Salmonella basics

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 FeverTyphoid 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 InfectionDr.T.V.Rao MD 4

Typhoid Mary Most Dangerous Woman in America

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Morphology of Salmonella

• Gram negative bacilli

• 1-3 / 0.5 microns,

• Motile by peritrichous flagella

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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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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

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Enrichment MediumLiquid 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

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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.

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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

SalmonellaAntigenic 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,

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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

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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

diagnosticDr.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.

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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

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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.

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Enteric FeverTyphoid

• Typhoid – caused by S.typhi• Paratyphoid Caused by

Paratyphi A,B,C• Typhoid --- Like Typhus• Infective dose ID50 / 107,

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Dr.T.V.Rao MD 22

Events in a Typical typhoid Fever

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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

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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 crucial

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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

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Complications of Enteric fever

• Intestinal perforation,• Hemorrhage,• Circulatory collapse.• Bronchitis Bronchopneumonia,• Meningitis,• Cholecystitis,• Arthritis,Periostitis / Nephritis,• Osteomyletis,

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Other complications• Causes relapses

in particular to patients treated with chloramphenicol.

• S.paratyphi produce septicemias.

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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

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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 stage

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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, FoodHIV patients potentially susceptible for Typhoid

disease.

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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.

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• 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

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Laboratory Diagnosis ofTyphoid 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 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%

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Castaneda’s method ofBlood 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.

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Salmonella on Mac Conkey's agar

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Salmonella on XLD agar

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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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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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Biochemical Characters

• Non Lactose fermenter,• Motile• Indole – MR + VP - Citrate +• Ferment Glu/Mal/Man• Do not ferment Lactose/Sucrose

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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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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

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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

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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).

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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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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

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

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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

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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

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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 TyphoralMutant S.typhi strain Ty 2 1a Lacking enzyme UDP

galctose 4 epimerase 10 to9Viable bacilliGiven orally 1 – 3 – 5 days

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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

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Treatment

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

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Other Drugs• Fluroquinolones

Ciprofloxacillin, Pefloxacillin Ofloxacillin

Ceftazidime Ceftriaxone / Cefotoxaime

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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.

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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.

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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

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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

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Diagnosis and Treatment

• Isolation by culturing

• Rarely need antibiotics.

• More frequent in Developed nations.

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Salmonella septicemias• S.cholera suis• Deep abscess,

Endocarditis• Isolation from

Blood and Pus.• Chloramphenicol

highly effective

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Programme created by Dr.T.V.Rao MD for Medical and Paramedical Students in the

Developing World

Emaildoctortvrao@gmail.com

Dr.T.V.Rao MD 71