title: Bacteriophage-based rapid and sensitive detection of Escherichia coli O157:H7 isolates from ground beef.
abstract: We investigated the efficacy of bacteriophage-based detection technology to detect Escherichia coli O157:H7 from ground beef. The assay involved a short enrichment period of 8 h followed by capture of the pathogen on O157-specific immunomagnetic beads. The captured cells were treated with O157-specific lytic bacteriophage, CSLO157. Upon phage-induced lysis, the enzyme adenylate kinase, which was released from the lysed cells, was measured in terms of relative light units using luciferin-luciferase assay. The plaque forming efficiency (e.g., phage susceptibility) and ability to capture cells with immunomagnetic beads were examined using an array of 74 E. coli O157:H7 isolates obtained from various clinical and foodborne 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 ∼ 10(7)pfu/mL (group I, n=15), and highly susceptible isolates, which generated high phage titer ∼ 10(9)pfu/mL (group II, n=38). We selected 15 isolates (9 from group I and 6 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. Pathogenic E. coli strains belonging to other serogroups (12 serogroups, 67 isolates) as well as nontarget microorganisms (n=18) were not lysed by the bacteriophage and hence were not detected. The method is high-throughput compatible, is rapid, and can provide live culture the following day by streaking an aliquot before phage lysis on conventional selective agar media.
authors.affiliation: Environmental Microbial and Food Safety Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, Maryland 20705-2350, USA.
authors.forename: Porteen
authors.initials: P
authors.lastname: Kannan
authors.forename: Ho Yao
authors.initials: HY
authors.lastname: Yong
authors.forename: Lucy
authors.initials: L
authors.lastname: Reiman
authors.forename: Claire
authors.initials: C
authors.lastname: Cleaver
authors.forename: Pradip
authors.initials: P
authors.lastname: Patel
authors.forename: Arvind A
authors.initials: AA
authors.lastname: Bhagwat
mesh_terms: D000818:Animals; D001435:Bacteriophages; D002417:Cattle; D015169:Colony Count, Microbial; D003470:Culture Media / Q000378:metabolism; D019453:Escherichia coli O157 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000821:virology; D005506:Food Contamination; D005516:Food Microbiology; D018189:Immunomagnetic Separation / Q000379:methods; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D003470:Culture Media