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  1. 1. Dr.Babasaheb N. Kumbhar M.V.Sc bobbyvph11@gmail.com ENTEROBACTERIACEAEENTEROBACTERIACEAE
  2. 2. EnterobacteriaceaeEnterobacteriaceae Habitat  digestive tube(colon) of human and animals  150 Species  Facultative anaerobes  Diarrheal illnesses  3 million death/year  4 billion infectoions/ worldwide
  3. 3. Enterobacteriaceae  Classification – more than15 different genera  Escherichia  Shigella  Edwardsiella  Salmonella  Citrobacter  Klebsiella  Enterobacter  Hafnia  Serratia
  4. 4. Enterobacteriaceae Proteus Providencia Morganella Yersinia Erwinia Pectinobacterium
  5. 5. Family Enterobacteriaceae (nonmotile) (nonmotile) True pathogen True pathogen True pathogen Certain E .coli strains can be considered true pathogens
  6. 6. Morphology and General Characteristics  Are facultative anaerobes  Gram-negative, non-sporing, rod shaped bacteria  If motile, motility is by peritrichous flagella(Except Shigella & Klebsiella)  Many are normal inhabitants of the intestinal tract of man and other animals  Some are enteric pathogens and others are urinary or respiratory tract pathogens  Differentiation is based on biochemical reactions and differences in antigenic structure
  7. 7. Morphology and Physiology Short gram-negative rods. Facultative anaerobes. Grow readily and rapidly on simple media. K. pneumoniae Klebsiella spp. have large capsule (form large and very mucoid colonies); those of Enterobacter have smaller capsule; the others produce diffusible slime layers (form circular, convex and smooth colonies).
  8. 8. ENTEROBACTERIACEAE PHYSIOLOGY  Glucose is fermented with strong acid formation and often gas  Reduce nitrates to nitrite  Do not liquify alginate  Oxidase negative  Basis for speciation within a family - differences in carbohydrate they ferment - variations in end-product production - variation in substrate utilization
  9. 9. Enterobacteriaceae Opportunistic pathogens Escherichia coli Klebsiella pneumoniae Enterobacter aerogenes Serratia marcescens Proteus spp. Providencia spp. Citrobacter spp. Obligate pathogens Salmonella spp. Shigella spp. Yersinia spp. Some E. coli strains Sepsis Meningitis UTI Diarrhea Pneumonia
  10. 10. Incidence of Enterobacteriaceae Associated with Bacteremia
  11. 11. Sites of Infections with Members of the Enterobacteriaceae
  12. 12. CULTURAL CHARACTERISTICS  On non differential or nonselective media blood agar or infusion agar - no species distinction - appear as moist, smooth, gray colonies  Selective media -To isolate Shigella and salmonella from fecal matter  Differential media – selectively inhibit gram-positive organisms and to separate enterics in broad categories
  13. 13. Modes of Infection • Contaminated food and water (Salmonella spp., Shigella spp., Yersinia enterocolitica, Escherichia coli O157:H7) • Endogenous (urinary tract infection, primary bacterial peritonitis, abdominal abscess) • Abnormal host colonization (nosocomial pneumonia) • Transfer between debilitated patients • Insect (flea) vector (unique for Yersinia pestis)
  14. 14. Urinary Tract Infection, Pneumonia • Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis • Pneumonia: Enterobacter spp., Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis • Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis • Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis Intestinal Infection • Shigella sonnei (serogroup D) • Salmonella serotype enteritidis • Salmonella serotype typhimurium • Shigella flexneri (serogroup B) • Escherichia coli O157:H7 • Yersinia enterocolitica
  15. 15. MacConkey (MAC) Agar
  16. 16. Eosin Methylene Blue (EMB) Agar (Levine)
  17. 17. Antigenic StructureAntigenic Structure – Most are motile byMost are motile by peritrichous flagellaperitrichous flagella ----HH antigens.antigens. – Capsule –Capsule – KK antigen (antigen ( ViVi for Salmonella).for Salmonella). – Cell envelope (wall)Cell envelope (wall) – LPS (endotoxin) –LPS (endotoxin) – OO antigen.antigen. – various outervarious outer membrane proteins.membrane proteins. – PiliPili - various antigen- various antigen types, some encodedtypes, some encoded by plasmidsby plasmids
  18. 18. Pathogenesis and Immunity Common virulence factors Type III secretion systems: possessed by some Enterobacteriaceae pathogens, e.g., E. coli, Yersinia, Salmonella, and Shigella; facilitate transport of bacterial virulence factors directly into host cells. Endotoxin (Lipid A of LPS) Capsule Antigenic phase variation Acquisition of growth factors (e.g. Fe) Resistance to serum killing Antimicrobial resistance
  19. 19. HE Agar: Growth of Enteric Pathogens and Commensals
  20. 20. Escherichia coliEscherichia coli  Toxins: two types of enterotoxin; Shiga-type toxin; Enteroaggregative ST-like toxin; Hemolysins; Endotoxin  Virulence factors that protect the bacteria from host defenses: Capsule/Iron capturing ability (enterochelin)
  21. 21. E. coli  May be hemolytic on CBA – more common in pathogenic strains  KEY tests for the normal strain:  TSI is A/A + gas  LIA K/K  Urea –  Indole +  Citrate –  Motility +  There is an inactive biotype that is anaerogenic, lactose –, and nonmotile.
  22. 22. EnteropathogenicEnteropathogenic E. coliE. coli  fever  infant diarrhea  vomiting  nausea  non-bloody stools  Destruction of surface microvilli loose attachment mediated by bundle forming pili (Bfp);  Stimulation of intracellular calcium level;  rearrangement of intracellular actin, EnterotoxigenicEnterotoxigenic E. coliE. coli  A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days.  Travellers diarrhea and diarrhea in children in developing countries  Transmission is via contaminated food or water.
  23. 23. E.coli-E.coli-Enteroinvasive (EIEC)Enteroinvasive (EIEC)  The organism attaches to the intestinal mucosa via pili  Outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa.  There is lateral movement of the organism from one cell to adjacent cells.  Symptoms- fever,severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous,& pus.  resembles shigellosis E.coli-E.coli-c. Enteropathogenic (EPEC)c. Enteropathogenic (EPEC)  Malaise and low grade fever diarrhea, vomiting, nausea, non- bloody stools  Bundle forming pili are involved in attachment to the intestinal mucosa.  This leads to changes in signal transduction in the cells, effacement of the microvilli, and to intimate attachment via a non- fimbrial adhesion called intimin.  This is a problem mainly in hospitalized infants and in day care centers.
  24. 24. Enterohemorrhagic (EHEC)Enterohemorrhagic (EHEC)  Hemorrhagic – bloody, copious diarrhea – few leukocytes – afebrile  hemolytic-uremic syndrome – hemolytic anemia – thrombocytopenia (low platelets) – kidney failure • Usually O157:H7 Transmission electron micrograph
  25. 25. EnteroaggregativeEnteroaggregative E. coliE. coli a cause of persistent, watery diarrhea with vomiting and dehydration in infants. That is autoagglutination in a ‘stacked brick’ arrangement. the bacteria adheres to the intestinal mucosa and elaborates enterotoxins (enteroaggregative heat-stable toxin, EAST). The result is mucosal damage, secretion of large amounts of mucus, and a secretory diarrhea.
  26. 26. ShigellaShigella
  27. 27. Shigella species  Shigella  Contains four species that differ antigenically and, to a lesser extent, biochemically.  S. dysenteriae (Group A)  S. flexneri (Group B)  S. boydii (Group C)  S. sonnei (Group D) bacillary dysentery, shigellosis, bloody feces, intestinal pain, pus  Biochemistry  TSI K/A with NO gas  LIA K/A  Urea –  Motility -  All ferment mannitol except S. dysenteriae  S. sonnei may show delayed lactose fermentation
  28. 28. Shiga toxinShiga toxin 1. Chromosomally encoded 2. Neurotoxic 3. Enterotoxic 4. Cytotoxic EnterotoxicityEnterotoxicity can make the disease clinically appear as a diarrheadiarrhea. The toxin inhibits protein synthesistoxin inhibits protein synthesis (acting on the 60S ribosome and lysing 28S rRNA).
  29. 29. Clinical significanceClinical significance man only "reservoir"man only "reservoir" mostly young childrenmostly young children – fecal to oral contactfecal to oral contact – children to adultschildren to adults transmitted by adult food handlerstransmitted by adult food handlers – unwashed handsunwashed hands
  30. 30. Diagnosis of Shigella infectionDiagnosis of Shigella infection  Specimen: stool.  Culture and Identification  Quick immunological methods: 1. Immunofluorescent “ball” test; 2. Coagglutination.
  31. 31. XLD Agar: Growth of Shigella and Proteus
  32. 32. Only a few types that are commonly associated with characteristic human diseases - 1.1. S. enteritidisS. enteritidis 2.2. S. cholerae-suisS. cholerae-suis 3.3. S. typhiS. typhi SalmonellaSalmonella several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia
  33. 33. Salmonella  Biochemistry  TSI K/A + gas and H2S: S. typhi produces only a small amount of H2S and no gas , and S. paratyphi A produces no H2S  LIA K/K with H2S with S. paratyphi A giving K/A results  Urea –  Motility +  Citrate +/-  Indole -  Virulence factors  Endotoxin – may play a role in intracellular survival  Capsule (for S. typhi and some strains of S. paratyphi)  Adhesions – both fimbrial and non-fimbrial
  34. 34. Salmonella typhiSalmonella typhi • The organism is transmitted from: 1. a human reservoir1. a human reservoir 2. in the water supply (if poor sanitary conditions)2. in the water supply (if poor sanitary conditions) 3. in contaminated food3. in contaminated food The antigenic structures of salmonellae used in serologic typingThe antigenic structures of salmonellae used in serologic typing
  35. 35. Virulence factorsVirulence factors  Endotoxin – may play a role in intracellular survival  Capsule (for S. typhi and some strains of S. paratyphi)  Adhesions – both fimbrial and non-fimbrial  Type III secretion systems and effector molecules – 2 different systems may be found: – One type is involved in promoting entry into intestinal epithelial cells – The other type is involved in the ability of Salmonella to survive inside macrophages  Outer membrane proteins - involved in the ability of Salmonella to survive inside macrophages  Flagella – help bacteria to move through intestinal mucous  Enterotoxin - may be involved in gastroenteritis  Iron capturing ability
  36. 36. XLD Agar: Appearance of Salmonella
  37. 37. DDiagnosisiagnosis  A. SpecimensA. Specimens  a) Enteric fever: blood, bone marrow, stool,a) Enteric fever: blood, bone marrow, stool, urine.urine.  b) Food poisoning: stool, vomitus, suspected food.b) Food poisoning: stool, vomitus, suspected food.  c) Septicemia: blood.c) Septicemia: blood.  B. Culture and identificationB. Culture and identification  C. Widal testC. Widal test
  38. 38. 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 antigens
  39. 39. 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 meningitis K.Pneumoniae On BA K.Pneumoniae on BA and MAC
  40. 40. ProteusProteus  General characteristics: “swarming” phenomenonGeneral characteristics: “swarming” phenomenon on nonselective agaron nonselective agar (P.vulgaris; P.mirabilis and(P.vulgaris; P.mirabilis and P.myxofaciens)P.myxofaciens)  P.vulgaris strains (OX-19, OX-K, OX-2)haveP.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test).common antigen with Rickettsia (Weil-Felix test).  urinary tract infections; food poisoningurinary tract infections; food poisoning..
  41. 41. Proteus spp. Some enteric bacteria are motile. Klebsiella species are not motile, while Proteus species move very actively by means of peritrichous flagella, resulting in "swarming" on solid medium. Some strains of E. coli produce hemolysis on blood plates.
  42. 42. CITROBACTERCITROBACTER Resident of soil, water, stool • C.freundii • UTI and bacteremia
  43. 43. SerratiaSerratia
  44. 44. Serratia  A free-living saprophyte  TSI A/A or K/A; +/- gas (does not ferment lactose)  LIA usually K/K  Citrate +  Motility +  Urea +/-  Has been found in RT and UT infections  Is resistant to many antimicrobics
  45. 45. Special Features: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-) Positive tes e.g. E. coli Negative test e.g. Klebsiella IIndole testndole test Positive Klebsiella, Enterobacter Negative E. coli Citrate Utilization Test Urease Test Positive test Negative test
  46. 46. MMR/R/VVP testP test Results Methyl Red test Voges-Proskauer test Red: Positive MR (E. coli) ellow or orange: Negative MR (Klebsiella) Pink: Positive VP (Klebsiella) No pink: Negative VP (E. coli)
  47. 47. GramGram stainstain OxidaseOxidase NitrateNitrate reductasereductase O/FO/F MacConMacCon keykey SSSS EMBEMB E. coliE. coli -ve rod-ve rod -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF MetallicMetallic sheensheen CitrobacterCitrobacter -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDark KlebsiellaKlebsiella -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDark EnterobacterEnterobacter -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDark SalmonellaSalmonella -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLF/NLF/ H2SH2S ColorlessColorless ShigellaShigella -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLFNLF ColorlessColorless ProteusProteus -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLF/NLF/ H2SH2S ColorlessColorless Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae
  48. 48. TSITSI IndoleIndole MRMR VPVP CitrateCitrate UreaseUrease MotilityMotility E. coliE. coli A/A/-A/A/- +ve+ve +ve+ve -ve-ve -ve-ve -ve-ve MotileMotile CitrobacterCitrobacter freundiifreundii A/A/-A/A/- +ve+ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotile KlebsiellaKlebsiella pneumoniaepneumoniae A/A/-A/A/- -ve-ve -ve-ve +ve+ve +ve+ve +ve+ve NonNon motilemotile EnterobacterEnterobacter cloacaecloacae A/A/-A/A/- -ve-ve -ve-ve +ve+ve +ve+ve +ve+ve MotileMotile SalmonellaSalmonella typhityphi A/Alk/+A/Alk/+ -ve-ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotile ShigellaShigella boydiiboydii A/Alk/-A/Alk/- -ve-ve +ve+ve -ve-ve -ve-ve -ve-ve NonNon motilemotile ProteusProteus mirabilismirabilis A/Alk/+A/Alk/+ -ve-ve +ve+ve -ve-ve +ve+ve +ve+ve MotileMotile SwarwingSwarwing Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae
  49. 49. Oxidase TestOxidase Test PositiveNegative Pseudomonas Enterobacteriacea e  Nitrate test: +ve further reduction to N2  Growth on cetrimide agar: Pale colonies with green pigmentation MacConkey’s agar & TSI Lactose fermenter Pink colonies on MacConkey & acidic butt and slant on TSI colorless colonies on MacConkey & acidic butt alkaline slant onTSI Lactose non-fermenter IMViC test & EMB IMViC ++ - - & black colonies with metalic shines on EMB E.coli IMViC - - ++ Klebsiella No H2S production (no blacking in TSI) H2S production (blacking in TSI) Shigella Urease production +ve Proteus -ve SS agar colorless colonies with black centers Salmonella  O/F test: O+ /F- Motility Not motile Motile