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Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and able to differentiate live cells from dead ones. Upon infection, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage ΦV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris . Once infected by the ΦV10 reporter phage, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo ® ). Enrichment assays using E. coli O157:H7 grown in LB broth with a reporter phage concentration of 1.76 × 10 2  pfu ml −1 are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23 × 10 3  pfu ml −1 of phage in selective culture enrichments of ground beef as a representative food matrix. Therefore we conclude that this NanoLuc reporter phage assay shows promise for detection of E. coli O157:H7 from food in a simple, fast and sensitive manner.

Ready-to-eat (RTE) meat and poultry product samples collected between 2005 and 2017 from RTE-producing establishments for the U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS) ALLRTE/RTEPROD_RAND (random) and RTE001/RTEPROD_RISK (risk-based) sampling projects were tested for Listeria monocytogenes (Lm). Data for 45,897 ALLRTE/RTEPROD_RAND samples collected from 3,607 distinct establishments and 112,347 RTE001/RTEPROD_RISK samples collected from 3,283 distinct establishments were analyzed for the presence of Lm. These data were also analyzed based upon the percentages of establishments with positive samples, annual production volume, sanitation control alternatives, geographic location, and season or month of sample collection. Results revealed low occurrence of Lm-positive samples from the random and risk-based sampling projects, with 152 (0.33%) positive samples for ALLRTE/RTEPROD_RAND and 403 (0.36%) positive samples for RTE001/RTEPROD_RISK. The percentage of positive samples significantly decreased over time, from about 0.7% in 2005 and 2006 to about 0.2% in 2017 (P < 0.05). From 2005 to 2017, 3.9% of establishments sampled under the ALLRTE/RTEPROD_RAND sampling project had at least one Lm-positive sample. Similarly, 10.0% of establishments sampled under the RTE001/RTEPROD_RISK sampling project had at least one positive sample. Samples positive for Lm were found in all geographic regions in all months. Thus, in 13 years of RTE product sampling in FSIS-regulated establishments (2005 through 2017), <0.4% of samples were positive for Lm in both risk-based and random sampling projects. The low prevalence of Lm in these products suggests that the combination of FSIS policies and industry practices may be effective for controlling Lm contamination. Information obtained from these sampling projects is relevant to the ongoing prevention of foodborne Lm illnesses from RTE meat and poultry products.

Ready-to-eat (RTE) meat and poultry product samples collected between 2005 and 2017 from RTE-producing establishments for the U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS) ALLRTE/RTEPROD_RAND (random) and RTE001/RTEPROD_RISK (risk-based) sampling projects were tested for Listeria monocytogenes (Lm). Data for 45,897 ALLRTE/RTEPROD_RAND samples collected from 3,607 distinct establishments and 112,347 RTE001/RTEPROD_RISK samples collected from 3,283 distinct establishments were analyzed for the presence of Lm. These data were also analyzed based upon the percentages of establishments with positive samples, annual production volume, sanitation control alternatives, geographic location, and season or month of sample collection. Results revealed low occurrence of Lm-positive samples from the random and risk-based sampling projects, with 152 (0.33%) positive samples for ALLRTE/RTEPROD_RAND and 403 (0.36%) positive samples for RTE001/RTEPROD_RISK. The percentage of positive samples significantly decreased over time, from about 0.7% in 2005 and 2006 to about 0.2% in 2017 (P < 0.05). From 2005 to 2017, 3.9% of establishments sampled under the ALLRTE/RTEPROD_RAND sampling project had at least one Lm-positive sample. Similarly, 10.0% of establishments sampled under the RTE001/RTEPROD_RISK sampling project had at least one positive sample. Samples positive for Lm were found in all geographic regions in all months. Thus, in 13 years of RTE product sampling in FSIS-regulated establishments (2005 through 2017), <0.4% of samples were positive for Lm in both risk-based and random sampling projects. The low prevalence of Lm in these products suggests that the combination of FSIS policies and industry practices may be effective for controlling Lm contamination. Information obtained from these sampling projects is relevant to the ongoing prevention of foodborne Lm illnesses from RTE meat and poultry products.

Bacteriophage ΦV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the ΦV10 genome is 39 104 bp long and contains 55 predicted genes. ΦV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage ε15 and a prophage from E. coli APEC O1. The attachment site of ΦV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. ΦV10 encodes an O-acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block ΦV10 superinfection, indicating that the O157 antigen is most likely the ΦV10 receptor.

Bacteriophages are obligate intracellular parasites that infect prokaryotes. Host specificity of phages varies as some phages can infect an entire genus of bacteria while some are species specific and others are strain specific. Phage host specificity has been exploited as a tool for the detection of pathogens using both whole phage in a reporter format or the use of their host specific binding proteins. This research focuses on the characterization and potential use of the temperate bacteriophage V10 for the detection of Escherichia coli O157:H7. The complete genome of V10, was sequenced and determined to be 39104bp containing 55 predicted genes. The predicted gene ORF 26 was tentatively identified to belong to the acyltransferase family associated with seroconversion. Primers were designed and ORF 26 was amplified from the phage genome and cloned into the expression plasmid pBAD-TOPO. Expression of ORF26 in O157:H7 was determined to confer V10 immunity using plaque assays. LPS was also isolated from the strain expressing ORF 26 and was found to inactivate V10. Further analysis of the LPS using nuclear magnetic resonance spectroscopy showed the acetylation of the O-antigen suggesting it is the receptor for V10. However the strains with the modified O-antigen are still detectable by commercial antibody-based O157 test kits. Reporter phage V10KanCobA was constructed by replacing the non-essential recET with a cobA-kanamycin gene cassette. E. coli O157:H7 infected with V10KanCobA and plated on plates containing kanamycin grow as colonies due to the integration of V10KanCobA into the chromosome. A two dimensional matrix was used with decreasing concentrations of V10KanCobA (107-104 pfu) and O157:H7 (106-10 2 cfu) and incubated for 1 h at room temperature. A 100 μl aliquot of each phage-bacteria mixture was spread on LBkm50 agar plates and incubated at 3°C overnight. The data showed a linear correlation with an R 2 value of 0.9866. The matrix analysis showed that as few as 10 4 pfu could detect approximately 105 cfu. However 10 7 pfu were required to detect as few as 102 cfu. The lysogen assay using V10KanCobA for the detection of E. coli O157:H7 is convenient, reproducible and inexpensive and when coupled with a selective enrichment has low limits of detection.

AbstractBacteriophage V10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the V10 genome is 39104bp long and contains 55 predicted genes. V10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage e15 and a prophage from E. coli APEC O1. The attachment site of V10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. V10 encodes an O-acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block V10 superinfection, indicating that the O157 antigen is most likely the V10 receptor.

Bacteriophage PhiV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the PhiV10 genome is 39 104 bp long and contains 55 predicted genes. PhiV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage epsilon15 and a prophage from E. coli APEC O1. The attachment site of PhiV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. PhiV10 encodes an O-acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block PhiV10 superinfection, indicating that the O157 antigen is most likely the PhiV10 receptor.

This chapter contains sections titled: Introduction Fundamentals of bacteriophage Phage‐based detection of pathogens Bacteriophage‐mediated biocontrol Conclusions