Large-scale implementation of standardized quantitative real-time PCR fecal source identification procedures in the Tillamook Bay Watershed

Abstract

Fecal pollution management remains one of the biggest challenges for water quality authorities worldwide. Advanced fecal pollution source identification technologies are now available that can provide quantitative information from many animal groups. As public interest in these methodologies grows, it is vital to use standardized procedures with clearly defined data acceptance metrics and conduct field studies demonstrating the use of these techniques to help resolve real-world water quality challenges. Here we apply recently standardized human-associated qPCR methods with custom data acceptance metrics (HF183/BacR287 and HumM2), along with established procedures for ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution sources to (i) demonstrate the feasibility of implementing standardized qPCR procedures in a large-scale field study, and (ii) characterize trends in fecal pollution sources in the research area. A total of 602 water samples were collected over a one-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries in the Tillamook Bay Watershed (OR, USA). Host-associated qPCR results were combined with high-resolution geographic information system (GIS) land use and general indicator bacteria ( E . coli ) measurements to elucidate water quality fecal pollution trends. Results demonstrate the feasibility of implementing standardized fecal source identification qPCR methods with established data acceptance metrics in a large-scale field study leading to new investigative leads suggesting that elevated E . coli levels may be linked to specific pollution sources and land use activities in the Tillamook Bay Watershed.

Microbial Indicators of Fecal Pollution: Recent Progress and Challenges in Assessing Water Quality

“…The value of fecal indicators as investigative tools to identify fecal pollution has been reaffirmed and expanded with the broad adoption of MST approaches, despite imperfect sensitivity and specificity [ 58 , 81 , 110 , 118 •, 119 ]. Also, the literature on many technical aspects of fecal indicators and their applications has notably matured, as demonstrated by the recent publication of several comprehensive reviews [ 2 •, 17 , 25 , 27 , 44 , 144 , 153 ].…”

“…Such approaches increase the complexity, expense, and time requirements for analysis, and physical removal of inhibitors through dilution or purification also reduces target DNA, providing diminishing returns to analytical sensitivity [ 147 , 148 ]. Complete abatement of qPCR inhibition is likely unrealistic; nevertheless, recent efforts to standardize qPCR procedures for water quality assessment suggest that a set of existing mitigation practices is sufficient to render matrix interference a manageable nuisance in most applications [ 81 , 144 , 149 •]. Increasing adoption of digital PCR (dPCR), a quantitative PCR approach robust to inhibition, will likely further alleviate the challenge of inhibition for routine molecular detection of fecal microbes [ 61 , 114 , 128 , 150 – 152 ].…”

“…Two toolbox constituents recently receiving much attention in the literature are pepper mild mottle virus (PMMoV), a plant virus infecting Capsicum species acquired by humans from dietary sources, and crAssphage, both viruses that hold promise as human-associated viral surrogates owing to their global distribution in sewage at densities typically much higher than other viruses [ 2 •, 71 , 74 , 78 , 108 – 110 ]. Nonetheless, Bacteroides HF183 and its variants have arguably consolidated their role as the default tool for human source tracking [ 43 ], featuring consistently high concentrations in sewage globally [ 83 •], frequent detection in surface waters [ 61 , 93 , 110 – 112 ], standardized protocols [ 81 , 113 ], and validated multiplex assays [ 89 , 114 ]. However, the diagnostic performance of HF183 and most other human-associated markers has typically been poor in highly contaminated settings in many low- and middle-income countries (LMIC) [ 58 , 69 , 70 , 72 , 75 , 115 ], with the exception of high sensitivity to child feces in urban Kenya [ 102 •].…”

“…Unlike human-associated markers, animal fecal markers (e.g., livestock-associated BacCow and canine-associated BacCan, both with Bacteroidales targets, and avian-associated GFD, which targets the genus Helicobacter ) have performed well in LMIC settings and have repeatedly identified livestock as major sources of fecal contamination and pathogens in the domestic environment, supporting recent calls for renewed emphasis on animal waste management [ 58 , 116 ]. Likewise, MST investigations can impact management and mitigation programs by determining that wildlife, livestock, or pets contributed substantially to fecal pollution in certain watersheds and beaches [ 81 , 117 •, 118 •, 119 ]. The forensic potential of MST was demonstrated during a 2019 Campylobacter outbreak in Norway, which was attributed to non-human sources, most likely horses, using a combination of FIB, MST, and direct pathogen detection [ 120 ].…”

Democratizing water monitoring: Implementation of a community-based qPCR monitoring program for recreational water hazards

“…Seven of these samples approached the LOD 95 of the Open qPCR thermocycler, and CBM partners detected one of these samples. This is a common issue in PCR based fecal source tracking studies, not just in CBM studies, and highlights the importance of ensuring a particular method fits the research question, especially in a CBM study [33]. Nonetheless, a recent study found that an HF183 gene copy number of 4200 HF183/100ml exceeds the USEPA benchmark risk of GI illness [10].…”