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

Email

doctortvrao@gmail.com

Dr.T.V.Rao MD 71