Evaluation of Bacteroides fragilis GB-124 bacteriophages as novel human-associated faecal indicators in the United States

Abstract

Phages infecting human-associated Bacteroides fragilis (GB-124 phages) have been employed in the European Union (EU) to identify human faecal pollution, but their utility for the United States was unclear. Primary sewage samples were collected seasonally from seven wastewater treatment plants (WWTP) across the continental United States, and more time-intensive sampling was conducted at local WWTPs. All samples were assayed for plaque-forming units (PFU) of GB-124 phages, somatic and FRNA-specific coliphages, as well as adenoviruses (by quantitative PCR [qPCR]). Animal faecal samples (>250) from 14 different species were tested for the presence of the three phage groups. GB-124 phages were consistently detected in sewage (10-10(2) PFU ml(-1) ), but not in animal faeces. While density estimates of both coliphages in sewage were approximately one order of magnitude higher than GB-124 phages, they were both randomly detected in animal faecal samples (10(2) -10(5) g(-1) dry weight). Stability of all three phages was inversely proportional to temperature; persistence was greatest at 5°C compared to 20 and 35°C, where no phages were detectable after a week. In summary, GB-124 phages appear to be a feasible alternative indicator organism and benefit from being sewage associated, while providing an inexpensive detection technique for infectious virions.

Significance and impact of the study: Bacteroides fragilis GB-124 phages appear to be restricted to human sewage sources in the United States, being absent from 264 animal faecal samples from 14 different species and present in approx. 90% (34/38) of primary sewage effluent samples collected across the country. Although somatic and F-specific coliphages were present in sewage samples at higher densities, unlike GB-124 phages, both coliphage types were also detected in animal faecal samples. Hence, GB-124 phages may prove to be a useful novel indicator group for human faecal pollution in the continental United States.

Keywords: GB-124 Bacteroides phage; adenovirus; human faecal indicator.