Enterobacteriaceae 120413233647-phpapp02

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Enterobacteriaceae 120413233647-phpapp02

  1. 1. Dr.T.V.Rao MD Dr.T.V.Rao MD 2/20/2014
  2. 2. Dr.T.V.Rao MD 2/20/2014
  3. 3. 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 2/20/2014
  4. 4. • Are facultative anaerobes • If motile, motility is by peritrichous flagella • 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
  5. 5. • Most grow well on a variety of lab media including a lot of selective and differential media originally developed for the selective isolation of enteric pathogens.  Most of this media is selective by incorporation of dyes and bile salts that inhibit G+ organisms and may suppress the growth of nonpathogenic species of Enterobacteriaceae.  Many are differential on the basis of whether or not the organisms ferment lactose and/or produce H2S.
  6. 6.  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 2/20/2014
  7. 7.  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 2/20/2014
  8. 8. Gram negative bacilli or coccobacilli Non-spore forming Colony morphology on BAP or CA of little value, as they look the same, except for Klebsiella Selective and differential media are used for initial colony evaluation (ex. MacConkey, HE, XLD agars)
  9. 9.  Some • • • • Enterobacteriaceae are true pathogens Salmonella spp. Shigella spp. Yersinia spp. Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)  Most members of the Enterobacteriaceae are opportunistic or cause secondary infections of wounds, the urinary and respiratory tracts, and the circulatory system e.g. E. coli.  Enterobacteriaceae divided into TWO main groups according to action on LACTOSE • Lactose Fermenters (LF)  E. coli, Citrobacter, Klebsiella, Enterobacter • Lactose Non-Fermenters (LNF)  Salmonella, Shigella, Proteus, Yersinia Dr.T.V.Rao MD 2/20/2014
  10. 10. Enterobacteriaceae Lactose fermenters E. coli, Citrobacter, Klebsiella, Enterobacter Non-lactose fermenter Salmonell, Shigella 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 Dr.T.V.Rao MD In addition to Triple Sugar 2/20/2014 Iron (TSI) agar
  11. 11. Oxidase Test Negative Positive Enterobacteriaceae Pseudomonas MacConkey’s agar & TSI  O/F test: O+/F Nitrate test: +ve further reduction to N2 Pink colonies on MacConkey colorless colonies on MacConkey & acidic butt and slant on TSI & acidic butt alkaline slant onTSI  Growth on cetrimide agar: Lactose non-fermenter Pale colonies with green pigmentation Lactose fermenter IMViC test & EMB IMViC ++ - & black colonies with metalic shines on EMB E.coli No H2S production (no blacking in TSI) IMViC - - ++ Shigella Urease production +ve Motility Not motile H2S production (blacking in TSI) -ve SS agar Motile colorless colonies with black centers Proteus Dr.T.V.Rao MD 2/20/2014 Salmonella
  12. 12.  MacConkey agar is selective & differential medium for Enterobacteriaceae MacConkey Agar Contains Bile salts Crystal violet Inhibit growth of G+ve bacteria Lactose Cause of differential Cause of selectivity Lactose feremnters Pink coloniesDr.T.V.Rao MD Neutral red pH indicator Acidic: Pink Lactose non feremnters colorless colonies 2/20/2014
  13. 13. Enterobacteriaceae Lactose Non-Fermenters Lactose Fermenters No acid Acid Neutral red Colorless colonies Pink colonies Escherichia coli Klebsiella spp Enterobacter spp Citrobacter spp Dr.T.V.Rao MD Salmonella spp Schigella spp Proteus spp 2/20/2014 Yersinina spp
  14. 14.   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 2 1 3 4 5 Flame & Cool Flame & Cool Dr.T.V.Rao MD 2/20/2014
  15. 15. Colorless colonies Uninoculated plate Lactose non feremters Salmonella, Shigella, Proteus Dr.T.V.Rao MD Pink colonies Lactose feremters E. coli, Citrobacter Klebsiella, Enterobacter 2/20/2014
  16. 16. Growth of Enterobacteriaceae on SS agar Both are lactose fermenters Both Salmonella sp. & Proteus product H2S Pseudomonas colonies are nearly colorless
  17. 17. Coli-type colonies are very dark, almost black e.g. E. coli
  18. 18. Principle Acidic pathway Glucose Or Acety methyl carbinol (ACETOIN) Mixed acids  pH less than 4.4 Barrit’s A Barrit;s B Methyl Red indicator Red color Neutral pathway VP positive MR positive Klebsiella Dr.T.V.Rao MD 2/20/2014 E. coli Pink color
  19. 19. Indole, Methyl Red, VogesProsakaur, Citrate (IMViC) Tests: • The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions • The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae. Dr.T.V.Rao MD 2/20/2014
  20. 20. Principle     Certain microorganisms can metabolize tryptophan by tryptophanase The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia The presence of indole is detected by addition of Kovac's reagent. Tryptophane amino acids Tryptophanase Indole + Pyurvic acid + NH3 Kovac’s Reagent Red color Dr.T.V.Rao MD organic layer` in upper 2/20/2014
  21. 21.  Method:  Inoculate tryptone water with the tested microorganism  Incubate at 37 C for 24 hours  After incubation interval, add 1 ml Kovacs reagent, shake the tube gently and read immediately Dr.T.V.Rao MD 2/20/2014
  22. 22. Negative test e.g. Klebsiella  Result:  A bright pink color in the top layer indicates the presence of indole  The absence of color means that indole was not produced i.e. indole is negative  Special Features:  Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-). Dr.T.V.Rao MD 2/20/2014 Positive test e.g. E. coli
  23. 23.  Inoculate the tested organism into One tube of MRVP broth  Incubate the tubes at 37 C for 24 hours  AFTER INCUBATION: Pour 1/3 of the suspension into a clean nonsterile tube:  Run the MR test in the tube with 2/3, and the VP test in the open tube with 1/3.  For methyl red: Add 6-8 drops of methyl red reagent.  For Voges-Proskauer: Add 12 drops of Barritt's A (-naphthol), mix, 4 drops of Barritt's B (40% KOH), mix  Let sit, undisturbed, for at least 1hour Dr.T.V.Rao MD 2/20/2014
  24. 24. Results Voges-Proskauer test Methyl Red test Pink: Positive VP (Klebsiella) Red: Positive MR (E. coli) Yellow or orange: Negative MR (Klebsiella) No pink: Negative VP (E. coli) Dr.T.V.Rao MD 2/20/2014
  25. 25. Principle: Citrate Citrate Utilization Test Pyruvate Na2CO3 CO2 + Na + H2O Alkaline,↑pH Simmone’s Citrate media Contains Citrate as a sole of C source Bromothymol blue Positive test Blue colour Positive test: Klebsiella, Enterobacter, Citrobacter Negative test: E. coli Dr.T.V.Rao MD 2/20/2014
  26. 26. Method  Streak a Simmon's Citrate agar slant with the organism  Incubate at 37 C for 24 hours. Dr.T.V.Rao MD 2/20/2014
  27. 27. Result  Examine for growth (+)  Growth on the medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue Positive Klebsiella, Enterobacter 2/20/2014 Dr.T.V.Rao MD Negative E. coli
  28. 28. Principle     Urea Urea agar contains urea and phenol red Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3 Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium. ↑ in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease Urease H2O CO2 + NH3 NH4 OH ↑ in pH Phenol Red Method  Streak a urea agar tube with the organism  incubate at 37 C for 24 h Dr.T.V.Rao MD 2/20/2014 Pink Positive test
  29. 29. Result   If color of medium turns from yellow to pink indicates positive test. Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h Positive test Dr.T.V.Rao MD 2/20/2014 Negative test
  30. 30. TSI contains  • Three different types of sugars   Lactose (10 part)  • Glucose (1 part) Sucrose (10 part) Phenol red (acidic: Yellow) TSI dispensed in tubes with equal butt & slant Principle   • To determine the ability of an organism to attack a specific carbohydrate incorporated into a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide production. Dr.T.V.Rao MD 2/20/2014
  31. 31. Method:  • Inoculate TSI medium with an organism by inoculating needle by stabbing the butt and streaking the slant • Incubate at 37 C for 24 hours Dr.T.V.Rao MD 2/20/2014
  32. 32. Reaction on TSI Butt color Slant color Yellow Red Yellow Red Yellow Yellow Red Red H2 S Negative Positive black in butt Negative Negative Result s Result Example A/Alk/(Glucose fermented) LNF e.g. Shigella A/Alk/+ (Glucose fermented with H2S) LNF e.g. Salmonella & Proteus A/A/(three sugars are fermented) LF e.g. E. coli, Klebsiella, Enterobacter Alk/Alk/(No action on sugars) Non fermenter e.g. Pseudomonas
  33. 33. Results Example Reaction on TSI Butt color Red Slant color Red H2S Negative Negative Yellow Yellow Yellow Red Red Yellow Positive black in butt Negative Result Alk/Alk/(No action on sugars) A/Alk/(Glucose fermented without H2S) A/Alk/+ (Glucose fermented with H2S) Non fermenter e.g. Pseudomonas LNF e.g. Shigella LNF e.g. Salmonella & Proteus LF e.g. E. coli, Klebsiella, Dr.T.V.Rao MDEnterobacter 2/20/2014 A/A/(three sugars are fermented)
  34. 34. Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae Gram stain Oxidase Nitrate reductase O/F MacCon key SS EMB E. coli -ve rod -ve +ve O+/F+ LF LF Metallic sheen Citrobacter -ve rods -ve +ve O+/F+ LF LF Dark Klebsiella -ve rods -ve +ve O+/F+ LF LF Dark Enterobacter -ve rods -ve +ve O+/F+ LF LF Dark Salmonella -ve rods -ve +ve O+/F+ NLF NLF/ H2S Colorless Shigella -ve rods -ve +ve O+/F+ NLF NLF Colorless Proteus -ve rods -ve +ve O+/F+ NLF NLF/ H2S Colorless Dr.T.V.Rao MD 2/20/2014
  35. 35. Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae TSI Indole MR VP Citrate Urease Motility E. coli A/A/- +ve +ve -ve -ve -ve Motile Citrobacter freundii A/A/- +ve +ve -ve +ve -ve Motile Klebsiella pneumoniae A/A/- -ve -ve +ve +ve +ve Non motile Enterobacter cloacae A/A/- -ve -ve +ve +ve +ve Motile Salmonella typhi A/Alk/+ -ve +ve -ve +ve -ve Motile Shigella boydii A/Alk/- -ve +ve -ve -ve -ve Non motile Proteus mirabilis A/Alk/+ -ve +ve -ve +ve +ve Motile Swarwing Dr.T.V.Rao MD 2/20/2014
  36. 36. Programme Created from Web Sources for skills in Identification of Enterobacteriaceae email  doctortvrao@gmail.com Dr.T.V.Rao MD 2/20/2014

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