SALMONELLA

Introduction
Bacilli that parasites large intestine of vertebrate
species.
Causes Infections in Humans and vertebrates.
Infections are:
Enteric Fever ( Typhoid fever or Paratyphoid fever)
Gastroenteritis
Septicemia and Carrier state
Important species: Salmonella.typhi – causative
agent of typhoid fever.
Contains more > 2,000 spp
 Typed on the basis of Serotyping, and species typing
Divided into two groups
1. Typhoidal group : Enteric fever group – typhoid and
paratyphoid bacilli.
2 Non typhoidal group: Food poisoning group. Prd
Gastroenteritis , septicemia or localised infections.  
Enteric Fever:
Typhoid Fever - Caused by Salmonella typhi
Paratyphoid fever – caused by Salmonella Paratyphi A,B,C.
Morphology 
Gram negative bacilli.
Motile by peritrichous flagella.
Do not form capsules or spores but may possess fimbriae.
Cultural Characters
 Aerobic / Facultatively anaerobic
 Grows on simple media – Nutrient agar
Temp 15 – 41ᵒC / 37ᵒC.
pH range : 6-8
Colonies appear as large 2 -3 mm, circular, low convex
and smooth. More translucent than coliform colonies.
 On MacConkey medium appear Colorless non lactose
fermenting colony.
On Deoxycholate citrate media and XLD(Xylose lysine
deoxycholate), colonies show black heads due to H2S
production.
Selective Medium - Wilson Blair Bismuth sulphide
medium. Produce Jet black colonies due to H2 S
production. S. paratyphi A and other species donot form
H2S and produce green colonies.
Enrichment Medium Liquid Medium - Selenite F medium
and tetrathionate broth.
Biochemical reactions
Ferment glucose , mannitol and maltose forming acid and
gas. S typhi do not form gas.
Lactose, sucrose and salicin are not fermented.
Indole negative, MR – positive, VP – negative, citrate –
positive.
S.typhi and some other salmonella do not grow on
Simmon’s citrate medium as they need tryptophan as
growth factor.
Urea not hydrolysed.
H2S is produced, except by S. paratyphi A and some
other species.
Pathogenicity
 Salmonella are strict parasites of humans or animals.
 Salmonella causes the following important clinical
syndromes in humans - Enteric fever, gastroenteritis/
food poisoning and Septicaemia with or without local
suppurative lesions..
 Enteric Fever
 The term enteric fever includes Typhoid caused by
S.typhi. Paratyphoid caused by S. paratyphi A,B and C.
 Infection is acquired through ingestion.
 On reaching the gut the bacilli attach themselves to the
Microvilli of the ileal mucosa and penetrate to the
Lamina propria and sub mucosa.
They are there phagocytosed by Polymorphs and
Macrophages. The ability to resist intracellular killing and
multiply in these cells is a measure of their virulence.
Then they enters the mesenteric lymph nodes where
they multiply via thoracic duct enter the Blood stream. 
Results in Bacteremia - bacteria then spread to Liver, Gall
bladder, Spleen, Bone marrow, Lymph nodes, Lungs, and
kidneys, where further multiplication takes place.
Towards the end of incubation period, severe bacteremia
occurs and clinical illness will starts. 
Bile is a good culture medium for bacilli ,it multiply
abundantly in Gall bladder and is discharged continuously
into the Intestine and infects payers patches and
Lymphoid follicles of the ileum.
These become Inflamed, Undergo necrosis, and slough off
leaving behind the characteristic Typhoid ulcers .
Ulceration of bowel leads to 2 major complications of the
disease – intestinal perforation and haemorrhage.
Clinical manifestation
Incubation period is usually 7 -14 days.
Onset is gradual with Head ache, malaise, anorexia,
coated tongue, Abdominal discomfort with either
constipation / diarrhea,
The typical feature is Step ladder type pyrexia with
relative bradycardia and toxemia.
A soft palpable spleen and Hepatomegaly is also common.
Rose spots that fade on pressure appear on the skin
during the second or third week .
Complications
Important complications are : Intestinal perforation,
hemorrhage and circulatory collapse.
Bronchitis and bronchopneumonia is always found.
Some may develop psychoses, deafness or meningitis.
Cholecystitis, arthritis, abscesses, periosteitis, nephritis,
hemolytic anemia, venous thromboses and peripheral
neuritis are other complications.
Osteomyelitis is a rare sequel.
Convalescence is slow. In some cases relapse occurs
during convalescence.
S.paratyphi A and B causes paratyphoid fever which
resembles typhoid fever but is generally milder.
Carriers
Source of infection is a patient or a carrier.
Patients who continue to shed typhoid bacilli in feces for
3 weeks to 3 months after clinical cure are called
convalescent carriers.
Persons who shed bacilli for more than 3 months but less
than 1 year are called temporary carriers.
Persons who shed bacilli for more than 1 year are called
chronic carriers.
About 2-4% patients become chronic carriers.
Development of carrier state is more common in women
and in the older age groups.
Bacilli persist in the gall bladder and kidney and are
eliminated in the feces or urine.
Food handlers or cooks who become carriers are particularly
dangerous. Best known carrier is Typhoid Mary – over a
period of 15 years caused atleast 7 outbreaks affecting over
200 persons.
S. paratyphi A will occurs only in human where as B can infect
animals and they may act as source of human diseases.
Typhoid occurs in 2 epidemiological types:
1. Endemic or residual typhoid that occurs through out the
year .
2. Epidemic typhoid which may occur in endemic or non
endemic areas. Typhoid epidemics are water, milk or food
borne.
Laboratory Diagnosis of Typhoid Fever
 1 Isolation of Bacilli.
2 Diagnosis for presence of Antibodies in serum.
1. Specimen: Blood is collected for culture. Urine and
feces also used for culturing. Serum used for Widal test.
Choice of specimen depends on duration of illness.
2. Blood Culture: Bacteriemia occurs early in the disease
and blood cultures are positive in 90 % of cases in the first
week of fever. Positive in 75% of cases in second week.
About 5-10 ml of blood is added in to a culture bottle
containing 50 - 100 ml of 0.5% bile broth. Blood contains
substances that inhibit the growth of the bacilli and
hence the broth have to be taken in sufficient quantity to
provide a 1:10 dilution of blood.
Addition of liquoid( sodium polyanethol sulphonate).
After overnight incubation at 37⁰C- subcultured on Mac
conkey agar – pale non lactose fermenting colonies –
confirmed by biochemical reactions.
Subculture repetition: if first culture is negative,
subcultures should be done and culture declared negative
only after incubation for 10 days.
To eliminate risk of contamination Castaneda’s method
can be employed : in this a double medium is used. The
bottle of bilebroth has an agar slant on one side.
After inoculation of blood, the bottle is incubated is
incubated in upright position. For subculture the bottle is
merely tilted so that the broth runs over the surface of
agar.
It is then reincubated in upright position. If bacteria is
present , colonies will appear on the slant.
Serotyping by slide agglutination method:
A loopful of growth from an agar slope is emulsified in 2
drops of saline on a slide. One act as a control. If
Salmonella typhi is suspected a drop of typhoid O
antiserum is added to one drop of bacterial culture and
look for agglutination after rocking. Fresh cultures should
be agglutinated with Vi antiserum or the bacterial culture
have to be heated for 20 minutes and then agglutinated
with O antiserum.
For identification of unusual Salmonella it can be send to
National salmonella reference centre.
3. Clot culture:
An alternative of blood culture.
5 ml blood of patient is taken in a test tube and allowed to
clot. Serum is pipetted out and used for Widal test. The clot
is broken with a sterile glass rod and added to a bottle of
bile broth.
Addition of streptokinase in bile broth facilitates the lysis of
clot. Clot culture yield a higher rate of isolation than blood
culture as the bactericidal action of serum is removed.
Another advantage is that a sample of serum also becomes
available.
4. Feces culture: Salmonella are shed in feces throughout
the course of disease and convalescence stage. Hence feces
culture is valuable as blood culture. But positive fecal
culture may occur in carrier as well as in patients.
Use of enrichment and selective media and repeated
sampling increase the rate of isolation.
Feces culture is valuable in patients on taking antibiotics
as the drug does not eliminate the bacteria from gut as
rapidly it does in blood.
Feces samples are plated on MA, DCA and Wilson blair
media.
On MA and DCA –pale colonies may be seen
On XLD- Pink coloured colonies with black centre can be
seen.
On wilson blair medium – S.typhi forms large black
colonies and S.paratyphi A forms green colonies due to the
absence of H2S production.
For enrichment specimens are inoculated onto selenite F
broth and tetrathionate broth and incubated for 12-18
hrs before subculture onto plates.
5. Urine culture
Salmonella are shed in urine irregularly or infrequently.
Hence urine culture is less useful than blood or feces
culture.
Clean voided urine sample is centrifuged and deposit is
inoculated on to selective and enrichment media.
6. Other samples: Bone marrow, Bile, CSF, sputum, roses
spots.
7. Serology
WIDAL Test – Tube agglutination test.
Test for the measurement of O and H antibodies for typhoid
and paratyphoid fever.
In Widal Test, two types of tubes were originally used:
(1)Dreyer’s tube (narrow tube with conical bottom) for H
agglutination.
(2) Felix tube (short round-bottomed tube) for O
agglutination.
Procedure
Take 4 sets of 7 test tubes. First set contains felix tube and
label all the tubes in that set as O. Remaining sets have
dryers tube and label each set as H, AH and BH respectively.
Pipette into the tube No.1 of all 4 sets 1.9 ml of normal
saline.
To each of the remaining tubes (2 to 7 in each row) add
1.0 ml of isotonic saline.
To the tube No.1 in each row add 0.1 ml of the serum
sample to be tested and mix well.
Transfer 1.0 ml of the diluted serum from tube no.1 to
tube no.2 in each sets and mix well.
Continue this serial dilution till tube no.6 in each set.
Discard 1.0 ml of the diluted serum from tube No.6 of
each set.
Tube No.7 in all the sets, serves as control with saline.
Now the dilution of the serum sample achieved in each
set is as follows: 1:20 1:40 1:80 1:160 1:320 1:640 
To all the tubes (1 to 7) of first set add one drop of O
antigen suspension and mix well.
To the all the tubes in 2nd , 3rd and 4th set H, AH and BH
antigens were added respectively.
Incubate the tubes at 37° C overnight and observe for
agglutination.
Control tubes were used to check for autoagglutination.
The agglutination titres of serum are read.
H agglutination leads to formation of loose, cotton-
woolly clumps, while O agglutination produces a disc like
or mat like pattern at the bottom of the tube.
In both supernatant will be clear.
Antigens used in the test are : H and O antigens of S.typhi
and AH and BH antigens for S.paratyphi A and B.
Interpretation: Antibody titre value of 1 : 80 for O
antigens and a titre of 1:120 for H antigens is considered
significant and usually suggests positive test for
Salmonella infection.
Prophylaxis
General measures:
Improvement in sanitation and provision of protected
water supply.
Vaccines:
TAB vaccine: Contains S.typhi 1000 million and
S,paratyphi A and B 750 million per ml killed by heating at
50- 60⁰C and preserved in 0.5 % phenol.  Injected
subcutaneously 0.5 ml at 4 – 6 weeks interval. Killed
vaccines do not provide cell mediated immunity.
Live oral vaccine: Typhoral vaccine –
Vi vaccine: injectable vaccine - Typhim Vi.
Treatment
Chloramphenicol was used in the earlier times for
treatment. But later becomes resistant. Then Ampicillin,
amoxycillin and cotrimoxazole were other drugs of choice
but current strains shows resistant towards these also.
At present ciprofloxacin and ceftriaxone are used for
treatment.
Salmonella food poisoning/ salmonella gastroenteritis:
This is generally a zoonotic disease – source of infection
being animal products.
Caused by any salmonella coming under non-typhoidal
groups.
Most imp one is S. typhimurium.
Source of infection: ingestion of contaminated food.
Frequent sources are poultry, meat, milk and milk
products. Egg and egg products are other major sources.
Clinically the disease develops after a short incubation
period of 24 hrs or less, with diarrhea, vomiting,
abdominal pain and fever.
It may vary from passage of loose stool to an acute
cholera like disease.
Usually subsides in 2-4 days but in some cases a more
prolonged enteritis develop , with passage of mucus and
pus in feces resembling dysentry.
In a few, typhoidal or septicemic type of fever may
develop.