E.coliCommensal, Pathogen,& Genetic tool                Dr.T.V.Rao MDDr.T.V.Rao MD
Dr.T.V.Rao MD   2
Enterobacteriaceae    Commonly present in large intestine    Non sporing , Non Acid fast, Gram – bacilli.    A complex ...
Characters of                 Enterobacteriaceae All Enterobacteriaceae      Gram-negative rods      Ferment glucose wi...
Classification of Enterobacteriaceae                                  Enterobacteriaceae                   Lactose ferment...
Escherichia coli    Named by Escherichia    Wide group of bacteria on basis of       Bio typing and Serotyping      Prod...
Classification Domain: Bacteria Kingdom: Bacteria Phylum: Proteobacteria Class: Gamma  Proteobacteria Order: Enteroba...
E.coli    Morphology Gram - ve Straight     rods,    1-3 X 0.4 -0.7 microns,    Appear in singles or in pairs,    Moti...
Enterobacteriaceae:      Genetic Properties Chromosomal DNA has 39-59% guanine-  plus-cytosine (G+C) content Escherichia...
E.coli                Cultural characters    Aerobic / Facultative Anaerobic    Grows between 10 – 40 c optimal at 37 c ...
E.coliDr.T.V.Rao MD     11
Identification of Enterobacteriaceae           Biochemical reactions Oxidase test   All members of Enterobacteriaceae ar...
E.coli        Biochemical Characters,   Glucose,Lactose,Mannitol,Maltose      fermented. with A/G   I,M,Vi,C tests.    Ind...
Identification of EnterobacteriaceaeDifferentiation between LF & NLF by Growth Method:             on MacConkey agar   M...
E.coli                Antigenic Structure    Somatic 0 170    Capsular K 100    Flagella H 75    Virulence factors    ...
Toxins and E.coli    E.coli produce Exotoxins    Hemolysins, Enterotoxins causes     Diarrheas,    Important toxins pro...
Toxins          Enterotoxins – produced by enterotoxigenic           strains of E. coli (ETEC). Causes a           moveme...
Mechanism of action of               Toxins    Increased cAMP     alters the activity of     sodium and     chloride     ...
Toxins in E.coli    Produce Enterotoxin L T and S T    Labile toxin 1956 De experiments in Rabbit     illeal loop causes...
E. coli toxins   • Both enterotoxins     are composed of     five beta subunits     (for binding) and     1 alpha subunit ...
Toxins E.coli                Labile toxin    Component A Activated to A1 and     A2    A1 Activates adenyl cyclase in th...
Toxins of E.coli                 Stable Toxin    ST A and ST B    ST A Acts by activation of Cyclic guano     sine monop...
E.coli a Complex Microbe    More than 700     serotypes of E. coli     have been     identified. The     different E. col...
Classification of E.coli   1.Enteropathogenic EPEC   2.Enterotoxigenic   ETEC   3.Enteroinvasive     EIEC   4.Enterohemorr...
Enteropathogenic E.coli    Causes diarrheal disease in children,    EPEC O26/O11    Produce Verocytotoxin    Infantile...
Enteropathogenic E. coli         destruction of surface microvilli• fever• diarrhea                         Gut lumen• vom...
Laboratory Diagnosis EPEC    Confirm with     Polyvalent sera    Test Sero groups     with polyvalent     and monovalent...
Enterotoxigenic E.coli    Produce Heat stable /Heat labile toxins    Adheres to epithelium of small intestine.    Prese...
Enterotoxigenic E. coli Heat labile toxin      like choleragen      Adenyl cyclase activated      cyclic AMP      sec...
Enterotoxigenic E.coli    Causes travelers diarrhea    Water contaminated with Human and Animal feces     predisposes.  ...
Treatment and Prophylaxis             in Travelers diarrhea    Doxycycline,    Trimethoprim,    Norfloxacillin    Flur...
Entero invasive E.coli    Some are non motile     strains,    Atypical resembles     like Shigella.    Clinically mild ...
Enteroinvasive E. coli (EIEC ) • Dysentery   - resembles shigellosis          Gut lumen                 33
Enterohemorrhagic E.coli    Produce Verocytotoxin    Mild diarrhea - can be fatal hemorrhagic     colitis. and uremic sy...
EHEC ( contd )    Culture    DNA detection     methods.    Cytotoxic effects     on Vero cells.    Detection with     ...
Dr.T.V.Rao MD   36
Enterohemorrhagic E.coli           can cause HUS    HUS develops when the toxin from     E. coli bacteria, known as Shiga...
Mechanism of HUS   As the inflammatory reaction process    accelerates, red blood cells are destroyed    and cellular deb...
Enteroaggresive E.coli                  EAEC    Can cause Diarrhea Detect     by Culture methods   • Brick-like aggregate...
E.coli resembles Shigella                  spp     The E. coli serotypes         that are responsible for         the rep...
Treatment – E.coli  Gastrointestinal disease Fluid replacement Antibiotics   not used usually unless   systemic infecti...
E. Coli leading cause of                    UTI Clinical significance   Is the leading cause    of urinary tract    infe...
Urinary Tract Infections E.coli produce urinary tract infection. Majority of UTI s are produce by E.coli. Instrumentati...
Facts on UTI Women suffer more than males Short urethra Pregnancy, Sexual  intercourse /Honey moon cystitis. Other facto...
Culturing for E.coli    Mid stream sample/semi quantitative     culturing (Kass et al ) >_ 1.00,000/ml of     urine. ( si...
Other infection with                       E.coli    Pyogenic infections.    Intraabdominal infections    Peritonitis. ...
Other Important E. coli                Infections     Neonatal meningitis – is the leading cause      of neonatal meningi...
E.coli Antimicrobial therapy- E. coli is usually  susceptible to a variety of chemotherapeutic  agents, though drug resis...
Escherichia coli as a                   Genetic tool.    The study of Escherichia coli and its plasmids and     bacteriop...
Why E.coli is preferred    E. coli cells only have about 4,400 genes     whereas the human genome project     has determi...
How Does Molecular                  Cloning Work?                              Cloning of any DNA sequence               ...
E.coli Preferred in         Genetic Engineering    It is a favorite organism for genetic     engineering as cultures of i...
 Programme Created by                     Dr.T.V.Rao MD for                Microbiologists, Medical and                Pa...
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E.coli infections

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E.coli infection

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E.coli infections

  1. 1. E.coliCommensal, Pathogen,& Genetic tool Dr.T.V.Rao MDDr.T.V.Rao MD
  2. 2. 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.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) Dr.T.V.Rao MD 4
  5. 5. Classification of Enterobacteriaceae Enterobacteriaceae Lactose fermenters Non-lactose fermenter E. coli, Citrobacter, Salmonell, 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 Dr.T.V.Rao MD 5
  6. 6. 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.Dr.T.V.Rao MD 6
  7. 7. Classification Domain: Bacteria Kingdom: Bacteria Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Escherichia Species: Escherichia coli (E. coli)
  8. 8. 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.Dr.T.V.Rao MD 8
  9. 9. Enterobacteriaceae: Genetic Properties Chromosomal DNA has 39-59% guanine- plus-cytosine (G+C) content Escherichia coli is the type genus and species of the Enterobacteriaceae Species of Enterobacteriaceae more closely related by evolutionary distance to Escherichia coli than to organisms of other families (Pseudomonadaceae, Aeromonadaceae)
  10. 10. 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.Dr.T.V.Rao MD 10
  11. 11. E.coliDr.T.V.Rao MD 11
  12. 12. Identification of Enterobacteriaceae Biochemical reactions Oxidase test  All members of Enterobacteriaceae are oxidase negative  Pseudomonas is oxidase positive O/F test  All members of Enterobacteriaceae are O+/F+  Pseudomonas is O+/F- Nitrate reductase  All members of Enterobacteriaceae are nitrate reductase positive  Pseudomonas is nitrate reductase negative
  13. 13. E.coli Biochemical Characters, Glucose,Lactose,Mannitol,Maltose fermented. with A/G I,M,Vi,C tests. Indole + Methyl Red + Voges Proskauer – ve I,M,Vi,C tests. Citrate –ve Urease not produced.Dr.T.V.Rao MD 13
  14. 14. Identification of EnterobacteriaceaeDifferentiation between LF & NLF by Growth Method: on MacConkey agar  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 Dr.T.V.Rao MD 14 Flame & Cool
  15. 15. 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 )Dr.T.V.Rao MD 15
  16. 16. 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 toxinsDr.T.V.Rao MD 16
  17. 17. 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 ADP- ribosylates Gs which stimulates adenylate cyclase to increase production of cAMPDr.T.V.Rao MD 17
  18. 18. Mechanism of action of Toxins  Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowelDr.T.V.Rao MD 18
  19. 19. Toxins in E.coli  Produce Enterotoxin L T and S T  Labile toxin 1956 De experiments in Rabbit illeal 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 cellsDr.T.V.Rao MD 19
  20. 20. E. coli toxins • Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity).Dr.T.V.Rao MD 20
  21. 21. 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 DiarrheaDr.T.V.Rao MD 21
  22. 22. 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 toxinDr.T.V.Rao MD 22
  23. 23. E.coli a Complex Microbe  More than 700 serotypes of E. coli have been identified. The different E. coli serotypes are distinguished by their “O” and “H” antigens on their bodies and flagella, respectively.Dr.T.V.Rao MD 23
  24. 24. Classification of E.coli 1.Enteropathogenic EPEC 2.Enterotoxigenic ETEC 3.Enteroinvasive EIEC 4.Enterohemorrhagic EHEC 5.Enteroaggresive EAECDr.T.V.Rao MD 24
  25. 25. 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 InstitutionsDr.T.V.Rao MD 25
  26. 26. Enteropathogenic E. coli destruction of surface microvilli• fever• diarrhea Gut lumen• vomiting• nausea• non-bloody stools (not generally seen as dysentery) 26
  27. 27. Laboratory Diagnosis EPEC  Confirm with Polyvalent sera  Test Sero groups with polyvalent and monovalent sera.  HEp2 – adherence.Dr.T.V.Rao MD 27
  28. 28. 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 CFA Mortality in children < 5 yearsDr.T.V.Rao MD 28
  29. 29. 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 29
  30. 30. Enterotoxigenic E.coli  Causes travelers diarrhea  Water contaminated with Human and Animal feces predisposes.  Laboratory Diagnosis Demonstration of Enterotoxin LT and ST Tissue culture tests, ELISA Passive agglutination tests. Animal experiments in Rabbit ileal loop test.Dr.T.V.Rao MD 30
  31. 31. Treatment and Prophylaxis in Travelers diarrhea  Doxycycline,  Trimethoprim,  Norfloxacillin  Fluroquinolones  Avoid contaminated food,  Safe protected water ,prefer bottled water,  Hot foods, Hot Drinks,  Boiled milkDr.T.V.Rao MD 31
  32. 32. Entero invasive E.coli  Some are non motile strains,  Atypical resembles like Shigella.  Clinically mild diarrhea  Sereny test positive animal Rabbit.  ELISADr.T.V.Rao MD 32
  33. 33. Enteroinvasive E. coli (EIEC ) • Dysentery - resembles shigellosis Gut lumen 33
  34. 34. Enterohemorrhagic E.coli  Produce Verocytotoxin  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 cookingDr.T.V.Rao MD 34
  35. 35. EHEC ( contd )  Culture  DNA detection methods.  Cytotoxic effects on Vero cells.  Detection with monovalent sera O157/H7Dr.T.V.Rao MD 35
  36. 36. Dr.T.V.Rao MD 36
  37. 37. 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.Dr.T.V.Rao MD 37
  38. 38. Mechanism of HUS  As the inflammatory reaction process accelerates, red blood cells are destroyed and cellular debris aggregates within the microvasculature while the body’s inherent clot breaking mechanisms are disrupted. The result is formation of micro thrombi within particularly susceptible organs such as the kidneys and brain. Because there exists no way to halt the progression of HUS,  The patients are supported with medical careDr.T.V.Rao MD 38
  39. 39. Enteroaggresive E.coli EAEC  Can cause Diarrhea Detect by Culture methods • Brick-like aggregates on cell surfaces • Mucus biofilm inhibits fluid absorption • Diarrhea  Detection of EnterotoxinDr.T.V.Rao MD 39
  40. 40. E.coli resembles Shigella spp  The E. coli serotypes that are responsible for the reports of contaminated foods and beverages are those that produce Shiga toxin, because the toxin is identical to that produced by another bacteria known as Shigella dysenteryDr.T.V.Rao MD 40
  41. 41. Treatment – E.coli Gastrointestinal disease Fluid replacement Antibiotics  not used usually unless systemic infections prevails  e.g. hemolytic-uremia syndrome 41
  42. 42. E. Coli leading cause of UTI Clinical significance  Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection).Dr.T.V.Rao MD 42
  43. 43. Urinary Tract Infections E.coli produce urinary tract infection. Majority of UTI s are produce by E.coli. Instrumentation, Prostatic enlargement, Urinary caliculi ,Pregnancy, increase the predisposition Asymptomatic Bacteriuria in pregnant women, Pyelonephritis, Dr.T.V.Rao MD 43
  44. 44. 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 urethra Dr.T.V.Rao MD 44
  45. 45. 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.Dr.T.V.Rao MD 45
  46. 46. Other infection with E.coli  Pyogenic infections.  Intraabdominal infections  Peritonitis. Abscess.  Septicemias  Produce Drug resistant infections.Dr.T.V.Rao MD 46
  47. 47. Other Important 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:Dr.T.V.Rao MD 47
  48. 48. E.coli Antimicrobial therapy- E. coli is usually susceptible to a variety of chemotherapeutic agents, though drug resistant strains are increasingly prevalent. It is essential to do susceptibility testing. Treatment of patients with EHEC infections is not recommended because it can increase the release of shiga-like toxins and actually trigger HUS
  49. 49. Escherichia coli as a Genetic tool.  The study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade.. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism.Dr.T.V.Rao MD 49
  50. 50. Why E.coli is preferred  E. coli cells only have about 4,400 genes whereas the human genome project has determined that humans contain approximately 30,000 genes. Also, bacteria, including E. coli, live their entire lifetime in a haploid state, with no second allele to mask the effects of mutations during protein engineering experiments.Dr.T.V.Rao MD 50
  51. 51. How Does Molecular Cloning Work?  Cloning of any DNA sequence involves the introduction of a foreign piece of DNA into an extra chromosomal element (cloning vector) of an organism which then produces copies of the vector as it replicates itself, thereby amplifying the DNA of interest. The whole process can be summarized in the following steps: fragmentation, ligation, transfection, screening/selection, andDr.T.V.Rao MD 51 conformation of insert.
  52. 52. E.coli Preferred in Genetic Engineering  It is a favorite organism for genetic engineering as cultures of it can be made to produce unlimited quantities of the product of an introduced gene. Several important drugs (insulin, for example) are now manufactured in E. coli. However, E. coli cannot attach sugars to proteins so proteins requiring such sugars have to be made in the cells of eukaryotes such as yeast cells and mammalian cells grown in cell culture.Dr.T.V.Rao MD 52
  53. 53.  Programme Created by Dr.T.V.Rao MD for Microbiologists, Medical and Paramedical Students in the Developing World  Email.com  doctortvrao@gmail.comDr.T.V.Rao MD 53

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