EnterobacteriaceaePAs & PHNs

P. K. OWIAFE

Outline • General characteristics

• Taxonomic Classification of enterobacteriaceae

• True pathogens among the entrobactriaceae

• Biochemical classification of enterobacteriaceae

• General identification of Gram negative rods

• The IMViC test

• The individual biochemical tests for enterobacteria

• Reaction on Triple Sugar Iron Agar

• The true pathogens

• Clinical manifestations, Lab diagnosis, treatment

Enterobacteriaceae

• Commonly present in large intestine

• Non-sporing, non-acid fast, Gram negative bacilli

• A complex family of organisms

• Many are normal habitats of the intestinal tract of man and other animals

• A few are highly pathogenic in the enteric and are urinary or respiratory tract pathogens

• Some commensals turn out to be pathogenic as in UTI after catheterization

• Differentiation is based on biochemical reactions and differences in antigenic structure

• Are facultative anaerobes

• Majority are motile by peritrichous flagella

• Most grow well on a variety of lab media including a lot of selective and differential media originally developed for the selective isolation of enteric pathogens

• Most of these media are selective by incorporation of dyes and bile salts that inhibit Gram+ organisms and may suppress the growth of non pathogenic Enterobacteriaceae

• Many are differential on the basis of whether or not the organisms ferment Lactose and/ or produce H2S

Enterobacteriaceae

Characters of Enterobacteriaceae

All Enterobacteriaceae• Gram negative rods• Ferment glucose with acid production• Reduce nitrates to nitrites• Oxidase negative• Facultative anaerobic• Motile except Shigella and Klebsiella• Non- capsulated except Klebsiella• Non fastidious• Grow on bile containing media(MacConkey agar)

TAXONOMICAL CLASSIFICATION of ENTEROBACTERIACEAE

• Tribe I: Escherichia

• Genus

• Escherichia

• Edwardsville

• Citrobacter

• Salmonella

• Shigella

• Tribe II: Klebsiella

• Genus

• Klebsiella

• Enterobacter

• Hafnia

• Serratia

• Tribe III: Proteae

• Genus

• Proteus

• Morganella

• Providencia

• Tribe IV: Erwinieae

• Genus

• Erwinia

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• Some Enterobacteriaceae are true pathogens

• Salmonella spp

• Shigella spp

• Yersinia spp

• Certain strains of E. coli (ETEC, EPEC, EIEC and EHEC)

• Most members of Enterobacteriaceae are opportunistic pathogens or cause 2’ry infection of the wound, UT and RT and the circulatory system eg E. coli

Enterobacteriaceae-True pathogens

Enterobacteriaceae

Opportunistic pathogens

Escherichia coli

Klebsiella pneumoniae

Enterobacter aerogenes

Serratia marcescens

Proteus spp.

Providencia spp.

Citrobacter spp.

Obligate pathogens

Salmonella spp.

Shigella spp.

Yersinia spp.

Some E. coli strains

Sepsis

Meningitis

UTI

Diarrhea

Pneumonia

• Enterobacteriaceae are divided into 2 main groups according to action on Lactose

Lactose fermenters (LF)- E.coli, Aeromonas, Klebsiella,

Enterobacter

Non Lactose fermenters (NLF) – Salmonella, Shigella, Proteus spp

and Yersinia

There are several selective and differential media used to distinguish between LFs and NLFs mainly:

MacConkey agar, EMB agar, SS agar, DCA including TSI agar

Classification of Enterobacteriaceae

IMViC TESTs

• Indole, Methyl Red, Voges-Proskauer, Citrate tests:

• The following 4 tests comprises of a series of important determinations that are collectively called the IMViC series of reactions

• These test allow for diffentiation of various members of Enterobacteriacea

IMViC: MRVP TEST

• Inoculate the test organism into one test tube of MRVP broth

• Incubate the tube overnight

• After incubation pour 1/3 of the suspension into a clean non sterile tube:• Run the MR test with 1/3 of the tube and then VP the open tube with

remaining 2/3• For MR test add 6-8 drops of Methyl red reagent

• For Voges- Proskauer: add 12 drops of Baritts A ( -naphthol),mix and add 4 drops of Baritt’s B (40% KOH) and mix

• Let it stand undisturbed for at least 1hr

Reaction Triple Sugar Iron Slant

Method:

• Inoculate TSI medium with suspected organism using a straight inoculating wire by stabbing the butt and streaking the slant

• Incubate at 35-37 C overnight

• Genus Escherichia named after Escherichia who was the first to describe the colon bacillus under the name Bacterium coli commune (1885)

Species:

• E.coli,

• E.fergusonii,

• E.hermanii,

• E.vulneris,

• E.blattae etc

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Escherichia coli

SHOWING GRAM NEGATIVE BACILLI AND PERITRICHOUS FLAGELLA

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ECOLI ON BLOOD AGAR

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E.COLI ON MACCONKEY AGAR

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BIOCHEMICAL REACTIONS

• Sugar fermentation – glucose, lactose, mannitol, maltose and many other sugars fermented with acid and gas production

• IMViC ++--

• Gelatin -, H2S -,

• urease -.

• 10% of E.coli strains

• are Lactose –ve (EIEC)

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ANTIGENIC STRUCTURE• O = somatic antigen

• H = flagellar antigen

• K = capsular antigen

• So far, >170 types of O, 100 types of H and 75 types of K have been identified

• Antigenic pattern of an organism based on these antigens is written as eg. O111:K58:H2, O54:K27:H41 etc

• K antigen is the acidic polysaccharide antigen located in the envelope or microcapsule (K for kapsel, german for capsule)

• It encloses the O antigen and renders the strain inagglutinable by the O antiserum

• It may also contribute to virulence by inhibiting phagocytosis

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TOXINS:

E.coli produce two kinds of exotoxins – haemolysins and enterotoxins

• It is not clear whether the haemolysin per se is a virulence determinant.

The enterotoxins have been identified into three distinct types:

• LT = heat labile toxin

• ST = heat stable toxin

• VT = serotoxin (also known as SLT = shiga like toxin)

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CLINICAL INFECTIONS

• URINARY TRACT INFECTIONS

• DIARRHEA

• PYOGENIC INFECTIONS

• SEPTICAEMIA

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URINARY TRACT INFECTIONS

• Community acquired: E.coli and other coliforms account for the large majority of naturally acquired UTIs

• Hospital acquired: Those acquired in the hospital, following instrumentation, are more often caused by other bacteria such as Pseudomonas and Proteus

• The E.coli serotypes commonly responsible for UTI are those normally found in the feces, O groups 1, 2, 4, 6, 7,etc.

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Asymptomatic bacteriuria:

• Observed in some pregnant women, it means urinary infection without any symptoms

• If it progresses, it may lead to symptomatic infection later in pregnancy, pyelonephritis and hypertension in the pregnant woman, as well as to prematurity and perinatal death of the fetus

Urinary tract infection can be:

• Ascending = via urethra

• Descending = haematogenous route

• Urine cultures constitute 40-70% of specimens sent to clinical microbe labs

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DIARRHEA CAUSING E.COLI

• EPEC = ENTEROPATHOGENIC E.COLI

• ETEC = ENTEROTOXIGENIC E.COLI

• EIEC = ENTEROINVASIVE E.COLI

• EHEC = ENTEROHAEMORRHAGIC E.COLI

• EAEC = ENTEROAGGREGATIVE E.COLI

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Shigella a Highly Infectious Bacteria

• Shigella is one of the most infectious of bacteria andingestion of as few as 100-200 organisms will causedisease

• Most individuals are infected with shigella when theyingest food or water contaminated with human fecalmaterial

• Shigella can survive up to 30 days in milk, eggs,cheese or shrimps

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Classification1. Shigella dysenteriae-12 serotypes:

most serious form of bacillary dysentery

2. Shigella flexneri -6 serotypes: shigellosis in underdeveloped countries

3. Shigella sonnei – 17 serotypes: shigellosis in developed countries

4. Shigella boydii -18 serotypes

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HABITAT AND TRANSMISSION•Shigella species are found only in the humanintestinal tract

•Carriers of pathogenic strains can excrete theorganism up to two weeks after infection andoccasionally for longer periods

• Shigella are killed by drying. Shigella aretransmitted by the fecal-oral route

• The highest incidence of Shigellosis occur in areasof poor sanitation and where water supplies arepolluted

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PATHOGENESIS• SOURCE : MAN: CASE OR CARRIER

• MODE OF SPREAD: CONTAMINATED FINGERS, FOOD, FLIES, FAECES, FOMITES

• PERSON TO PERSON TRANSMISSION

• INFECTIVE DOSE: 10-100 VIABLE BACILLI

• HIGHEST CONCENTRATION IN STOOL DURING EARLY/ACUTE INFECTION 103 TO 109 VIABLE BACILLI PER GRAM OF STOOL

• POST CONVALESCENT SHEDDING : LOW COUNTS 102 TO 103

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PATHOGENICITYShigella dysentery’s form a powerful exotoxin, it isassociated with epidemics of bacillary dysentery

In man, shigellosis begins with symptoms of acute gastro-enteritis which is accompanied by abdominal pain anddiarrhea

As it progresses, diarrhea becomes more frequent and isusually accompanied with colicky pain

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Clinical Syndromes (Shigellosis)

• Ranges from asymptomatic infection to severe bacillary dysentery

• Two-stage disease: watery diarrhea changing to dysentery with frequent small stools with blood and mucus, tenesmus, cramps, fever

Early stage:

• Watery diarrhea attributed to the enterotoxic activity of Shiga toxin

• Fever attributed to neurotoxic activity of toxin

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Clinical Features• FEVER

• BLOODY DIARRHOEA

• ABDOMINAL CRAMPS

• TENESMUS

• MUCUS , PUS

• CONVULSIONS

• MILD INFECTION :WATERY STOOL

• BACTEREMIA - RARE

• REITER,S SYNDROME

• HAEMOLYTIC – URAEMIC SYNDROME

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Methods to Diagnose Shigellosis

• Shigellosis can be correctly diagnosed in most patients on the basis of fresh blood in the stool. Neutrophils in faecal smears is also a strongly suggestive sign

• Nonetheless, watery, mucoid diarrhea may be the only symptom of many S. sonnei infections, and any clinical diagnosis should be confirmed by cultivation of the etiologic agent from stools

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Epidemiology• HUMAN

• FECAL – ORAL ROUTE

• ( water ,, food ,, faeces ,, flies )

• PERSON – PERSON CONTACT

• CHIILDHOOD

• ID : 10 - 100 ORGANISMS

• HIGH INFECTIVITY

• IP - 1– 4 DAYS

• SOURCE - CASES ,, CARRIERS

• DAYCARE CENTERS,, MENTAL INST.

• TRAVEL ,, HOMOSEXEUAL

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Yet No Licenced Vaccine

• Currently, no licensed vaccines targeting Shigella or ETEC exist; however, vaccines against both bacteria are in development

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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 FeverTyphoid Fever• Caused by Salmonella typhi, and other Groups named 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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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, DCA, SS. Produce Jet black colonies

H2 S produced by Salmonella typhi

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SalmonellaAntigenic Structure

• H – Flagellar antigens

• O – Somatic antigen

• Vi – Surface antigen in some species only(S.typhi)

• H antigens also called flagellar antigens, heat labile protein

• Boiling destroys antigenicity

• When mixed with Antiserum produces agglutination as fluffy clumps

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Salmonella Antigen (Vi)• Many strains in S. typhi cover the O antigens-

prevents agglutination

• Resembles K antigens

• Destroyed after boiling at 60ºC / 1 hour

• Vi is 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

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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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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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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

• Incubation 7 -14, ( 3-56 days )

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Clinical manifestation

• Head ache, malaise,anorexia ,coated tongue

• Abdominal discomfort,

• Constipation / Diarrhea

• Step ladder type fever,

• Relative bradycardia,

• A soft palpable spleen

• Hepatomegaly

• Rose spots appear

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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 the rise

• 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

• S.typhi and S.paratyphi in humans

• S.para B in Animals,

• Typhoid spread through

Water, Milk, Food

HIV patients potentially susceptible to Typhoid disease.

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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 methodsDetection of antigen in Blood and Urine

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Salmonella on Mac Conkey's agar

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Salmonella on XLD agar

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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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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 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• 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 infected67

• The Widal test should be interpreted in the light of baseline titres 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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Vaccines

•An Injectable 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

Cotrimoxazole

Chloramphenical resistance /Mexico

Kerala

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Other Drugs

• Fluroquinolones

Ciprofloxacillin, Pefloxacillin Ofloxacillin

Ceftazidime Ceftriaxone / Cefotoxaime

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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 Faecal droppings.

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Yersinia

• Genus Yersinia has 11 species; 3 species are important human pathogens

• Yersinia enterocolitica

• Yersinia pestis

• Yersinia pseudotuberculosis