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

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
Microbiology

3. General Information Most common GNR in clinical specimens
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

4. Host – Enteric Interactions
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

5. Host – Enteric Interactions
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

6. Host – Enteric Interactions
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

7. 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

8. Isolation – MacConkey Agar (Pink)
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

9. Isolation – EMB Agar (Pink)
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

10. Isolation – SS Agar (Pink)
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

11. Isolation – HE Agar (Green)
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

12. Isolation – XLD Agar (Red)
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

13. Other Isolation Media MacConkey Sorbitol – E. coli O157:H7
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

14. Main Characteristics GNR Glucose “F” Oxidase negative
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

15. Biochemical Tests: CHO Utilization: Glucose
GNR: Enterobacteriaceae
Biochemical Tests: CHO Utilization: Glucose
“I” or “N”
“O”
“F”
Microbiology

16. Biochemical Tests: CHO Utilization: Lactose
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

17. Biochemical Tests: Kligler’s Iron Agar
GNR: Enterobacteriaceae
Biochemical Tests: Kligler’s Iron Agar
Contains CHOs: Glucose & Lactose (10x)
Glucose “F” & Lactose “F”
Uninoculated
Glucose “F”
Glucose “F” & Lactose “F” H2S +
Glucose & lactose nonfermenter
Glucose “F” H2S +
Microbiology

18. ONPG
Detects delayed lactose F Organisms that have beta-galactosidase but not the permease enzyme
Negative
Positive
Microbiology

19. Biochemical Tests: Other Carbohydrates
GNR: Enterobacteriaceae
Biochemical Tests: Other Carbohydrates
Microbiology

20. Biochemical Tests: Indole Production
GNR: Enterobacteriaceae
Biochemical Tests: Indole Production
Negative
Positive
Microbiology

21. Biochemical Tests: Spot Indole
GNR: Enterobacteriaceae
Biochemical Tests: Spot Indole
Positive
Microbiology

22. Biochemical Tests: Citrate Utilization
GNR: Enterobacteriaceae
Biochemical Tests: Citrate Utilization
Utilize citrate as a sole carbon source
Negative
Positive
Microbiology

23. Biochemical Tests: Urease Production
GNR: Enterobacteriaceae
Biochemical Tests: Urease Production
Hydrolysis of Urea = increased pH
Negative
Positive
Strongly Positive
Microbiology

24. Biochemical Tests: Decarboxylation of specific amino acid
GNR: Enterobacteriaceae
Biochemical Tests: Decarboxylation of specific amino acid
Media contains one of the following amino acids: arginine, lysine or ornithine
Decarboxylase + = alkaline product = dark purple
Inoculate & overlay with oil
Microbiology

25. Biochemical Tests: Ornithine Decarboxylase
GNR: Enterobacteriaceae
Biochemical Tests: Ornithine Decarboxylase
ODC & MIO agar: must be a glucose “F”
Only Glucose “F”
Negative
Positive
Microbiology

26. Biochemical Tests: Lysine Iron Agar
GNR: Enterobacteriaceae
Biochemical Tests: Lysine Iron Agar
Organism must ferment glucose; detects deamination or decarboxylation of lysine & H2S production
LDC (-), LDA (-) Only Glucose “F”
LDA+
LDC+
Uninoculated
LDC (-), LDA (-) H2S + Only Glucose “F”
LDA+ & H2S +
LDC+ H2S +
Microbiology