Gram-negative rods: Enterobacteriaceae Part I

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Gram-negative rodsEnterobacteriaceae Part I

• Karen Honeycutt, M.Ed., MT(ASCP)SM
• CLS 418 Clinical Microbiology I
• Student Laboratory Session

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Differentiation of Major GNR Genera (there are
many!)

• Utilization of Glucose
• Ferment F, oxidize O or inactive I or N
• Oxidase
• Positive or negative
• Ability to grow on MacConkey agar
• Some GNRs that grow on BAP are unable to grow on
  MacConkey agar

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General Information
GNR Enterobacteriaceae

• Most common GNR in clinical specimens
• Widely dispersed in nature, soil, human GI tract
• Certain Enterobacteriaceae are endemic to a
  particular hospital environment, for example
• Klebsiella sp., Enterobacter sp., Serratia sp.
  and other opportunistic pathogenic enterics

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Host Enteric Interactions
GNR Enterobacteriaceae

• Normal flora of GI tract, skin of GU tract
• Colonization of skin mucous membranes
  hospitalized patients
• Potential source for nosocomial infections
• Overt or primary pathogens always considered
  pathogenic or source of infection, for example
• Shigella, Salmonella, Yersinia, E. coli O157H7

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Host Enteric Interactions
GNR Enterobacteriaceae

• Opportunistic pathogens
• Immunosuppressed, debilitated
• Can be passed from person to person
• Endogenous strains for host can cause infection
  in normally sterile sites or if overgrown (normal
  flora bacterial balance disrupted.antibiotics)
• Nosocomial infections often after an invasive
  procedure

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Host Enteric Interactions
GNR Enterobacteriaceae

• Endotoxic shock
• Endotoxin lipopolysaccharide contained within
  cell wall
• Bacteria are lysed exposing endotoxin
  (antibiotics, immune system-complement, aging
  bacterial cells,)
• Septic shock fever, leukopenia, capillary
  hemorrhage, hypotension, circulatory collapse

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Isolation
GNR Enterobacteriaceae

• Growth on BAP at 24 hrs, ambient air, CO2 or
  anaerobically (facultative anaerobe)
• In general colony morphology large, gray, beta-
  or nonhemolytic(normally larger than Staph,
  Strep, Enterococci)

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Isolation MacConkey Agar (Pink)
GNR Enterobacteriaceae

• Selective (gnrs) differential mediumGeneral
  purpose
• Lactose F pinkLactose NF clear
• pH indicator in agarLactose F pH pink
  color

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Isolation EMB Agar (Pink)
GNR Enterobacteriaceae

• Selective (gnrs) differential medium - General
  purpose
• Lactose /or sucrose F pinkE. coli metallic
  green sheenNL NSF clear colonies

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Isolation SS Agar (Pink)
GNR Enterobacteriaceae

• Selective (gnrs) differential medium
• Stool cultures - Lactose F H2S
  productionLactose F pink coloniesLactose NF
  colorless coloniesH2S black ppt.

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Isolation HE Agar (Green)
GNR Enterobacteriaceae

• Selective (gnrs) differential medium
• Stool cultures - Lactose sucrose F H2S
  Lactose /or sucrose F yellow/orange
  coloniesLactose sucrose NF colorless or
  blue-green H2S black ppt.

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Isolation XLD Agar (Red)
GNR Enterobacteriaceae

• Selective (gnrs) differential medium
• Stool cultures Lactose, sucrose xylose F
  H2S
• Lactose /or sucrose /or xylose F yellow
• Lactose, sucrose xylose NF colorless/red
• H2S black ppt.

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Other Isolation Media
GNR Enterobacteriaceae

• MacConkey Sorbitol E. coli O157H7
• CIN or Yersinia selective
• Gram-negative broth enrichment
  mediumselectively enriched to promote Salmonella
  Shigella (cost effective?)
• Phosphate bufferd saline cold
  enrichmentYersinia entercolitica will grow in
  4C

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Main Characteristics
GNR Enterobacteriaceae

• GNR
• Glucose F
• Oxidase negative
• Reduce nitrate to nitrite NO3 to NO2
• Facultative anaerobes
• Able to grow on MacConkey agar

Catalase positive most GNRs are , therefore
not routinely performed unless CAT (-) GNR is
suspected
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Biochemical Tests CHO Utilization Glucose
GNR Enterobacteriaceae
I or N
O
F
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Biochemical Tests CHO Utilization Lactose
GNR Enterobacteriaceae

• Disaccharide glucose galactose
• Beta-galactoside permease Beta-galactosidase
• LF have both enzymesNLF have neitherSlow LF
  have beta-galactosidase but lack the permease
  enzyme

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Biochemical Tests Kliglers Iron Agar
GNR Enterobacteriaceae
Contains CHOs Glucose Lactose (10x)
Glucose F Lactose F
Uninoculated
Glucose F
Glucose F Lactose F H2S
Glucose lactose nonfermenter
Glucose F H2S
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ONPG

• Detects delayed lactose F Organisms that have
  beta-galactosidase but not the permease enzyme

Negative
Positive
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Biochemical Tests Other Carbohydrates
GNR Enterobacteriaceae
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Biochemical Tests Indole Production
GNR Enterobacteriaceae
Negative
Positive
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Biochemical Tests Spot Indole
GNR Enterobacteriaceae
Positive
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Biochemical Tests Citrate Utilization
GNR Enterobacteriaceae
Utilize citrate as a sole carbon source
Positive
Negative
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Biochemical Tests Urease Production
GNR Enterobacteriaceae
Hydrolysis of Urea increased pH
Positive
Negative
Strongly Positive
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Biochemical Tests Decarboxylation of specific
amino acid
GNR Enterobacteriaceae
Media contains one of the following amino acids
arginine, lysine or ornithine
Decarboxylase alkaline product dark purple
Inoculate overlay with oil
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Biochemical Tests Ornithine Decarboxylase
GNR Enterobacteriaceae
ODC MIO agar must be a glucose F
Only Glucose F
Positive
Negative
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Biochemical Tests Lysine Iron Agar
GNR Enterobacteriaceae
Organism must ferment glucose detects
deamination or decarboxylation of lysine H2S
production
LDC (-), LDA (-) Only Glucose F
LDA
LDC
Uninoculated
LDA H2S
LDC (-), LDA (-) H2S Only Glucose F
LDC H2S