This document provides information about Salmonella, which causes typhoid fever and other infections. It discusses that Salmonella is a gram-negative bacillus that exists in over 2,000 serotypes. Typhoid fever is specifically caused by Salmonella typhi and paratyphoid fevers are caused by Salmonella paratyphi A, B, or C. The document outlines the pathogenesis of typhoid fever and how the bacteria spreads. It also discusses methods for diagnosing typhoid fever through blood and stool cultures as well as the Widal test to detect antibodies. Complications, treatment, epidemiology, and the importance of sanitation in preventing transmission are summarized.

1. Salmonella
basics
Dr.T.V.Rao MD
Dr.T.V.RaoMD 1

2. Salmonella
Dr.T.V.RaoMD 2
• Causes Infections in Humans and
vertebrates,
• Enteric Fever ( Typhoid fever )
• Gastroenteritis
• Septicemias,
• Carrier state.

3. Salmonella
Dr.T.V.RaoMD 3
• A Very complex group
• Contains more > 2,000 spp
• Typed on the basis of Serotyping, and
species typing
• Divided into two groups
1 Enteric fever group
2 Food poisoning group – Septicemias.

4. Enteric Fever
Typhoid Fever
Dr.T.V.RaoMD 4
• Caused by Salmonella typhi, and other
Groups called as Paratyphi A, B, C
• Salmonella typhi - Causes Typhoid
• Salmonella Paratyphi A,B,C Causes
Paratyphoid fevers.
• Food Poison group
• Spread from Animals – Humans
• Causes Gastroenteritis – Septicemias,
Localized Infection

5. Typhoid Mary Most Dangerous
Woman in America
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6. Morphology of Salmonella
• Gram negative
bacilli
• 1-3 / 0.5
microns,
• Motile by
peritrichous
flagella
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7. Bacteriology –Typhoid fever
• The Genus
Salmonella belong to
Enterobacteriaceae
• Facultative anaerobe
• Gram negative bacilli
• Distinguished from
other bacteria by
Biochemical and
antigen structure
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8. Cultural Characters
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• Aerobic / Facultatively anaerobic
• Grows on simple media – Nutrient agar,
• Temp 15 – 41ºc / 37º c
• Colonies appear as large 2 -3 mm, circular, low
convex,
• On MacConkey medium appear
Colorless ( NLF )
Selective Medium - Wilson Blair Bismuth sulphide
medium. Produce Jet black colonies
H2 S produced by Salmonella typhi

9. Enrichment Medium
Dr.T.V.RaoMD 9
Liquid Medium
• Selenite F medium
• Tetrathionate broth
• Above medium are used for
isolation of Salmonella from
contaminated specimens
• Particularly stool specimens..

10. Identifying Enteric Organisms
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• Isolates which are Non lactose fermenting
• Motile, Indole positive
• Urease negative
• Ferment Glucose,Mannitol,Maltose
• Do not ferment Lactose, Sucrose
• Typhoid bacilli are anaerogenic
• Some of the Paratyphoid form acid and gas
• Further identification done by slide agglutination
tests

11. Biochemical Characters
• Glucose ,Mannitol ,Maltose produce A/G
• Salmonella typhi do not produce gas
• Lactose/Salicin/sucrose not fermented.
• Indole –
• Methyl Red +
• V P -
• Citrate +
• Urea –
• H2S – produced by Salmonella typhi
• Paratyphi A do not produce H2S
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12. Resistance of Salmonella
• 55º c – 1 hour
• 60º c – 15 MT
• Boiling ,Chlorination,
Pasteurization Destroy the
Bacilli.
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13. Antigenic structure of
Salmonella
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• Two sets of antigens
• Detection by serotyping
• 1 Somatic or 0 Antigens contain long
chain polysaccharides ( LPS ) comprises
of heat stable polysaccharide commonly.
• 2 Flagellar or H Antigens are strongly
immunogenic and induces antibody formation
rapidly and in high titers following infection or
immunization. The flagellar antigen is of a dual
nature, occurring in one of the two phases.

14. Salmonella
Antigenic Structure
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• H – Flegellar antigens
• O – Somatic antigen,
• Vi – Surface antigen in some species only
• H antigens also called flegellar antigens,
heat labile protein,
• Boiling destroys antigenicity
• When mixed with Antiserum produces
agglutination and fluffy clumps are produced
• H antigens are strongly immunogenic Induces
antibodies rapidly,

15. Antigens – Salmonella ( cont )
Dr.T.V.RaoMD 15
• O Antigens
• Forms integral part of Cell wall,
• Like Endotoxin
• 0 Antigens unaffected by boiling.
• When mixed with antiserum produce chalky
clumps are formed, take more time reaction, at
high temp 50º – 55º c
• O antigens are less immunogenic. than H
antigens

16. Antigen (Vi) – Salmonella ( contd )
• Vi antigens
• Many strains in S.typhi covers the O antigens-
prevents agglutination.
• Resembles like K antigens
• Destroyed after boiling at 60º c / 1 hour.
• Vi a polysaccharide
• Acts as virulence factor, protects the bacilli
against Phagocytosis and activity of
Complement
• Poorly immunogenic
• Low titer of antibodies are produced, Not
diagnostic Dr.T.V.RaoMD 16

17. Classification of Salmonella
• Classified on the basis of
Kauffmann-White Scheme
• Structure of 0 and H antigens
are taken into consideration,
• More than 2000 species
characterized.
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18. Kauffmann – White scheme
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• Serotype 0 antigens H antigens
Phase 1 2
1.Typhi 9,12,(Vi) d 1,2
2 Paratyphi A 1,2.12 a -
3 Paratyphi B 1,4,5,12 b 1,2
4 Typhimuruim 1,4,5,12 I 1,7
5 Enteritidis 1,9,12 g m 1,2

19. Antigenic Variation in
Salmonella
• May be phenotypic / Genotypic
• H to O = loss of Flagella
May be phase variation from I
to II
V to W variation
S to R variation
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20. Pathogenicity
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• Salmonella are definite parasites to
humans.
• Eg S.typhi.
• S.paratyphi A, B ,C
• Other groups Salmonella
• The important clinical syndromes
1.Enteric fever, Septicemias,
gastroenteritis.

21. Enteric Fever
Typhoid
• Typhoid – caused by S.typhi
• Paratyphoid Caused by
Paratyphi A,B,C
• Typhoid --- Like Typhus
• Infective dose ID50 / 107,
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22. Dr.T.V.RaoMD 22

23. Events in a Typical typhoid Fever
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24. Pathology and Pathogenesis
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• Bacilli enter through ingestion,
• Bacilli attach to Microvilli,ileal mucosa,
penetrate to Lamina propria and sub
mucosa
• Phagocytosis by Polymorphs and
Macrophages
• Enters the mesenteric lymph nodes
• Enter the thoracic duct – Blood stream

25. Pathology and Pathogenesis
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• Bacteremia Spread to Liver, Gall
bladder, Spleen, Bone marrow,
Lymph nodes, Lungs, Multiply in
kidneys
Once again spill into Blood
stream
Causes clinical illness.

26. Pathology and Pathogenesis
• Multiply abundantly in Gall bladder,
• Bile rich source of Bacteria
• Spill into Intestine, infects payers patches,
Lymph follicles
• Inflammation – Undergo necrosis, Slough
off
• Typhoid ulcers
• Typhoid ulcers can cause perforation and
hemorrhage
• Duration of Illness 3 – 4 weeks
• Incubation 7 -14, D
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27. Immunity in Typhoid
• Typhoid
bacilli are
Intracellular
pathogens
• Cell mediated
immunity is
crucial Dr.T.V.RaoMD 27

28. Clinical manifestation
• Head ache, malise,anorexia ,coated
tongue
• Abdominal discomfort,
• Constipation / Diarrhea
• Step ladder type fever,
• Relative bradycardia,
• A soft palpable spleen
• Hepatomegaly
• Rose spots appea
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.V.Rao MD 28

29. Events in a Typical typhoid Fever
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30. Rashes in Typhoid
• May present with
rash, rose spots 2 -4
mm in diameter
raised discrete
irregular blanching
pink maculae's found
in front of chest
• Appear in crops of up
to a dozen at a time
• Fade after 3 – 4 days
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31. Complications of Enteric
fever
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• Intestinal perforation,
• Hemorrhage,
• Circulatory collapse.
• Bronchitis Bronchopneumonia,
• Meningitis,
• Cholecystitis,
• Arthritis,Periostitis / Nephritis,
• Osteomyletis,

32. Other complications
• Causes relapses
in particular to
patients treated
with
chloramphenicol.
• S.paratyphi
produce
septicemias.
Dr.T.V.RaoMD 32

33. Epidemiology
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• Developed countries - Controlled.
• Water supply/ Sanitation /Economically
poor.
• S.typhi and S.paratyphi are prevalent in
India
• Previously Typhi are more common
Paratyphoid A on raise.
• Age 5 – 20 years, Sanitation

34. Epidemiology
Dr.T.V.RaoMD 34
• Sanitation has great role
• Source an active patient or a Carrier shed
the Bacilli.
• Who are carriers.
Convalescent carrier 3 weeks to 3
months
Temporary carrier
Chronic carrier
3 months to 1 year
> 1 year,
Women attain more carrier stage

35. Epidemiology (Contd)
Dr.T.V.RaoMD 35
• Bacilli persist in the Gall bladder and kidney
• Food handlers spread the infection
• Cooks great role
• S.typhi and S.paratyphi in humans
• S.para B in Animals,
• Typhoid spread through
Water, Milk, Food
HIV patients potentially susceptible for Typhoid
disease.

36. Typhoid Mary
• A famous example is
“Typhoid” Mary
Mallon, who was a
food handler
responsible for
infecting at least 78
people, killing 5.
These highly
infectious carriers
pose a great risk to
public health.
Dr.T.V.RaoMD 36

37. • Diagnosis is made by any blood, bone
marrow or stool cultures and with the
Widal test (demonstration of salmonella
antibodies against antigens O-somatic and
H-flagellar ). In epidemics and less
wealthy countries, after excluding malaria,
dysentery or pneumonia, a therapeutic trial
time with chloramphenicol is generally
undertaken while awaiting the results of
Widal test and cultures of the blood and
Dr.T.V.RaoMD 37
stool.
How we Diagnose Typhoid
Fever

38. Laboratory Diagnosis of
Typhoid Fever
• 1 Isolation of Bacilli. A Gold standard
• 2 Diagnosis for presence of
Antibodies,
• Positive Blood culture – A gold
standard
• Isolation from Feces and Urine ?
• Detection of Antibodies Inconclusive.
• Newer methods
Detection of antD
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39. Blood Culture
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1 st week Positive in 90 %
2 nd week Positive in 75 %
3 rd week Positive in 60 %
> 3 weeks positive in 25 %
Draw 5 – 10 cc of Blood by venipuncture.
ADD to 50 -100 ml of Bile broth.
Incubate at 37 c /Subculture in MacConkey
At regular intervals

40. Blood Cultures in Typhoid
Fevers
• Bacteremia occurs
early in the disease
• Blood Cultures are
positive in
1st week in 90%
2nd week in 75%
3rd week in 60%
4th week and later in
25%
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41. Castaneda’s method of
Blood Culture
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• Double medium used Solid/Liquid medium
in the same Bottle.
• Bottle contains Bile broth/agar slant,
• For subculture the bottle is merely tilted.
• A subculture into MacConkey at regular
intervals,
• Reduces the chances of contamination
• Increases the chances of isolation.

42. Salmonella on Mac Conkey's
agar
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43. Salmonella on XLD agar
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44. Clot culture
• Clot cultures are
more productive in
yielding better
results in isolation.
• A blood after
clotting, the clot is
lysed with
Streptokinase ,but
expensive to
perform in
developing
countries.
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45. Bactek and Radiometric based
methods are in recent use
• Bactek methods in
isolation of
Salmonella is a rapid
and sensitive method
in early diagnosis of
Enteric fever.
• Many Microbiology
Diagnostic
Laboratories are
upgrading to Bactek
methods
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46. Biochemical Characters
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• Non Lactose fermenter,
• Motile
• Indole – MR + VP - Citrate +
• Ferment Glu/Mal/Man
• Do not ferment Lactose/Sucrose

47. Slide agglutination tests
• In slide agglutination
tests a known serum
and unknown culture
isolate is mixed,
clumping occurs
within few minutes
• Commercial sera are
available for detection
of A, B,C1,C2,D, and
E.
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48. Culturing other Specimens
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• Feces Enrichment in Tetrathionate
broth and Selenite broth
• Culturing in MacConkey/DCA/Wilson
Blair medium – Large black colonies.
• Urine Culture – positive in 25 %
• Other samples
Bone Marrow,Bile,CSF/Sputum

49. Serology
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• WIDAL Test – Tube agglutination test.
• Detects O and H antibodies
• Diagnosis of Typhoid and Paratyphoid
• Testing for H agglutinins in Dryers tubes, a
narrow tube floccules at the bottom
• Testing for O agglutinins in Felix tubes,
Chalky
• Incubated at 37º c overnight

50. Widal Test
• In 1896 Widal A professor of
pathology and internal
medicine at the University of
Paris (1911–29), he
developed a procedure for
diagnosing typhoid fever
based on the fact that
antibodies in the blood of an
infected individual cause the
bacteria to bind together into
clumps (the Widal reaction).
Dr.T.V.RaoMD 50

51. WIDAL Test
land Mark In Diagnosis
• The Widal test is an old
serologic assay for detecting
IgM and IgG antibodies to
the O and H antigens of
Salmonella. The test is
unreliable, but is widely used
in developing countries
because of its low cost.
Newer serologic assays are
somewhat more sensitive
and specific than the Widal
test, but are infrequently
available.
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52. Widal test
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• S.typhi O and H tubes
• Paratyphi A/B H agglutinins only
• Common antigens O in all Factor sharing
12
• Significance
• I st week negative.
• Titers raise in 2nd week Raise of titers
diagnostic

53. Widal Test
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• Single test not diagnostic.
• Paired samples tests
• Diagnostic.
O > 1 in 80
H > 1in 160
H agglutinins appear first
False positives in Unapparent infection,
Immunization
Previously infected

54. Widal test
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• Anamnestic response previous
infection and responding to
unrelated infection
• Other Diagnostic tests
CIE and ELISA
Detection of Circulating antigens
Co agglutination test.

55. Limitation of Widal Test
• The Widal test is
time consuming and
often times when
diagnosis is reached
it is too late to start
an antibiotic
regimen.
• In spite of several
limitation many
Physicians depend
on Widal Test Dr.T.V.RaoMD 55

56. • The Widal test should be interpreted
in the light of baseline titers in a
healthy local population. This is
especially important when there is a
high local prevalence of non-typhoid
salmonellosis.
The Widal test may be falsely positive
in patients who have had previous
vaccination or infection with S typhi.
False Positive and Negative
Reactions with WIDAL Test
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57. False Positive and Negative
Reactions with WIDAL Test
• Widal titers have also been reported in
association with the
dysgammaglobulinaemia of chronic active
hepatitis and other autoimmune
diseases.64 '8 '9 False negative results
may be associated with early treatment,
with "hidden organisms" in bone and
joints, and with relapses of typhoid fever.
Occasionally the infecting strains are
poorly immunogeD
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58. Diagnosis of Carriers and
Environments
• Fecal carriers by
isolation from
specimens. or
Bile aspirated.
• Sewer swabs
• Bacteriophage
typing
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59. Prophylaxis
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• TAB vaccine
S.typhi 1,000 millions
S Paratyphi A,B 750 millions.
Injected subcutaneously 0.5 ml
at 4 – 6 weeks.
Live Oral Vaccine Typhoral
Mutant S.typhi strain Ty 2 1a Lacking enzyme
UDP galctose 4 epimerase 10 to9
Viable bacilli
Given orally 1 – 3 – 5 days

60. Vaccines
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• An Inject able vaccine Typhium Vi
• Contains purified Vi polysaccharide
antigen from S.typhi strain Ty2
• A single dose, subcutaneous route
• Given to children > 5 years
• Immunity lasts for 2- 3 years.
• Follow a booster

61. Treatment
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• Chloramphenicol 1948 /1970 resistance.
• Other Important drugs
Ampicillin Amoxicillin,
Furazolidine
Cotromoxazole
Chloramphenical resistance /Mexico
Kerala

62. Other Drugs
• Fluroquinolones
Ciprofloxacillin,
Pefloxacillin
Ofloxacillin
Ceftazidime
Ceftriaxone /
Cefotoxaime
Dr.T.V.RaoMD 62

63. Coalition against Typhoid
• Since May 2011, the
Coalition against Typhoid
(CaT) has featured
monthly articles in the
WHO’s Global
Immunization Newsletters
(GIN). The articles,
written by CaT members
from around the world,
highlight important work
being done to accelerate
adoption of typhoid
vaccines. Dr.T.V.RaoMD 63

64. Salmonella Gastroenteritis
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• Zoonotic disease
• S.enteritidis
• S.typhimurium
• S.halder
• S. agana
• S.indiana
• Contaminated poultry, Meat Milk, Milk products.
• Enters the shells of the Intact eggs – Chicken
feed, and Fecal droppings.

65. Nontyphoidal Salmonella
• General Incubation: 6 hrs-10 days; Duration: 2-7 days
• Infective Dose = usually millions to billions of cells
• Transmission occurs via contaminated food and water
• Reservoir:
a) multiple animal reservoirs
b) mainly from poultry and eggs (80% cases from eggs)
c)fresh produce and exotic pets are also a source of contamination
(> 90% of reptile stool contain salmonella bacterium); small turtles
ban.
• General Symptoms: diarrhea with fever, abdominal cramps, nausea
and sometimes vomiting Dr.T
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66. Nontyphoidal Salmonella:
Gastroenteritis
• Incubation: 8-48 hrs ; Duration: 3-7 days for
diarrhea & 72 hrs. for fever
• Inoculum: large
• Limited to GI tract
• Symptoms include: diarrhea, nausea, abdominal
cramps and fevers of 100.5-102.2ºF. Also
accompanied by loose, bloody stool; Pseudo
appendicitis (rare)
• Stool culture will remain positive for 4-5 weeks
• < 1% will become carD
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67. Nontyphoidal Salmonella:
Bacteremia and Endovascular Infections
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• 5% develop septicemia; 5-10% of septicemia patients
develop localized infections
• Endocarditis: Salmonella often infect vascular sites;
preexisting heart valve disease risk factor
• Arteritis: Elderly patients with a history of back/chest +
prolonged fever or abdominal pain proceeding
gastroenteritis are particularly at risk.
- Both are rare, but can cause complications that may lead
to death

68. Salmonella Gastroenteritis
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• Can occur as cross infection
• 24 hours
• Manifest with Diarrhea, omitting
• Abdominal pain mucous and blood in
stools
• Last for 2 – 4 days
• Some times may lead to septicemias

69. Diagnosis and Treatment
• Isolation by
culturing
• Rarely need
antibiotics.
• More frequent in
Developed
nations.
Dr.T.V.RaoMD 69

70. Salmonella septicemias
• S.cholera suis
• Deep abscess,
Endocarditis
• Isolation from
Blood and Pus.
• Chloramphenicol
highly effective
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71. Programme created by Dr.T.V.Rao MD for
Medical and Paramedical Students in the
Developing World
Dr.T.V.RaoMD 71
Email
doctortvrao@gmail.com