A reporter bacteriophage - beta-galactosidase assay for detection of generic Escherichia coli from beef carcasses

Recent outbreaks of foodborne illness during the past decade have highlighted the need for fundamental changes in meat inspection programs. The Hazard Analysis Critical Control Point (HACCP) program is a method for evaluating and controlling the safety and quality of all processed food. Central to the HACCP plan is the requirement for rapid and reliable microbiological detection methods. This thesis describes the development and characterization of a reporter bacteriophage - beta-galactosidase assay for detection and enumeration of generic Escherichia coli from beef carcasses. Generic E. coli testing is often employed as an indicator of fecal contamination. Two bacteriophages capable of growing on many serotypes of E. coli were characterized according to their morphological, host range, and genetic properties. The host range genes of one of these bacteriophages, bacteriophage AR1, were cloned into a plasmid and this host range plasmid was used to create several T4 bacteriophages that had an expanded host range by growing the T4 bacteriophages on cells harboring the host range plasmid and allowing for homologous recombination between the plasmid and the T4 bacteriophages. The expanded host range T4 bacteriophages were further genetically modified to carry the E. coli beta-galactosidase (lacZ) gene. These reporter bacteriophages were used in the reporter - bacteriophage, beta-galactosidase assay. The sensitivity of the assay for E. coli in pure suspensions was determined. The specificity of the assay was determined with E. coli isolates from beef and with other bacterial genera. The reporter bacteriophage beta-galactosidase assay was capable of detecting 5 x 102 CFU/ml of E. coli without an enrichment step if a chemiluminescent substrate for beta-galactosidase was employed. However, the assay could only detect 5 of 19 E. coli isolates from beef carcasses. None of the non E. coli bacteria tested were detected by the assay. A most probable number method (MPN) with three parallels, based on the use of the lacZ reporter bacteriophages is described. The lacZ reporter bacteriophages were used to differentiate between positive and negative samples in the MPN. The MPN method is sensitive, and enables the enumeration of 10' CFU/ml of E. coli cells in broth culture within 8 hours.