Occurrence, fate, and particle association of the potential fecal indicator plasmid pBI143 in wastewater from a small urban system

• pBI143 consistently exceeded crAssphage concentrations in wastewater. • pBI143 has a faster decay rate than crAssphage in wastewater. • Greater log reduction values for pBI143 indicate higher removal during wastewater treatment compared to crAssphage. As fecal contamination continues to be a global problem, reliable microbial fecal indicators are essential for detecting and tracking human fecal contamination in wastewater and contaminated environmental waters. This study assessed the performance of the cryptic plasmid pBI143 as a fecal contamination marker by comparing its occurrence, treatment removal, and decay behavior with those of crAssphage, a widely recognized human-associated bacteriophage. Over a seven-month monitoring period, pBI143 was consistently detected at higher concentrations (mean concentrations of 9.24 log 10 copies/L) than crAssphage across untreated influent and multiple stages of the wastewater treatment process. Quantitative analyses revealed that pBI143 exhibited greater log reduction values through treatment (up to 2.5 log 10 reductions), suggesting a higher degree of removal compared to crAssphage. Controlled decay experiments in raw wastewater further demonstrated that pBI143 undergoes faster degradation, with higher k values than those observed for crAssphage. The consistently high concentrations of pBI143 in untreated wastewater highlight its sensitivity for detecting fecal pollution, while its higher removal rates and faster decay suggest it may serve as a more conservative indicator for treatment performance and recent contamination events. In contrast, the greater environmental persistence of crAssphage may make it more suitable for detecting historical or distal contamination sources. Together, these findings indicate that pBI143 could complement existing viral and bacterial indicators to provide a more nuanced understanding of fecal pollution dynamics. Further research is warranted to evaluate pBI143 across diverse geographic regions, environmental conditions, and contamination scenarios to fully establish its applicability as a standardized tool for fecal contamination monitoring. Wastewater samples were collected over 7 months and showed that pBI143 consistently exceeded crAssphage concentrations in wastewater. Greater log reduction values for pBI143 indicate higher removal during wastewater treatment compared to crAssphage. Overall, these findings underscore the unique potential of pBI143 as both a fecal indicator and a reliable marker for population normalization.