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The Enterobacteriaceae Basic Properties Dr. John R. Warren Department of Pathology Northwestern University Feinberg School of Medicine June 2007.

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Presentation on theme: "The Enterobacteriaceae Basic Properties Dr. John R. Warren Department of Pathology Northwestern University Feinberg School of Medicine June 2007."— Presentation transcript:

1 The Enterobacteriaceae Basic Properties Dr. John R. Warren Department of Pathology Northwestern University Feinberg School of Medicine June 2007

2 Characteristics of the Enterobacteriaceae Gram-negative rods Glucose is fermented with strong acid formation and often gas Cytochrome oxidase activity is negative Nitrate is reduced to nitrite

3 Gram’s Stain for Bacterial Morphology Crystal violet binds to cell wall peptidoglycan with Gram’s iodine as a mordant Safranin 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- acetone Thin 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 solution After 1 min thoroughly rinse with cold tap water Flood smear with Gram’s iodine for 1 min Rinse smear with acetone-alcohol decolorizer until no more crystal violet in rinse effluent Rinse with cold tap water Flood smear with safranin (regular Gram’s stain) or basic fuchsin (enhanced Gram’s stain) Rinse with cold tap water Dry smear in slide rack Microscopically examine stained smear using oil- immersion light microscopy

6 Glucose Fermentation Oxidation-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 NADH 2 by accepting electrons during glycolytic conversion of glucose to pyruvate NADH 2 in turn reduces pyruvate with oxidation of NADH 2 to NAD which supports continued anaerobic glycolysis, and generation from pyruvate of alcohols, carboxylic acids, and CO 2 gas End products of glucose fermentation: organic acids and CO 2 gas Fermentation 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 culture The optimal plate medium for a spot cytochrome oxidase test is a trypticase soy agar (TSA) containing 5% sheep blood Bacterial 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 product Streak a small portion of bacterial colony to filter paper soaked with a 1% solution of TPDD If the streak mark turns purple in 10 sec or less, the spot oxidase test is interpreted as positive

9 Nitrate Reduction Enterobacteriaceae extract oxygen from nitrate (NO 3 ) producing nitrite (NO 2 ) NO 2 detected by reaction with α- naphthylamine and sulfanilic acid producing a red colored complex Absence of red color indicates either no reduction of NO 3 or reduction to products other than NO 2 (denitrification) Confirmation of true negative test: addition of zinc ions which reduce NO 3 to NO 2 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) 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)

11 Enterobacteriaceae: Major Genera Escherichia Shigella Salmonella Edwardsiella Citrobacter Yersinia Klebsiella Enterobacter Serratia Proteus Morganella Providencia

12 Enterobacteriaceae: Microbiological Properties Gram-negative and rod shaped (bacilli) Ferment rather than oxidize D-glucose with acid and (often) gas production Reduce nitrate to nitrite Grow readily on 5% sheep blood or chocolate agar in carbon dioxide or ambient air Grow anaerobically (facultative anaerobes)

13 Enterobacteriaceae: Microbiological Properties Catalase positive and cytochrome oxidase negative Grow readily on MacConkey (MAC) and eosin methylene blue (EMB) agars Grow readily at 35 o C except Yersinia (25 o - 30 o C) Motile by peritrichous flagella except Shigella and Klebsiella which are non-motile Do 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 patients Insect (flea) vector (unique for Yersinia pestis)

16 Enterobacteriaceae: Types of Infectious Disease Intestinal (diarrheal) infection Extraintestinal infection Urinary tract (primarily cystitis) Respiratory (nosocomial pneumonia) Wound (surgical wound infection) Bloodstream (gram-negative bacteremia) Central nervous system (neonatal meningitis)

17 Enterobacteriaceae: Urinary Tract Infection, Pneumonia Urinary tract infection: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis Pneumonia: Enterobacter spp., Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis

18 Enterobacteriaceae: Wound Infection, Bacteremia Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis

19 Enterobacteriaceae: Nosocomial Infections in the United States 1986-1989 and 1990-1996 1 Escherichia coli27,871 (13.7%) Enterobacter spp.12,757 (6.2%) Klebsiella pneumoniae11,015 (5.4%) Proteus mirabilis 4,662 (2.3%) Serratia marcescens 3,010 (1.5%) Citrobacter spp. 2,912 (1.4%) 1 Enteric Reference Laboratory, Centers for Disease Control and Prevention

20 Enterobacteriaceae: Intestinal Infection Shigella sonnei (serogroup D) Salmonella serotype Enteritidis Salmonella serotype Typhimurium Shigella flexneri (serogroup B) Escherichia coli O157:H7 Yersinia enterocolitica

21 Triple Sugar Iron (TSI) Agar Yeast extract0.3% (% = grams/100 mL) Beef extract0.3% Peptone1.5% Proteose peptone0.5% Total Protein = 2.6% Lactose 1.0% Sucrose 1 1.0% Glucose0.1% Carbohydrate = 2.1% 1 Absent in Kligler Iron Agar

22 Triple Sugar Iron (TSI) Agar Ferrous sulfate Sodium thiosulfate Sodium chloride Agar (1.2%) Phenol red pH = 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 protein Black deep, purple slant: acid deep due to glucose fermentation with H 2 S production, no lactose or sucrose fermentation Yellow deep and slant: acid deep and slant due to glucose as well as lactose and/or sucrose fermentation Black deep and yellow or black slant: acid deep and slant with glucose and lactose and/or sucrose fermentation with H 2 S production Fracturing or lifting of agar from base of culture tube: CO2 production


25 TSI Reactions of the Enterobacteriaceae A/A + g = acid/acid plus gas (CO 2 ) A/A = acid/acid A/A + g, H 2 S = acid/acid plus gas, H 2 S Alk/A = alkaline/acid Alk/A + g = alkaline/acid plus gas Alk/A + g, H 2 S = alkaline/acid plus gas, H 2 S Alk/A + g, H 2 S (w) = alkaline/acid plus gas, H 2 S (weak)

26 A/A + g Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Enterobacter aerogenes Enterobacter cloacae Serratia marcescens 1, 2 1 Non-lactose, sucrose fermenter 2 55% + g

27 A/A Serratia marcescens 1, 2 Yersinia enterocolitica 2 1 45% of strains 2 Non-lactose, sucrose fermenter

28 A/A + g, H 2 S Citrobacter freundii Proteus vulgaris 1 1 Non-lactose, sucrose fermenter

29 Alk/A Shigella Providencia

30 Alk/A + g Salmonella serotype Paratyphi A

31 Alk/A + g, H 2 S Salmonella (most serotypes) Proteus mirabilis Edwardsiella tarda

32 Alk/A + g, H 2 S (w) Salmonella serotype Typhi

33 MacConkey (MAC) Agar Peptone1.7% Polypeptone0.3% Lactose 1 1.0% Bile salts 2 0.15% Crystal violet 2 Neutral red 3 Sodium chloride0.5% Agar 1.35% pH=7.1 1 Differential medium for lactose fermentation 2 Inhibit gram positives and fastidious gram-negatives; MAC agar selective for gram-negatives 3 Red color at pH < 6.8



36 Eosin Methylene Blue (EMB) Agar (Levine) Peptone1.0% Lactose 1 0.5% Eosin y 2 Methylene blue 2 Agar pH = 7.2 1 Modified formula also contains sucrose (0.5%) 2 Inhibit gram-positives and fastidious gram-negatives; selective for gram-negatives. Eosin y and methylene blue form a precipitate 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 wall Presence of β-galactosidase: Hydrolysis of β-galactoside bond (lactose  glucose + 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+) pitted Klebsiella 1 Red colonies, (A/A, ONPG+) mucoid Enterobacter Red colonies (A/A, ONPG+) Citrobacter 2 Red or colorless (A/A or Alk/A, ONPG+) colonies Serratia Colorless colonies (A/A, ONPG+) 1 K. pneumoniae, indole –, K. oxytoca, indole + 2 C. freundii, indole – and H 2 S +, C. koseri, indole + and H2S –

41 Differential Reactions of the Enterobacteriaceae by TSI, ONPG, and MAC ShigellaColorless Colonies (Alk/A; ONPG – A, B, and C 1 ; ONPG + D 1 ) SalmonellaColorless Colonies (Alk/A + H2S; ONPG –) ProteusColorless Colonies (Alk/A + H2S 2 ; ONPG –) Edwardsiella tardaColorless Colonies (Alk/A + H2S; ONPG–) YersiniaColorless Colonies (A/A, ONPG +) 1 Shigella A, B, and C, ornithine –; Shigella D, ornithine + 2 Proteus mirabilis. P. vulgaris sucrose + with A/A + H2S on TSI

42 Differential Reactions of the Enterobacteriaceae by EMB Escherichia coliColonies with metallic green sheen KlebsiellaColonies with precipitate (ppt) and mucoid appearance EnterobacterColonies with ppt CitrobacterColonies with/without ppt SerratiaColonies without ppt ShigellaColonies without ppt SalmonellaColonies without ppt ProteusColonies without ppt YersiniaColonies without ppt

43 ONPG Reaction and Lactose Fermentation (Lac) ONPG Lac Escherichia coli + + Shigella sonnei + – Citrobacter + +/– Yersinia enterocolitica + – Klebsiella + + Serratia marcescens + –

44 Xylose Lysine Deoxycholate (XLD) Agar: Composition Xylose0.35% Lysine0.5% Lactose0.75% Sucrose0.75% Sodium chloride0.5% Yeast extract0.3% Sodium deoxycholate0.25% Sodium thiosulfate Ferric ammonium citrate Agar1.35% Phenol red pH = 7.4

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 are lysine-positive coliforms that are also lactose and sucrose fermenters. The high lactose and sucrose concentrations result in strong acid production, which quenches amines produced by lysine decarboxylation. Colonies and agar appear bright yellow. Neither Escherichia coli nor 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.



53 Hektoen Enteric (HE) Agar: Composition Peptone1.2% Yeast extract0.3% Bile salts0.9% Lactose1.2% Sucrose1.2% Salicin0.2% Sodium chloride0.5% Ferric ammonium citrate Acid fuchsin Thymol blue Agar1.4% pH = 7.6

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.


56 Salmonella-Shigella Agar Beef extract0.5% Peptone0.5% Bile salts0.85% Sodium citrate0.85% Brilliant green dyeTrace Lactose1.0% Sodium thiosulfate0.85% Ferric citrate0.1% Neutral red Agar1.4% pH = 7.4

57 Salmonella-Shigella (SS) Agar Bile salts, citrates, and brilliant green dye inhibit gram-positives and most gram-negative coliforms Lactose the sole carbohydrate Sodium thiosulfate a source of sulfur for H2S production Salmonella forms transparent colonies with black centers Shigella forms transparent colonies without blackening Lactose 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 Specimens Feces 1 : MAC or EMB + XLD &/or SS or HE 2 Sputum and Urine 1 : MAC or EMB Wound 3 :MAC or EMB Peritoneal and pleural fluid 4 : MAC or EMB Subculture of blood positive for gram-negative’s in broth culture 4 : MAC or EMB CSF, pericardial fluid, synovial fluid, bone marrow 5 : Not required 1 Heavy population of commensal bacteria 2 Utilized with enrichment broth containing selenite or mannitol to differentially inhibit enteric commensals 3 Commensal bacteria (skin) and frequent polymicrobial etiology 4 Possible polymicrobial etiology (normally sterile fluids) 5 Normally sterile, unimicrobial etiology predominant

59 Selectivity of Differential Agars for Salmonella 1 and Shigella 2 HE or SS agar (absence of lactose fermentation 1,2, H 2 S production 1 ) XLD agar (absence of lactose fermentation 1,2, H 2 S production 1, lysine decarboxylation 1 ) MAC or EMB agar (absence of lactose fermentation 1,2 ) TSI agar (glucose fermentation 1,2, absence of lactose fermentation 1,2, H 2 S production 1 ) 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 of Diagnostic 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, 9 th Edition, ASM Press, 2007: Farmer, J.J., III, Boatwright, K.D., and Janda J.M. Chapter 42. Enterobacteriaceae: Introduction and Identification

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