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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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