Occurrence of novel human tomato brown rugose fruit virus and conventional microbial source tracking genetic markers in a Hawaiian coupled stream-beach system

Fecal contamination of coastal waters threatens human and ecosystem health. Microbial source tracking (MST) methods offer a strategy to identify sources of fecal contamination through the measurement of genetic markers associated with a particular animal host. In this study, we measured fecal indicator bacteria (FIB) and employed MST methods to evaluate the sources of fecal contamination in a coupled stream-beach system in Kailua Bay, Hawai'i where residents adjacent to the shoreline use onsite cesspools for sewage management. In a baseline campaign, we measured enterococci concentrations in surface water samples from the stream and beach (<i>n</i> = 36). Results indicated that the stream contained enterococci in exceedance of the state standard (50% of samples) and therefore represented a potential source of contamination to the coastal ocean. To identify potential fecal sources, five MST genetic markers - three indicative of human feces (HF183/BacR287, CPQ_056, and ToBRFV), one of dog feces (DG3), and one of avian feces (GFD) - were measured alongside enterococci concentrations and environmental parameters (water temperature, salinity, tidal stage, and rainfall) in stream and beach water samples from longitudinal (<i>n</i> = 78) and spatial (<i>n</i> = 25) sampling campaigns. During the two-week longitudinal campaign, detections were observed for the avian marker (78% of samples positive), human marker ToBRFV (40%), and dog marker (10%), while HF183/BacR287 and CPQ_056 were not detected. Marker detection frequency varied by sampling location, with GFD most frequently detected in the stream and ToBRFV most frequently detected at the site adjacent to cesspools. In the spatial campaign, enterococci concentrations significantly decreased along the stream towards the beach (<i>p</i> < 0.001) but similar trends were not observed for MST markers. The occurrence of human, avian, and canine MST genetic markers in this study confirms these are important sources of fecal contamination in the Kailua Bay area. This study is the first to implement the RNA-based ToBRFV digital PCR assay in tropical coastal waters.