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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL COMMUNITIES AND INTERACTIONS AND THEIR IMPACT ON FOOD SAFETY

Location: Molecular Characterization of Foodborne Pathogens

Title: Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette

Authors
item Applegate, Bruce -
item Rosenfield, Carla -
item Martinez, Marcela -
item Zhu, Frank -
item Farrokhzad, Khashayer -
item Paoli, George

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 14, 2014
Publication Date: April 9, 2014
Citation: Applegate, B., Rosenfield, C., Martinez, M., Zhu, F., Farrokhzad, K., Paoli, G. 2014. Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette. Meeting Abstract. 1-2.

Technical Abstract: Bacteriophage and reporter phage are used for typing and/or detection of pathogens. The temperate tailed phage fV10 has been utilized for phage-typing E. coli O157:H7. By modifying fV10 to transduce kanamycin resistance and the a luxCDABE cassette, we developed a reporter bacteriophage (fV10-lux) producing detectable light from the infected host. Using fV10-lux, clinical, foodborne, and environmental isolates of Shiga toxin-producing E. coli (STEC) were screened to determine the specificity of the system. Freezer stocks were allowed to recover then 1mL aliquots were inoculated with fV10-lux and incubated for 16 hours (37 degrees C) followed by measurement using a Zylux luminometer. Of the O157:H7 strains (260) tested, 233 gave a positive result. We are currently confirming the 27 negative results. Non-O157 STEC (52) all tested negative. To determine the applicability of using fV10-lux during selective enrichment of ground beef, serial dilutions of E. coli O157:H7 were performed in a standard selective enrichment protocol containing ground meat and modified tryptic soy broth in a 1:4 ratio. After one hour incubation (37 degrees C) fV10-lux phage (105 pfu) were added to the dilutions. Luminescence was monitored every hour for 24 hours as described above to determine the time to and limit of detection. High concentrations of cells (107 cfu/g) were detected within one hour, concentrations of approximately 10 cfu/g at 12 hours and below 10 cfu/g at 16 hours. After 24 hours positive samples could be detected visually. This approach offers a potential low cost detection protocol for O157:H7 which can be used during selective enrichment.

Last Modified: 10/21/2014
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