Bacteriophage-based bioluminescent bioreporter for the detection of Escherichia coli 0157:H7.
The rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this study, the lux reporter genes were used in a new bioassay that allows pathogen monitoring without multiple sample manipulations or the addition of exogenous substrate. A recombinant phage specific for Escherichia coli 0157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli 0157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP0-luxl to detect several strains of E. coli 0157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PPOI-luxl and E. coli 0157:H7, and luminescence was monitored. Reporter phages induced light in bioreporter cells within I h when exposed to 10(4) CFU/ml of E. coli 0157:H7 and were able to detect 10 CFU/ml in pure culture with a preincubation step (total detection time, 4 h). The detection method was also applied to contaminated apple juice and was able to detect 10(4) CFU/ml of E. coli 0157:H7 in 2 h after a 6-h preincubation.
Brigati
Jennifer R
JR
The Center for Environmental Biotechnology, The Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996, USA.
Ripp
Steven A
SA
Johnson
Courtney M
CM
Iakova
Polina A
PA
Jegier
Patricia
P
Sayler
Gary S
GS
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D048908:Aliivibrio fischeri / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001435:Bacteriophages; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000235:genetics* / Q000821:virology; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006714:Homoserine / Q000031:analogs & derivatives; D006801:Humans; D007783:Lactones; D008163:Luminescent Measurements; D009876:Operon; D011994:Recombinant Proteins; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D007783:Lactones; C085427:LuxI protein, Bacteria; C091088:N-octanoylhomoserine lactone; D011994:Recombinant Proteins; D014157:Transcription Factors; C088386:homoserine lactone; D006714:Homoserine; D015107:4-Butyrolactone