enterobacteriaceae...dr bhat

8/12/2019 Enterobacteriaceae...Dr Bhat

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Dr Vivek Bhat


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Enterobacteriaceae

Classificationmore than15 different genera Escherichia

Shigella

Edwardsiella Salmonella

Citrobacter

Klebsiella

Enterobacter Hafnia

Serratia


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Enterobacteriaceae

Proteus

Providencia

Morganella

Yersinia

Erwinia

Pectinobacterium


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Enterobacteriaceae

Morphology and General Characteristics

Gram-negative, nonsporing rod shapedbacteria

Oxidase Ferment glucose and may or may not produce

gas in the process (aerogenic vsanaerogenic)

Reduce nitrate to nitrite (are a few exceptions)


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Enterobacteriaceae

Are facultative anaerobes

If motile, motility is by peritrichous flagella

Many are normal inhabitants of the intestinal

tract of man and other animals Some are enteric pathogens and others are

urinary or respiratory tract pathogens

Differentiation is based on biochemical

reactions and and differences in antigenicstructure


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Enterobacteriaceae

Most grow well on a variety of lab mediaincluding a lot of selective and differentialmedia originally developed for the theselective isolation of enteric pathogens.

Most of this media is selective by incorporation ofdyes and bile salts that inhibit G+ organisms andmay suppress the growth of nonpathogenicspecies of Enterobacteriaceae.

Many are differential on the basis of whether or notthe organisms ferment lactose and/or produceH2S.


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Enterobacteriaceae

On BA they all produce similar colonies thatare relatively large and dull gray. They mayor may not be hemolytic.

The three most useful media for screeningstool cultures for potential pathogens are TSI,LIA, and urea or phenylalanine agar.

The antigenic structure is used to differentiate

organisms within a genus or species. Threemajor classes of antigens are found:


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Enterobacteriaceae

Somatic O antigens these are the heat stablepolysaccharide part of the LPS. Variation fromsmooth to rough colonial forms is accompanied byprogressive loss of smooth O Antigen.

Flagellar Hantigensare heat labile Envelope or capsule K antigens overlay the

surface O antigen and may block agglutination byO specific antisera. Boiling for 15 minutes willdestroy the K antigen and unmask O antigens. T K

antigen is called the Vi (virulence) antigen inSalmonella.


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Antigenic structure of

Enterobacteriaceae


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Enterobacteriaceae

Escherichia coli

Normal inhabitant of the G.I. tract.

Some strains cause various forms of

gastroenteritis. Is a major cause of urinary tract infection and

neonatal meningitis and septicemia.

May have a capsule.

Biochemistry Most are motile.


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E. coli

May be hemolytic on BAmore common inpathogenic strains/

Colonies on MALF.

KEY tests for the normal strain: (IMViC : ++--)

TSI is A/A + gas

LIA K/K

Urea

Indole +

Citrate

Motility +

There is an inactive biotype that is anaerogenic,lactose, and nonmotile.


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Escherichia coli

ANTIGENIC STRUCTURE : Three antigensthe somatic antigen O, the capsular antigen K

and flagellar antigen H.

So far, some 170 types of O antigens, 100 K Ags, and 75 H Ags.

The K Ag is the acidic polysaccharide antigen located in the

envelope or microcapsule ( K for kapsel = capsule). Most of the E.

coli found in human intestine do not have K antigen.

VIRULENCE FACTORS:

The somatic lipopolysaccharide surface O antigen has endotoxic,anti-phagocytic and anti complement activities.

Fimbriae also promote virulenceimp in UTIs.


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E. colivirulence.

E. coli produces 2 types of exotoxins; Hemolysins and Enterotoxins.

Hemolysins do not appear to be relevant in pathogenesis.

Enterotoxins are imp in pathogenesis of diarrhea.

3 distinct types of enterotoxins are identified --- LT, ST, VT

E. coli LT

resembles the cholera toxin in its structure, Ag properties & mode ofaction.

It is a complex of polypeptide subunits-each unit containing onesubunit A( A = active) and 5 subunits B (B= binding)

The toxin binds to the Gm1ganglioside receptors on intestinal epth cellsby means of subunit B, foll by actvation of subunit A to A1 and A2. TheA1 fragment activates adenyl cyclase in the enterocyte to form cAMP,leading to outflow of water and electrolytes into gut lumen , withconsequent diarrhea. Though the Mech of action of CT (cholera toxin)and LT is similar, the CT is 100 times more potent than LT.


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E. Colivirulence

TheSTsof E. coli are LMW polypeptides are poorly antigenic.

2 types are known ST1(STA) and ST2(STB)

Acts by activation of cGMP in the intestine leading to rapid accumulation

of fluids.

E.coli Verotoxin or VT - cytotoxic effect on vero -monkey kidney cells

Also called Shiga like toxin (SLT)

Cytoxicity in vero cells and enterotoxicity .

A and B subunits.


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Various types of E. coli


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Clinical Infections .E. coli

URINARY TRACT INFECTIONS

E. coli & other coliforms account for the large majority of communityacquired UTIs.

Serotypes commonly responsible are those commonly found in

fecesO groups 1,2 ,4, 6, 7 etc

Inf of the lower UT seem to be ascending infections caused by

fecal coliforms, pyelonephritis is by hematogenous inf.

Collection of urineMSU

Processing of specimen

Colony countsignificant bacteriuria

UTI screening testsGreiss nitrate test, Catalase test, Microscopy,dip slide culture methods


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E. coliinfections

Neonatal meningitis is the leading cause ofneonatal meningitis and septicemia with a highmortality rate. Usually caused by strains with the K1capsular antigen.

Pyogenic infections : Intra-abdominal infections,

such as peritonitis and abscesses resulting fromspillage of bowel contents.

Septicemia and sepsis syndrome.

Gastroenteritis there are several distinct types of

E. coli that are involved in different types ofgastroenteritis: enterotoxigenic E. coli (ETEC),enteroinvasive E. coli (EIEC), enteropathogenic E.coli(EPEC), enteroaggregative E. coli(EAEC),

& enterohemorrhagic E. coli(EHEC).


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E.coli.Diarrheacont

EPEC Bundle forming pili are involved in attachment to theintestinal mucosa. This leads to changes in signal transduction in

the cells, effacement of the microvilli, and to intimate attachment via

a non-fimbrial adhesion called intimin. The exact mode of

pathogenesis is unclear, but diarrhea with large amounts of mucous

without blood or pus occurs along with vomiting, malaise and lowgrade fever. This is a problem mainly in hospitalized infants and in

day care centers. The diagnosis of EPEC diarrhea is relatively easy

during outbreaks but difficult is sporadic cases. EPEC polyvalent or

monovalent sera is used to test colonies growing on MA ( 10 or

more colonies are to be tested). EPEC sera may be difficult to

obtain. Some serotypes are O26; O55, O111 etc.


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E. Coli diarrheacont

ETEC

Is a common cause of travelersdiarrhea and diarrhea in children indeveloping countries. The organism attaches to the intestinal

mucosa via colonization factors and then liberates enterotoxin. The

disease is characterized by a watery diarrhea, nausea, abdominal

cramps and low-grade fever for 1-5 days. Transmission is via

contaminated food or water . O6, O8, O15, O25, O27, O167 can beenterotoxigenic strains. Toxin production has to be supplemented by

fimbrial adhesion to intestinal mucosa mediated by fimbrial or

colonization factor antigens (CFA I, II, III, IV). Diagnosis of ETEC

may be by ELISA , DNA probes, passive agglutination tests etc.


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E. Coli ..diarrhea cont

EIECThe organism attaches to the intestinal mucosa via pili andouter membrane proteins are involved in direct penetration, invasionof the intestinal cells, and destruction of the intestinal mucosa.There is lateral movement of the organism from one cell to adjacentcells. Symptoms include fever, severe abdominal cramps, malaise,

and watery diarrhea followed by scanty stools containing blood,mucous, and pus. The organism and the clinical disease resembleShigellosis in many respects. Many of the strains may be non motile,NLF, etc. EIEC strains usually belong to serogroups O28, O112,O124, O136, O143, O114, O152, O154. Diagnosis is by Serenystest ( purulent conjunctivitis and sever keratitis when fresh

suspension is instilled into eyes of guinea pig) or by detectingplasmid codes for outer membrane antigens ( virulence markerantigens- VMA) by ELISA ( VMAELISA test)


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E. coli...diarrheacont

EHECThe organism attaches via pili to the intestinal mucosa andliberates the shiga-like toxin called verotoxin. The symptoms startwith a watery diarrhea that progresses to bloody diarrhea withoutpus and crampy abdominal pain with no fever or a low-grade fever.This may progress to fatal hemorrhagic colitis and hemolytic-uremicsyndrome that is characterized by low platelet count, hemolytic

anemia, and kidney failure. The primary target for VT appears to bevascular endothelial cells. This is most often caused by serotypesO157:H7. This strain of E. coli can be differentiated from otherstrains of E. coli by the fact that it does not ferment sorbitol in 48hours (other strains do). A sorbitol-Mac (SMAC) plate (containssorbitol instead of lactose) is used to selectively isolate thisorganism. Confirm that the isolate is E. coli O1547:H7 usingserological testing and confirm production of the shiga-like toxinbefore reporting out results.

Source of EHEC is contamination by human or animal feces directlyor indirectly.cont


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E. Coli diarrhea cont

EHEC cont : Changing lifestyles and eating habits with growing

popularity of fast foods have led to in EHEC infections. One study

implicated salad vegetables radish and alfalfa sprouts in an

outbreak. Proper washing and cooking is imp. Lab diagnosis may

be made by demonstration of the bacilli or VT in feces directly or

culture. DNA probes are useful for VT detection.

EAEC :

Mucous associated auto agglutinins cause aggregation of the

bacteria at the cell surface and result in the formation of a mucous

biofilm. The organisms attach via pili and liberate a cytotoxin distinct

from, but similar to the ST and LT enterotoxins liberated by ETEC.

Symptoms include watery diarrhea, vomiting, dehydration

and occasional abdominal pain.


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Klebsiella

Non motile capsulated rods that grow well on ordinary mediaforming large dome shaped, mucoid, LF colonies.

Short, plump, straight rods about 1-- 2 x 0.50.8 mm in size.

They are classified into 4 spp based on biochemical reactions andinto over 80 serotypes based on capsular (K) antigens.

K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca.

KLEBSIELLA PNEUMONIA ; (Friedlandersbacillus)

First isolated by Friedlander (1883) from fatal cases of pneumonia.

IMViC = --++. Urease +

Ferments glucose, lactose, sucrose, mannitol (acid + gas.)

It has become a very imp cause of Nosocomial infections , evenreplacing E. coli in some centers.


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Klebsiella cont

Klebsiella pneumonia is a serious disease with high case fatality. Itoccurs in middle aged or older persons who have medical problemssuch as like alcoholism, chronic bronchopulmonary disease ordiabetes mellitus. The disease is characterized by massive mucoidinflammatory exudate of lobar or lobular distribution , involving oneor more lobes of the lung. Necrosis and abscess formation are more

frequent. Serotypes 1, 2and 3 are usually responsible forpneumonia.

Klebsiella also causes UTI, pyogenic infections such as abscesses,meningitis and septicemia.

K. ozaenae is associated with ozena, a disease characterized by

foul smelling nasal discharge . ( capsular types 36) K. rhinoscleromatis causes rhinoscleroma , a chronic granulomatous

hypertrophy of the nose; the bacilli are seen intracellularlyin the lesions.


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Enterobacter

They are motile, capsulated, LF which are indole and MR negativeand VP and citrate + ve.

Two imp clinically relevant isolates are E. cloacae andE. aerogenes.

They are normally found in feces, sewage, soil and water and rarely

in urine, pus and other pathological materials. They may beresponsible for hospital infections.

SERRATIA: It forms a pink, red or magenta, non diffusible pigmentcalled prodigiosin. S. marcescens is of medical imp; it is

pleomorphic, with minute, coccobacillary, and normal bacillaryforms. It is a saprophyte found in water, soil, food. It is beingassociated with Nosocomial infections, rarely with meningitis,endocarditis, septicemia, peritonitis, resp inf etc.Multiple drug resistance is common in hospital strains.


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Proteeae

Proteus bacilli constituting the tribe Proteeae are NLF and so do notstrictly belong to the group of coliformbacilli.

However they are also intestinal commensals and opportunisticpathogens like the coliforms.

The name Proteus refers to their pleomorphism, after the Greek God

Proteus who could assume any shape. The tribe Proteeae is classified into 3 generaProteus, Morganella

and Providencia.

Characteristic feature of Proteeae is that they all produce theenzyme Phenyl alanine deaminase ( PPA +ve)

They are generally Gram ve, noncapsulated, pleomorphic, motilerods.

MR+; VP-; resistant to KCN; degrade Tyrosine.

Fail to acidify lactose, dulcitol.


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Proteeaecont

Proteus bacilli possess somatic O and flagellar H antigens. (H=Hauch = film of breath; O = Ohne= no - film of breath)

Weil Felix reaction- non motile strains OX2, OX19, OXK.

They are usually opportunistic pathogens, commonly responsible

for UTI and septic infections, often Nosocomial.

CULTURE :

Colonies of Proteus bacilli have a characteristic putrefactive odor

fishy. Pr. mirabilis and Pr. vulgaris show swarming.

Swarming may be inhibited by : conc of agar ( 6%); chloralhydrate ( 1: 500); Sodium azide ( 1: 500); Alcohol (6%),

sulphonamide, surface active agents or boric acid(1:1000).

Use of CLED agar to prevent swarming.


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Proteeae

The genus Morganella has only one spp. (M. morganii). It does notswarm on BA. It sometimes causes UTIs and nosocomial wound

infections.

The genus providencia contains 3 spp seen in clinical infections

Prov. alcalifacients

Prov. stuartii Prov. rettgirii

These may be seen in UTIs, infections of wounds, burns , and blood.


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TEST Pr.mirabilis

Pr.vulgaris

Morg.morganii

Prov. alc-alifaciens

Prov.

stuartii

Prov.rettgeri

Urease + + + -- +

Ornithinedecarboxylase

+ -- + -- -- --

Indole -- + + + + +

Fermentation of

mannitol

-- -- -- +

Fermentation of

trehalose

-- -- + --

Biochemicals features of Proteeae


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Flagella (H Antigen)

Capsule (K Antigen)

LPS (O Antigen)

Structure of the E. coliCell

Outer membrane

Inner membrane


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The life cycle of E. coliO157:H7

5-10% prevalence in animals

40% prevalence in farms


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Enterobacteriaceae Antigenic Structure

Gram positive cell wall vs


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Gram positive cell wall vs

Gram negative cell wall and outer

membrane

Priming for


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LPS

B-cells

T-cells

Stem cells

Vascular cells

Macrophages/ monocytes

Granulocytes

?

Proliferation

INF

IL-2

Proliferation

Immunoglobulin

IL-1

IL-6Adhesion molecules

TNF-

IL-1

IL-6

IL-8

PAF

O2--Radicals

O2--Radicals

Adhesion molecules

phagocytosis

Direct actions

Stimulation of

additional cells

Recruitment of

additional mediators

(e.g. complement

factors, clotting cascade)

Fever

Hypotension

TachycardiaTachypnea

Neutropenia

etc.

Multi-organ failure

Death


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MacConkey Agar plate

Lactose fermentation


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Klebsiella pneumoniae

pneumonia


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Lactose positive Klebsiellasp

Di Diff i A i i bi l


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Disc Diffusion Antimicrobial

Susceptibility Testing


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Urease test


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E d t i ( tt h d t ll)


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Endotoxin (attached to cell)

LPS, in the outer leaflet of Gram negative bacteria

Lipid A is toxicif organisms enter bloodstream Massive immune cell infiltration

Activation of coagulation


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enterobacteriaceae...dr bhat

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