The Enterobacteriaceae Basic Properties

1
The EnterobacteriaceaeBasic 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
Grams Stain for Bacterial Morphology

• Crystal violet binds to cell wall peptidoglycan
  with Grams iodine as a mordant
• Safranin or basic fuchsin counterstains bacterial
  cells decolorized by alcohol-acetone

4
Grams 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
Grams Stain Procedure

• Flood surface of smear with crystal violet
  solution
• After 1 min thoroughly rinse with cold tap water
• Flood smear with Grams 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 Grams stain)
  or basic fuchsin (enhanced Grams 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 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 a-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 a-naphthylamine and
  sulfanilic acid

10
Enterobacteriaceae Genetic Properties

• Chromosomal DNA has 39-59 guanine-plus-cytosine
  (GC) 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 O157H7)
• 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. 2,912 (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 O157H7
• 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 1.0
• 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 amines 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

24
(No Transcript)
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 salts2 0.15
• Crystal violet2
• Neutral red3
• Sodium chloride 0.5
• Agar 1.35
• pH7.1
• 1Differential medium for lactose fermentation
• 2Inhibit gram positives and fastidious
  gram-negatives MAC agar selective for
• gram-negatives
• 3Red color at pH lt 6.8

34
(No Transcript)
35
(No Transcript)
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

37
(No Transcript)
38
(No Transcript)
39
Bacterial Utilization of Lactose

• Presence of ß-galactoside permease Transport of
  ß-galactoside (lactose) across the bacterial cell
  wall
• Presence of ß-galactosidase Hydrolysis of
  ß-galactoside bond (lactose?glucose galactose)
• ONPG Orthonitrophenyl-ß-D-galacto-
• pyranoside

40
Differential Reactions of the Enterobacteriaceae
by TSI, ONPG, and MAC

• Escherichia coli Red
  colonies,
• (A/A, ONPG) pitted
• Klebsiella1 Red colonies,
• (A/A, ONPG) mucoid
• Enterobacter Red colonies
• (A/A, ONPG)
• Citrobacter2 Red or colorless
• (A/A or Alk/A, ONPG)
  colonies
• Serratia Colorless colonies
• (A/A, ONPG)
• 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

• Xylose 0.35
• Lysine 0.5
• Lactose 0.75
• Sucrose 0.75
• Sodium chloride 0.5
• Yeast extract 0.3
• Sodium deoxycholate 0.25
• Sodium thiosulfate
• Ferric ammonium citrate
• Agar 1.35
• 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.

47
(No Transcript)
48
(No Transcript)
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.

51
(No Transcript)
52
(No Transcript)
53
Hektoen Enteric (HE) Agar Composition

• Peptone 1.2
• Yeast extract 0.3
• Bile salts 0.9
• Lactose 1.2
• Sucrose 1.2
• Salicin 0.2
• Sodium chloride 0.5
• Ferric ammonium citrate
• Acid fuchsin
• Thymol blue
• Agar 1.4
• 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.

55
(No Transcript)
56
Salmonella-Shigella Agar

• Beef extract 0.5
• Peptone 0.5
• Bile salts 0.85
• Sodium citrate 0.85
• Brilliant green dye Trace
• Lactose 1.0
• Sodium thiosulfate 0.85
• Ferric citrate 0.1
• Neutral red
• Agar 1.4
• 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

• Feces1 MAC or EMB XLD /or SS or HE2
• Sputum and Urine1 MAC or EMB
• Wound3MAC or EMB
• Peritoneal and pleural fluid4 MAC or EMB
• Subculture of blood positive for gram-negatives
  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.
• Konemans 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