Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream

Abstract

Fecal indicator bacteria (FIB) are the basis for water quality regulations and are considered proxies for waterborne pathogens when conducting human health risk assessments. The direct detection of pathogens in water and simultaneous identification of the source of fecal contamination are possible with microarrays, circumventing the drawbacks to FIB approaches. A multigene target microarray was used to assess the prevalence of waterborne pathogens in a fecally impaired mixed-use watershed. The results indicate that fecal coliforms have improved substantially in the watershed since its listing as a 303(d) impaired stream in 2002 and are now near United States recreational water criterion standards. However, waterborne pathogens are still prevalent in the watershed, as viruses (bocavirus, hepatitis E and A viruses, norovirus, and enterovirus G), bacteria (Campylobacter spp., Clostridium spp., enterohemorrhagic and enterotoxigenic Escherichia coli, uropathogenic E. coli, Enterococcus faecalis, Helicobacter spp., Salmonella spp., and Vibrio spp.), and eukaryotes (Acanthamoeba spp., Entamoeba histolytica, and Naegleria fowleri) were detected. A comparison of the stream microbial ecology with that of sewage, cattle, and swine fecal samples revealed that human sources of fecal contamination dominate in the watershed. The methodology presented is applicable to a wide range of impaired streams for the identification of human health risk due to waterborne pathogens and for the identification of areas for remediation efforts.IMPORTANCE The direct detection of waterborne pathogens in water overcomes many of the limitations of the fecal indicator paradigm. Furthermore, the identification of the source of fecal impairment aids in identifying areas for remediation efforts. Multitarget gene microarrays are shown to simultaneously identify waterborne pathogens and aid in determining the sources of impairment, enabling further focused investigations. This study shows the use of this methodology in a historically impaired watershed in which total maximum daily load reductions have been successfully implemented to reduce risk. The results suggest that while the fecal indicators have been reduced more than 96% and are nearing recreational water criterion levels, pathogens are still detectable in the watershed. Microbial source tracking results show that additional remediation efforts are needed to reduce the impact of human sewage in the watershed.

Keywords: TMDL; fecal organisms; indicator bacteria; microarray; microbial source tracking; waterborne pathogen.

FIG 1

Prickett Creek watershed boundary and land use, sampling sites, NPDES outlets, and water quality parameters at time of sample collection. Map created using ArcGIS version 10.3 (Environmental Systems Research Institute, Redlands, CA).

FIG 2

Comparisons of qPCR-based concentrations and normalized fluorescence values for different gene targets on the microarray.

FIG 3

Overview of the association among fecal and stream samples by NMDS using the rRNA gene data. Plot stress, 0.052; S, human sewage; C, cattle feces; P, swine feces. Numbers 1 to 8 are the stream identifiers. Numbers 10, 20, 30, 50, 70, 90, and 100 indicate the percentages of nucleic acids from that source group added to the whole-genome amplification.

FIG 4

Heat map indicating relative concentrations of pathogens, antibiotic resistance genes, and source tracking markers and the clustering of different samples. Cluster analysis was done by the Bray-Curtis similarity measure and the paired group algorithm, resulting in a coefficient of correlation of 0.95. Numbers at the nodes represent the percentages of the time the branch topology was generated during 1,000 bootstrap analyses. S, human sewage; C, cattle feces; P, swine feces; W, PCR-grade water; mixed, 50% S plus 10% C plus 10% P plus 10% sheep feces plus 20% PCR-grade water. Numbers 1 to 8 are the stream identifiers.