E coli, KlebsiellaE coli, Klebsiella and and EnterobacterEnterobacter

Prof M.I.N. MateeProf M.I.N. Matee

School of MedicineSchool of Medicine

MUCHSMUCHS

Escherichia coliEscherichia coli Normal inhabitant of the G.I. tractNormal inhabitant of the G.I. tract Some strains cause various forms of Some strains cause various forms of

gastroenteritis.gastroenteritis.

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

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

Biochemical reactionsBiochemical reactions

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

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

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.

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 IgM

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.

Colicines/Bacteriocines

• Produced by many Gram -ves

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

E. coliE. coli toxins toxins

Virulence factorsVirulence factorsToxinsToxins

Enterotoxins – produced by enterotoxigenic strains of Enterotoxins – produced by enterotoxigenic strains of E. coliE. coli (ETEC). Causes a movement of water and ions from the (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two tissues to the bowel 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 gangliosides on the epithelial cells of the small intestine where it on the epithelial cells of the small intestine where it stimulates production of cAMP. stimulates production of cAMP.

Increased cAMP alters the activity of sodium and chloride Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transporters producing an ion imbalance that results in fluid transport into the bowel.transport into the bowel.

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.

Urinary tract infectionsUrinary tract infections

E coliE coli is the most common cause of UTI is the most common cause of UTI Account for 90% of cases in young womenAccount for 90% of cases in young women

SymptomsSymptoms Frequency, dysuria, hematuria, pyuriaFrequency, dysuria, hematuria, pyuria

UTI may lead to bacteremia and sepsisUTI may lead to bacteremia and sepsis

Nephropathogenic E coli produces Nephropathogenic E coli produces hemolysinhemolysin

E coli associated diarrhoeal diseasesE coli associated diarrhoeal diseases

Enterotoxigenic Enterotoxigenic E. coliE. coli Heat labile toxinHeat labile toxin

like choleragenlike choleragen Adenyl cyclase activated Adenyl cyclase activated cyclic AMP cyclic AMP secretion water/ionssecretion water/ions

Heat stable toxinHeat stable toxin Guanylate cyclase activated Guanylate cyclase activated cyclic GMPcyclic GMP uptake water/ionsuptake water/ions

A.Enterotoxigenic E coli (A.Enterotoxigenic E coli ( ETEC) ETEC) Common cause for Common cause for travelerstravelers diarrhoea diarrhoea, and , and

watery diarrhoea in watery diarrhoea in children.children. Colonisation factor facilitates the attachment to Colonisation factor facilitates the attachment to the intestinal epithelium.the intestinal epithelium. Some ETEC produces heat labile exotoxin LT and Some ETEC produces heat labile exotoxin LT and

heat stable or either of the toxinsheat stable or either of the toxins

B.Enteropathogenic E coli B.Enteropathogenic E coli (EPEC)(EPEC) Important cause of diarrhoea inImportant cause of diarrhoea in infantsinfants of of

developing countries.developing countries. Adhere to mucosal cells in small bowel,loss Adhere to mucosal cells in small bowel,loss of of

microvilli, NONINVASIVEmicrovilli, NONINVASIVE enter to cell body. enter to cell body. result in watery result in watery

diarrhoeadiarrhoea.. Self limiting ,can be chronic.Self limiting ,can be chronic.

Normally do not produce toxinsNormally do not produce toxins

C. Enterohemorrhagic E coli (C. Enterohemorrhagic E coli ( EHEC) EHEC) Produce verotoxin which has similarities Produce verotoxin which has similarities

to Shiga toxinto Shiga toxin Associated with hemorrhagic Associated with hemorrhagic

colitis, severe form of diarrhoea.colitis, severe form of diarrhoea.

Hemolytic uremic syndrome Disease can Hemolytic uremic syndrome Disease can be prevented by thorough cookingbe prevented by thorough cooking..

Escherichia coli O157:H7Escherichia coli O157:H7

This strain produces a powerful toxin, verotoxin, which can This strain produces a powerful toxin, verotoxin, which can cause severe illness and death.cause severe illness and death.

E coli O157:H7 does not ferment sorbitol and is negative on E coli O157:H7 does not ferment sorbitol and is negative on sorbitol MacConkey agarsorbitol MacConkey agar

Other Shiga toxin producing serotypes (e.g. O111 and O26) Other Shiga toxin producing serotypes (e.g. O111 and O26) are also in the family of enterohemorrhagic are also in the family of enterohemorrhagic E. coliE. coli and can and can cause similar disease. cause similar disease.

Hemorrhagic colitis – preventable by thorough cooking of meatHemorrhagic colitis – preventable by thorough cooking of meat

Hemorrhagic colitis(HC) or bloody diarrhea and Hemorrhagic colitis(HC) or bloody diarrhea and abdominal crampsabdominal crampsLasts for 5-10 days usuallyLasts for 5-10 days usually

Infection may proceed to Hemolytic Uremic Infection may proceed to Hemolytic Uremic Syndrome (HUS), a severe cytopathic attack on Syndrome (HUS), a severe cytopathic attack on the kidneys requiring intensive care and dialysis. the kidneys requiring intensive care and dialysis.

Red blood cells are destroyed requiring blood Red blood cells are destroyed requiring blood tranfusiontranfusion

D.Enteroinvasive E coli (EIEC) • Produces disease similar to shigellosis. •In adults this has been isolated with Shigella

•Commonly affect children in developing countries, and travelers. Disease is due to invasion into mucosal cells of the intestinemultiply inside the cells and destruction /inflammation/ulceration diarrhoea with blood

EIEC are nonlactose fermenter,or late lactose fermenter and non motile.

E. E. Enteroaggregative E coliEnteroaggregative E coli (EAEC) (EAEC)

Produce acute/chronic diarrhoea in persons in Produce acute/chronic diarrhoea in persons in developing countries.developing countries.

Sepsis When normal host defense is poor, Sepsis When normal host defense is poor, sepsis can happen. Common in new born babies sepsis can happen. Common in new born babies whose IgM level is low.whose IgM level is low.

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

Meningitis

• E coli and Gp.B Strept. are the leading causes for meningitis in infants. •K1 antigen is responsible for meningitis K1 cross reacts with the Gp.B capsular polysaccharides of N meningitides.

PneumoniaPneumonia

25% of gram -ve pneumonia with 50% 25% of gram -ve pneumonia with 50% mortalitymortality

Usually broncho pneumoniaUsually broncho pneumonia

High level of resistance to Ampicillin High level of resistance to Ampicillin /Cotrimoxazole/Cotrimoxazole

Klebsiella sppKlebsiella spp

Morphology and culture Morphology and culture characteristicscharacteristics

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

Culture characteristicsCulture characteristics N.agarN.agar – mucoid, circular, convex, small – mucoid, circular, convex, small

coloniescolonies MacConkeyMacConkey – mucoid, rose pink – mucoid, rose pink

Biochemical reactionsBiochemical reactions

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

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

K pneumoniae and K oxytoca Hospital acquired infections due to K rhinoscleromatis produces rhinoscleroma,condition with destructive granuloma of the nose and pharynx.

K ozenae – progressive atrophy of nasal mucous membrane

KlebsiellaKlebsiella

K pneumoniaeK pneumoniae

Present in respiratory tract and feces of Present in respiratory tract and feces of about 5% of about 5% of

normal individuals.normal individuals.

Can cause bacterial pneumonia (3%).Can cause bacterial pneumonia (3%).

Produce extensive hemorrhagic Produce extensive hemorrhagic necrotising consolidation of lungs.necrotising consolidation of lungs.

Enterobacter aerogenusEnterobacter aerogenus CapsulatedCapsulated Free living in the intestineFree living in the intestine Cause UTI and sepsis.Cause UTI and sepsis.

MotileMotile Citrate +Citrate + Ornithine decarboxylaseOrnithine decarboxylase Produce gas from glucoseProduce gas from glucose Ferments lactoseFerments lactose VP + (like Klebsiella)VP + (like Klebsiella)

Laboratory identification Laboratory identification teststests

Diagnosis of Bacterial Diagnosis of Bacterial InfectionInfection

culture

on plates or in broth

identification by biochemical or serological tests on pure growth

from single colony

microscopy

Decolorise CounterstainStain

unstained or stained with e.g. Gram stain

sensitivities

Serodiagnosis DNA technologies

by disc diffusion methods,

breakpoints or MICs

Gram negative rodsGram negative rods

Lactose fermentor on MCALactose fermentor on MCA

Lactose positive Lactose positive KlebsiellaKlebsiella sp sp

E. coliE. 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.

Indole productionIndole production

• Degradation of tryptophan by tryptophanase to produce indole• Red colour develops after adding a solution containing p-dimethylaminobenzaldehyde (Kovacs or Erlichs)

NegativePositive

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.

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

ONPG testONPG test

• Determines presence of -galactosidase• Utilizes o-nitrophenyl-d--galactosidase• Differentiates late lactose fermenting organisms

Positive Negative

Methyl RedMethyl Red

This is a qualitative test of the acidity This is a qualitative test of the acidity produced by the growth of a bacterium in produced by the growth of a bacterium in phosphate –buffered glucose peptone waterphosphate –buffered glucose peptone water

E coliE coli produces a pH of about 5, and hence a red produces a pH of about 5, and hence a red colour is produced after addition of methyl red.colour is produced after addition of methyl red.

Enterobacter aerogenesEnterobacter aerogenes the pH never drops so the pH never drops so low, it appears yellow after addition of methyl red.low, it appears yellow after addition of methyl red.

Voges-ProskauerVoges-Proskauer

This test for production of This test for production of acetylmethylcarbinol acetylmethylcarbinol (AMC)(AMC) from from carbohydrates by bacteria in glucose carbohydrates by bacteria in glucose phosphate peptone waterphosphate peptone water

Red colour is produced after addition of Red colour is produced after addition of alpha naphthol and KOHalpha naphthol and KOH

Klebsiella Klebsiella ++E coliE coli - -

MR/VP continuedMR/VP continuedReading Results: Reading Results:

MR— a + result is red (indicating pH below 6) and a – result is yellow MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production)(indicating no acid production)

VP—A + result is red after VP reagents are added (indicating the VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change.presence of acetoin) and a – result is no color change.

Methyl Red: left – and right + VP: left + and right –

IMViC testIMViC test

IMViC test is a group of four biochemical IMViC test is a group of four biochemical tests collectively used for primary tests collectively used for primary identification of enteric bacteriaidentification of enteric bacteria IndoleIndole Methyl redMethyl red Voges proskuerVoges proskuer Citrate utilization testCitrate utilization test

Indole (IMViC tests)Indole (IMViC tests)

• • E. coli E. coli (pink/red) +(pink/red) +

• • E. coli E. coli (left side) –(left side) –

• • Kovac’s reagentKovac’s reagent

detects if tryptophandetects if tryptophan

has been hydrolyzedhas been hydrolyzed

to indol/tryptophanaseto indol/tryptophanase

Methyl Red (MR) (IMViC tests)Methyl Red (MR) (IMViC tests)

EnterobacterEnterobacteraerogenes aerogenes (left) –(left) –

E. coli E. coli (bright red) +(bright red) +

Reagent: Methyl redReagent: Methyl red

indicator identifies pHindicator identifies pHchange due to mixedchange due to mixedacid fermentationacid fermentation

Voges – Proskauer (VP)Voges – Proskauer (VP)(IMViC tests)(IMViC tests)

Enterobacter aerogenes Enterobacter aerogenes +(left)+(left)

E. coli E. coli – (right)– (right)

Red colour is produced Red colour is produced after addition of alpha after addition of alpha naphthol and KOHnaphthol and KOH

Klebsiella Klebsiella ++E coliE coli - -

Citrate utilizationCitrate utilization

• Ability to use sodium citrate as sole source of carbon

• Medium has sodium citrate, ammonium salt, bromothymol blue

• Positive test - deep blue colour in 24-48hrs

• Indicates citrate utilization

• Production of alkaline productsE. coli (left green) –

• Enterobacter aerogenes(right royal blue) +

Positive Negative

Treatment of enteric bacteriaTreatment of enteric bacteria

No specific treatment is availableNo specific treatment is available

A number of antibiotics could be usedA number of antibiotics could be used

Marked antibiotic resistance due to Marked antibiotic resistance due to plasmidsplasmids May involve surgical correctionMay involve surgical correction Treatment of shock Treatment of shock Environmental sanitationEnvironmental sanitation