Biochemical Tests in Enterobacteriaceae

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Biochemical Tests in Enterobacteriaceae

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Biochemical Tests in Enterobacteriaceae

  1. 1. Biochemical Tests Enterobacteriaceae Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Tests To KnowCommon Study Tests Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production Dr.T.V.Rao MD 2
  3. 3. Initial Grouping of the Enterobacteriaceae (VP=Voges Proskauer, PDA=Phenylalanine Deaminase)GENERA VP PDAKlebsiella POSITIVE NEGATIVEEnterobacter POSITIVE NEGATIVESerratia POSITIVE NEGATIVEHafnia POSITIVE NEGATIVEPantoea POSITIVE NEGATIVE Dr.T.V.Rao MD 3
  4. 4. Initial Grouping of the EnterobacteriaceaeGENERA VP PDAProteus1 NEGATIVE POSITIVEMorganella NEGATIVE POSITIVEProvidencia NEGATIVE POSITIVE1Proteus mirabilis: 50% of MD Dr.T.V.Rao strains VP positive 4
  5. 5. Initial Grouping of the EnterobacteriaceaeGENERA VP PDAEscherichia NEGATIVE NEGATIVEShigella NEGATIVE NEGATIVEEdwardsiella NEGATIVE NEGATIVESalmonella NEGATIVE NEGATIVECitrobacter NEGATIVE NEGATIVEYersinia NEGATIVE NEGATIVE Dr.T.V.Rao MD 5
  6. 6. Initial Grouping of the Enterobacteriaceae1GENERA INDOLE CITRATEEscherichia POSITIVE NEGATIVE 2Shigella POSITIVE NEGATIVE 3Yersinia POSITIVE NEGATIVEEdwardsiella POSTIVE NEGATIVE1 VP negative, PDA negative2 Shigella groups A, B, and C variably positivefor indole production (25-50%), group DShigella negative. Dr.T.V.Rao MD 63 Yersinia enterocolitica 50% positive
  7. 7. Initial Grouping of the Enterobacteriaceae 1GENERA INDOLE CITRATESalmonella NEGATIVE POSITIVE2Citrobacter NEGATIVE POSITIVE1 VP negative, PDA negative2 Salmonella serotype Paratyphi A and Typhinegative Dr.T.V.Rao MD 7
  8. 8. Key Characteristics of the Enterobacteriaceae TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AREcoli A/A + +   +    + + +/ /  + /     +       Shi Ak/A- AC          + Shi Ak/D A +      + + + Ed Ak/ A + + +     Sal Ak/ A + + + + + + +/  /     +  + Cit A/A +/ +/ Ak/ + + + + AYer A/A +    +/    Dr.T.V.Rao MD +/  RT (1)  +  8(1) RT=room temperature
  9. 9. Key Characteristics of the Enterobacteriaceae TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR      Kle A/Apne + + + + + +     Kle A/Aoxy + + + + + + +     En A/Aaer + + + + + + +    En A/A +/cloa + + + + + + +     Serr A/A(1) + + + + + + +      Haf Ak/ A + + + + + +Pan A/A + /+  +/ /+ +/ /+ /+     Alk/ A(1) Produces DNase, lipase, and gelatinase Dr.T.V.Rao MD 9
  10. 10. Key Characteristics of the Enterobacteriaceae TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR  + +  + + +s  + Prot Ak/ +/ +/mir Aa  +/ +  + /+ + + +s   Prot A/Avulg  +   +  + + +  +Mor Ak/ A     + + + + +   Pro Ak/v As = swarming motility Dr.T.V.Rao MD 10
  11. 11. Biochemical Characteristics of Escherichia coli and Shigella E. coli E. coli O157:H7 ShigellaTSI A/Ag A/Ag Alk/ALactose + + –ONPG + + –/+1Sorbitol + – +/–Indole + + +/–Methyl re + + +VP – – –Citrate – – –Lysine + + –Motility + + Dr.T.V.Rao MD – 11
  12. 12. Biochemical Characteristics of Salmonella Most Serotypes Typhi Paratyphi ATSI Alk/A Alk/A Alk/AH2S (TSI) + + (weak) –Citrate + – –Lysine + + –Ornithine + – +Dulcitol + – +Rhamnose + – +Indole – – –Methyl red + + +VP – – – Dr.T.V.Rao MD 12
  13. 13. IMViC ReactionsI = Indole production from tryptophanM = methyl red test in which acidification of glucose broth (pH<4.4) due to formation of mixed carboxylic acids (lactic, acetic, formic) from pyruvate results in pH indicator methyl red turning redVi = positive Voges-Proskauer test due to formation of acetoin from pyruvate in glucose brothC = ability to utilize citrate as single carbon source Dr.T.V.Rao MD 13
  14. 14. Indole ReactionEnterobacteriaceae that possesstryptophanase can utilize tryptophan bydeamination and hydrolytic removal of theindole side chain.Free indole is detected by p-dimethylamino-benzaldehyde, whose aldehyde group reactswith indole forming a red-colored complex.Production of indole from tryptophan is animportant biochemical property of Escherichiacoli, many strains of group A, B, and CShigella, Edwardsiella tarda, Klebsiellaoxytoca, and Proteus vulgaris. Dr.T.V.Rao MD 14
  15. 15. Indole TestHow to Perform Test: Inoculate Tryptone broth with inoculating loop.Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species’ ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one.Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color.Reading Results: Kovac’s reagent reacts with indole and Dr.T.V.Rao MD 15 creates a red color at the top part of the test tube.
  16. 16. Reading the Result Indole Dr.T.V.Rao MD 16
  17. 17. Methyl Red/Voges Proskauer (MR/VP)How to Perform Tests: Inoculate 2 glucose broths with inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube.Properties they test for: Both tests are used to help differentiate species of the family Enterobacteriaceae. MR—tests for acid end products from glucose fermentation. VP—tests for acetoin production from glucose fermentation.Media and Reagents Used: Glucose Broth Methyl Red indicator for acid Voges Proskauer reagents—A: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water. Dr.T.V.Rao MD 17
  18. 18. Voges-Proskauer ReactionAcetoin and butylene glycol aredetected by oxidation to diacteyl at analkaline pH, and the addition of -naphthol which forms a red-coloredcomplex with diacetyl.The production of acetoin and butyleneglycol by glucose fermentation is animportant biochemical property usedfor the identification ofKlebsiella, Enterobacter, and Serratia. Dr.T.V.Rao MD 18
  19. 19. MR/VP continuedReading Results: MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production) VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change. Methyl Red: left – and right + MD Dr.T.V.Rao VP: left + and right – 19
  20. 20. Citrate UtilizationCitrate is utilized by several of theEnterobacteriaceae as a singlecarbon source. To test this abilitybacteria are incubated in mediumthat contains only citrate as asource of carbon. Ammonium phosphate is availableas a nitrogen source. Dr.T.V.Rao MD 20
  21. 21. Citrate TestHow to Perform Test: Inoculate slant with inoculating loop.Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source.Media and Reagents Used: Simmon’s Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator—bromthymol blue. Dr.T.V.Rao MD 21
  22. 22. Citrate Test Reading Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a – result remains greenLeft positive and right negative. Dr.T.V.Rao MD 22
  23. 23. IMViC Reactions I M Vi CEscherichia coli + + – –Edwardsiella tarda + + – –Proteus vulgaris + + – –Klebsiella pneumoniae – – + +Klebsiella oxytoca + – + +Enterobacter spp. – – + +Serratia marcescens – – + +Citrobacter freundii – + – +Citrobacter koseri + + Dr.T.V.Rao MD – + 23
  24. 24. Urease-Producing EnterobacteriaceaeProteusMorganellaProvidencia rettgeriKlebsiella pneumoniaeKlebsiella oxytocaEnterobacter cloacaeYersinia enterocolitica Dr.T.V.Rao MD 24
  25. 25. Urea HydrolysisHow to Perform Test: Inoculate Urea broth with inoculating loop.Property it tests for: This test is done to determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease.Media and Reagents Used: Urea broth contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator.Dr.T.V.Rao MD 25
  26. 26. Urease Test Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made. Dr.T.V.Rao MD 26
  27. 27. Reactions for Identification of Genera and Species1Decarboxylation of amino acidsMotilityUrease activityHydrogen sulfide (H2S) production1Voges-Proskauer, phenylalaninedeaminase, indole, and citrate reactions areuseful to both cluster Enterobacteriaceaeand identify to genus and species. Dr.T.V.Rao MD 27
  28. 28. Amino Acid DecarboxylationEnterobacteriaceae containdecarboxylases with substratespecificity for amino acids, and aredetected using Moeller decarboxylasebroth overlayed with mineral oil foranaerobiosis.Moeller broth contains glucose forfermentation, peptone and beef extract,an amino acid, pyridoxal, and the pHindicator bromcresol purple. Dr.T.V.Rao MD 28
  29. 29. Amino Acid DecarboxylationIf an Enterobacteriaceae contains amino acid decarboxylase, amines produced by decarboxylase action cause an alkaline pH, and bromcresol purple turns purple.Lysine, ornithine, and arginine areutilized. A base broth without aminoacid is included in which glucosefermentation acidifies the broth, turningthe bromcresol purple yellow. Dr.T.V.Rao MD 29
  30. 30. Amino Acid Decarboxylation 1 Lysine → Cadaverine Ornithine → Putrescine Arginine → Citrulline → Ornithine → Putrescine1Conversionof arginine to citrulline is a dihydrolase reaction Dr.T.V.Rao MD 30
  31. 31. Amino Acid DecarboxylationTube Amino Acid Color InterpretationBase None Yellow Broth acidified1 1 Lysine Purple Positive 2 Ornithine Yellow Negative 3 Arginine Yellow Negative1Indicates organism is a viable glucose fermenter, and pH of broth medium sufficiently acidified to activate decarboxylase enzymes. Dr.T.V.Rao MD 31
  32. 32. Amino Acid DecarboxylationDecarboxylation patterns are essentialfor the genus identification ofKlebsiella, Enterobacter, Escherichia,and Salmonella.Decarboxylation patterns are alsoessential for the species identificationof Enterobacter aerogenes,Enterobacter cloacae, Proteus mirabilis,and Shigella sonnei. Dr.T.V.Rao MD 32
  33. 33. Amino Acid Decarboxylation Lys Orn ArgKlebsiella + – –Enterobacter +/– + +/–Escherichia + +/– –/+Salmonella + + + Dr.T.V.Rao MD 33
  34. 34. Amino Acid Decarboxylation Lys Orn ArgE. aerogenes + + –E. cloacae – + +P. Mirabilis – + –P. vulgaris – – –Shigella D – + –Shigella A-C – – _ Dr.T.V.Rao MD 34
  35. 35. H2S-Producing EnterobacteriaceaeSalmonellaEdwardsiellaCitrobacterProteus Dr.T.V.Rao MD 35
  36. 36. Hydrogen Sulfide (H2S)In presence of H+ and a sulfur source(sodium thiosulfate, sulfur-containingamino acids and proteins) manyEnterobacteriaceae produce thecolorless gas H2S.For detection of H2S a heavy-metal (ironor lead) compound is present thatreacts with H2S to form black-coloredferrous sulfide. Dr.T.V.Rao MD 36
  37. 37. Systems for H2S Detection1Lead acetate paperSIM tube (peptonized iron)Hektoen and SS2 agar (ferric ammonium citrate)XLD3 agar (ferric ammonium citrate)Triple-sugar-iron agar (ferrous sulfate)1In order of decreasing sensitivity2Salmonella-Shigella3Xylose-lysine-deoxycholate Dr.T.V.Rao MD 37
  38. 38. Bacterial MotilityMany but not all Enterobacteriaceae demonstrate flagellar motility.Motility can be measured by use of <0.4% semisolid (soft) agar or microscopic examination of drops of broth containing bacteria and ―hanging‖ from cover slips.Shigella and Klebsiella are non- motile, and Yersinia is non-motile at 35oC but motile at 22o-25oC. Dr.T.V.Rao MD 38
  39. 39. Motility AgarsSulfide-indole-motility (SIM) is asemisolid motility agar that containspeptonized iron for detection of H2Sand tryptophan for indole production.Pure motility agar lacks an H2Sindicator and tryptophan for indoleproduction, and contains tetrazoliumsalts that are reduced to red formazancomplexes to enhance visualassessment of motility. Dr.T.V.Rao MD 39
  40. 40. Additional Biochemical Reactions for the Enterobacteriaceae1 Fermentation of mannitol, dulcitol, salicin, adonitol, inositol, sorbitol, arabinose, raffinose, rhamnose, maltose, xylose, trehalose, cellobiose, alpha- methyl –D-glucoside, erythritol, melibiose, arabitol, glycerol, mucate, and mannoseUtilization of malonate, acetate, and tartrateGelatin hydrolysis, esculin hydrolysis, lipase, and DNaseGrowth in KCNYellow pigment1JJ Farmer, Enterobacteriaceae: Introduction and Identification, ASM Manual, 8th Edition (2003). Dr.T.V.Rao MD 40
  41. 41. Programme Created for Medical and Paramedical students in Microbiology Email doctortvrao@gmail.com Dr.T.V.Rao MD 41

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