Gram-negative rods:
Enterobacteriaceae
Part I
Karen Honeycutt, M.Ed., MT(ASCP)SM
CLS 418 Clinical Microbiology I
Student Laboratory Session
Microbiology
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
Microbiology
GNR: Enterobacteriaceae
General Information
• 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
Microbiology
GNR: Enterobacteriaceae
Host – Enteric Interactions
• 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 O157:H7
Microbiology
GNR: Enterobacteriaceae
Host – Enteric Interactions
• 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
Microbiology
GNR: Enterobacteriaceae
Host – Enteric Interactions
• 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
Microbiology
GNR: Enterobacteriaceae
Isolation
• 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)
Microbiology
GNR: Enterobacteriaceae
Isolation – MacConkey Agar (Pink)
• Selective (gnrs) &
differential medium
General purpose
– Lactose F = pink
Lactose NF = clear
– pH indicator in agar
Lactose F = pH = pink color
Microbiology
GNR: Enterobacteriaceae
Isolation – EMB Agar (Pink)
• Selective (gnrs) & differential medium General purpose
– Lactose &/or sucrose F = pink
E. coli = metallic green sheen
NL & NSF = clear colonies
Microbiology
GNR: Enterobacteriaceae
Isolation – SS Agar (Pink)
• Selective (gnrs) & differential medium
Stool cultures - Lactose “F”& H2S production
Lactose F = pink colonies
Lactose NF = colorless colonies
H2S + = black ppt.
Microbiology
GNR: Enterobacteriaceae
Isolation – HE Agar (Green)
• Selective (gnrs) & differential medium
Stool cultures - Lactose & sucrose “F” & H2S
Lactose &/or sucrose F = yellow/orange colonies
Lactose & sucrose NF = colorless or blue-green
H2S + = black ppt.
Microbiology
GNR: Enterobacteriaceae
Isolation – XLD Agar (Red)
• 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.
Microbiology
GNR: Enterobacteriaceae
Other Isolation Media
• MacConkey Sorbitol – E. coli O157:H7
• CIN or Yersinia selective
• Gram-negative broth – enrichment medium
selectively enriched to promote Salmonella &
Shigella (cost effective?)
• Phosphate bufferd saline – cold enrichment
Yersinia entercolitica will grow in 4˚C
Microbiology
GNR: Enterobacteriaceae
Main Characteristics
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
Microbiology
GNR: Enterobacteriaceae
Biochemical Tests:
CHO Utilization: Glucose
“I” or “N”
Microbiology
“O”
“F”
GNR: Enterobacteriaceae
Biochemical Tests:
CHO Utilization: Lactose
• Disaccharide: glucose +galactose
Beta-galactoside permease
Beta-galactosidase
• LF: have both enzymes
NLF: have neither
Slow LF: have beta-galactosidase but
lack the permease enzyme
Microbiology
GNR: Enterobacteriaceae
Biochemical Tests:
Kligler’s Iron Agar
Contains CHOs: Glucose & Lactose (10x)
Uninoculated
Glucose &
lactose
nonfermenter
Microbiology
Glucose “F”
Glucose “F” &
Lactose “F”
Glucose “F”
H2S +
Glucose “F” &
Lactose “F”
H2S +
ONPG
• Detects delayed lactose F
Organisms that have beta-galactosidase but
not the permease enzyme
Negative
Microbiology
Positive
GNR: Enterobacteriaceae
Biochemical Tests:
Other Carbohydrates
Microbiology
GNR: Enterobacteriaceae
Biochemical Tests:
Indole Production
Positive
Microbiology
Negative
GNR: Enterobacteriaceae
Biochemical Tests: Spot Indole
Positive
Microbiology
GNR: Enterobacteriaceae
Biochemical Tests: Citrate Utilization
Utilize citrate as a sole carbon source
Negative
Microbiology
Positive
GNR: Enterobacteriaceae
Biochemical Tests: Urease
Production
Hydrolysis of Urea = increased pH
Negative
Microbiology
Positive
Strongly Positive
GNR: Enterobacteriaceae
Biochemical Tests: Decarboxylation
of specific amino acid
Media contains one of
the following amino
acids: arginine, lysine
or ornithine
Inoculate &
overlay with
oil
Microbiology
Decarboxylase + =
alkaline product =
dark purple
GNR: Enterobacteriaceae
Biochemical Tests: Ornithine
Decarboxylase
ODC & MIO agar: must be a glucose “F”
Only
Glucose
“F”
Negative
Microbiology
Positive
GNR: Enterobacteriaceae
Biochemical Tests: Lysine Iron Agar
Organism must ferment glucose; detects deamination or
decarboxylation of lysine & H2S production
LDC (-),
LDA (-)
Only Glucose “F”
Uninoculated
Microbiology
LDC (-),
LDA (-)
H2S +
Only Glucose “F”
LDA+
LDC+
LDC+
H2S +
LDA+ &
H2S +