Enterobacteriaceae...Dr Bhat
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Transcript of 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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