Student Research: Nicola Koren Josephs

MS, , 2004
Faculty Advisor: John Meschke

An Evaluation of the Utility of F+RNA Coliphage for Source Tracking Pathogens in Environmental Waters


Abstract

The ability to identify the source of a waterborne contaminant is an important risk assessment tool, and a variety of microbial fingerprinting methods have been proposed for this purpose. F+RNA bacteriophages, a class of viruses that infect coliform bacteria possessing the F plasmid and expressing F-pili, are considered indicators of faecal contamination and potentially a method of tracking its origin. Prior studies have found general associations between the four F+RNA phage genotypes and faecal origin: gourp I has been found in human and animal wastes, groups II & III in human sources, and group IV with animal contamination. However, to date no biological mechanism has been proposed to account for these general associations. We hypothesize that the association between phage type and faecal source is due to differences in efficiency of phage infection fo E. coli that express different F plasmid genes and that he type of F-plasmis expressed is faeces type-specific. To evaluate the utility of F+RNA phages as a source tracking tool, ~1,000 spatially and temporally distinct E. coli isolates of known origin (human, non-human mammalian and avian) are examined for the presence of one or more of the five groups of pili-encoding plasmids and for their susceptibility to prototype strains of coliphage. Phage susceptibility is determined by spotting each phage type on an agar plate or an E. coli isolate. After overnight incubation plates are examined for lysis zones to assess the isolates' susceptibility to each phage strain. Two PCR primer sets were designed that screen E. coli for the presence of pilin encoding region found in the traA gene of the F plasmid's transfer region, and ~100 positive plasmid positive isolates were sequenced to distinguish between the 5 pili variants in order to investigate the biological mechanism which may be responsible for the selective infectivity of F+RNA phage groups and to determine the percent of different species that are expected to be susceptible to F+RNA phages. This study showed that a large % of environmental E. coli have F-like plasmids that should be susceptible to phages. The plasmid type carriage rates were different between different animal species, which may indicate a biologically plausible reason for faeces specificity of F+RNA phages.