Transcript E coli
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 Morphology 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 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 and travelers. in developing countries, 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 Morphology 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 infec 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 identification by biochemical or serological tests on pure growth from single colony culture 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 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 production • Degradation of tryptophan by tryptophanase to produce indole • Red colour develops after adding a solution containing pdimethylaminobenzaldehyde (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 A K K BUTT - A A A A 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: 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. Methyl Red: left – and right + VP: 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 References: www.slideshare.net