Salmonella

basics
Dr.T.V.Rao MD

Dr.T.V.Rao MD

Salmonella
Causes Infections in Humans and vertebrates, Enteric Fever ( Typhoid fever ) Gastroenteritis Septicemias, Carrier state.
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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.
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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
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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 41c / 37 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 Medium

Liquid Medium
Selenite F medium Tetrathionate broth Above medium are used for isolation of Salmonella from contaminated specimens Particularly stool specimens..
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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 + VP Citrate + Urea H2S produced by Salmonella typhi Paratyphi A do not produce H2S Dr.T.V.Rao MD

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Resistance of Salmonella 55 c 1 hour 60 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.
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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,
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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 50 55 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 antigensprevents agglutination. Resembles like K antigens Destroyed after boiling at 60 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 Dr.T.V.Rao MD diagnostic

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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
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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 Fever Typhoid Typhoid caused by S.typhi Paratyphoid Caused by Paratyphi A,B,C Typhoid --- Like Typhus 7, Infective dose ID50 / 10
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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.

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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 26 Incubation 7 -14, Dr.T.V.Rao MD days ) ( 3-56

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 Dr.T.V.Rao Rose spots appear MD

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Events in a Typical typhoid Fever

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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, Food HIV 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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How we Diagnose Typhoid Fever

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 37 Dr.T.V.Rao MD stool.

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
Dr.T.V.Rao MD Detection of antigen in Blood and Urine 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
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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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Castanedas 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.
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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 Dr.T.V.Rao MD 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 37 c overnight
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Widal Test
In 1896 Widal A professor of pathology and internal medicine at the University of Paris (191129), 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 160 H agglutinins appear first False 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.
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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

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False Positive and Negative Reactions with WIDAL Test

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.
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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. MD Dr.T.V.Rao 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 Typhoral Mutant S.typhi strain Ty 2 1a Lacking enzyme UDP galctose 4 epimerase 10 to9 Viable bacilli Given 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 WHOs 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

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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 65 and sometimes vomiting Dr.T.V.Rao MD

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 100.5-102.2F. Also accompanied by loose, bloody stool; Pseudo appendicitis (rare) Stool culture will remain positive for 4-5 weeks Dr.T.V.Rao < 1% will become carriers MD

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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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