The Application of Bacteriophage for the Elimination of Pathogenic Bacteria in Compost

The demand for organically grown produce is rapidly rising in the United States, resulting in a resurgence of natural fertilizers, such as animal waste, being used in agriculture. Currently, there are guidelines set by the USDA for the proper processing of fecal materials through composting; however food-borne illnesses associated with fresh-produce have become more and more common. Research is being conducted toward developing more effective treatments of fecal waste to reduce the survival of harmful bacteria. One possible form of treatment is through the use of bacteriophages that are able to \u27seek and destroy\u27 specific bacteria. The objectives of this study were to: 1) isolate and characterize strains of bacteriophage capable of lysing Salmonella spp. and Escherichia coli O157:H7, 2) to develop and optimize a cocktail of these bacteriophages, and 3) to reduce pathogens in compost by administering bacteriophage to autoclaved and non-autoclaved composts inoculated with either Salmonella or E. coli O157:H7. In the characterization and isolation of bacteriophage study, thirty-four Salmonella-specific and forty-two E. coli O157:H7-specific bacteriophages were isolated from raw sewage using an enrichment method. Ten phage strains for each pathogen were selected for use in optimization studies in liquid media. Phages exhibited little to no effectiveness in 0.85% saline or SM buffer, but were highly active in tryptic soy broth (TSB). Electron microscopy and restriction digest analysis were used to characterize the selected phages. A five-phage cocktail was developed and tested against five strains of Salmonella enterica encompassing four serovars: Typhimurium, Poona, Newport and Enteritidis. The cocktail was effective at preventing the growth of Salmonella Typhimurium 8243 indefinitely in TSB. Treatments of phage cocktail suppressed the growth of other strains initially, but had little effect on preventing the development of phage resistance. For the compost study, both autoclaved and non-autoclaved dairy compost was inoculated with 105 CFU/g of either Salmonella Typhimurium or E. coli O157:H7 and held for 24 h followed by phage treatment. Reductions of 2.34, 2.41 and 2.56 logs were observed in non-autoclaved compost inoculated with Salmonella Typhimurium 8243 at phage multiplicity of infections (MOIs) of 1, 10 and 50, respectively. An identical study using E. coli O157:H7 strain 0923-21 had reductions of 0.17, 1.21 and 1.5 logs with MOIs of 1, 10 and 50, respectively. The impact of water activity in compost on pathogen reductions by phages was further studied. Compost prepared at water activity levels of 0.9, 0.95 and 0.98 yielded no reductions as a result of phage treatment in any of the trials. Non-autoclaved compost inoculated with Salmonella had reductions of 2 to 3 logs when the water activity was close to 1. Non-autoclaved compost inoculated with E. coli O157:H7 had a one log decrease after phage treatment. Only slight reductions in E. coli O157:H7 and Salmonella were detected in the autoclaved compost (aw=0.999) as a result of 2 to 3 logs of bacterial growth prior to phage treatment. The results indicate that specific bacteriophage cocktails are effective at reducing pathogen populations in compost under certain conditions, such as an appropriate ratio of phage to bacteria and high water activity. This suggests that phage treatment may be useful as a supplement to composting for the further elimination of pathogenic bacteria in animal waste