e coli lectur revised alpana

E .coliE .coli

ByBy

DR Alpana vermaDR Alpana verma

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IntroductionIntroduction

The family Enterobacteriaceae is the largest, most heterogeneous The family Enterobacteriaceae is the largest, most heterogeneous collection of medically important gram-negative rods. collection of medically important gram-negative rods. A total of more than 40 genera and 150 species and subspecies have A total of more than 40 genera and 150 species and subspecies have been described. been described. These genera have been classified based on biochemical properties, These genera have been classified based on biochemical properties, antigenic structure, and nucleic acid hybridization and sequencing.antigenic structure, and nucleic acid hybridization and sequencing.Despite the complexity of this family, almost 20 species are responsible Despite the complexity of this family, almost 20 species are responsible for more than 95% of the infections .for more than 95% of the infections .Enterobacteriaceae are ubiquitous organisms, found worldwide in soil, Enterobacteriaceae are ubiquitous organisms, found worldwide in soil, water, and vegetation, and are part of the normal intestinal flora of most water, and vegetation, and are part of the normal intestinal flora of most animals, including humans. animals, including humans. These bacteria cause a variety of human diseases, including 30% to These bacteria cause a variety of human diseases, including 30% to 35% of all septicemias, more than 70% of urinary tract infections (UTIs), 35% of all septicemias, more than 70% of urinary tract infections (UTIs), and many intestinal infections. and many intestinal infections. Some organisms (e.g., Some organisms (e.g., Salmonella typhi, ShigellaSalmonella typhi, Shigella species, species, Yersinia Yersinia pestispestis) are always associated with disease, whereas others (e.g., ) are always associated with disease, whereas others (e.g., Escherichia coli, Klebsiella pneumoniae, Proteus mirabilisEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis) are members ) are members of the normal commensal flora that can cause opportunistic infections. of the normal commensal flora that can cause opportunistic infections.

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Common Medically Important Common Medically Important EnterobacteriaceaeEnterobacteriaceae

Citrobacter freundii, Citrobacter koseriCitrobacter freundii, Citrobacter koseri Enterobacter aerogenes, Enterobacter cloacaeEnterobacter aerogenes, Enterobacter cloacae Escherichia coliEscherichia coli Klebsiella pneumoniae, Klebsiella oxytocaKlebsiella pneumoniae, Klebsiella oxytoca Morganella morganiiMorganella morganii Proteus mirabilis, Proteus vulgarisProteus mirabilis, Proteus vulgaris Salmonella entericaSalmonella enterica Serratia marcescensSerratia marcescens Shigella sonnei, Shigella flexneriShigella sonnei, Shigella flexneri Yersinia pestis, Yersinia enterocolitica,Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosisYersinia pseudotuberculosis

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Figure 31-1 Sites of infections with common members of the Enterobacteriaceae listed in order of prevalence.

© 2005 Elsevier

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Physiology & StructurePhysiology & Structure

Members of the Enterobacteriaceae family are moderately sized (0.3 to Members of the Enterobacteriaceae family are moderately sized (0.3 to 1.0 × 1.0 to 6.0 μm) gram-negative rods .1.0 × 1.0 to 6.0 μm) gram-negative rods .They share a common antigen They share a common antigen (enterobacterial common antigen),(enterobacterial common antigen), are are either nonmotile or motile with peritrichous flagella), and do not form either nonmotile or motile with peritrichous flagella), and do not form spores. spores. All members can grow rapidly, aerobically and anaerobically (facultative All members can grow rapidly, aerobically and anaerobically (facultative anaerobes), on a variety of nonselective (e.g., blood agar) and selective anaerobes), on a variety of nonselective (e.g., blood agar) and selective (e.g., MacConkey agar) media. (e.g., MacConkey agar) media. The Enterobacteriaceae have simple nutritional requirements, ferment The Enterobacteriaceae have simple nutritional requirements, ferment glucose, reduce nitrate, and are catalase positive and glucose, reduce nitrate, and are catalase positive and oxidase oxidase negative.negative. The absence of cytochrome oxidase activity is an important The absence of cytochrome oxidase activity is an important characteristic, because it can be measured rapidly with a simple test characteristic, because it can be measured rapidly with a simple test and is used to distinguish the Enterobacteriaceae from many other and is used to distinguish the Enterobacteriaceae from many other fermentative and nonfermentative gram-negative rods. fermentative and nonfermentative gram-negative rods. A few exceptions to these rules exist (e.g., A few exceptions to these rules exist (e.g., Plesiomonas shigelloidesPlesiomonas shigelloides is is oxidase positive; oxidase positive; Klebsiella granulomatisKlebsiella granulomatis cannot be cultured on cannot be cultured on traditional media). traditional media). Characteristics of the organisms' colonies on different media have been Characteristics of the organisms' colonies on different media have been used to identify common members of the family Enterobacteriaceae. used to identify common members of the family Enterobacteriaceae.

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Physiology & StructurePhysiology & StructureFor example, the ability to For example, the ability to ferment lactoseferment lactose has been used has been used to differentiate lactose-fermenting strains (e.g., to differentiate lactose-fermenting strains (e.g., Escherichia, Escherichia, Klebsiella, Enterobacter, Citrobacter,Klebsiella, Enterobacter, Citrobacter, and and SerratiaSerratia spp.) spp.) from strains that do not ferment lactose or do so slowly from strains that do not ferment lactose or do so slowly (e.g., (e.g., Proteus, Salmonella, Shigella,Proteus, Salmonella, Shigella, and and YersiniaYersinia spp.). spp.). Resistance to bile salts in some selective media has been Resistance to bile salts in some selective media has been used to separate enteric pathogens (e.g., used to separate enteric pathogens (e.g., Shigella, Shigella, SalmonellaSalmonella) from commensal organisms that are inhibited ) from commensal organisms that are inhibited by bile salts (e.g., gram-positive and some gram-negative by bile salts (e.g., gram-positive and some gram-negative bacteria present in the gastrointestinal tract). bacteria present in the gastrointestinal tract). Some Enterobacteriaceae have prominent capsules (e.g., Some Enterobacteriaceae have prominent capsules (e.g., Klebsiella,Klebsiella, some some EnterobacterEnterobacter and and EscherichiaEscherichia strains), strains), The heat-stable The heat-stable lipopolysaccharide (LPS)lipopolysaccharide (LPS) is the major cell is the major cell wall antigen and consists of three components: the wall antigen and consists of three components: the outermost somatic O polysaccharide, a core polysaccharide outermost somatic O polysaccharide, a core polysaccharide common to all Enterobacteriaceae (enterobacterial common common to all Enterobacteriaceae (enterobacterial common antigen), and lipid Aantigen), and lipid A

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Antigenic structure of Enterobacteriaceae.

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© 2005 Elsevier

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Escherichia coliEscherichia coli

Normal inhabitant of the G.I. tractNormal inhabitant of the G.I. tract Some strains cause various Some strains cause various

forms of gastroenteritis.forms of gastroenteritis.

Is a major cause of urinary tract Is a major cause of urinary tract infection and neonatal meningitis infection and neonatal meningitis and septicemia.and septicemia.

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Morphology and culture Morphology and culture characteristicscharacteristics

MorphologyMorphology Gram negative rodsGram negative rods Non-spore former, non-caspulated and motileNon-spore former, non-caspulated and motile

Culture characteristicsCulture characteristics N.agarN.agar – circular, convex, small colonies – circular, convex, small colonies MacConkeyMacConkey mediummedium– rose pink– rose pink Eosin Methylene blueEosin Methylene blue – Metallic sheen – Metallic sheen

coloniescolonies

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Escherichia coliEscherichia coli

The genus The genus EscherichiaEscherichia consists of five species, of consists of five species, of which which E. coliE. coli is the most common and clinically is the most common and clinically most important. most important. This organism is associated with a variety of This organism is associated with a variety of diseases, including sepsis, UTIs, meningitis, and diseases, including sepsis, UTIs, meningitis, and gastroenteritis. gastroenteritis. As expected, the multitude of strains capable of As expected, the multitude of strains capable of causing disease is reflected in the antigenic causing disease is reflected in the antigenic diversity of the bacteria. diversity of the bacteria. Many O, H, and K antigens have been described, Many O, H, and K antigens have been described, and they are used to classify the isolates for and they are used to classify the isolates for epidemiologic purposes. epidemiologic purposes. Specific antigenic serogroups are also associated Specific antigenic serogroups are also associated with greater virulence. with greater virulence.

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DiagnosisDiagnosis

Laboratory identification Laboratory identification teststests

1111

1212

Gram negative Rods of E.coli

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Typical Peritrichous E.coli

EMB agar—Selective AND Differential

Eosine and methylene blue block are dyes that block the growth of gram (+) bacteria.

Why are gram (-) bacteria more resistant?

E. coli forms blue-black colonies with a green metalic sheen. (due to lactose fermentation)

E. aerogenes forms large mucoid pink to purple colonies with no metallic sheen.

Salmonella large forms pink colonies.

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MacConkey Agar plateMacConkey Agar plateLactose fermentationLactose fermentation

Biochemical reactionsBiochemical reactions

IMViC: ++--IMViC: ++--

TSI: Acid butt/acid slant with gas TSI: Acid butt/acid slant with gas productionproduction

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• • E. coli (pink/red) +• • E. coli (left side) –

• • Kovac’s reagent• detects if tryptophan• has been hydrolyzed• to indol/tryptophanase

Indole (IMViC tests)

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Enterobacteraerogenes (left) –

E. coli (bright red) +

Reagent: Methyl red

indicator identifies pHchange due to mixedacid fermentation

Methyl Red (MR) (IMViC tests)

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Enterobacter aerogenes +(left)

E. coli – (right)

Barritt’s reagent Tests for acetoin, precursor to 2,3 butanediol fermentation

Voges – Proskauer (VP)(IMViC tests)

2020

• • E. coli (left green) –

• • Enterobacter aerogenes• (right royal blue) +

Reagent: Bromothymolblue indicator

tests for ability to use citrate assole carbon source/citrate

permease

Citrate (IMViC tests)

Triple Sugar Iron Agar (TSI):Triple Sugar Iron Agar (TSI):

Purpose:Purpose: To differentiate To differentiate bacteria based on their ability bacteria based on their ability to ferment glucose, lactose to ferment glucose, lactose and/or sucrose, and to and/or sucrose, and to reduce sulfur to hydrogen reduce sulfur to hydrogen sulfide. sulfide.

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INTERPRETATION OF TUBES ABOVEINTERPRETATION OF TUBES ABOVE

TUBE 1TUBE 1(UNINOCULATED)(UNINOCULATED)

TUBE 2 TUBE 2 TUBE 3 TUBE 3 TUBE 4 TUBE 4 TUBE 5 TUBE 5

SLANT SLANT -- AA AA KK KK

BUTT BUTT -- AA AA AA AA

HYDROGENHYDROGENSULFIDE SULFIDE

-- -- -- ++ ++

GAS GAS -- -- ++ -- --

A=Acidic K=AlkalineA=Acidic K=Alkaline 2222

Antigenic structureAntigenic structure

Has O, H, and K antigens. Has O, H, and K antigens.

K1 has a strong association with virulence, K1 has a strong association with virulence, particularly meningitis in neonates.particularly meningitis in neonates.

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Antigenic structure.

Complex

•Lipopolisaccharides/Somatic or O antigen

• Heat stable.more than 150 types

•Most external in the cell wall •detected by bacterial agglutination

•Antibody produced is predominantly IgM2424

Capsular/K antigenCapsular/K antigen Sometimes external to O antigen but Sometimes external to O antigen but

not alwaysnot always Can be polysaccharides or proteinCan be polysaccharides or protein

Flagella /H antigenFlagella /H antigen Heat and alcohol labile.Heat and alcohol labile.

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Colicines/Bacteriocines

• Produced by many Gram -ves

• Virus like bactericidal substance •Active against some other bacteria of similar or closely related species

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E. coliE. coli toxins toxins Virulence factorsVirulence factors

ToxinsToxins Enterotoxins – produced by enterotoxigenic Enterotoxins – produced by enterotoxigenic

strains of strains of E. coliE. coli (ETEC). Causes a movement (ETEC). Causes a movement of water and ions from the tissues to the bowel of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two resulting in watery diarrhea. There are two types of enterotoxin:types of enterotoxin:

LT – is heat labile and binds to specific GmLT – is heat labile and binds to specific Gm11 gangliosides on the epithelial cells of the gangliosides on the epithelial cells of the small intestine where it stimulates small intestine where it stimulates production of cAMP. production of cAMP. Increased cAMP alters the activity of Increased cAMP alters the activity of sodium and chloride transporters producing sodium and chloride transporters producing an ion imbalance that results in fluid an ion imbalance that results in fluid transport into the bowel.transport into the bowel.

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E. coliE. coli toxins toxinsST – is heat stable and binds to ST – is heat stable and binds to

specific receptors to stimulate the specific receptors to stimulate the production of cGMP with the same production of cGMP with the same results as with LT.results as with LT.

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Clinical DiseasesClinical Diseases

SepticaemiaSepticaemia Typically, septicemia caused by gram-negative rods such as Typically, septicemia caused by gram-negative rods such as E. coliE. coli

originates from infections in the urinary or gastrointestinal tract (e.g., originates from infections in the urinary or gastrointestinal tract (e.g., intestinal perforation leading to an intraabdominal infection). intestinal perforation leading to an intraabdominal infection).

The mortality associated with The mortality associated with E. coliE. coli septicemia is high for patients in septicemia is high for patients in whom immunity is compromised or the primary infection is in the whom immunity is compromised or the primary infection is in the abdomen or central nervous system (CNS).abdomen or central nervous system (CNS).

Urinary Tract Infection Urinary Tract Infection Most gram-negative rods that produce UTIs originate in the colon, Most gram-negative rods that produce UTIs originate in the colon,

contaminate the urethra, ascend into the bladder, and may migrate contaminate the urethra, ascend into the bladder, and may migrate to the kidney or prostate. to the kidney or prostate.

Although most strains of Although most strains of E. coliE. coli can produce UTIs, disease is more can produce UTIs, disease is more common with certain specific serogroups. common with certain specific serogroups.

These bacteria are particularly virulent because of their ability to These bacteria are particularly virulent because of their ability to produce adhesins), which bind to cells lining the bladder and upper produce adhesins), which bind to cells lining the bladder and upper urinary tract (preventing the elimination of the bacteria in voided urinary tract (preventing the elimination of the bacteria in voided urine), and hemolysin HlyA, which lyses erythrocytes and other cell urine), and hemolysin HlyA, which lyses erythrocytes and other cell types (leading to cytokine release and stimulation of an inflammatory types (leading to cytokine release and stimulation of an inflammatory response).response).

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Neonatal Meningitis Neonatal Meningitis E. coliE. coli and group B streptococci cause the majority of CNS and group B streptococci cause the majority of CNS

infections in infants younger than 1 month. infections in infants younger than 1 month. Approximately 75% of the Approximately 75% of the E. coliE. coli strains possess the K1 capsular strains possess the K1 capsular

antigen. antigen. This serogroup is also commonly present in the gastrointestinal This serogroup is also commonly present in the gastrointestinal

tracts of pregnant women and newborn infants. tracts of pregnant women and newborn infants. However, the reason this serogroup has a predilection for causing However, the reason this serogroup has a predilection for causing

disease in newborns is not understood. disease in newborns is not understood.

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Gastroenteritis Gastroenteritis The strains of The strains of E. coliE. coli that cause gastroenteritis are subdivided into that cause gastroenteritis are subdivided into

the following five major groups: enteropathogenic (EPEC), the following five major groups: enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic (EHEC), enteroinvasive enterotoxigenic (ETEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), and enteroaggregative (EAEC) (EIEC), and enteroaggregative (EAEC) E. coliE. coli (. (. EPECEPEC

o EnteropathogenicEnteropathogenic E. coliE. coli was the first was the first E. coliE. coli associated associated with diarrheal disease and remains a major cause of infant with diarrheal disease and remains a major cause of infant diarrhea in impoverished countries. diarrhea in impoverished countries.

o Disease is rare in older children and adults, presumably Disease is rare in older children and adults, presumably because they have developed protective immunity. because they have developed protective immunity.

o Although specific O serogroups have been associated with Although specific O serogroups have been associated with outbreaks of EPEC diarrhea in nurseries, the serotyping of outbreaks of EPEC diarrhea in nurseries, the serotyping of the the E. coliE. coli isolated in random or endemic disease is isolated in random or endemic disease is discouraged except in epidemiologic investigations.discouraged except in epidemiologic investigations.

o Disease is characterized by bacterial attachment to epithelial Disease is characterized by bacterial attachment to epithelial cells of the small intestine, with subsequent effacement cells of the small intestine, with subsequent effacement (destruction) of the microvillus (destruction) of the microvillus (attachment/effacement (attachment/effacement [A/E] histopathology).[A/E] histopathology).

o The genes responsible for the "locus of enterocyte The genes responsible for the "locus of enterocyte effacement (LEE)" reside on a pathogenicity island. effacement (LEE)" reside on a pathogenicity island. 3131


o This island of more than 40 genes mediates attachment and This island of more than 40 genes mediates attachment and destruction of the host mucosal surface. destruction of the host mucosal surface.

o EPEC strains form microcolonies on the epithelial cell EPEC strains form microcolonies on the epithelial cell surface with the bacteria attached to the host cells by means surface with the bacteria attached to the host cells by means of cuplike pedestals. of cuplike pedestals.

o Initially a loose attachment mediated by bundle-forming pili Initially a loose attachment mediated by bundle-forming pili occurs, followed by active secretion of proteins by the occurs, followed by active secretion of proteins by the bacterial type III secretion system into the host epithelial cell. bacterial type III secretion system into the host epithelial cell.

o One protein, One protein, translocated intimin receptor (Tir),translocated intimin receptor (Tir), is inserted is inserted into the epithelial cell membrane (this process is mediated by into the epithelial cell membrane (this process is mediated by two other secreted proteins) and functions as a receptor for two other secreted proteins) and functions as a receptor for an outer membrane bacterial adhesin, an outer membrane bacterial adhesin, intimin.intimin.

o The watery diarrhea characteristic of this disease results The watery diarrhea characteristic of this disease results from malabsorption caused by microvilli destruction.from malabsorption caused by microvilli destruction.

ETEC ETEC o Disease caused by Disease caused by enterotoxigenicenterotoxigenic E. coliE. coli is seen most is seen most

commonly in developing countries (an estimated 650 million commonly in developing countries (an estimated 650 million cases per year), although almost 80,000 cases are estimated cases per year), although almost 80,000 cases are estimated to occur annually in travelers from the United States. to occur annually in travelers from the United States.

o Infections are observed in either young children in Infections are observed in either young children in developing countries or travelers to these areas. developing countries or travelers to these areas. 3232

Gastroenteritis Caused by Gastroenteritis Caused by Escherichia coliEscherichia coli Organism Site of Action Disease Pathogenesis

Enteropathogenic E. coli (EPEC) Small intestine Infant diarrhea in underdevelopedcountries; watery diarrhea andvomiting, nonbloody stools

Plasmid-mediated A/Ehistopathology with disruption of normal microvillus structure resulting in malabsorption and diarrhea

Enterotoxigenic E. coli (ETEC) Small intestine Traveler's diarrhea; infant diarrheain developing countries; watery diarrhea, vomiting, cramps, nausea, low-grade fever

Plasmid-mediated, heat-stable and/or heat-labile enterotoxins that stimulate hypersecretion of fluids and electrolytes

Enterohemorrhagic E. coli (EHEC) Large intestine Initial watery diarrhea, followed bygrossly bloody diarrhea (hemorrhagic colitis) with abdominal cramps; little or no fever; may progress to hemolytic uremic syndrome (HUS)

Mediated by cytotoxic Shiga toxins (Stx-1, Stx-2), which disrupt protein synthesis; A/E lesions with destruction of intestinal microvillus resulting in decreased absorption

Enteroinvasive E. coli (EIEC) Large intestine Disease in underdeveloped countries; fever, cramping, watery diarrhea; may progress to dysentery with scant, bloody stools

Plasmid-mediated invasion and destruction of epithelial cells lining colon

Enteroaggregative E. coli (EAEC) Small intestine Infant diarrhea in underdeveloped countries; traveler's diarrhea; persistent watery diarrhea with vomiting, dehydration, and low-grade fever

Plasmid-mediated aggregative adherence of rods ("stacked bricks") with shortening of microvilli, mononuclear infiltration, and hemorrhage; decreased fluid absorption

A/E, Attachment/effacement. 3333


o The inoculum for disease is high, so infections are primarily The inoculum for disease is high, so infections are primarily acquired through consumption of fecally contaminated food acquired through consumption of fecally contaminated food or water. or water.

o Person-to-person spread does not occur. Person-to-person spread does not occur. o ETEC produce two classes of enterotoxins: heat-labile toxins ETEC produce two classes of enterotoxins: heat-labile toxins

(LT-I, LT-II) and heat-stabile toxins (STa and STb). (LT-I, LT-II) and heat-stabile toxins (STa and STb). o Whereas LT-II is not associated with human disease, LT-I is Whereas LT-II is not associated with human disease, LT-I is

functionally and structurally similar to cholera toxin (see functionally and structurally similar to cholera toxin (see Chapter 32) and is associated with human disease. ) and is associated with human disease.

o This toxin consists of one A subunit and five identical B This toxin consists of one A subunit and five identical B subunits. The B subunits bind to the same receptor as subunits. The B subunits bind to the same receptor as cholera toxin (GM1 gangliosides), as well as other surface cholera toxin (GM1 gangliosides), as well as other surface glycoproteins on epithelial cells in the small intestine. glycoproteins on epithelial cells in the small intestine.

o After endocytosis, the A subunit of LT-I translocates across After endocytosis, the A subunit of LT-I translocates across the membrane of the vacuole. the membrane of the vacuole.

o The A subunit has adenosine diphosphate (ADP)-The A subunit has adenosine diphosphate (ADP)-ribosyltransferase activity and interacts with a membrane ribosyltransferase activity and interacts with a membrane protein (Gs) that regulates adenylate cyclase. protein (Gs) that regulates adenylate cyclase.

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o The net effect of this interaction is an increase in cyclic The net effect of this interaction is an increase in cyclic adenosine monophosphate (cAMP) levels, with enhanced adenosine monophosphate (cAMP) levels, with enhanced secretion of chloride and a decreased absorption of sodium secretion of chloride and a decreased absorption of sodium and chloride. and chloride.

o These changes are manifested in a watery diarrhea. These changes are manifested in a watery diarrhea. o Exposure to the toxin also stimulates prostaglandin secretion Exposure to the toxin also stimulates prostaglandin secretion

and production of inflammatory cytokines, resulting in further and production of inflammatory cytokines, resulting in further fluid loss. fluid loss.

o STa, but not STb, is associated with human disease. STa, but not STb, is associated with human disease. o STa is a small, monomeric peptide that binds to the STa is a small, monomeric peptide that binds to the

transmembrane guanylate cyclase receptor, leading to an transmembrane guanylate cyclase receptor, leading to an increase in the level of cyclic guanosine monophosphate and increase in the level of cyclic guanosine monophosphate and subsequent hypersecretion of fluids. subsequent hypersecretion of fluids.

o Genes for LT-I and STa are present on a transferable Genes for LT-I and STa are present on a transferable plasmid, which can also carry the genes for adhesins (CFA/I, plasmid, which can also carry the genes for adhesins (CFA/I, CFA/II, CFA/III). CFA/II, CFA/III).

o The colonization factors are fimbriae that recognize specific The colonization factors are fimbriae that recognize specific host glycoprotein receptors (define the host specificity). Both host glycoprotein receptors (define the host specificity). Both the toxin and colonization factors are required for disease to the toxin and colonization factors are required for disease to develop.develop.

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o Secretory diarrhea caused by ETEC develops after a 1- to 2-Secretory diarrhea caused by ETEC develops after a 1- to 2-day incubation period and persists for an average of 3 to 4 day incubation period and persists for an average of 3 to 4 days. days.

o The symptoms (watery diarrhea and abdominal cramps; The symptoms (watery diarrhea and abdominal cramps; nausea and vomiting are less commonly observed) are nausea and vomiting are less commonly observed) are similar to those of cholera but are milder. similar to those of cholera but are milder.

o Neither histologic changes of the intestinal mucosa nor Neither histologic changes of the intestinal mucosa nor inflammation is observed.inflammation is observed.

o Disease mediated by heat-labile toxin is indistinguishable Disease mediated by heat-labile toxin is indistinguishable from that mediated by heat-stable toxin. from that mediated by heat-stable toxin.

o Toxin production is not associated with specific serogroups, Toxin production is not associated with specific serogroups, so culture combined with immunoasays for the detection of so culture combined with immunoasays for the detection of the heat-labile and heat-stable toxins must be performed.the heat-labile and heat-stable toxins must be performed.

o Commercial assays have been developed that detect toxin in Commercial assays have been developed that detect toxin in cell cultures, but these tests are primarily used in reference cell cultures, but these tests are primarily used in reference labs.labs.

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EHEC EHEC o EnterohemorrhagicEnterohemorrhagic E. coliE. coli strains are the most common strains are the most common

strains producing disease in developed countries. It is strains producing disease in developed countries. It is estimated that these bacteria cause 73,000 infections and 60 estimated that these bacteria cause 73,000 infections and 60 deaths each year in the United States. deaths each year in the United States.

o The ingestion of fewer than 100 bacteria can produce The ingestion of fewer than 100 bacteria can produce disease. The severity of the disease caused by EHEC disease. The severity of the disease caused by EHEC ranges from mild, uncomplicated diarrhea to ranges from mild, uncomplicated diarrhea to hemorrhagic hemorrhagic colitiscolitis with severe abdominal pain, bloody diarrhea, and little with severe abdominal pain, bloody diarrhea, and little or no fever. or no fever.

o More than 50 serogroups of EHEC have been isolated; More than 50 serogroups of EHEC have been isolated; however, the majority that cause human disease in the however, the majority that cause human disease in the United States are believed to be serotype O157 : H7. United States are believed to be serotype O157 : H7.

o Hemolytic uremic syndrome (HUS), a disorder characterized Hemolytic uremic syndrome (HUS), a disorder characterized by acute renal failure, thrombocytopenia, and by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, is a complication in 5% microangiopathic hemolytic anemia, is a complication in 5% to 10% of infected children younger than 10 years. to 10% of infected children younger than 10 years.

o EHEC disease is most common in the warm months, and the EHEC disease is most common in the warm months, and the highest incidence is in children younger than 5 years. highest incidence is in children younger than 5 years.

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o Most cases of disease have been attributed to the Most cases of disease have been attributed to the consumption of undercooked ground beef or other meat consumption of undercooked ground beef or other meat products, water, unpasteurized milk or fruit juices (e.g., cider products, water, unpasteurized milk or fruit juices (e.g., cider made from apples contaminated with feces from cattle), made from apples contaminated with feces from cattle), uncooked vegetables, and fruits. uncooked vegetables, and fruits.

o Initially, a nonbloody diarrhea with abdominal pain develops Initially, a nonbloody diarrhea with abdominal pain develops in patients after 3 to 4 days of incubation. in patients after 3 to 4 days of incubation.

o Vomiting is observed in approximately half the patients. Vomiting is observed in approximately half the patients. o Within 2 days of onset, disease in 30% to 65% of patients Within 2 days of onset, disease in 30% to 65% of patients

progresses to a bloody diarrhea with severe abdominal pain.progresses to a bloody diarrhea with severe abdominal pain.o Complete resolution of symptoms typically occurs after 4 to Complete resolution of symptoms typically occurs after 4 to

10 days in most untreated patients; however, HUS is a 10 days in most untreated patients; however, HUS is a serious complication, particularly in young children. serious complication, particularly in young children.

o Death can occur in 3% to 5% of patients with HUS, and Death can occur in 3% to 5% of patients with HUS, and severe sequelae (e.g., renal impairment, hypertension, CNS severe sequelae (e.g., renal impairment, hypertension, CNS manifestations) can occur in as many as 30% of patients.manifestations) can occur in as many as 30% of patients.

o EHEC strains express a Shiga toxin (i.e., Stx-1, Stx-2, or EHEC strains express a Shiga toxin (i.e., Stx-1, Stx-2, or both), induce A/E lesions on epithelial cells, and possess a both), induce A/E lesions on epithelial cells, and possess a 60-MDa plasmid that carries genes for other virulence 60-MDa plasmid that carries genes for other virulence factors. factors.

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o Stx-1 is essentially identical to the Shiga toxin produced by Stx-1 is essentially identical to the Shiga toxin produced by Shigella dysenteriae;Shigella dysenteriae; Stx-2 has 60% homology. Stx-2 has 60% homology.

o Both toxins are acquired by lysogenic bacteriophages. Both Both toxins are acquired by lysogenic bacteriophages. Both have one A subunit and five B subunits, with the B subunits have one A subunit and five B subunits, with the B subunits binding to a specific glycolipid on the host cell binding to a specific glycolipid on the host cell (globotriaosylceramide, GB3). (globotriaosylceramide, GB3).

o A high concentration of GB3 receptors is found in the A high concentration of GB3 receptors is found in the intestinal villus and renal endothelial cells. intestinal villus and renal endothelial cells.

o After the A subunit is internalized, it is cleaved into two After the A subunit is internalized, it is cleaved into two molecules, and the A1 fragment binds to 28S ribosomal molecules, and the A1 fragment binds to 28S ribosomal ribonucleic acid (rRNA) and disrupts protein synthesis.ribonucleic acid (rRNA) and disrupts protein synthesis.

o Destruction of the intestinal villus results in decreased Destruction of the intestinal villus results in decreased absorption with a relative increase in fluid secretion. absorption with a relative increase in fluid secretion.

o HUS has been preferentially associated with the production HUS has been preferentially associated with the production of Stx-2, which has been shown to destroy glomerular of Stx-2, which has been shown to destroy glomerular endothelial cells. endothelial cells.

o Damage to the endothelial cells leads to platelet activation Damage to the endothelial cells leads to platelet activation and thrombin deposition, which in turn results in decreased and thrombin deposition, which in turn results in decreased glomerular filtration and acute renal failure. glomerular filtration and acute renal failure.

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o The Shiga toxins also stimulate expression of inflammatory The Shiga toxins also stimulate expression of inflammatory cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin [IL]-6), which among other effects enhance expression of [IL]-6), which among other effects enhance expression of GB3.GB3.

o Two approaches have been used to detect EHEC: culture Two approaches have been used to detect EHEC: culture and toxin detection. In contrast with most and toxin detection. In contrast with most E. coli,E. coli, many O157 many O157 strains do not ferment sorbitol. strains do not ferment sorbitol.

o Sorbitol-containing MacConkey agar (S-MAC) has been Sorbitol-containing MacConkey agar (S-MAC) has been used to screen stool specimens for sorbitol-negative used to screen stool specimens for sorbitol-negative (colorless), gram-negative bacteria that are then confirmed (colorless), gram-negative bacteria that are then confirmed by serogrouping and biochemical tests to be O157 by serogrouping and biochemical tests to be O157 E. coli.E. coli.

o The limitations to this approach are some strains of O157 The limitations to this approach are some strains of O157 and many other EHEC serotypes ferment sorbitol and toxin and many other EHEC serotypes ferment sorbitol and toxin production is not assessed. production is not assessed.

o The preferred method to detect EHEC is to culture stool The preferred method to detect EHEC is to culture stool specimens on nonselective MacConkey agar and then to specimens on nonselective MacConkey agar and then to assay isolated colonies for toxin production by commercially assay isolated colonies for toxin production by commercially available immunoassays. available immunoassays.

o Unfortunately, the direct detection of toxin in stool samples is Unfortunately, the direct detection of toxin in stool samples is currently too insensitive to recommend. currently too insensitive to recommend.

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EIEC EIEC o EnteroinvasiveEnteroinvasive E. coliE. coli strains are rare in the United States strains are rare in the United States

and uncommon in developing countries. and uncommon in developing countries. o Pathogenic strains are primarily associated with a few Pathogenic strains are primarily associated with a few

restricted O serotypes: O124, O143, and O164. restricted O serotypes: O124, O143, and O164. o The strains are closely related by phenotypic and pathogenic The strains are closely related by phenotypic and pathogenic

properties to properties to Shigella.Shigella. o The bacteria are able to invade and destroy the colonic The bacteria are able to invade and destroy the colonic

epithelium, producing a disease characterized initially by epithelium, producing a disease characterized initially by watery diarrhea. watery diarrhea.

o A minority of patients progress to the dysenteric form of A minority of patients progress to the dysenteric form of disease, consisting of fever, abdominal cramps, and blood disease, consisting of fever, abdominal cramps, and blood and leukocytes in stool specimens. and leukocytes in stool specimens.

o A series of bacterial genes carried on a plasmid mediate A series of bacterial genes carried on a plasmid mediate invasion invasion ((pInvpInv genes)genes) into the colonic epithelium. into the colonic epithelium.

o The bacteria then lyse the phagocytic vacuole and replicate The bacteria then lyse the phagocytic vacuole and replicate in the cell cytoplasm. in the cell cytoplasm.

o Movement within the cytoplasm and into adjacent epithelial Movement within the cytoplasm and into adjacent epithelial cells is regulated by formation of actin tails (similar to that cells is regulated by formation of actin tails (similar to that observed with observed with ListeriaListeria). ).

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o This process of epithelial cell destruction with inflammatory This process of epithelial cell destruction with inflammatory infiltration can progress to colonic ulceration. infiltration can progress to colonic ulceration.

o Detection of EIEC strains is restricted to research Detection of EIEC strains is restricted to research laboratories. laboratories.

o Although immunoassays and nucleic acid based assays Although immunoassays and nucleic acid based assays have been developed for detecting invasion-related factors, have been developed for detecting invasion-related factors, the usefulness of these assays is limited by the fact the the usefulness of these assays is limited by the fact the genes for these virulence factors reside on a large plasmid genes for these virulence factors reside on a large plasmid that is rapidly lost in vitro. that is rapidly lost in vitro.

EAEC EAEC o EnteroaggregativeEnteroaggregative E. coliE. coli strains have been implicated as a strains have been implicated as a

cause of persistent, watery diarrhea with dehydration in cause of persistent, watery diarrhea with dehydration in infants in developing countries and in travelers to these infants in developing countries and in travelers to these countries. countries.

o The persistence of these bacteria is associated with chronic The persistence of these bacteria is associated with chronic diarrhea and growth retardation in children. diarrhea and growth retardation in children.

o The bacteria are characterized by their autoagglutination in a The bacteria are characterized by their autoagglutination in a "stacked-brick" arrangement. "stacked-brick" arrangement.

o This process is mediated by bundle-forming fimbriae This process is mediated by bundle-forming fimbriae (aggregative adherence fimbriae I and II), which are carried (aggregative adherence fimbriae I and II), which are carried on a plasmid. on a plasmid. 4242


o EAEC stimulate secretion of mucus, which traps the bacteria EAEC stimulate secretion of mucus, which traps the bacteria in a biofilm overlying the epithelium of the small intestine. in a biofilm overlying the epithelium of the small intestine. Shortening of the microvilli, mononuclear infiltration, and Shortening of the microvilli, mononuclear infiltration, and hemorrhage are then observed. hemorrhage are then observed.

o A cytotoxin has not been demonstrated but is likely to be A cytotoxin has not been demonstrated but is likely to be present. Diagnosis of these infections is primarily restricted present. Diagnosis of these infections is primarily restricted to research laboratories.to research laboratories.

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Treatment of Treatment of E.coliE.coli related diarrhoea related diarrhoea

1st Line1st Line

NitrofurantoinNitrofurantoin

Nalidixic acidNalidixic acid

NorfloxacinNorfloxacin

Ampicillin Ampicillin

CotrimoxazoleCotrimoxazole

2nd line2nd line

Ciprofloxacin/Ceftriaxone/CefuroximeCiprofloxacin/Ceftriaxone/Cefuroxime

GentamicinGentamicin

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Summary of Summary of Escherichia coliEscherichia coli

Physiology and StructurePhysiology and Structure Gram-negative, facultative anaerobic rods Gram-negative, facultative anaerobic rods Fermenter; oxidase negative Fermenter; oxidase negative Outer membrane makes the organisms susceptible to drying Outer membrane makes the organisms susceptible to drying Lipopolysaccharide consists of outer somatic O polysaccharide, core Lipopolysaccharide consists of outer somatic O polysaccharide, core

polysaccharide (common antigen), and lipid A (endotoxin) polysaccharide (common antigen), and lipid A (endotoxin)

VirulenceVirulence Refer to Refer to Boxes 31-2 and and 31-3 Endotoxin Endotoxin Permeability barrier of outer membrane Permeability barrier of outer membrane Adhesins (e.g., colonization factor antigen, Dr adhesins) Adhesins (e.g., colonization factor antigen, Dr adhesins) Exotoxins (e.g., heat-stabile and heat-labile enterotoxins, Shiga Exotoxins (e.g., heat-stabile and heat-labile enterotoxins, Shiga

toxins) toxins) Invasive capacity Invasive capacity

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EpidemiologyEpidemiology Most common aerobic, gram-negative rods in the gastrointestinal Most common aerobic, gram-negative rods in the gastrointestinal

tract tract Most infections are endogenous (patient's normal microbial flora) Most infections are endogenous (patient's normal microbial flora) Strains causing gastroenteritis are generally acquired exogenously Strains causing gastroenteritis are generally acquired exogenously

DiseasesDiseases Bacteremia (most commonly isolated gram-negative rod in the Bacteremia (most commonly isolated gram-negative rod in the

United States) United States) Urinary tract infection (most common cause of bacterial UTIs); Urinary tract infection (most common cause of bacterial UTIs);

limited to bladder (cystitis) or can spread to kidneys (pyelonephritis) limited to bladder (cystitis) or can spread to kidneys (pyelonephritis) or prostate (prostatitis) or prostate (prostatitis)

At least five different pathogenic groups cause gastroenteritis At least five different pathogenic groups cause gastroenteritis (EPEC, ETEC, EHEC, EIEC, EAEC); most cause diseases in (EPEC, ETEC, EHEC, EIEC, EAEC); most cause diseases in developing countries, although EHEC is an important cause of developing countries, although EHEC is an important cause of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in the United States the United States

Neonatal meningitis (usually with strains carrying the K1 capsular Neonatal meningitis (usually with strains carrying the K1 capsular antigen) antigen)

Intraabdominal infections (associated with intestinal perforation) Intraabdominal infections (associated with intestinal perforation)

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DiagnosisDiagnosis Organisms grow rapidly on most culture media Organisms grow rapidly on most culture media Enteric pathogens with the exception of EHEC are detected only in Enteric pathogens with the exception of EHEC are detected only in

reference or research laboratories reference or research laboratories Treatment, Prevention, and ControlTreatment, Prevention, and Control

Enteric pathogens are treated symptomatically unless disseminated Enteric pathogens are treated symptomatically unless disseminated disease occurs disease occurs

Antibiotic therapy is guided by in vitro susceptibility tests Antibiotic therapy is guided by in vitro susceptibility tests Appropriate infection-control practices are used to reduce the risk of Appropriate infection-control practices are used to reduce the risk of

nosocomial infections (e.g., restricting use of antibiotics, avoiding nosocomial infections (e.g., restricting use of antibiotics, avoiding unnecessary use of urinary tract catheters) unnecessary use of urinary tract catheters)

Maintenance of high hygienic standards to reduce the risk of Maintenance of high hygienic standards to reduce the risk of exposure to gastroenteritis strains exposure to gastroenteritis strains

Proper cooking of beef products to reduce risk of EHEC infectionsProper cooking of beef products to reduce risk of EHEC infections

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e coli lectur revised alpana

Technology

family enterobacteriaceae

enterobacteriaceae family

common members

important gramnegative

core polysaccharide

macconkey agar media

physiology structure

organisms colonies