Gram-negative rods: Enterobacteriaceae Part I - PowerPoint PPT Presentation

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Gram-negative rods: Enterobacteriaceae Part I

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Gram-negative rods: Enterobacteriaceae Part I Karen Honeycutt, M.Ed., MT(ASCP)SM CLS 418 Clinical Microbiology I Student Laboratory Session Differentiation of Major ... – PowerPoint PPT presentation

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Title: Gram-negative rods: Enterobacteriaceae Part I


1
Gram-negative rodsEnterobacteriaceae Part I
  • Karen Honeycutt, M.Ed., MT(ASCP)SM
  • CLS 418 Clinical Microbiology I
  • Student Laboratory Session

2
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

3
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

4
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

5
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

6
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

7
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)

8
Isolation MacConkey Agar (Pink)
GNR Enterobacteriaceae
  • Selective (gnrs) differential mediumGeneral
    purpose
  • Lactose F pinkLactose NF clear
  • pH indicator in agarLactose F pH pink
    color

9
Isolation EMB Agar (Pink)
GNR Enterobacteriaceae
  • Selective (gnrs) differential medium - General
    purpose
  • Lactose /or sucrose F pinkE. coli metallic
    green sheenNL NSF clear colonies

10
Isolation SS Agar (Pink)
GNR Enterobacteriaceae
  • Selective (gnrs) differential medium
  • Stool cultures - Lactose F H2S
    productionLactose F pink coloniesLactose NF
    colorless coloniesH2S black ppt.

11
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.

12
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.

13
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

14
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
15
Biochemical Tests CHO Utilization Glucose
GNR Enterobacteriaceae
I or N
O
F
16
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

17
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
18
ONPG
  • Detects delayed lactose F Organisms that have
    beta-galactosidase but not the permease enzyme

Negative
Positive
19
Biochemical Tests Other Carbohydrates
GNR Enterobacteriaceae
20
Biochemical Tests Indole Production
GNR Enterobacteriaceae
Negative
Positive
21
Biochemical Tests Spot Indole
GNR Enterobacteriaceae
Positive
22
Biochemical Tests Citrate Utilization
GNR Enterobacteriaceae
Utilize citrate as a sole carbon source
Positive
Negative
23
Biochemical Tests Urease Production
GNR Enterobacteriaceae
Hydrolysis of Urea increased pH
Positive
Negative
Strongly Positive
24
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
25
Biochemical Tests Ornithine Decarboxylase
GNR Enterobacteriaceae
ODC MIO agar must be a glucose F
Only Glucose F
Positive
Negative
26
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
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