1. The Enterobacteriaceae Basic Properties.

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)
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 NADH2 by accepting electrons during glycolytic conversion of glucose to pyruvate
NADH2 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 gas
End products of glucose fermentation: organic acids and CO2 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 (NO3) producing nitrite (NO2)
NO2 detected by reaction with α- naphthylamine and sulfanilic acid producing a red colored complex
Absence 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) 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 35oC except Yersinia (25o-30oC)
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 1990-19961
Escherichia coli 27,871 (13.7%)
Enterobacter spp. 12,757 (6.2%)
Klebsiella pneumoniae 11,015 (5.4%)
Proteus mirabilis 4,662 (2.3%)
Serratia marcescens 3,010 (1.5%)
Citrobacter spp ,912 (1.4%)
1Enteric 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 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 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 H2S 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 H2S 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 (CO2)
A/A = acid/acid
A/A + g, H2S = acid/acid plus gas, H2S
Alk/A = alkaline/acid
Alk/A + g = alkaline/acid plus gas
Alk/A + g, H2S = alkaline/acid plus gas, H2S
Alk/A + g, H2S (w) = alkaline/acid plus gas, H2S (weak)

26. A/A + g Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca
Enterobacter aerogenes
Enterobacter cloacae
Serratia marcescens1, 2
1Non-lactose, sucrose fermenter
255% + g

27. A/A Serratia marcescens1, 2 Yersinia enterocolitica2 145% of strains
2Non-lactose, sucrose fermenter

28. A/A + g, H2S Citrobacter freundii Proteus vulgaris1
1Non-lactose, sucrose fermenter

29. Alk/A
Shigella
Providencia

30. Alk/A + g
Salmonella serotype Paratyphi A

31. Alk/A + g, H2S Salmonella (most serotypes) Proteus mirabilis
Edwardsiella tarda

32. Alk/A + g, H2S (w)
Salmonella serotype Typhi

33. MacConkey (MAC) Agar Peptone 1.7% Polypeptone 0.3% Lactose1 1.0%
Bile salts %
Crystal violet2
Neutral red3
Sodium chloride 0.5%
Agar %
pH=7.1
1Differential medium for lactose fermentation
2Inhibit gram positives and fastidious gram-negatives; MAC agar selective for
gram-negatives
3Red color at pH < 6.8

36. Eosin Methylene Blue (EMB) Agar (Levine)
Peptone 1.0%
Lactose1 0.5%
Eosin y2
Methylene blue2
Agar
pH = 7.2
1Modified formula also contains sucrose (0.5%)
2Inhibit 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 Red colonies,
(A/A, ONPG+) mucoid
Enterobacter Red colonies
(A/A, ONPG+)
Citrobacter Red or colorless
(A/A or Alk/A, ONPG+) colonies
Serratia Colorless colonies
1K. 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 on
TSI

42. Differential Reactions of the Enterobacteriaceae by EMB
Escherichia coli Colonies with metallic green sheen
Klebsiella Colonies with precipitate (ppt)
and mucoid appearance
Enterobacter Colonies with ppt
Citrobacter Colonies with/without ppt
Serratia Colonies without ppt
Shigella Colonies without ppt
Salmonella Colonies without ppt
Proteus Colonies without ppt
Yersinia Colonies 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
Lactose %
Sucrose %
Sodium chloride %
Yeast extract %
Sodium deoxycholate %
Sodium thiosulfate
Ferric ammonium citrate
Agar %
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
Peptone %
Yeast extract %
Bile salts %
Lactose %
Sucrose %
Salicin %
Sodium chloride %
Ferric ammonium citrate
Acid fuchsin
Thymol blue
Agar %
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 extract %
Peptone %
Bile salts %
Sodium citrate %
Brilliant green dye Trace
Lactose %
Sodium thiosulfate %
Ferric citrate %
Neutral red
Agar %
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
Feces1: MAC or EMB + XLD &/or SS or HE2
Sputum and Urine1: MAC or EMB
Wound3:MAC or EMB
Peritoneal and pleural fluid4: MAC or EMB
Subculture of blood positive for gram-negative’s in broth culture4: MAC or EMB
CSF, pericardial fluid, synovial fluid, bone marrow5: Not required
1Heavy population of commensal bacteria
2Utilized with enrichment broth containing selenite or mannitol to
differentially inhibit enteric commensals
3Commensal bacteria (skin) and frequent polymicrobial etiology
4Possible 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 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, 9th
Edition, ASM Press, 2007:
Farmer, J.J., III, Boatwright, K.D., and Janda J.M. Chapter 42. Enterobacteriaceae: Introduction and Identification