Presentation on theme: "The Enterobacteriaceae Basic Properties"— Presentation transcript:
1 The Enterobacteriaceae Basic Properties Dr. John R. WarrenDepartment of PathologyNorthwestern University Feinberg School of MedicineJune 2007
2 Characteristics of the Enterobacteriaceae Gram-negative rodsGlucose is fermented with strong acid formation and often gasCytochrome oxidase activity is negativeNitrate is reduced to nitrite
3 Gram’s Stain for Bacterial Morphology Crystal violet binds to cell wall peptidoglycan with Gram’s iodine as a mordantSafranin or basic fuchsin counterstains bacterial cells decolorized by alcohol-acetone
4 Gram’s Stain for Bacterial Morphology Thick cell-wall peptidoglycan layer of gram-positive bacteria strongly binds crystal violet and resists decolorization by alcohol-acetoneThin cell-wall peptidoglycan layer of gram-negative bacteria located beneath a thick lipid-rich outer membrane weakly binds crystal violet that is readily removed by alcohol-acetone decolorization
5 Gram’s Stain Procedure Flood surface of smear with crystal violet solutionAfter 1 min thoroughly rinse with cold tap waterFlood smear with Gram’s iodine for 1 minRinse smear with acetone-alcohol decolorizer until no more crystal violet in rinse effluentRinse with cold tap waterFlood smear with safranin (regular Gram’s stain) or basic fuchsin (enhanced Gram’s stain)Dry smear in slide rackMicroscopically examine stained smear using oil-immersion light microscopy
6 Glucose FermentationOxidation-reduction of glucose in the absence of molecular oxygen (anaerobic glycolysis)Energy from hydrolysis of chemical bonds in anaerobic glycolysis captured as high energy phosphate bonds of adenosine triphosphate (ATP)NAD is reduced to NADH2 by accepting electrons during glycolytic conversion of glucose to pyruvateNADH2 in turn reduces pyruvate with oxidation of NADH2 to NAD which supports continued anaerobic glycolysis, and generation from pyruvate of alcohols, carboxylic acids, and CO2 gasEnd products of glucose fermentation: organic acids and CO2 gasFermentation detected by acidification of glucose-containing broth (color change in broth or agar medium containing pH indicators), and (for aerogenic species) production of gas (fractures in agar, gas bubbles in inverted Durham tube)pH indicators: phenol red (yellow at acid pH), methyl red (red at acid pH), neutral red (red at acid pH), bromcresol purple (yellow at acid pH)
7 Spot Cytochrome Oxidase Test The spot cytochrome oxidase test is the first test performed with gram-negative bacteria recovered in cultureThe optimal plate medium for a spot cytochrome oxidase test is a trypticase soy agar (TSA) containing 5% sheep bloodBacterial colonies should be 18 to 24 hr old
8 Spot Cytochrome Oxidase Test In a positive test, bacterial cytochrome oxidase oxidizes the colorless reduced substrate tetramethyl-p-phenylenediamine dihydrochloride (TPDD) forming a dark purple oxidized indophenol productStreak a small portion of bacterial colony to filter paper soaked with a 1% solution of TPDDIf the streak mark turns purple in 10 sec or less, the spot oxidase test is interpreted as positive
9 Nitrate ReductionEnterobacteriaceae extract oxygen from nitrate (NO3) producing nitrite (NO2)NO2 detected by reaction with α-naphthylamine and sulfanilic acid producing a red colored complexAbsence of red color indicates either no reduction of NO3 or reduction to products other than NO2 (denitrification)Confirmation of true negative test: addition of zinc ions which reduce NO3 to NO2 producing a red color in the presence of α-naphthylamine and sulfanilic acid
10 Enterobacteriaceae: Genetic Properties Chromosomal DNA has 39-59% guanine-plus-cytosine (G+C) contentEscherichia coli is the type genus and species of the EnterobacteriaceaeSpecies of Enterobacteriaceae more closely related by evolutionary distance to Escherichia coli than to organisms of other families (Pseudomonadaceae, Aeromonadaceae)
11 Enterobacteriaceae: Major Genera EscherichiaShigellaSalmonellaEdwardsiellaCitrobacterYersiniaKlebsiellaEnterobacterSerratiaProteusMorganellaProvidencia
12 Enterobacteriaceae: Microbiological Properties Gram-negative and rod shaped (bacilli)Ferment rather than oxidize D-glucose with acid and (often) gas productionReduce nitrate to nitriteGrow readily on 5% sheep blood or chocolate agar in carbon dioxide or ambient airGrow anaerobically (facultative anaerobes)
13 Enterobacteriaceae: Microbiological Properties Catalase positive and cytochrome oxidase negativeGrow readily on MacConkey (MAC) and eosin methylene blue (EMB) agarsGrow readily at 35oC except Yersinia (25o-30oC)Motile by peritrichous flagella except Shigella and Klebsiella which are non-motileDo not form spores
14 Enterobacteriaceae: Natural Habitats Environmental sites (soil, water, and plants)Intestines of humans and animals
15 Enterobacteriaceae: 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 patientsInsect (flea) vector (unique for Yersinia pestis)
21 Triple Sugar Iron (TSI) Agar Yeast extract 0.3% (% = grams/100 mL)Beef extract 0.3%Peptone 1.5%Proteose peptone 0.5%Total Protein = 2.6%Lactose %Sucrose1 1.0%Glucose 0.1%Carbohydrate = 2.1%1Absent in Kligler Iron Agar
22 Triple Sugar Iron (TSI) Agar Ferrous sulfateSodium thiosulfateSodium chlorideAgar (1.2%)Phenol redpH = 7.4
23 TSI Reactions of the Enterobacteriaceae Yellow deep, purple slant: acid deep due to glucose fermentation , no lactose or sucrose fermentation with alkaline slant due to production of amine’s from proteinBlack deep, purple slant: acid deep due to glucose fermentation with H2S production, no lactose or sucrose fermentationYellow deep and slant: acid deep and slant due to glucose as well as lactose and/or sucrose fermentationBlack deep and yellow or black slant: acid deep and slant with glucose and lactose and/or sucrose fermentation with H2S productionFracturing or lifting of agar from base of culture tube: CO2 production
25 TSI Reactions of the Enterobacteriaceae A/A + g = acid/acid plus gas (CO2)A/A = acid/acidA/A + g, H2S = acid/acid plus gas, H2SAlk/A = alkaline/acidAlk/A + g = alkaline/acid plus gasAlk/A + g, H2S = alkaline/acid plus gas, H2SAlk/A + g, H2S (w) = alkaline/acid plus gas, H2S (weak)
26 A/A + g Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Enterobacter aerogenesEnterobacter cloacaeSerratia marcescens1, 21Non-lactose, sucrose fermenter255% + g
33 MacConkey (MAC) Agar Peptone 1.7% Polypeptone 0.3% Lactose1 1.0% Bile salts %Crystal violet2Neutral red3Sodium chloride 0.5%Agar %pH=7.11Differential medium for lactose fermentation2Inhibit gram positives and fastidious gram-negatives; MAC agar selective forgram-negatives3Red color at pH < 6.8
36 Eosin Methylene Blue (EMB) Agar (Levine) Peptone 1.0%Lactose1 0.5%Eosin y2Methylene blue2AgarpH = 7.21Modified formula also contains sucrose (0.5%)2Inhibit gram-positives and fastidious gram-negatives; selectivefor gram-negatives. Eosin y and methylene blue form aprecipitate at acid pH; differential for lactose fermentation
39 Bacterial Utilization of Lactose Presence of β-galactoside permease: Transport of β-galactoside (lactose) across the bacterial cell wallPresence of β-galactosidase: Hydrolysis of β-galactoside bond (lactoseglucose + galactose)ONPG: Orthonitrophenyl-β-D-galacto-pyranoside
40 Differential Reactions of the Enterobacteriaceae by TSI, ONPG, and MAC Escherichia coli Red colonies,(A/A, ONPG+) pittedKlebsiella Red colonies,(A/A, ONPG+) mucoidEnterobacter Red colonies(A/A, ONPG+)Citrobacter Red or colorless(A/A or Alk/A, ONPG+) coloniesSerratia Colorless colonies1K. pneumoniae, indole –, K. oxytoca, indole +2C. freundii, indole – and H2S +, C. koseri, indole + and H2S –
41 Differential Reactions of the Enterobacteriaceae by TSI, ONPG, and MAC Shigella Colorless Colonies(Alk/A; ONPG – A, B, and C1; ONPG + D1)Salmonella Colorless Colonies(Alk/A + H2S; ONPG –)Proteus Colorless Colonies(Alk/A + H2S2; ONPG –)Edwardsiella tarda Colorless Colonies(Alk/A + H2S; ONPG–)Yersinia Colorless Colonies(A/A, ONPG +)1Shigella A, B, and C, ornithine –; Shigella D, ornithine +2Proteus mirabilis. P. vulgaris sucrose + with A/A + H2S onTSI
42 Differential Reactions of the Enterobacteriaceae by EMB Escherichia coli Colonies with metallic green sheenKlebsiella Colonies with precipitate (ppt)and mucoid appearanceEnterobacter Colonies with pptCitrobacter Colonies with/without pptSerratia Colonies without pptShigella Colonies without pptSalmonella Colonies without pptProteus Colonies without pptYersinia Colonies without ppt
45 XLD Agar: Growth of Salmonella Salmonella selective due to bile salt.Xylose fermentation (except Salmonella serotype Paratyphi A) acidifies agar activating lysine decarboxylase. With xylose depletion fermentation ceases, and colonies of Salmonella (except S. Paratyphi A) alkalinize the agar due to amines from lysine decarboxylation.Xylose fermentation provides H+ for H2S production (except S. Paratyphi A).
46 XLD Agar: Appearance of Salmonella Ferric ammonium citrate present in XLD agar reacts with H2S gas and forms black precipitates within colonies of Salmonella.Agar becomes red-purple due to alkaline pH produced by amines.Back colonies growing on red-purple agar-presumptive for Salmonella.
49 XLD Agar: Growth of Escherichia coli and Klebsiella pneumoniae Escherichia coli and Klebsiella pneumoniae arelysine-positive coliforms that are also lactoseand sucrose fermenters. The high lactose andsucrose concentrations result in strong acidproduction, which quenches amines producedby lysine decarboxylation. Colonies and agarappear bright yellow. Neither Escherichia colinor Klebsiella pneumoniae produce H2S.
50 XLD Agar: Growth of Shigella and Proteus Shigella species do not ferment xylose, lactose, and sucrose, do not decarboxylate lysine, and do not produce H2S. Colonies appear colorless.Proteus mirabilis ferments xylose, and thereby provides H+ for H2S production. Colonies appear black on an agar unchanged in color (Proteus deaminates rather than decarboxylates amino acids). Proteus vulgaris ferments sucrose, and colonies appear black on a yellow agar.
54 HE Agar: Growth of Enteric Pathogens and Commensals High bile salt concentration inhibits growth of gram-positive and gram-negative intestinal commensals, and thereby selects for pathogenic Salmonella (bile-resistant growth) present in fecal specimens.Salmonella species as non-lactose and non-sucrose fermenters that produce H2S form colorless colonies with black centers.Shigella species (non-lactose and non-sucrose fermenters, no H2S production) form colorless colonies.Lactose and sucrose fermenters (E. coli, K. pneumoniae) form orange to yellow colonies due to acid production.
57 Salmonella-Shigella (SS) Agar Bile salts, citrates, and brilliant green dye inhibit gram-positives and most gram-negative coliformsLactose the sole carbohydrateSodium thiosulfate a source of sulfur for H2S productionSalmonella forms transparent colonies with black centersShigella forms transparent colonies without blackeningLactose fermentative Enterobacteriaceae produce pink to red colonies with bile precipitate for strong lactose fermenters
58 Use of Selective-Differential Agars for Recovery of the Enterobacteriaceae from Different Types of SpecimensFeces1: MAC or EMB + XLD &/or SS or HE2Sputum and Urine1: MAC or EMBWound3:MAC or EMBPeritoneal and pleural fluid4: MAC or EMBSubculture of blood positive for gram-negative’s in broth culture4: MAC or EMBCSF, pericardial fluid, synovial fluid, bone marrow5: Not required1Heavy population of commensal bacteria2Utilized with enrichment broth containing selenite or mannitol todifferentially inhibit enteric commensals3Commensal bacteria (skin) and frequent polymicrobial etiology4Possible polymicrobial etiology (normally sterile fluids)5Normally sterile, unimicrobial etiology predominant
59 Selectivity of Differential Agars for Salmonella1 and Shigella2 HE or SS agar (absence of lactose fermentation1,2, H2S production1)XLD agar (absence of lactose fermentation1,2, H2S production1, lysine decarboxylation1)MAC or EMB agar (absence of lactose fermentation1,2)TSI agar (glucose fermentation1,2, absence of lactose fermentation1,2, H2S production1)Descending Order of Selectivity for Salmonella and Shigella
60 Recommended Reading Winn, W., Jr., Allen, S., Janda, W., Koneman, E., Procop, G., Schrenckenberger, P., Woods, G.Koneman’s Color Atlas and Textbook ofDiagnostic Microbiology, Sixth Edition,Lippincott Williams & Wilkins, 2006:Chapter 5. Medical Bacteriology: Taxonomy, Morphology, Physiology, and Virulence.Chapter 6. The Enterobacteriaceae.
61 Recommended Reading Murray, P., Baron, E., Jorgensen, J., Landry, M., Pfaller, M.Manual of Clinical Microbiology, 9thEdition, ASM Press, 2007:Farmer, J.J., III, Boatwright, K.D., and Janda J.M. Chapter 42. Enterobacteriaceae: Introduction and Identification