Transcript The Enterobacteriaceae

The Enterobacteriaceae
Basic Properties
Dr. John R. Warren
Department of Pathology
Northwestern University
Feinberg School of Medicine
June 2007
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
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 alcoholacetone
Gram’s Stain for Bacterial
Morphology
• Thick cell-wall peptidoglycan layer of grampositive bacteria strongly binds crystal violet
and resists decolorization by alcoholacetone
• Thin cell-wall peptidoglycan layer of gramnegative bacteria located beneath a thick
lipid-rich outer membrane weakly binds
crystal violet that is readily removed by
alcohol-acetone decolorization
Gram’s Stain Procedure
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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 oilimmersion light microscopy
Glucose Fermentation
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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)
Spot Cytochrome Oxidase Test
• The spot cytochrome oxidase test is
the first test performed with gramnegative 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
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
Nitrate Reduction
• Enterobacteriaceae extract oxygen from
nitrate (NO3) producing nitrite (NO2)
• NO2 detected by reaction with αnaphthylamine 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 αnaphthylamine and sulfanilic acid
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)
Enterobacteriaceae: Major
Genera
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Escherichia
Shigella
Salmonella
Edwardsiella
Citrobacter
Yersinia
Klebsiella
Enterobacter
Serratia
Proteus
Morganella
Providencia
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)
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 (25o30oC)
• Motile by peritrichous flagella except
Shigella and Klebsiella which are non-motile
• Do not form spores
Enterobacteriaceae: Natural
Habitats
• Environmental sites (soil, water, and
plants)
• Intestines of humans and animals
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)
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)
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
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
Enterobacteriaceae: Nosocomial
Infections in the United States
1986-1989 and 1990-19961
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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
Enterobacteriaceae: Intestinal
Infection
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Shigella sonnei (serogroup D)
Salmonella serotype Enteritidis
Salmonella serotype Typhimurium
Shigella flexneri (serogroup B)
Escherichia coli O157:H7
Yersinia enterocolitica
Triple Sugar Iron (TSI) Agar
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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
Triple Sugar Iron (TSI) Agar
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Ferrous sulfate
Sodium thiosulfate
Sodium chloride
Agar (1.2%)
Phenol red
pH = 7.4
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
TSI Reactions of the
Enterobacteriaceae
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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)
A/A + g
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Escherichia coli
Klebsiella pneumoniae
Klebsiella oxytoca
Enterobacter aerogenes
Enterobacter cloacae
Serratia marcescens1, 2
1Non-lactose,
255%
+g
sucrose fermenter
A/A
• Serratia marcescens1, 2
• Yersinia enterocolitica2
145% of strains
2Non-lactose, sucrose fermenter
A/A + g, H2S
• Citrobacter freundii
• Proteus vulgaris1
1Non-lactose, sucrose fermenter
Alk/A
• Shigella
• Providencia
Alk/A + g
• Salmonella serotype Paratyphi A
Alk/A + g, H2S
• Salmonella (most serotypes)
• Proteus mirabilis
• Edwardsiella tarda
Alk/A + g, H2S (w)
• Salmonella serotype Typhi
MacConkey (MAC) Agar
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Peptone
Polypeptone
Lactose1
Bile salts2
Crystal violet2
Neutral red3
Sodium chloride
Agar
pH=7.1
1Differential
1.7%
0.3%
1.0%
0.15%
0.5%
1.35%
medium for lactose fermentation
2Inhibit
gram positives and fastidious gram-negatives; MAC agar selective for
gram-negatives
3Red
color at pH < 6.8
Eosin Methylene Blue (EMB)
Agar (Levine)
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Peptone
1.0%
Lactose1
0.5%
Eosin y2
Methylene blue2
Agar
pH = 7.2
1Modified
2Inhibit
formula also contains sucrose (0.5%)
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
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-galactopyranoside
Differential Reactions of the
Enterobacteriaceae by TSI,
ONPG, and MAC
• Escherichia coli
(A/A, ONPG+)
• Klebsiella1
(A/A, ONPG+)
• Enterobacter
(A/A, ONPG+)
• Citrobacter2
(A/A or Alk/A, ONPG+)
• Serratia
(A/A, ONPG+)
Red colonies,
pitted
Red colonies,
mucoid
Red colonies
Red or colorless
colonies
Colorless colonies
pneumoniae, indole –, K. oxytoca, indole +
2C. freundii, indole – and H S +, C. koseri, indole + and H2S –
2
1K.
Differential Reactions of the
Enterobacteriaceae by TSI,
ONPG, and MAC
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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 +)
A, B, and C, ornithine –; Shigella D, ornithine +
2Proteus mirabilis. P. vulgaris sucrose + with A/A + H2S on
TSI
1Shigella
Differential Reactions of the
Enterobacteriaceae by EMB
• Escherichia coli
• Klebsiella
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Enterobacter
Citrobacter
Serratia
Shigella
Salmonella
Proteus
Yersinia
Colonies with metallic green
sheen
Colonies with precipitate (ppt)
and mucoid appearance
Colonies with ppt
Colonies with/without ppt
Colonies without ppt
Colonies without ppt
Colonies without ppt
Colonies without ppt
Colonies without ppt
ONPG Reaction and Lactose
Fermentation (Lac)
ONPG
Escherichia coli
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Shigella sonnei
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Citrobacter
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Yersinia enterocolitica
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Klebsiella
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Serratia marcescens
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Lac
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Xylose Lysine Deoxycholate
(XLD) Agar: Composition
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Xylose
Lysine
Lactose
Sucrose
Sodium chloride
Yeast extract
Sodium deoxycholate
Sodium thiosulfate
Ferric ammonium citrate
Agar
Phenol red
pH = 7.4
0.35%
0.5%
0.75%
0.75%
0.5%
0.3%
0.25%
1.35%
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).
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.
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.
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.
Hektoen Enteric (HE) Agar:
Composition
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Peptone
Yeast extract
Bile salts
Lactose
Sucrose
Salicin
Sodium chloride
Ferric ammonium citrate
Acid fuchsin
Thymol blue
Agar
pH = 7.6
1.2%
0.3%
0.9%
1.2%
1.2%
0.2%
0.5%
1.4%
HE Agar: Growth of Enteric
Pathogens and Commensals
• High bile salt concentration inhibits growth of grampositive and gram-negative intestinal commensals,
and thereby selects for pathogenic Salmonella (bileresistant 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.
Salmonella-Shigella Agar
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Beef extract
Peptone
Bile salts
Sodium citrate
Brilliant green dye
Lactose
Sodium thiosulfate
Ferric citrate
Neutral red
Agar
pH = 7.4
0.5%
0.5%
0.85%
0.85%
Trace
1.0%
0.85%
0.1%
1.4%
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
Use of Selective-Differential Agars for
Recovery of the Enterobacteriaceae from
Different Types of Specimens
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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
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
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.
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