E coli, Klebsiella and Enterobacter Prof M.I.N. Matee School of Medicine MUCHS

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.

Morphology and culture characteristics Gram negative rods Non-spore former, non-caspulated and motile Culture characteristics N.agar – circular, convex, small colonies MacConkey medium– rose pink Eosin Methylene blue – Metallic sheen colonies

Biochemical reactions IMViC: ++-- TSI: Acid butt/acid slant with gas production

Antigenic structure Has O, H, and K antigens. K1 has a strong association with virulence, 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 antigen Flagella /H antigen Sometimes external to O antigen but not always Can be polysaccharides or protein Flagella /H antigen 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. coli toxins Virulence factors Toxins Enterotoxins – produced by enterotoxigenic strains of E. coli (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two types of enterotoxin: LT – is heat labile and binds to specific Gm1 gangliosides on the epithelial cells of the small intestine where it stimulates production of cAMP. Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.

E. coli toxins ST – is heat stable and binds to specific receptors to stimulate the production of cGMP with the same results as with LT.

Urinary tract infections E coli is the most common cause of UTI Account for 90% of cases in young women Symptoms Frequency, dysuria, hematuria, pyuria UTI may lead to bacteremia and sepsis Nephropathogenic E coli produces hemolysin

E coli associated diarrhoeal diseases

Enterotoxigenic E. coli Heat labile toxin like choleragen Adenyl cyclase activated cyclic AMP secretion water/ions Heat stable toxin Guanylate cyclase activated cyclic GMP uptake water/ions

A.Enterotoxigenic E coli ( ETEC) Common cause for travelers diarrhoea, and watery diarrhoea in children. Colonisation factor facilitates the attachment to the intestinal epithelium. Some ETEC produces heat labile exotoxin LT and heat stable or either of the toxins

B.Enteropathogenic E coli (EPEC) Important cause of diarrhoea in infants of developing countries. Adhere to mucosal cells in small bowel,loss of microvilli, NONINVASIVE enter to cell body. result in watery diarrhoea. Self limiting ,can be chronic. Normally do not produce toxins

C. Enterohemorrhagic E coli ( EHEC) Produce verotoxin which has similarities to Shiga toxin Associated with hemorrhagic colitis, severe form of diarrhoea. Hemolytic uremic syndrome Disease can be prevented by thorough cooking.

Escherichia coli O157:H7 This strain produces a powerful toxin, verotoxin, which can cause severe illness and death. E coli O157:H7 does not ferment sorbitol and is negative on sorbitol MacConkey agar Other Shiga toxin producing serotypes (e.g. O111 and O26) are also in the family of enterohemorrhagic E. coli and can cause similar disease. Hemorrhagic colitis – preventable by thorough cooking of meat

Hemorrhagic colitis(HC) or bloody diarrhea and abdominal cramps Lasts for 5-10 days usually Infection may proceed to Hemolytic Uremic Syndrome (HUS), a severe cytopathic attack on the kidneys requiring intensive care and dialysis. Red blood cells are destroyed requiring blood tranfusion

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 intestine multiply inside the cells and destruction /inflammation/ulceration diarrhoea with blood EIEC are nonlactose fermenter,or late lactose fermenter and non motile.

E. Enteroaggregative E coli (EAEC) Produce acute/chronic diarrhoea in persons in developing countries. Sepsis When normal host defense is poor, sepsis can happen. Common in new born babies whose IgM level is low.

Treatment of E.coli related diarrhoea 1st Line Nitrofurantoin Nalidixic acid Norfloxacin Ampicillin Cotrimoxazole 2nd line Ciprofloxacin/Ceftriaxone/Cefuroxime Gentamicin

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.

Pneumonia 25% of gram -ve pneumonia with 50% mortality Usually broncho pneumonia High level of resistance to Ampicillin /Cotrimoxazole

Klebsiella spp

Morphology and culture characteristics Gram negative rods Non-spore former, caspulated and non-motile Culture characteristics N.agar – mucoid, circular, convex, small colonies MacConkey – mucoid, rose pink

Biochemical reactions IMViC: --++ TSI: Acid butt/acid slant with gas production

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

Klebsiella K pneumoniae Present in respiratory tract and feces of about 5% of normal individuals. Can cause bacterial pneumonia (3%). Produce extensive hemorrhagic necrotising consolidation of lungs.

Enterobacter aerogenus Capsulated Free living in the intestine Cause UTI and sepsis. Motile Citrate + Ornithine decarboxylase Produce gas from glucose Ferments lactose VP + (like Klebsiella)

Laboratory identification tests

Diagnosis of Bacterial Infection microscopy unstained or stained with e.g. Gram stain Stain Decolorise Counterstain culture identification by biochemical or serological tests on pure growth from single colony on plates or in broth sensitivities by disc diffusion methods, breakpoints or MICs Serodiagnosis DNA technologies

Gram negative rods

Lactose fermentor on MCA

Lactose positive Klebsiella sp

E. coli

EMB is both selective and differential

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

Triple Sugar Iron Agar (TSI): Purpose: To differentiate bacteria based on their ability to ferment glucose, lactose and/or sucrose, and to reduce sulfur to hydrogen sulfide.

INTERPRETATION OF TUBES ABOVE TUBE 1 (UNINOCULATED) TUBE 2 TUBE 3 TUBE 4 TUBE 5 SLANT - A K BUTT HYDROGEN SULFIDE + GAS A=Acidic K=Alkaline

ONPG test Determines presence of -galactosidase Utilizes o-nitrophenyl-d--galactosidase Differentiates late lactose fermenting organisms Positive Negative

Methyl Red This is a qualitative test of the acidity produced by the growth of a bacterium in phosphate –buffered glucose peptone water E coli produces a pH of about 5, and hence a red colour is produced after addition of methyl red. Enterobacter aerogenes the pH never drops so low, it appears yellow after addition of methyl red.

Voges-Proskauer This test for production of acetylmethylcarbinol (AMC) from carbohydrates by bacteria in glucose phosphate peptone water Red colour is produced after addition of alpha naphthol and KOH Klebsiella + E coli -

MR/VP continued Reading Results: VP: left + and right – MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production) VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change. VP: left + and right – Methyl Red: left – and right +

IMViC test IMViC test is a group of four biochemical tests collectively used for primary identification of enteric bacteria Indole Methyl red Voges proskuer Citrate utilization test

Indole (IMViC tests) • E. coli (pink/red) + • E. coli (left side) – • Kovac’s reagent detects if tryptophan has been hydrolyzed to indol/tryptophanase

Methyl Red (MR) (IMViC tests) Enterobacter aerogenes (left) – E. coli (bright red) + Reagent: Methyl red indicator identifies pH change due to mixed acid fermentation

Voges – Proskauer (VP) (IMViC tests) Enterobacter aerogenes +(left) E. coli – (right) Red colour is produced after addition of alpha naphthol and KOH Klebsiella + E coli -

Citrate 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 products E. coli (left green) – • Enterobacter aerogenes (right royal blue) + Positive Negative

Treatment of enteric bacteria No specific treatment is available A number of antibiotics could be used Marked antibiotic resistance due to plasmids May involve surgical correction Treatment of shock Environmental sanitation

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