vi antiserum

35. Antibiotic sensitivity
Many strains are sensitive to chloramphenicol,
Ampicillin, Tetracycline and Cotrimoxazole.
However, resistance to individual drugs
depends on serotype, phagetype and country
of origin.
Chloramphenicol was considered to be the
drug of choice

36. Antibiotic sensitivity contd…
Ciprofloxacin is considered as a 1
st
line
choice for treatment of typhoid fever.
In case of Nalidixic acid resistant (NAR)
strain, ciprofloxacin should be given in
higher in dose for longer period or third
generation cephalosporin should be
administered.

37. Gastroenteritis
Common serotypes S. Enteriditis and S.Typhimurium
Symptoms appear within 48 hours of ingesting
contaminated food and water.
Characterized by nausea, vomiting and diarrhea usu.
non-bloody.
Fever and abdominal cramps are common.
Self limiting within 48-72 hours and doesn’t require
treatment except in children and debilitated adults.
For unknown reasons, it is found in persons who carry
HLA-B 27 histocompatibility marker.

38. Salmonella gastroenteritis contd…
TTSS- Type III Secretion System

39. Salmonella gastroenteritis contd…

40. Salmonella gastroenteritis contd…

41. Salmonella gastroenteritis contd…

42. Salmonella gastroenteritis contd…

43. Prevention
Proper sewage disposal
Correct handling of food
Good personal hygiene

44. Immunisation
 Several vaccines are in use
1.Killed S. Typhi vaccine
 TAB vaccine containing S. Typhi, S. Paratyphi A and B
2. Live oral vaccine (Ty 21a)
 Oral administration of avirulent mutant strain of S. Typhi, Gal E
mutant lacking UDP-galactose-4-epimerase
 Mutant initiate infection in the intestine but self-destructs after
4-5 cell divisions and can’t produce any illness.
 Three doses on alternate days to children.
3.Purified Vi polysaccharide vaccine( typhim-Vi)
 Single dose
 Intramuscularly