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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL FOOD SAFETY OF FRESH AND FRESH-CUT PRODUCE Title: Bacteriophage-based rapid and sensitive detection of Escherichia coli O157:H7 isolates from ground beef

Authors
item Kannan, Porteen -
item Yong, Ho Yao -
item Reiman, Lucy -
item Cleaver, Claire -
item Patel, Pradip -
item BHAGWAT, ARVIND

Research conducted cooperatively with:
item

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 10, 2010
Publication Date: December 1, 2010
Citation: Kannan, P., Yong, H., Reiman, L., Cleaver, C., Patel, P., Bhagwat, A.A. 2010. Bacteriophage-based rapid and sensitive detection of Escherichia coli O157:H7 isolates from ground beef. Foodborne Pathogens and Disease. 12:1151-1158.

Interpretive Summary: Conventional methods to detect human pathogens may take up to one week to accurately predict the presence of human pathogens. Considering the urgency in food-borne outbreaks, polymerase chain reaction (PCR)-based detection assays have been developed. However, these methods do not always generate live cultures of the contaminating pathogen which are needed to perform the follow-up studies. Alternatives to PCR-based are urgently needed while retaining the benefits of molecular methods. A detection system based on viruses of E. coli O157:H7 was evaluated. This method required less than 12 hours to detect E. coli O157:H7 from beef and generated a live culture the following day. The method is compatible with future high throughput sample analyses requirements. Detection of human pathogens from foods is a crucial step in ensuring food safety. Both the food industry and consumers will benefit from the results of this research

Technical Abstract: We investigated efficacy of bacteriophage-based detection technology to detect Escherichia coli O157:H7 from ground beef. The assay involved 8 h enrichment of cold stressed beef samples in presence of antimicrobials followed by capture of the pathogen on O157:H7-specific immunomagnetic beads and specific lysis of cells using O157:H7-specific bacteriophage. Upon phage induced lysis, enzyme adenylate kinase was released from lysed cells was measured in terms of ‘relative light units’ using luciferin-luciferase assay. Initially we examined plaque forming efficiency (e.g. phage susceptibility) and binding ability to immunomagnetic beads with the array of 74 E. coli O157:H7 isolates obtained from various clinical and food-borne samples. Immunmagnetic beads successfully captured all 74 isolates, however only 53 isolates showed susceptibility toward the bacteriophage. Susceptible isolates were further classified into two broad groups, moderately sensitive isolates which generated phage titer ~107 pfu/ml (Group I, n=15) and highly susceptible isolates which generated high phage titer ~109 pfu/ml (Group II, n= 38). We selected 15 isolates (nine from Group I and six from Group II) and individually spiked beef samples (ca. 3 – 8 cells/25 g beef) to evaluate the bacteriophage-based detection system. Eight out of nine isolates from Group I and all six isolates from Group II were successfully detected. The method is specific to O157:H7 serogroup as pathogenic E. coli strains belonging to other serogroups (12 serogroups, 67 isolates) as well as non-target microorganisms (n= 18) were not lysed by the bacteriophage. The method is high throughput compatible, rapid and able to provide live culture the following day on conventional selective agar media.

Last Modified: 7/28/2014
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