Isolation and characterisation of bacteriophages for the biocontrol of Enterococcus faecalis and Escherichia coli on surfaces and in milk

Bacteriophages have been used to treat bacterial infections since their discovery almost 100 years ago. Furthermore, phages have been increasingly investigated for the biocontrol of bacterial pathogens in a diverse range of environmental applications. The major aims of this research were to isolate and characterise bacteriophages for the biocontrol of two organisms, Enterococcus faecalis and Escherichia coli, on surfaces and in milk. While normally harmless, both organisms are capable of causing disease. E. faecalis and E. coli are commonly isolated from foods of animal origin including dairy products. Furthermore, E. faecalis and E. coli contaminated surfaces have been indirectly linked to cases of human illness. While many studies have investigated using phages to control E. coli O157 on surfaces and in foods, fewer studies have considered other serotypes. In contrast, there are no studies reporting the use of phages in the biocontrol of E. faecalis. In the current study, phages infecting E. faecalis and E. coli were isolated from environmental sources in Victoria, Australia, including sewage and landfill leachate. Selected isolates were characterised by biological and genetic means. Four enterococcal phages, designated фSUT1, фSUT3, фSUT4 and фSUT6 were found by electron microscopy to belong to the Siphoviridae. Three coliphages, фEC6, фEC9 and фEC11, were morphologically distinct. The coliphage isolates possessed a wider host range compared to the enterococcal phages, including pathogenic strains of E. coli. The ability of the фSUT phages to control their enterococcal hosts on a range of surfaces commonly found in hospitals was assessed. фSUT1 achieved significant reductions in E. faecalis on all surfaces following 120 minutes of incubation at ambient temperature. The remaining phages were less effective in reducing their respective hosts on all surfaces. Phage cocktails comprising the фEC phages were used to similarly disinfect glass slides and stainless steel coupons contaminated with one of four strains of E. coli. When applied at a concentration of 108 PFU/mL, the phage cocktails eliminated each strain within 10 minutes of application. At lower concentrations, the effect was largely dose and time-dependent. Given the association of E. faecalis and E. coli with dairy products it was determined that milk, and in particular raw milk, could be a potential novel application for phage biocontrol. While the microbiota of raw milk has been studied extensively worldwide, there is little published information regarding the quality of raw milk produced in Australia. This study found Australian raw milk to contain pathogens including Salmonella spp., as well as indicators of faecal contamination including coliforms, Enterococcus spp. and E. coli. There was considerable variation in the microbiological quality of raw milk samples obtained from three sources indicating that a wider study of Australian milk over time could be beneficial. Given the incidence of Enterococcus spp. and E. coli in raw milk in the current study, the ability of phages to control these organisms in raw milk was investigated. фSUT1 and фSUT4 were selected to control two strains of E. faecalis in UHT and pasteurized milk. The two phages varied considerably in their ability to reduce their respective hosts. фSUT4 initially inhibited the growth of a vancomycin-resistant Enterococcus (VRE) in UHT milk but growth resumed over time. While фSUT1 significantly reduced E. faecalis in UHT milk at 10 and 25°C, a complete reduction was not achieved. Both phages were less effective in reducing their hosts in pasteurized milk, although phage titres remained stable. The фEC phages were combined in a cocktail to control the growth of four strains of E. coli in raw milk. The cocktail eliminated E. coli K12, G106 and O127:H6 in raw milk at both 5-9°C and 25°C. In milk contaminated with E. coli O5:NM, the phage cocktail achieved a complete reduction initially but E. coli increased at both 5-9°C and 25°C over time. The results of the raw milk studies were promising but further investigations should be conducted to determine the effect of differences in milk composition and microbiota on the efficacy of the phages. The findings from this research suggested that phage biocontrol of E. faecalis, which has not been previously reported, can be successful if the appropriate phages are used. Furthermore, the coliphages described were effective in reducing E. coli, including pathogenic strains, on surfaces and in milk. Therefore, further study of these phages in a wider range of applications is warranted.