E coli, Klebsiella  and  Enterobacter Prof M.I.N. Matee School of Medicine MUCHS
Escherichia coli <ul><ul><li>Normal inhabitant of the G.I. tract </li></ul></ul><ul><ul><li>Some strains cause various for...
Morphology and culture characteristics <ul><li>Morphology </li></ul><ul><ul><li>Gram negative rods </li></ul></ul><ul><ul>...
<ul><li>Biochemical reactions </li></ul><ul><li>IMViC: ++-- </li></ul><ul><li>TSI: Acid butt/acid slant with gas productio...
Antigenic structure <ul><li>Has O, H, and K antigens.  </li></ul><ul><li>K1 has a strong association with virulence, parti...
<ul><li>Antigenic structure . </li></ul><ul><li>Complex </li></ul><ul><li>Lipopolisaccharides/ Somatic or O antigen </li><...
<ul><li>Capsular/K antigen </li></ul><ul><ul><li>Sometimes external to O antigen but not always </li></ul></ul><ul><ul><li...
<ul><li>Colicines/Bacteriocines </li></ul><ul><li>Produced by  many  Gram -ves </li></ul><ul><li>Virus like bactericidal s...
E. coli  toxins <ul><ul><li>Virulence factors </li></ul></ul><ul><ul><ul><li>Toxins </li></ul></ul></ul><ul><ul><ul><ul><l...
E. coli  toxins <ul><ul><ul><ul><ul><li>ST – is heat stable and binds to specific receptors to stimulate the production of...
Urinary tract infections <ul><li>E coli  is the most common cause of UTI </li></ul><ul><ul><li>Account for 90% of cases in...
<ul><li>E coli associated diarrhoeal diseases </li></ul>
Enterotoxigenic  E. coli   <ul><li>Heat labile toxin </li></ul><ul><ul><ul><ul><li>like choleragen </li></ul></ul></ul></u...
<ul><li>A.Enterotoxigenic E coli (  ETEC) </li></ul><ul><ul><li>Common cause for  travelers  diarrhoea , and  watery diarr...
<ul><li>B.Enteropathogenic E coli  (EPEC) </li></ul><ul><ul><li>Important cause of diarrhoea in   infants   of  </li></ul>...
<ul><li>C. Enterohemorrhagic E coli (  EHEC) </li></ul><ul><ul><li>Produce verotoxin which has similarities to Shiga toxin...
Escherichia coli O157:H7 <ul><li>This strain produces a powerful toxin, verotoxin, which can cause severe illness and deat...
<ul><li>Hemorrhagic colitis(HC) or bloody diarrhea and abdominal cramps </li></ul><ul><li>Lasts for 5-10 days usually </li...
<ul><li>D. Enteroinvasive E coli  (EIEC) </li></ul><ul><li>Produces disease  similar to shigellosis . </li></ul><ul><li>In...
<ul><li>E.  Enteroaggregative E coli  (EAEC) </li></ul><ul><li>Produce acute/chronic diarrhoea in persons in developing  c...
Treatment of  E.coli  related diarrhoea   <ul><li>1st Line </li></ul><ul><li>Nitrofurantoin </li></ul><ul><li>Nalidixic ac...
<ul><li>Meningitis </li></ul><ul><li>E coli  and  Gp.B Strept . are the leading causes for  </li></ul><ul><li>meningitis i...
Pneumonia <ul><li>25% of gram -ve pneumonia with 50% mortality </li></ul><ul><li>Usually broncho pneumonia </li></ul><ul><...
Klebsiella spp
Morphology and culture characteristics <ul><li>Morphology </li></ul><ul><ul><li>Gram negative rods </li></ul></ul><ul><ul>...
<ul><li>Biochemical reactions </li></ul><ul><li>IMViC: --++ </li></ul><ul><li>TSI: Acid butt/acid slant with gas productio...
K pneumoniae   and  K oxytoca   Hospital acquired infections due to  K rhinoscleromatis  produces rhinoscleroma, condition...
<ul><li>Klebsiella </li></ul><ul><li>K pneumoniae </li></ul><ul><li>Present in respiratory tract and feces of about 5% of ...
<ul><li>Enterobacter aerogenus </li></ul><ul><li>Capsulated </li></ul><ul><li>Free living in the intestine </li></ul><ul><...
<ul><li>Laboratory identification tests </li></ul>
Diagnosis of Bacterial Infection culture on plates or in broth  identification by biochemical or serological tests on pure...
Gram negative rods
Lactose fermentor on MCA
Lactose positive  Klebsiella  sp
E. coli
 
Indole production <ul><li>Degradation of tryptophan by tryptophanase to produce indole </li></ul><ul><li>Red colour develo...
<ul><li>Triple Sugar Iron Agar (TSI): </li></ul><ul><ul><li>Purpose:  To differentiate bacteria based on their ability to ...
A=Acidic K=Alkaline - - + - - GAS  + + - - - HYDROGEN SULFIDE  A A A A - BUTT  K K A A - SLANT  TUBE 5  TUBE 4  TUBE 3  TU...
ONPG test <ul><li>Determines presence of   -galactosidase </li></ul><ul><li>Utilizes o-nitrophenyl-d-  -galactosidase </...
Methyl Red <ul><li>This is a qualitative test of the acidity produced by the growth of a bacterium in phosphate –buffered ...
Voges-Proskauer <ul><li>This test for production of acetylmethylcarbinol  (AMC)  from carbohydrates by bacteria in glucose...
MR/VP continued <ul><li>Reading Results:  </li></ul><ul><ul><li>MR— a + result is red (indicating pH below 6) and a – resu...
IMViC test <ul><li>IMViC test is a group of four biochemical tests collectively used for primary identification of enteric...
Indole (IMViC tests) <ul><li>•  E. coli  (pink/red) + </li></ul><ul><li>•  E. coli  (left side) – </li></ul><ul><li>•  Kov...
Methyl Red (MR) (IMViC tests) <ul><li>Enterobacter </li></ul><ul><li>aerogenes  (left) – </li></ul><ul><li>E. coli  (brigh...
Voges – Proskauer (VP) (IMViC tests) <ul><li>Enterobacter aerogenes  +(left) </li></ul><ul><li>E. coli  – (right) </li></u...
Citrate utilization <ul><li>Ability to use sodium citrate as sole source of carbon </li></ul><ul><li>Medium has sodium cit...
Treatment of enteric bacteria <ul><li>No specific treatment is available </li></ul><ul><li>A number of antibiotics could b...
 
 
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  • EMB is both selective and differential
  • E coli, klebsiella, enterobacter lecture notes

    1. 1. E coli, Klebsiella and Enterobacter Prof M.I.N. Matee School of Medicine MUCHS
    2. 2. Escherichia coli <ul><ul><li>Normal inhabitant of the G.I. tract </li></ul></ul><ul><ul><li>Some strains cause various forms of gastroenteritis. </li></ul></ul><ul><ul><li>Is a major cause of urinary tract infection and neonatal meningitis and septicemia. </li></ul></ul>
    3. 3. Morphology and culture characteristics <ul><li>Morphology </li></ul><ul><ul><li>Gram negative rods </li></ul></ul><ul><ul><li>Non-spore former, non-caspulated and motile </li></ul></ul><ul><li>Culture characteristics </li></ul><ul><ul><li>N.agar – circular, convex, small colonies </li></ul></ul><ul><ul><li>MacConkey medium – rose pink </li></ul></ul><ul><ul><li>Eosin Methylene blue – Metallic sheen colonies </li></ul></ul>
    4. 4. <ul><li>Biochemical reactions </li></ul><ul><li>IMViC: ++-- </li></ul><ul><li>TSI: Acid butt/acid slant with gas production </li></ul>
    5. 5. Antigenic structure <ul><li>Has O, H, and K antigens. </li></ul><ul><li>K1 has a strong association with virulence, particularly meningitis in neonates. </li></ul>
    6. 6. <ul><li>Antigenic structure . </li></ul><ul><li>Complex </li></ul><ul><li>Lipopolisaccharides/ Somatic or O antigen </li></ul><ul><ul><li>Heat stable.more than 150 types </li></ul></ul><ul><ul><li>Most external in the cell wall </li></ul></ul><ul><ul><li>detected by bacterial agglutination </li></ul></ul><ul><ul><li>Antibody produced is predominantly IgM </li></ul></ul>
    7. 7. <ul><li>Capsular/K antigen </li></ul><ul><ul><li>Sometimes external to O antigen but not always </li></ul></ul><ul><ul><li>Can be polysaccharides or protein </li></ul></ul><ul><li>Flagella /H antigen </li></ul><ul><ul><li>Heat and alcohol labile. </li></ul></ul>
    8. 8. <ul><li>Colicines/Bacteriocines </li></ul><ul><li>Produced by many Gram -ves </li></ul><ul><li>Virus like bactericidal substance </li></ul><ul><li>Active against some other bacteria of similar </li></ul><ul><li>or closely related species </li></ul>
    9. 9. E. coli toxins <ul><ul><li>Virulence factors </li></ul></ul><ul><ul><ul><li>Toxins </li></ul></ul></ul><ul><ul><ul><ul><li>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: </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>LT – is heat labile and binds to specific Gm 1 gangliosides on the epithelial cells of the small intestine where it stimulates production of cAMP. </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel. </li></ul></ul></ul></ul></ul>
    10. 10. E. coli toxins <ul><ul><ul><ul><ul><li>ST – is heat stable and binds to specific receptors to stimulate the production of cGMP with the same results as with LT. </li></ul></ul></ul></ul></ul>
    11. 11. Urinary tract infections <ul><li>E coli is the most common cause of UTI </li></ul><ul><ul><li>Account for 90% of cases in young women </li></ul></ul><ul><li>Symptoms </li></ul><ul><ul><li>Frequency, dysuria, hematuria, pyuria </li></ul></ul><ul><li>UTI may lead to bacteremia and sepsis </li></ul><ul><li>Nephropathogenic E coli produces hemolysin </li></ul>
    12. 12. <ul><li>E coli associated diarrhoeal diseases </li></ul>
    13. 13. Enterotoxigenic E. coli <ul><li>Heat labile toxin </li></ul><ul><ul><ul><ul><li>like choleragen </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Adenyl cyclase activated </li></ul></ul></ul></ul><ul><ul><ul><ul><li>cyclic AMP </li></ul></ul></ul></ul><ul><ul><ul><ul><li>secretion water/ions </li></ul></ul></ul></ul><ul><li>Heat stable toxin </li></ul><ul><ul><li>Guanylate cyclase activated </li></ul></ul><ul><ul><li>cyclic GMP </li></ul></ul><ul><ul><li>uptake water/ions </li></ul></ul>
    14. 14. <ul><li>A.Enterotoxigenic E coli ( ETEC) </li></ul><ul><ul><li>Common cause for travelers diarrhoea , and watery diarrhoea in children. </li></ul></ul><ul><li>Colonisation factor facilitates the attachment to the intestinal epithelium. </li></ul><ul><ul><li>Some ETEC produces heat labile exotoxin LT and heat stable or either of the toxins </li></ul></ul>
    15. 15. <ul><li>B.Enteropathogenic E coli (EPEC) </li></ul><ul><ul><li>Important cause of diarrhoea in infants of </li></ul></ul><ul><ul><li>developing countries. </li></ul></ul><ul><ul><li>Adhere to mucosal cells in small bowel,loss of microvilli, NONINVASIVE </li></ul></ul><ul><ul><li>enter to cell body. result in watery diarrhoea . </li></ul></ul><ul><ul><li>Self limiting ,can be chronic. </li></ul></ul><ul><li>Normally do not produce toxins </li></ul>
    16. 16. <ul><li>C. Enterohemorrhagic E coli ( EHEC) </li></ul><ul><ul><li>Produce verotoxin which has similarities to Shiga toxin </li></ul></ul><ul><ul><li>Associated with hemorrhagic colitis, severe form of diarrhoea. </li></ul></ul><ul><ul><li>Hemolytic uremic syndrome Disease can be prevented by thorough cooking . </li></ul></ul>
    17. 17. Escherichia coli O157:H7 <ul><li>This strain produces a powerful toxin, verotoxin, which can cause severe illness and death. </li></ul><ul><li>E coli O157:H7 does not ferment sorbitol and is negative on sorbitol MacConkey agar </li></ul><ul><li>Other Shiga toxin producing serotypes (e.g. O111 and O26) are also in the family of enterohemorrhagic E. coli and can cause similar disease. </li></ul><ul><li>Hemorrhagic colitis – preventable by thorough cooking of meat </li></ul>
    18. 18. <ul><li>Hemorrhagic colitis(HC) or bloody diarrhea and abdominal cramps </li></ul><ul><li>Lasts for 5-10 days usually </li></ul><ul><li>Infection may proceed to Hemolytic Uremic Syndrome (HUS), a severe cytopathic attack on the kidneys requiring intensive care and dialysis. </li></ul><ul><li>Red blood cells are destroyed requiring blood tranfusion </li></ul>
    19. 19. <ul><li>D. Enteroinvasive E coli (EIEC) </li></ul><ul><li>Produces disease similar to shigellosis . </li></ul><ul><li>In adults this has been isolated with Shigella </li></ul><ul><li>Commonly affect children in developing countries , </li></ul><ul><li>and travelers. </li></ul><ul><li>Disease is due to invasion into mucosal cells of the intestine </li></ul><ul><li>multiply inside the cells and destruction /inflammation/ulceration </li></ul><ul><li>diarrhoea with blood </li></ul><ul><li>EIEC are nonlactose fermenter,or late lactose fermenter </li></ul><ul><li>and non motile. </li></ul>
    20. 20. <ul><li>E. Enteroaggregative E coli (EAEC) </li></ul><ul><li>Produce acute/chronic diarrhoea in persons in developing countries. </li></ul><ul><li>Sepsis When normal host defense is poor, sepsis can happen. Common in new born babies whose IgM level is low. </li></ul>
    21. 21. Treatment of E.coli related diarrhoea <ul><li>1st Line </li></ul><ul><li>Nitrofurantoin </li></ul><ul><li>Nalidixic acid </li></ul><ul><li>Norfloxacin </li></ul><ul><li>Ampicillin </li></ul><ul><li>Cotrimoxazole </li></ul><ul><li>2nd line </li></ul><ul><li>Ciprofloxacin/Ceftriaxone/Cefuroxime </li></ul><ul><li>Gentamicin </li></ul>
    22. 22. <ul><li>Meningitis </li></ul><ul><li>E coli and Gp.B Strept . are the leading causes for </li></ul><ul><li>meningitis in infants. </li></ul><ul><li>K1 antigen is responsible for meningitis </li></ul><ul><li>K1 cross reacts with the Gp.B capsular </li></ul><ul><li>polysaccharides of N meningitides . </li></ul>
    23. 23. Pneumonia <ul><li>25% of gram -ve pneumonia with 50% mortality </li></ul><ul><li>Usually broncho pneumonia </li></ul><ul><li>High level of resistance to Ampicillin /Cotrimoxazole </li></ul>
    24. 24. Klebsiella spp
    25. 25. Morphology and culture characteristics <ul><li>Morphology </li></ul><ul><ul><li>Gram negative rods </li></ul></ul><ul><ul><li>Non-spore former, caspulated and non-motile </li></ul></ul><ul><li>Culture characteristics </li></ul><ul><ul><li>N.agar – mucoid, circular, convex, small colonies </li></ul></ul><ul><ul><li>MacConkey – mucoid, rose pink </li></ul></ul>
    26. 26. <ul><li>Biochemical reactions </li></ul><ul><li>IMViC: --++ </li></ul><ul><li>TSI: Acid butt/acid slant with gas production </li></ul>
    27. 27. 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
    28. 28. <ul><li>Klebsiella </li></ul><ul><li>K pneumoniae </li></ul><ul><li>Present in respiratory tract and feces of about 5% of </li></ul><ul><li>normal individuals. </li></ul><ul><li>Can cause bacterial pneumonia (3%). </li></ul><ul><li>Produce extensive hemorrhagic necrotising consolidation of lungs. </li></ul>
    29. 29. <ul><li>Enterobacter aerogenus </li></ul><ul><li>Capsulated </li></ul><ul><li>Free living in the intestine </li></ul><ul><li>Cause UTI and sepsis. </li></ul><ul><ul><li>Motile </li></ul></ul><ul><ul><li>Citrate + </li></ul></ul><ul><ul><li>Ornithine decarboxylase </li></ul></ul><ul><ul><li>Produce gas from glucose </li></ul></ul><ul><ul><li>Ferments lactose </li></ul></ul><ul><ul><li>VP + (like Klebsiella) </li></ul></ul>
    30. 30. <ul><li>Laboratory identification tests </li></ul>
    31. 31. Diagnosis of Bacterial Infection culture on plates or in broth identification by biochemical or serological tests on pure growth from single colony microscopy Decolorise Counterstain Stain unstained or stained with e.g. Gram stain sensitivities Serodiagnosis DNA technologies by disc diffusion methods, breakpoints or MICs
    32. 32. Gram negative rods
    33. 33. Lactose fermentor on MCA
    34. 34. Lactose positive Klebsiella sp
    35. 35. E. coli
    36. 37. Indole production <ul><li>Degradation of tryptophan by tryptophanase to produce indole </li></ul><ul><li>Red colour develops after adding a solution containing p-dimethylaminobenzaldehyde (Kovacs or Erlichs) </li></ul>Negative Positive
    37. 38. <ul><li>Triple Sugar Iron Agar (TSI): </li></ul><ul><ul><li>Purpose: To differentiate bacteria based on their ability to ferment glucose, lactose and/or sucrose, and to reduce sulfur to hydrogen sulfide. </li></ul></ul>
    38. 39. A=Acidic K=Alkaline - - + - - GAS + + - - - HYDROGEN SULFIDE A A A A - BUTT K K A A - SLANT TUBE 5 TUBE 4 TUBE 3 TUBE 2 TUBE 1 (UNINOCULATED) INTERPRETATION OF TUBES ABOVE
    39. 40. ONPG test <ul><li>Determines presence of  -galactosidase </li></ul><ul><li>Utilizes o-nitrophenyl-d-  -galactosidase </li></ul><ul><li>Differentiates late lactose fermenting organisms </li></ul>Positive Negative
    40. 41. Methyl Red <ul><li>This is a qualitative test of the acidity produced by the growth of a bacterium in phosphate –buffered glucose peptone water </li></ul><ul><ul><li>E coli produces a pH of about 5, and hence a red colour is produced after addition of methyl red. </li></ul></ul><ul><ul><li>Enterobacter aerogenes the pH never drops so low, it appears yellow after addition of methyl red. </li></ul></ul>
    41. 42. Voges-Proskauer <ul><li>This test for production of acetylmethylcarbinol (AMC) from carbohydrates by bacteria in glucose phosphate peptone water </li></ul><ul><li>Red colour is produced after addition of alpha naphthol and KOH </li></ul><ul><ul><ul><li>Klebsiella + </li></ul></ul></ul><ul><ul><ul><li>E coli - </li></ul></ul></ul>
    42. 43. MR/VP continued <ul><li>Reading Results: </li></ul><ul><ul><li>MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production) </li></ul></ul><ul><ul><li>VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change. </li></ul></ul>Methyl Red: left – and right + VP: left + and right –
    43. 44. IMViC test <ul><li>IMViC test is a group of four biochemical tests collectively used for primary identification of enteric bacteria </li></ul><ul><ul><li>Indole </li></ul></ul><ul><ul><li>Methyl red </li></ul></ul><ul><ul><li>Voges proskuer </li></ul></ul><ul><ul><li>Citrate utilization test </li></ul></ul>
    44. 45. Indole (IMViC tests) <ul><li>• E. coli (pink/red) + </li></ul><ul><li>• E. coli (left side) – </li></ul><ul><li>• Kovac’s reagent </li></ul><ul><li>detects if tryptophan </li></ul><ul><li>has been hydrolyzed </li></ul><ul><li>to indol/tryptophanase </li></ul>
    45. 46. Methyl Red (MR) (IMViC tests) <ul><li>Enterobacter </li></ul><ul><li>aerogenes (left) – </li></ul><ul><li>E. coli (bright red) + </li></ul><ul><li>Reagent: Methyl red </li></ul><ul><li>indicator identifies pH </li></ul><ul><li>change due to mixed </li></ul><ul><li>acid fermentation </li></ul>
    46. 47. Voges – Proskauer (VP) (IMViC tests) <ul><li>Enterobacter aerogenes +(left) </li></ul><ul><li>E. coli – (right) </li></ul><ul><li>Red colour is produced after addition of alpha naphthol and KOH </li></ul><ul><ul><ul><li>Klebsiella + </li></ul></ul></ul><ul><ul><ul><li>E coli - </li></ul></ul></ul>
    47. 48. Citrate utilization <ul><li>Ability to use sodium citrate as sole source of carbon </li></ul><ul><li>Medium has sodium citrate, ammonium salt, bromothymol blue </li></ul><ul><li>Positive test - deep blue colour in 24-48hrs </li></ul><ul><li>Indicates citrate utilization </li></ul><ul><li>Production of alkaline products </li></ul><ul><li>E. coli (left green) – </li></ul><ul><li>• Enterobacter aerogenes </li></ul><ul><li>(right royal blue) + </li></ul>Positive Negative
    48. 49. Treatment of enteric bacteria <ul><li>No specific treatment is available </li></ul><ul><li>A number of antibiotics could be used </li></ul><ul><li>Marked antibiotic resistance due to plasmids </li></ul><ul><ul><li>May involve surgical correction </li></ul></ul><ul><ul><li>Treatment of shock </li></ul></ul><ul><ul><li>Environmental sanitation </li></ul></ul>
    49. 52. Thank you
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