Development of green fluorescent protein-expressing bacterial strains and evaluation for potential use as positive controls in sample analyses.
Strains of enterohemorrhagic Escherichia coli O157:H7 and Salmonella Typhimurium were engineered to express the gene for a modified green fluorescent protein (GFP) and were evaluated for potential use as positive controls in sample analyses. The strains fluoresced when observed as colonies with a handheld UV lamp or as individual cells under a fluorescent microscope. The strains maintained their fluorescence following growth in three series of transfer experiments including 8 to 11 passages from broth to broth and twice for 15 consecutive transfers from broth onto Trypticase soy agar plates. Cultures also maintained stability in the ability to fluoresce when agar plates were refrigerated (4 degrees C) for up to 12 days. Growth characteristics of the GFP-positive strains were comparable to those of corresponding control strains. The GFP-positive strains were successfully identified using rapid diagnostic methods and were differentiated from their corresponding non-GFP strains by pulsed-field gel electrophoresis but not by repetitive extragenic palindromic PCR. The GFP-positive and the control strains were recovered successfully from individually inoculated food samples (Feta cheese, raw shrimp, cooked shrimp, and cooked crawfish). However, in one Feta cheese sample and one raw shrimp sample inoculated with combined GFP-positive and GFP-negative cultures, colonies of the GFP-positive strains were not observed under UV light; fluorescing cells in one of the inoculated samples (raw shrimp) were revealed by microscopy. In general, the isolates from the inoculated foods were GFP positive by microscopic examination; the pure isolates could also be restreaked onto Trypticase soy agar, and colonies could be visually examined under UV light. Because GFP strains are not known to occur naturally in the environment, the use of the Salmonella GFP-positive strain may offer advantages as a positive control even when distinct and rare serotypes are available. The GFP-positive E. coli O157:H7 strain may also prove beneficial for use as a positive control strain for sample analyses.
Noah
Charles W
CW
Denver District Office, Food and Drug Administration, Denver, Colorado 80225, USA. cnoah@ora.fda.gov
Shaw
Christine I
CI
Ikeda
Jack S
JS
Kreuzer
Karen S
KS
Sofos
John N
JN
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002611:Cheese / Q000382:microbiology*; D003470:Culture Media; D019453:Escherichia coli O157 / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D005453:Fluorescence; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D008856:Microscopy, Fluorescence; D012486:Salmonella typhimurium / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D012758:Shellfish / Q000382:microbiology*; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003470:Culture Media