Human-specific bacteriophages in sediments : a novel approach to waterborne hazard identification

Climate change has already led to an increase in the frequency and intensity of storm events in many parts of the world. With further increases predicted, and growing evidence of the link between extreme precipitation and waterborne disease, it is important to elucidate the role of sediments in pathogen transmission. Intense rainfall can trigger discharges from combined sewer overflows, increase surface and subsurface faecal inputs and can re-suspend microorganisms already present in sediments. Previous research has shown that sediments can act as environmental reservoirs of numerous waterborne pathogens, including enteric viruses and antibiotic resistant bacteria. Sediments are therefore a growing public health concern and are an increasing focus of human health protection strategies. However, further research is needed in order to elucidate the behaviour of microorganisms within these matrices. Routine monitoring for pathogens within sediments has not to date been considered feasible in many parts of the world and although low-cost microbial tools to detect faecal pollution of human origin, such as those that detect phages infecting Bacteroides fragilis (GB-124), have shown promise in many situations, they have seldom been applied to sediment matrices. Sediments might offer a more sensitive and longer-term assessment of contamination sources and hazards to health, compared with analysis of overlying waters from the same location. This study therefore sought to determine an effective elution method to extract GB-124 phages from sediments and to use it in an intensive six month investigation of the River (Ouse) catchment in Southeast England. The results (ANOVA with post-hoc Tukey) revealed that a low-cost elution method, involving 10% beef extract, provided the most effective means of recovering GB-124 phage from a range of river sediments (66% recovery). GB124 phages were subsequently enumerated in sediment and overlying water collected from 25 sites across the study catchment, along with somatic coliphage (SC), faecal coliforms (FC), and intestinal enterococci (IE). Physicochemical data were also collected. Analyses revealed evidence of faecal contamination at all sites, and human contamination at 13 of the 25 sites. Whilst levels of microorganism in the water and sediment were significantly correlated: GB-124 (p= 0.015); SC (p= 0.000); FC (p= 0.018); and IE (p= 0.038), importantly GB-124, SC, FC and IE (p= 0.00) were detected at significantly higher levels (Mann-Whitney) in the sediment samples. Significant correlations (p= <0.01) were also observed between levels of FC, IE and SC and sediment temperature, but not between water temperature and any of the parameters in the water column. Interestingly, GB-124 phage showed no significant correlation with the non human-specific parameters (SC, FC, and IE) in the sediment matrices, which were found to co-correlate with one another (p= 0.00). The findings suggest that the application of low-cost monitoring approaches to analyse river sediments may not only provide a better assessment of dominant pollution sources than grab samples of overlying water (due to the higher levels and incidence of phages in sediments), but that they may also provide a better indication of potential risks to health from human enteric viruses. As such, the findings of this study add significantly to the body of extant knowledge relating to the behaviour of GB-124 phages in the environment and further support their use for microbial source tracking (MST) and as a potential component of quantitative microbial risk assessment (QMRA) studies.