Bacteriophage Lysis of Enterococcus Host Strains: A Tool for Microbial Source Tracking?

Abstract

This paper describes the isolation of Enterococcus host strains, for potential use as simple bacteriophage (phage)-based microbial source tracking (MST) tools. Presumptive Enterococcus host strains were isolated from cattle feces, raw municipal wastewater, agricultural runoff, and waters impacted by farms or wastewater treatment works (WWTW) in southern England, United Kingdom (UK). All enterococcal host strains (n = 390) were first screened for their ability to detect phage in samples of raw municipal wastewater and fecal material from cattle, pigs, and sheep. Host strains that detected phage (n = 147) were ranked according to both their specificity to a particular fecal source and also the number of phages (expressed as plaque-forming units, PFU) that they detected per milliliter of sample. Host strains that demonstrated host specificity and which detected phages at levels greater than 100 PFU/mL (n = 29) were further tested using additional fecal samples of human and nonhuman origin. The specificity and sensitivity of the enterococcal host strains were found to vary, ranging from 44 to 100% and from 17 to 83%, respectively. Most notably, seven strains exhibited 100% specificity to either cattle, human, or pig samples. Isolates exhibiting specificity to cattle were identified as belonging to the species Enterococcus casseliflavus , Enterococcus mundtii , or Enterococcus gallinarum , while human and pig isolates were members of either Enterococcus faecium or Enterococcus faecalis . The high specificity of phages infecting Enterococcus hosts and the simplicity and relatively low cost of the approach collectively indicate a strong potential for using this method as a tool in MST.

Enterococci in the Environment

“…Bacteriophages that infect various Enterococcus spp. ("enterophage") from different sources (i.e., raw sewage, cow manure, and environmental waters) were recently described (31,223,268,279). However, the effect of enterophage on bacterial survival was not tested directly, since the main objective of these works was to examine the utility of enterophage as a microbial source tracking marker.…”

“…However, the effect of enterophage on bacterial survival was not tested directly, since the main objective of these works was to examine the utility of enterophage as a microbial source tracking marker. Nonetheless, the relatively high concentrations of enterophage that specifically infects E. casseliflavus, E. mundtii, or E. gallinarum from cow fecal slurry (10 4 to 10 5 PFU/100 ml) and E. faecalis or E. faecium (ϳ10 3 PFU/100 ml) from raw sewage (268) indicate that, at least in these instances, lysis by enterophage can be a predatory factor on populations of enterococci.…”

Bacteriophage removal in a full-scale membrane bioreactor (MBR) – Implications for wastewater reuse

“…Indeed, phages may be a more appropriate indicator of the presence of enteric viruses in water and wastewaters than the bacterial indicators that continue to be widely used (Jofre et al, 1986;Gantzer et al, 1998;Purnell et al, 2011;Ebdon et al, 2012;Jofre et al, 2014) et al (2013) demonstrated that reductions in levels of somatic coliphages and F-RNA specific phages were 2.7 and 1.7 log higher as a result of MBR treatment than by conventional activated sludge treatment. Zanetti et al (2010) observed that, despite the smaller diameter of F-RNA specific phages (21-30nm), their levels in the permeate were lower than those of somatic coliphages (30-100nm).…”

Bioaccumulation and persistence of faecal bacterial and viral indicators in Mytilus edulis and Crassostrea gigas

“…A dosing volume of 2.5 ml for each of the six microorganisms commonly observed in faecally impacted waters (E. coli, faecal coliforms, intestinal enterococci, somatic coliphages, F-RNA coliphages and bacteriophages infecting B. fragilis GB124) was added to experimental tanks (volume of water in each tank was 10 litres) containing C. gigas (15)(16)(17)(18) live oysters) or M. edulis (30 live mussels).…”

“…Enteric viruses have been detected in shellfish harvested from areas classified as 'Category A' status according to EU standards 11 , suggesting that the current hygiene classifications for shellfisheries, based on levels of bacterial indicators, may not offer an indication of risk of enteric viral pathogens. Several studies have proposed bacteriophages as alternative indicators of enteric viral contamination 2,7,8,15,18,24 . In this study, the parameters investigated in C. gigas and M. edulis were E. coli, faecal coliforms, intestinal enterococci, somatic 4 coliphages and F-RNA coliphages.…”