Enumeration of salmonella and shigella

1. ENUMERATION OF SALMONELLA
AND SHIGELLA
Chapter 25

2. Introduction
 Salmonellosis is a bacterial disease commonly manifested by an acute
enterocolitis, with sudden onset of headache, abdominal pain,
diarrhea, nausea and sometimes vomiting.
 Deaths are uncommon, except in the very young, in the very old, the
debilitated and immunosuppressed. However, morbidity and
associated costs of salmonellosis may be high.
 Salmonellosis is classified as a food borne disease, because
contaminated food, mainly of animal origin, is the predominant mode
of transmission.
 Epidemiologically, Salmonella gastroenteritis's may occur in small
outbreaks in the general population.

3.  However, large outbreaks in hospitals, institutions for children,
restaurants are not uncommon and usually arise from food
contaminated at its source, or, less often, during handling by an ill
person or a carrier, but person-to-person spread can occur.
 They cause illnesses in humans and many animals, such as typhoid fever
and enteritis. Salmonella (e.g. Salmonella enterica subsp. enterica serovar
enteritidis) can cause diarrhea.
 According to the World Health Organization over 16 million people
worldwide are infected with typhoid fever each year, with 500,000 to
600,000 of these cases proving to be fatal.

4. • A large outbreak of Shigella sonnei gastroenteritis occurred in Murcia
Region (Southeast Spain) in the winter of 1995–1996. More than 200
people were affected.
• Epidemiological investigations implicated a regionally manufactured
fresh pasteurized milk cheese as the vehicle of infection.
• The dispersed sale of the cheese resulted in a regional dissemination of
the organism and people were affected in eight townships.

5. • The higher susceptibility in young children of contracting
Shigellosis and typhoid fever in addition to the high
prevalence of Salmonella and Shigella—found to grow
rapidly in liquid infant formula—has focused the attention of
the scientific community to study the survival capabilities of
these organisms in foods.
• In addition, the wide distribution of this commodity
throughout the world creates the risk of a bioterrorism attack
directed against the infant population.

6. Transmission and Source of Infection of
Salmonella
 Salmonella infections are zoonotic
 Salmonella is usually transmitted to humans by eating foods
contaminated with animal faeces.
 Contaminated foods usually look and smell normal.
 Foods of animal origin, such as beef, poultry, milk, or eggs, but any
food, including vegetables, may become contaminated.
 Food may also become contaminated by the hands of an infected food
handler who did not wash hands with soap after using the bathroom.
 Salmonella may also be found in the feaces of some pets, especially
those with diarrhoea, and people can become infected if they do not
wash their hands after contact with pets or pet feaces.

7. Salmonella
Salmonella spp. do not ferment lactose.
Most species of Salmonella are motile with peritrichous flagella.
Some Salmonellae have capsular antigens; that of S. Typhi is referred to
as Vi antigen.
Groups and species of Salmonella are identified by serologic analysis of
O and H antigens (> 2,500 serotypes). Classification of salmonellae is
traditionally based on serogrouping and serotyping (e.g. S. typhimurium,
which is reclassified as S. enterica together with most human pathogens
by analysis of DNA homology). The correct name for S. typhi is S.
enterica, serovar. Typhi or S. Typhi. They can be identified by
biochemical tests and serogrouping, with follow-up serotyping
confirmation.

8. Salmonella
Epidemiology
S. Typhi and S. Paratyphi are primarily infective for humans.
Other salmonellae are chiefly pathogenic in animals (poultry, pigs,
rodents, cattle, pets etc.) that constitute the reservoir for human
infection.
Humans usually become infected by ingestion of contaminated food or
drink (mean infective dose: 106
-108
, but that of S. typhi is lower). In
children, infections can result from direct fecal-oral spread.
The most common sources of human infections: poultry, eggs, dairy
products, and foods prepared on contaminated work surfaces. However,
the major source of infection for enteric fever is the carriers (convalescent
or healthy permanent).

9. Salmonella
Pathogenesis and Immunity
Invasion
Acid tolerance response (ATR) gene protects the organism from
gastric acid.
The bacteria invade into (by inducing membrane ruffling) and multiply
in the M cells and enterocytes of the small intestine. They can also be
transported across the enterocytes and released into the blood and
lymphatic circulation.
Inflammatory response confines the infection to the GI tract in non-
typhoid salmonellosis.
Survival in macrophages
Salmonellae are facultative intracellular pathogen.

10. Salmonella
Clinical diseases
1. Enteritis
Incubation period: 6-48 hours.
Symptoms: nausea, headache, vomiting, non-bloody profuse diarrhea,
with few leukocytes in the stools. Low-grade fever, abdominal cramp,
myalgia, and headache are also common.
Episode resolves in 2-7 days.
Inflammatory lesions of the small and large intestine are present. Stool
cultures remain positive for several weeks after clinical recovery.

11. Salmonella
Clinical diseases
2. Bacteremia
Most common causal species: S. Choleraesuis, S Typhi and S. Paratyphi.
Symptoms: like sepsis caused by other gram-negative bacteria. 10% of
patients may have localized suppurative infections, e.g., osteomyelitis,
endocarditis, arthritis, etc.
High risk population: pediatric and geriatric patients; AIDS patients.

12. Salmonella
Clinical diseases
3. Enteric fever (typhoid fever)
Causal species: S. Typhi, S. Paratyphi A, S. Schottmuelleri,
and S. Hirschfeldii.
Mouth small intestine lymphatics and bloodstream
infect liver, spleen and bone marrow
multiply and pass into the blood second and heavier
bacteremia onset of clinical illness
colonization of gallbladder invasion of the
intestine typhoid ulcers and severe illness.
Chronic carriers (1%-5% of patients): bacteria persist in the
gallbladder and the biliary tract for more than one year.

13. Symptoms: incubation time: 10-14 days. Gradually increasing fever,
malaise, headache, myalgias, and anorexia, which persist for a week
or longer.
In severe cases: intestinal hemorrhage and perforation.
Principal lesions: hyperplasia and necrosis of lymphoid tissue,
hepatitis, focal necrosis of the liver, and inflammation of the
gallbladder, periosteum, lungs and other organs.

14. Salmonella
Treatment
 Enteric fever and bacteremia require antibiotic treatment:
chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole.
Surgical drainage of metastatic abscesses may be required.
 Salmonella enterocolitis needs only supportive therapy (antibiotic
treatment may prolong the symptoms and excretion of the
salmonellae). Drugs to control hypermotility of the gut should be
avoided because it is easy to transform a trivial gastroenteritis into
a life-threatening bacteremia by paralyzing the bowel.
 Chronic carriers of S. Typhi may be cured by antibiotics alone or
combined with cholecystectomy.

15. Salmonella
Prevention and control
Sanitary measures.
Carriers must not be allowed to work as food handlers.
Strict hygienic precautions for food handling.
Vaccines against S. Typhi:
Purified Vi antigen
Oral, live attenuated vaccine.

16. Shigella
S. dysenteriae, S. flexneri , S. sonnei , & S. boydii: bacillary dysentery
> 45 O serotypes; have no H antigen; do not ferment lactose.
Pathogenesis and Immunity
Shigellosis is primarily a pediatric disease, and is restricted to the GI tract.
Mean infective dose: 103
.
Mouth colon invade M cells and subsequently spread to mucosal
epithelial cells cause microabscess in the wall of colon and terminal
ileum necrosis of the mucous membrane, superficial ulceration,
bleeding, and formation of pseudomembrane.
Shiga toxin
An A-B toxin inhibiting protein synthesis.
Damages intestinal epithelium and glomerular endothelial cells (associated
with HUS) .

17. Internalized shigellae induce
apoptosis of macrophage and
release of the bacteria
Attracted by the
cytokines
released by
macrophage
Destablize the
intestinal wall
Activates the invasion genes on
the virulence plasmid
M cell

18. Shigella
Clinical diseases
Incubation period: 1-3 days
Sudden onset of abdominal pain, fever and watery diarrhea
number of stools increase, less liquid, often contain mucus
and blood, rectal spasms with resulting lower abdominal pain
(tenesmus) symptoms subside spontaneously in 2-5 days in
adult cases, but loss of water and electrolytes frequently occur in
children and the elderly a small number of patients remain
chronic carriers.
Some cases were accompanied by hemolytic uremic syndrome
(HUS).

19. Shigella
Laboratory diagnosis
Specimens: fresh stool, mucus flecks, and rectal swabs. Large
numbers of fecal leukocytes and some RBC may often be seen
microscopically.
Culture: differential and selective media as used for salmonellae.
Treatment
Antibiotic treatment: chloramphenicol, ampicillin, tetracycline, and
trimethoprim-sulfamethoxazole. Drug resistance is common.
Opiates should be avoided.

20. Shigella
Prevention and control
Humans are the only reservoir for shigellae.
Transmission of shigellae: water, food, fingers, feces, and flies.
Most cases occur in children under 10 years of age.
Prevention and control of dysentery:
1. Sanitary control of water, food and milk; sewage
disposal; and fly control.
2. Isolation of patients and disinfection of excreta.
3. Detection of subclinical cases and carriers.

21. Isolation and enumeration principle
Pre-enrichment
Twenty-five (25) grams or ml of sample is added to 225 ml of
buffered peptone water and incubated at 37°C for 24 hours.
Selective Enrichment
Transfer one ml portion from pre-enrichment step to each 10 mL of
selenite eosine broth and tetrathionate broth and incubated at 37°C
for overnight.
Selective plating
Then the contents of both tubes were mixed and a loopful was
streaked on to the xylose lysine deoxycolate agar (XLDA), and
bismuth sulphite agar (BSA) plate and Hektoen enteric agar (HEA).
These plates were incubated at 37°C for 24 hrs. The incubation may
be continued up to 72 hrs before report as nil.

22. Xylose lysine deoxycolate (XLDA) agar
 Lactose, Sucrose, and Xylose are the fermentable carbohydrates present
and phenol red is used as the pH indicator.
 Bacteria that ferment none of these sugars, e.g., Shigella, appear as red,
translucent colonies
 Yellow colonies indicate a rapid fermentation of lactose and acid pH, as
demonstrated by E. coli
 Organisms that ferment xylose as well as decarboxylate lysine exhaust
the xylose rapidly and the lysine reaction causes a pH reversal to the
alkaline reaction similar to Shigella
 Sodium thiosulfate and ferric ammonium citrate are indicators of H2S
production only when alkaline conditions exist

23. • Salmonella will, therefore, form red colonies with black
centre in 24 hrs
• Sodium deoxycolate is added to inhibit gram-positive
growth and to retard the growth of many strains of coli
forms
Pink and black color colony of Salmonella on XLD agar

24. Bismuth sulphite agar (BSA)
• Brown, grey, or black colonies; sometimes they have a metallic
sheen.
• In this medium freshly precipitated bismuth sulphite acts together
with brilliant green as a selective agent by suppressing the growth
of coliforms, whilst permitting the growth of Salmonellae.
• Sulphur compounds provide a substrate for hydrogen sulphide
production, whilst the metallic salts in the medium stain the
colony and surrounding medium black or brown in the presence
of hydrogen sulphide.

25. Black color colony of Salmonella on BSA

26. Hektoen Enteric Agar (HEA)
 HEA is used for isolating and differentiating enteric pathogens such as
Salmonella, Shigella and other Gram-negative Enterobacteriaceae.
 The nutrients for growth are provided by the meat, peptone and yeast
extract.
 The increased content of the peptone and the three fermentable
carbohydrates (lactose, sucrose, salicin) as sources of carbon and energy
reduce the inhibitory action of the bile salts on Salmonella and Shigella
spp.
 Bromo-thymol blue and acid fuchsin are pH indicators.

27.  Sodium thiosulphate provides sulphur and ferric ammonium citrate is
the indicator for H2S production.
 H2S positive colonies are blue-green to blue colonies with or without
black center
Blue-green to bluish colony of Salmonella on HEA

28. RAMBACH Agar (chromogenic medium)
for Salmonella
• Differential diagnostic culture medium for identifying non-typhi
Salmonella in foodstuffs and clinical samples
• Sodium deoxycolate inhibits the accompanying Gram-positive flora.
RAMBACH Agar enables species of Salmonella to be differentiated
unambiguously from other bacteria
• Propylene glycol to the culture medium. Salmonellae form acid with
propylene glycol, so that, in combination with a pH indicator, the
colonies have a characteristic red color
• Differentiate coliforms from Salmonellae, the medium contains a
chromogen indicating the presence of ß-galactosidase

29. Coliform microorganisms grow as blue-green or blue-violet colonies. Other
Enterobacteriaceae and Gram-negative bacteria, such as Proteus,
Pseudomonas, Shigella, S. typhi and S. parathyphi grow as colorless to yellow
colonies.
Red color colony of Salmonella on RAMBACH agar

30. Triple Sugar Iron (TSI) Agar
• Ferrous sulfate
• Sodium thiosulfate
• Sodium chloride
• Agar (1.2%)
• Phenol red
• pH = 7.4

31. TSI Reactions of the Enterobacteriaceae
• Yellow deep, purple slant: acid deep due to glucose fermentation,
no lactose or sucrose fermentation with alkaline slant due to
production of amine’s from protein
• Black deep, purple slant: acid deep due to glucose fermentation
with H2S production, no lactose or sucrose fermentation
• Yellow deep and slant: acid deep and slant due to glucose as well
as lactose and/or sucrose fermentation
• Black deep and yellow or black slant: acid deep and slant with
glucose and lactose and/or sucrose fermentation with H2S
production
• Fracturing or lifting of agar from base of culture tube: CO2
production

32. Triple sugar iron test
Reaction Fermentation
Acid butt (yellow), alkaline Slant (red) Glucose fermented
Acid throughout medium, butt and slant yellow both
fermented
Lactose or Sucrose or both fermented
Gas bubbles in butt, medium sometimes split Aerogenic culture
Blackening of the butt Hydrogen sulphide Produced
Alkaline slant and butt (medium entirely red) None of the three sugars fermented

33. Biochemical reactions of Salmonella and Shigella
Test or substrate
Result
Salmonella
species
reaction
Shigella
species
reaction
Positive Negative
Glucose (TSI) Yellow Butt Red Butt + -
Lysine decarboxylase (LIA) Purple butt Yellow butt + -
H2S (TSI) Blackening No blackening + -
Urease Purple-red color No color change - -
Phenol red dulcitol broth Yellow color and/ or gas No gas; no color
change
+ -
KCN broth Growth No growth - +
Malonate broth Blue color No color change - +
Indole test Violet color at surface Yellow color at
surface
- ±
Phenol red lactose broth Yellow color and/ or gas No gas; no color
change
- -
Phenol red sucrose broth Yellow color and/ or gas No gas; no color
change
- -
Voges-Proskauer test Pink-to-red color No color change - -
Methyl red test Diffuse red color Diffuse yellow color + +
Citrate utilization + +
p-phenyl Pyruvic acid - -

34. Urease Test
 Ability of microorganism to degrade urea in the presence Urease
enzyme produced by bacteria and is indicated by phenol red
indicator which undergoes color change from peach to pink.
 If on streaking a Urease agar slant and on incubating it at 37°C for
24 hrs color changes to pink the bacteria is Urease positive