ENTEROBACTERIACEAE basics

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ENTEROBACTERIACEAE basics

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ENTEROBACTERIACEAE basics

  1. 1. ENTEROBACTERIACEAE basics Chapter I Dr.T.V.Rao MD4/16/2012 Dr.T.V.Rao MD 1
  2. 2. Bacteria Gram Gram positive negative Cocci Bacilli Cocci Rods4/16/2012 Dr.T.V.Rao MD 2
  3. 3. Enterobacteriaceae• Commonly present in large intestine• Non sporing , Non Acid fast, Gram – bacilli.• A complex family of organisms,• Some are non pathogenic• A few are highly Pathogenic,• Some commensals turn out to be pathogenic. as in UTI after catheterization.4/16/2012 Dr.T.V.Rao MD 3
  4. 4. Characters of Enterobacteriaceae• All Enterobacteriaceae – Gram-negative rods – Ferment glucose with acid production – Reduce nitrates into nitrites – Oxidase negative• Facultative anaerobic• Motile except Shigella and Klebsiella• Non-capsulated except Klebsiella• Non-fastidious• Grow on bile containing media (MacConkey agar) 4/16/2012 Dr.T.V.Rao MD 4
  5. 5. Basic Tests ……• Basic characters• Catalase +• Oxidase -• Reduce nitrates,• All are Gram negative and non spore forming.• Wide diversity / Antigenic heterogeneity,4/16/2012 Dr.T.V.Rao MD 5
  6. 6. Classification of Enterobacteriaceae Enterobacteriaceae Lactose fermenters Non-lactose fermenter E. coli, Citrobacter, Salmonella, Shigella Klebsiella, Enterobacter Proteus, YersiniaThere are several selective and differential media used to isolate distinguishes between LF & LNFThe most important media are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella Shigella (SS) agar In addition to Triple Sugar Iron (TSI) agar 4/16/2012 Dr.T.V.Rao MD 6
  7. 7. Differentiation between LF & NLF by Growth on MacConkey agar  MacConkey agar is selective & differential medium for Enterobacteriaceae MacConkey Agar ContainsBile salts Crystal violet Lactose Neutral red pH indicatorInhibit growth of G+ve bacteria Cause of differential Acidic: PinkCause of selectivity Lactose feremnters Lactose non feremnters 4/16/2012 Dr.T.V.Rao MD colorless colonies Pink colonies
  8. 8. Basic Classification• I Lactose fermenters E.coli, Escherichia Klebsiella,• II Late Lactose fermenters, Shigella sonnei,• III Non Lactose fermenters, Salmonella Shigella,Commonly tested with MacConkey mediumMany are commensals Lactose fermentersAlso called as coli forms , Enteric Bacilli4/16/2012 Dr.T.V.Rao MD 8
  9. 9. Enterobacteriaceae Taxonomical• Tribe I Escherichia Genus – 1 Escherichia, 2 Edwardsville 3 Citrobacter 4 Salmonella 5 Shigella4/16/2012 Dr.T.V.Rao MD 9
  10. 10. Enterobacteriaceae.• Tribe II; Klebsiella Genus 1. Klebsiella 2.Enterobacter, 3.Hafnia 4 Serratia Tribe III ; Proteeae Genus 1,Proteus 2,Morganella 3 . Providencia4/16/2012 Dr.T.V.Rao MD 10
  11. 11. Classification of Enterobacteriaceae ( Contd )• Tribe IV; Erwinieae Genus 1. Erwinia4/16/2012 Dr.T.V.Rao MD 11
  12. 12. Identification of EnterobacteriaceaeDifferentiation between LF & NLF by Growth on MacConkey agar • Method: – MacConkey agar is inoculated with tested organism using streak plate technique – Incubate the plate in incubator at 37 C/24 hrs • Results: – LF organism appears as pink colonies (e.g. E. coli) – NLF organism appears as colorless colonies (e.g. Shigella) Flame & Cool 1 2 3 4 Flame & Cool 5 4/16/2012 Dr.T.V.Rao MD 12 Flame & Cool
  13. 13. Highly Pathogenic Enterobacteriaceae• Salmonella• Shigella• All are Lactose – non fermenters,• Produce colorless colonies on MacConkey medium• LF also called as Para colons,4/16/2012 Dr.T.V.Rao MD 13
  14. 14. Escherichia coli• Named by Escherichia• Wide group of bacteria on basis of Bio typing and Serotyping Produce infections in Humans and Animals Detection of E.coli in water indicates pollution and contamination.4/16/2012 Dr.T.V.Rao MD 14
  15. 15. 4/16/2012 Dr.T.V.Rao MD 15
  16. 16. E.coli• Morphology Gram - ve Straight rods,• 1-3 X 0.4 -0.7 microns,• Appear in singles or in pairs,• Motile by peritrichate flagella.• Very few strains non motile• Not spore forming, Non acid fast.4/16/2012 Dr.T.V.Rao MD 16
  17. 17. E.coli Cultural characters• Aerobic / Facultative Anaerobic• Grows between 10 – 40 c optimal at 37 c• Grown in simple medium• Produce Large grayish ,Thick white , moist smooth opaque colonies• May contain capsule.• On MacConkey medium Produce Bright pink Lactose fermenters.4/16/2012 Dr.T.V.Rao MD 17
  18. 18. E.coli on Blood Agar• Many pathogenic strains are haemolytic on blood agar.4/16/2012 Dr.T.V.Rao MD 18
  19. 19. E.coli Biochemical Characters,• Glucose,Lactose,Mannitol,Maltose fermented. with A/G Indole + Methyl Red + Voges Proskauer – I,M,Vi,C tests. Citrate – Urease not produced.4/16/2012 Dr.T.V.Rao MD 19
  20. 20. E.coli Antigenic Structure• Somatic 0 170• Capsular K 100• Flagella H 75• Virulence factors Surface Antigens Toxins O Endotoxic activity K protects against the phagocytosis Fimbriae promote virulence ( important in UTI )4/16/2012 Dr.T.V.Rao MD 20
  21. 21. Virulence Factors• Two types of virulence factors in E.coli• Surface antigens and Toxins• The somatic lipopolysaccharide surface O antigen has endotoxic activity and protects from phagocytosis and bactericidal effects of complement .4/16/2012 Dr.T.V.Rao MD 21
  22. 22. Fimbriae• Fimbriae also promote virulence• Present in large numbers causing mannose sensitive Haemagglutination• Colonisation factor antigens is enterotoxigenic E.coli• E.coli produce two kinds of Exotoxins hemolysins and enterotoxins4/16/2012 Dr.T.V.Rao MD 22
  23. 23. Toxins and E.coli• E.coli produce Exotoxins• Hemolysins, Enterotoxins causes Diarrheas,• Important toxins produces.• Heat labile HL Heat stable HS Vero toxins VT Like Shigella toxins4/16/2012 Dr.T.V.Rao MD 23
  24. 24. 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:4/16/2012 Dr.T.V.Rao MD 24
  25. 25. Toxins• LT – is heat labile and binds to specific Gm1 gangliosides on the epithelial cells of the small intestine where it ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP.4/16/2012 Dr.T.V.Rao MD 25
  26. 26. Toxins• Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.4/16/2012 Dr.T.V.Rao MD 26
  27. 27. Toxins in E.coli• Produce Enterotoxin L T and S T• Labile toxin 1956 De experiments in Rabbit ileal loop causes outpouring of fluids• E.coli Labile toxin like Cholera toxin• L T contains component A and B• A = Active B= Binding• B causes Binding with Gm I Ganglioside receptor on Intestinal epithelial cells4/16/2012 Dr.T.V.Rao MD 27
  28. 28. E. coli toxins• Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity).4/16/2012 Dr.T.V.Rao MD 28
  29. 29. Toxins E.coli Labile toxin• Component A Activated to A1 and A2• A1 Activates adenyl cyclase in the enterocytes to form cyclic adenosine 5 monophosphate• Causes to increase outflow of water and electrolytes in the gut lumen causes Diarrhea4/16/2012 Dr.T.V.Rao MD 29
  30. 30. Toxins of E.coli Stable Toxin• ST A and ST B• ST A Acts by activation of Cyclic guano sine monophosphate.( C GMP )• Causes fluid accumulation in Intestine.• E.coli ( Some ) produce Verocytotoxin causes cytotoxicity to Vero cells.• Acts like Shigella dysentery toxin4/16/2012 Dr.T.V.Rao MD 30
  31. 31. E. coli infections • Neonatal meningitis – is the leading cause of neonatal meningitis and septicemia with a high mortality rate. – Usually caused by strains with the K1 capsular antigen. • Gastroenteritis – there are several distinct types of E. coli that are involved in different types of gastroenteritis: – enterotoxigenic E. coli (ETEC), – enteroinvasive E. coli (EIEC), – enteropathogenic E. coli (EPEC) , – enteroaggregative E. coli (EAEC), and – enterohemorrhagic E. coli (EHEC).4/16/2012 Dr.T.V.Rao MD 31
  32. 32. Classification of E.coli1.Enteropathogenic EPEC2.Enterotoxigenic ETEC3.Enteroinvasive EIEC4.Enterohemorrhagic EHEC5.Enteroaggresive EAEC4/16/2012 Dr.T.V.Rao MD 32
  33. 33. Enteropathogenic E.coli• Causes diarrheal disease in children,• EPEC O26/O11• Produce Verocytotoxin• Infantile enteritis, Involves upper part of Intestine• Brush border of the intestine is lost• Intimacin – EPEC adhesion factor.• Frequent in summer months• Poor hygiene predisposes.• Out breaks in Institutions4/16/2012 Dr.T.V.Rao MD 33
  34. 34. Enteropathogenic E.coli• Causes diarrheal disease in children,• EPEC O26/O11• Produce Verocytotoxin• Infantile enteritis, Involves upper part of Intestine• Brush border of the intestine is lost• Intimacin – EPEC adhesion factor.• Frequent in summer months• Poor hygiene predisposes.• Out breaks in Institutions4/16/2012 Dr.T.V.Rao MD 34
  35. 35. EPEC• EPEC are identified by serotyping by their O and B antigens• Diagnosis is difficult during sporadics• Routine culture is done for isolation• The EPEC fails to ferment sorbitol• Causes the disruption of brush border4/16/2012 Dr.T.V.Rao MD 35
  36. 36. Laboratory Diagnosis EPEC• Confirm with Polyvalent sera• Test Sero groups with polyvalent and monovalent sera.• HEp2 – adherence.4/16/2012 Dr.T.V.Rao MD 36
  37. 37. Enterotoxigenic E.coli• Common and causes endemics in developing countries in all age groups• May be mild watery diarrhoea to fatal conditions• Fimbrial colonization factor antigens4/16/2012 Dr.T.V.Rao MD 37
  38. 38. Enterotoxigenic E.coli•Produce Heat stable /Heat labile toxins•Adheres to epithelium of small intestine.•Present with Nausea, Vomiting and Lose stool•H L like cholera toxin•Causes accumulation of fluids•Adhesive factors Fimbriae specific receptor in the intestinal epithelium CFAMortality in children < 5 years4/16/2012 Dr.T.V.Rao MD 38
  39. 39. ETEC• Causes travelers diarrhea• Water contaminated with Human and Animal feces predisposes.• Laboratory Diagnosis Demonstration of Enterotoxin LT and STTissue culture tests,ELISAPassive agglutination tests.Animal experiments in Rabbit ileal loop test.4/16/2012 Dr.T.V.Rao MD 39
  40. 40. Treatment and Prophylaxis in Travelers diarrhea• Doxycycline,• Trimethoprim,• Norfloxacillin• Fluroquinolones• Avoid contaminated food,• Safe protected water ,prefer bottled water,• Hot foods, Hot Drinks,• Boiled milk4/16/2012 Dr.T.V.Rao MD 40
  41. 41. Treatment and Prophylaxis in Travelers diarrhea• Doxycycline,• Trimethoprim,• Norfloxacillin• Fluroquinolones• Avoid contaminated food,• Safe protected water ,prefer bottled water,• Hot foods, Hot Drinks,• Boiled milk4/16/2012 Dr.T.V.Rao MD 41
  42. 42. Enteroinvasive E.coli• Resembles Shigella in many respects• Non lactose fermenter and non motile• They invade the intestinal epithelium• Penetrate HeLa cells in tissue culture• May produce mild diarrhoea to frank dysentery• Sereny test4/16/2012 Dr.T.V.Rao MD 42
  43. 43. Entero invasive E.coli• Some are non motile strains,• Atypical resembles like Shigella.• Clinically mild diarrhea• Sereny test positive animal Rabbit.• ELISA4/16/2012 Dr.T.V.Rao MD 43
  44. 44. Enterohemorrhagic E.coli• Produce Verocytotoxin or shiga like toxin• Mild diarrhea - can be fatal hemorrhagic colitis. and uremic syndrome.• Present in Human and Animal feces.• Hemorrhagic complication with O157 in Japan and USA.• Salads vegetables, Radish Proper cooking4/16/2012 Dr.T.V.Rao MD 44
  45. 45. 4/16/2012 Dr.T.V.Rao MD 45
  46. 46. Enterohemorrhagic E.coli• The primary target for VT appear to be vascular endothelial cells• This may contribute to HUS with characteristic renal lesion is capillary micro angiopathy• The typical EHEC serotype is 0 157 ; H7• The disease may manifest as food poisoning• Occurs due to contamination of feces of 464/16/2012 Dr.T.V.Rao MD humans or animals
  47. 47. Enterohemorrhagic E.coli can cause HUS • HUS develops when the toxin from E. coli bacteria, known as Shiga-like toxin (SLT) enters the circulation by binding to special receptors. These Shiga-toxin receptors, known as Gb3 receptors , are probably heterogeneously distributed in the major body organs allowing disparate thrombotic (blood clotting) impacts in different HUS victims, although the greatest receptor concentration appears to be in the kidneys, especially in children.4/16/2012 Dr.T.V.Rao MD 47
  48. 48. EHEC ( E.coli )• Culture• DNA detection methods.• Cytotoxic effects on Vero cells.• Detection with monovalent sera O157/H74/16/2012 Dr.T.V.Rao MD 48
  49. 49. Enteroaggresive E.coli• They appear aggregated in s stacked brick formation Hep-2 cell• They produce persistent diarrheal• They produce weight heat stable enterotoxin called as low molecular heat stable enterotoxin4/16/2012 Dr.T.V.Rao MD 49
  50. 50. Enteroaggresive E.coli EAEC• Stool Culture methods• Detection of Enterotoxin4/16/2012 Dr.T.V.Rao MD 50
  51. 51. Treatment – E.coli Gastrointestinal disease• Fluid replacement• Antibiotics – not used usually unless systemic infections prevails –e.g. hemolytic-uremia4/16/2012 syndrome Dr.T.V.Rao MD 51
  52. 52. E. coli – Clinical significance • Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis4/16/2012 (kidney Dr.T.V.Rao MD 52 infection).
  53. 53. Ascending urinary tract infection4/16/2012 Dr.T.V.Rao MD 53
  54. 54. Urinary Tract Infections• E.coli produce urinary tract infection.• Majority of UTI s are produce by E.coli.• Instrumentation, Prostatic enlargement, Urinary caliculi,Pregnancy,• Asymptomatic Bacteriuria in pregnant women,• Pyelonephritis, 4/16/2012 Dr.T.V.Rao MD 54
  55. 55. Facts on UTI• Women suffer more than males Short urethra Pregnancy, Sexual intercourse /Honey moon cystitis.• Other factors Urethral obstruction, Urinary stones Congenital malformations Neurological disorders, Catheterization , CystoscopyUsually cystitis is produced from fecal strains entering urethra4/16/2012 Dr.T.V.Rao MD 55
  56. 56. Culturing for E.coli• Mid stream sample/semi quantitative culturing (Kass et al ) >_ 1.00,000/ml of urine. ( significant Bacteriuria )• Urine should not be kept in wards for > 2 hours and to be preserved at 4 c• Culture by standard loop method.• Fixed volume cultured on MacConkey agar Lactose fermenters I M Vi C• Antibiotic sensitivity tested.4/16/2012 Dr.T.V.Rao MD 56
  57. 57. Other Bacteria and UTI• Majority of UTI are caused by E.coli• Other agents which cause UTI,1 Staphylococcus2.Proteus.3.Pseudomonas,4.Klebsilella spp,Citrobacter,5.Enteococcus.4/16/2012 Dr.T.V.Rao MD 57
  58. 58. Other infection with E.coli• Pyogenic infections.• Intraabdominal infections• Peritonitis. Abscess.• Septicemias• Produce Drug resistant infections.4/16/2012 Dr.T.V.Rao MD 58
  59. 59. 4/16/2012 Dr.T.V.Rao MD 59
  60. 60. Klebsiella, Enterobacter, Serratia & Hafnia sp.• Usually found in intestinal tract• Wide variety of infections, primarily pneumonia, wound, and UTI• General characteristics: – Some species are non-motile – Simmons citrate positive – H2S negative – Phenylalanine deaminase negative – Some weakly urease positive – MR negative; VP positive4/16/2012 Dr.T.V.Rao MD 60
  61. 61. Klebsiella species• Usually found in GI tract• Four major species• K. pneumoniae is mostly commonly isolated species – Possesses a polysaccharide capsule, which protects against phagocytosis and antibiotics AND makes the colonies moist and mucoid – Has a distinctive “yeasty” odor – Frequent cause of nosocomial pneumonia4/16/2012 Dr.T.V.Rao MD 61
  62. 62. Klebsiella• Klebsiella is a genus of non-motile, Gram- negative, oxidase-negative, rod-shaped bacteria with a prominent polysaccharide- based capsule. It is named after the German microbiologist Edwin Klebs (1834–1913). Frequent human pathogens, Klebsiella organisms can lead to a wide range of disease states, notably pneumonia, urinary tract infections, septicaemia, and Dr.T.V.Raotissue infections. 624/16/2012 soft MD
  63. 63. Klebsiella• The genus was originally divided into 3 main species based on biochemical reactions. Today, 7 species with demonstrated similarities in DNA homology are known. These are (1) Klebsiella pneumoniae, (2) Klebsiella ozaenae, (3) Klebsiella rhinoscleromatis, (4) Klebsiella oxytoca, (5) Klebsiella planticola, (6) Klebsiella terrigena, and (7) Klebsiella ornithinolytica4/16/2012 Dr.T.V.Rao MD 63
  64. 64. Klebsiella• K pneumoniae is the most medically important species of the group. K oxytoca and K rhinoscleromatis have also been demonstrated in human clinical specimens. In recent years, klebsiellae have become important pathogens in nosocomial infections.4/16/2012 Dr.T.V.Rao MD 64
  65. 65. Klebsiella• Klebsiella – NF of GI tract, but potential pathogen in other areas – TSI A/A + gas – LIA K/K – Urea + – Citrate + – MR-, VP+ – Motility - – Has both O and K antigens4/16/2012 Dr.T.V.Rao MD 65
  66. 66. Klebsiella species• Usually found in GI tract• Four major species• K. pneumoniae is mostly commonly isolated species – Possesses a polysaccharide capsule, which protects against phagocytosis and antibiotics AND makes the colonies moist and mucoid – Has a distinctive “yeasty” odor – Frequent cause of nosocomial pneumonia4/16/2012 Dr.T.V.Rao MD 66
  67. 67. Klebsiella species (cont’d) – Significant biochemical reactions • Lactose positive • Most are urease positive • Non-motile4/16/2012 Dr.T.V.Rao MD 67
  68. 68. Klebsiella – Virulence factors • Capsule • Adhesions • Iron capturing ability – Clinical significance • Causes pneumonia, mostly in immunocompromised hosts. – Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia) • A major cause of nosocomial infections such as septicemia and meningitis4/16/2012 Dr.T.V.Rao MD 68
  69. 69. Enterobacter species• Comprised of 12 species; E. cloacae and E. aerogenes are most common• Isolated from wounds, urine, blood and CSF• Major characteristics – Colonies resemble Klebsiella – Motile – MR negative; VP positive4/16/2012 Dr.T.V.Rao MD 69
  70. 70. Serratia species• Seven species, but S. marcescens is the only one clinically important• Frequently found in nosocomial infections of urinary or respiratory tracts• Implicated in bacteremic outbreaks in nurseries, cardiac surgery, and burn units• Fairly resistant to antibiotics4/16/2012 Dr.T.V.Rao MD 70
  71. 71. Serratia species (cont’d)• Major characteristics – Ferments lactose slowly – Produce characteristic pink pigment, especially when cultures are left at room temperature S. marscens on nutrient agar →4/16/2012 Dr.T.V.Rao MD 71
  72. 72. Hafnia species• Hafnia alvei is only species• Has been isolated from many anatomical sites in humans and the environment• Occasionally isolated from stools• Delayed citrate reaction is major characteristic4/16/2012 Dr.T.V.Rao MD 72
  73. 73. Other Enterobacteriaceae• Proteus, Providencia, and Morganella – Are all part of the NF of the GI tract (except Providencia). – All motile, with Proteus swarming – PA + – Lysine deamination + (LIA R/A) – Urea + for most, strongly + for Proteus – TSI variable (know the reactions for each in the lab!) – Indole – only P. mirabilis is -4/16/2012 Dr.T.V.Rao MD 73
  74. 74. Proteus, Morganella & Providencia species• All are normal intestinal flora• Opportunistic pathogens• Deaminate phenylalanine• All are lactose negative4/16/2012 Dr.T.V.Rao MD 74
  75. 75. Proteus species• P. mirabilis and P. vulgaris are widely recognized human pathogens• Isolated from urine, wounds, and ear and bacteremic infections• Both produce swarming colonies on non-selective media and have a distinctive “burned chocolate” odor• Both are strongly urease positive• Both are phenylalanine deaminase positive4/16/2012 Dr.T.V.Rao MD 75
  76. 76. Proteus species (cont’d)• A exhibits characteristic “swarming”• B shows urease positive on right4/16/2012 Dr.T.V.Rao MD 76
  77. 77. Morganella species• Morganella morganii is only species• Documented cause of UTI• Isolated from other anatomical sites• Urease positive• Phenylalanine deaminase positive4/16/2012 Dr.T.V.Rao MD 77
  78. 78. Providencia species• Providencia rettgeri is pathogen of urinary tract and has caused nosocomial outbreaks• Providenicia stuartii can cause nosocomial outbreaks in burn units and has been isolated from urine• Both are phenylalanine deaminase positive4/16/2012 Dr.T.V.Rao MD 78
  79. 79. Citrobacter species• Citrobacter freundii associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses)• Ferments lactose and hydrolyzes urea slowly• Resembles Salmonella sp.4/16/2012 Dr.T.V.Rao MD 79
  80. 80. • Programme Created by Dr.T.V.Rao MD for benefit of Medical and Paramedical Students as e-learning Resource • Email • doctortvrao@gmail4/16/2012 Dr.T.V.Rao MD 80

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