Download The Enterobacteriaceae

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 Gram-negative rods
 Glucose is fermented with strong acid formation
and often gas
 Cytochrome oxidase activity is negative
 Nitrate is reduced to nitrite
 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
 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 gramnegative bacteria located beneath a thick
lipid-rich outer membrane weakly binds
crystal violet that is readily removed by
alcohol-acetone decolorization
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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
 Oxidation-reduction of glucose in the absence of molecular oxygen
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(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)
 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
 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
 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
 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)
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Escherichia
Shigella
Salmonella
Edwardsiella
Citrobacter
Yersinia
Klebsiella
Enterobacter
Serratia
Proteus
Morganella
Providencia
 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)
 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
 Environmental sites (soil, water, and plants)
 Intestines of humans and animals
 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)
 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)
 Urinary tract infection: Escherichia coli,
Klebsiella pneumoniae, Enterobacter spp., and
Proteus mirabilis
 Pneumonia: Enterobacter spp., Klebsiella
pneumoniae, Escherichia coli, and Proteus
mirabilis
 Wound Infection: Escherichia coli, Enterobacter
spp., Klebsiella pneumoniae, and Proteus
mirabilis
 Bacteremia: Escherichia coli, Enterobacter spp.,
Klebsiella pneumoniae, and Proteus mirabilis
 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
 Shigella sonnei (serogroup D)
 Salmonella serotype Enteritidis
 Salmonella serotype Typhimurium
 Shigella flexneri (serogroup B)
 Escherichia coli O157:H7
 Yersinia enterocolitica
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
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 Ferrous sulfate
 Sodium thiosulfate
 Sodium chloride
 Agar (1.2%)
 Phenol red
 pH = 7.4
 Yellow deep, purple slant: acid deep due to glucose fermentation
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, 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
 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)
 Escherichia coli
 Klebsiella pneumoniae
 Klebsiella oxytoca
 Enterobacter aerogenes
 Enterobacter cloacae
 Serratia marcescens1, 2
1Non-lactose,
255%
+g
sucrose fermenter
 Serratia marcescens1, 2
 Yersinia enterocolitica2
145%
of strains
2Non-lactose, sucrose fermenter
 Citrobacter freundii
 Proteus vulgaris1
1Non-lactose,
sucrose fermenter
 Shigella
 Providencia
 Salmonella serotype Paratyphi A
 Salmonella (most serotypes)
 Proteus mirabilis
 Edwardsiella tarda
 Salmonella serotype Typhi
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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
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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
 Presence of β-galactoside permease: Transport of
β-galactoside (lactose) across the bacterial cell
wall
 Presence of β-galactosidase: Hydrolysis of βgalactoside bond (lactoseglucose + galactose)
 ONPG: Orthonitrophenyl-β-D-galactopyranoside
 Escherichia coli
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(A/A, ONPG+)
Klebsiella1
(A/A, ONPG+)
Enterobacter
(A/A, ONPG+)
Citrobacter2
(A/A or Alk/A, ONPG+)
Serratia
(A/A, ONPG+)
1K.
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
 Shigella
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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
 Escherichia coli
 Klebsiella
(ppt)
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Enterobacter
Citrobacter
Serratia
Shigella
Salmonella
Proteus
Yersinia
Colonies with metallic green
sheen
Colonies with precipitate
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
Escherichia coli
+
Shigella sonnei
+
Citrobacter
+
Yersinia enterocolitica +
Klebsiella
+
Serratia marcescens
+
Lac
+
–
+/–
–
+
–
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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%
 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).
 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 agarpresumptive for Salmonella.
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.
 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.
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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%
 High bile salt concentration inhibits growth of gram-
positive 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.
 Beef extract
 Peptone
 Bile salts
 Sodium citrate
 Brilliant green dye
 Lactose
 Sodium thiosulfate
 Ferric citrate
0.5%
0.5%
0.85%
0.85%
Trace
1.0%
0.85%
0.1%
 Neutral red
 Agar
 pH = 7.4
1.4%
 Bile salts, citrates, and brilliant green dye inhibit
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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
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
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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
 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
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.
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