Salmonellabasics
Dr.T.V.Rao MD
Dr.T.V.Rao MD 1
Salmonella• Causes Infections in Humans and
vertebrates,
• Enteric Fever ( Typhoid fever )
• Gastroenteritis
• Septicemias,
• Carrier state.
Dr.T.V.Rao MD 2
Salmonella
• 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.
Dr.T.V.Rao MD 3
Enteric Fever
Typhoid Fever• 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
Dr.T.V.Rao MD 4
Typhoid Mary Most Dangerous
Woman in America
Dr.T.V.Rao MD 5
Morphology of Salmonella
• Gram negative
bacilli
• 1-3 / 0.5
microns,
• Motile by
peritrichous
flagella
Dr.T.V.Rao MD 6
Bacteriology –Typhoid fever
• The Genus
Salmonella belong to
Enterobacteriaceae
• Facultative anaerobe
• Gram negative bacilli
• Distinguished from
other bacteria by
Biochemical and
antigen structure
Dr.T.V.Rao MD 7
Cultural Characters • 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
Dr.T.V.Rao MD 8
Enrichment Medium
Liquid Medium
• Selenite F medium
• Tetrathionate broth
• Above medium are used for
isolation of Salmonella from
contaminated specimens
• Particularly stool specimens..Dr.T.V.Rao MD 9
Identifying Enteric Organisms
• 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
Dr.T.V.Rao MD 10
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 H2SDr.T.V.Rao MD 11
Resistance of Salmonella
• 55º c – 1 hour
• 60º c – 15 MT
• Boiling ,Chlorination,
Pasteurization Destroy the
Bacilli.
Dr.T.V.Rao MD 12
Antigenic structure of
Salmonella• 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.
Dr.T.V.Rao MD 13
Salmonella
Antigenic Structure• 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,
Dr.T.V.Rao MD 14
Antigens – Salmonella ( cont )
• 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
Dr.T.V.Rao MD 15
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.Rao MD 16
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.Dr.T.V.Rao MD 17
Kauffmann – White scheme
• 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
Dr.T.V.Rao MD 18
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 Dr.T.V.Rao MD 19
Pathogenicity
• 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.
Dr.T.V.Rao MD 20
Enteric Fever
Typhoid
• Typhoid – caused by S.typhi
• Paratyphoid Caused by
Paratyphi A,B,C
• Typhoid --- Like Typhus
• Infective dose ID50 / 107,
Dr.T.V.Rao MD 21
Dr.T.V.Rao MD 22
Events in a Typical typhoid Fever
Dr.T.V.Rao MD 23
Pathology and Pathogenesis
• 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
Dr.T.V.Rao MD 24
Pathology and Pathogenesis
• Bacteremia Spread to Liver, Gall
bladder, Spleen, Bone marrow,
Lymph nodes, Lungs, Multiply in
kidneys
Once again spill into Blood
stream
Causes clinical illness.Dr.T.V.Rao MD 25
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, ( 3-56 days )Dr.T.V.Rao MD 26
Immunity in Typhoid
• Typhoid
bacilli are
Intracellular
pathogens
• Cell mediated
immunity is
crucialDr.T.V.Rao MD 27
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 appear Dr.T.V.Rao MD 28
Events in a Typical typhoid Fever
Dr.T.V.Rao MD 29
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
Dr.T.V.Rao MD 30
Complications of Enteric
fever• Intestinal perforation,
• Hemorrhage,
• Circulatory collapse.
• Bronchitis Bronchopneumonia,
• Meningitis,
• Cholecystitis,
• Arthritis,Periostitis / Nephritis,
• Osteomyletis,Dr.T.V.Rao MD 31
Other complications
• Causes relapses
in particular to
patients treated
with
chloramphenicol.
• S.paratyphi
produce
septicemias.Dr.T.V.Rao MD 32
Epidemiology
• 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
Dr.T.V.Rao MD 33
Epidemiology• 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 3 months to 1 year
Chronic carrier > 1 year,
Women attain more carrier stageDr.T.V.Rao MD 34
Epidemiology (Contd)
• 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.
Dr.T.V.Rao MD 35
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.Rao MD 36
• 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
stool.
How we Diagnose Typhoid
Fever
Dr.T.V.Rao MD 37
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 methodsDetection of antigen in Blood and UrineDr.T.V.Rao MD 38
Blood Culture
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
Dr.T.V.Rao MD 39
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%
Dr.T.V.Rao MD 40
Castaneda’s method of
Blood Culture
• 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.
Dr.T.V.Rao MD 41
Salmonella on Mac Conkey's
agar
Dr.T.V.Rao MD 42
Salmonella on XLD agar
Dr.T.V.Rao MD 43
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.Dr.T.V.Rao MD 44
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
Dr.T.V.Rao MD 45
Biochemical Characters
• Non Lactose fermenter,
• Motile
• Indole – MR + VP - Citrate +
• Ferment Glu/Mal/Man
• Do not ferment Lactose/Sucrose
Dr.T.V.Rao MD 46
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.
Dr.T.V.Rao MD 47
Culturing other Specimens
• 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
Dr.T.V.Rao MD 48
Serology
• 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
Dr.T.V.Rao MD 49
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.Rao MD 50
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.
Dr.T.V.Rao MD 51
Widal test
• 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
Dr.T.V.Rao MD 52
Widal Test• 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
Dr.T.V.Rao MD 53
Widal test
• Anamnestic response previous
infection and responding to
unrelated infection
• Other Diagnostic tests
CIE and ELISA
Detection of Circulating antigens
Co agglutination test.Dr.T.V.Rao MD 54
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.Rao MD 55
• 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
Dr.T.V.Rao MD 56
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 immunogenic.Dr.T.V.Rao MD 57
Diagnosis of Carriers and
Environments• Fecal carriers by
isolation from
specimens. or
Bile aspirated.
• Sewer swabs
• Bacteriophage
typing
Dr.T.V.Rao MD 58
Prophylaxis • 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
Dr.T.V.Rao MD 59
Vaccines
• 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
Dr.T.V.Rao MD 60
Treatment
• Chloramphenicol 1948 /1970 resistance.
• Other Important drugs
Ampicillin
Amoxicillin,
Furazolidine
Cotromoxazole
Chloramphenical resistance /Mexico
Kerala
Dr.T.V.Rao MD 61
Other Drugs
• Fluroquinolones
Ciprofloxacillin,
Pefloxacillin
Ofloxacillin
Ceftazidime
Ceftriaxone /
Cefotoxaime
Dr.T.V.Rao MD 62
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.Rao MD 63
Salmonella Gastroenteritis
• 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.
Dr.T.V.Rao MD 64
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.V.Rao MD 65
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 carriersDr.T.V.Rao MD 66
Nontyphoidal Salmonella:
Bacteremia and Endovascular Infections
• 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
Dr.T.V.Rao MD 67
Salmonella Gastroenteritis
• 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
Dr.T.V.Rao MD 68
Diagnosis and Treatment
• Isolation by
culturing
• Rarely need
antibiotics.
• More frequent in
Developed
nations.
Dr.T.V.Rao MD 69
Salmonella septicemias
• S.cholera suis
• Deep abscess,
Endocarditis
• Isolation from
Blood and Pus.
• Chloramphenicol
highly effective
Dr.T.V.Rao MD 70
Programme created by Dr.T.V.Rao MD for
Medical and Paramedical Students in the
Developing World
Dr.T.V.Rao MD 71