Impact of a Wastewater Treatment Plant on Enterococci Species Distribution in Southwestern Puerto Rico

Enterococci are widely used indicators of fecal contamination because they originate in the gastrointestinal tracts of warm-blooded animals, and species-level identification can support source attribution. This study evaluated the temporal abundance and species composition of enterococci in Quebrada Mondongo, southwestern Puerto Rico, a stream influenced by wastewater treatment plant (WWTP) effluent and nonpoint-source inputs. Five sampling campaigns for species distribution and fourteen for population quantification were conducted over approximately one year at the WWTP effluent discharge and at upstream and downstream stations. Enterococci concentrations exceeded the regulatory threshold for surface waters. Among the confirmed isolates, E. faecium dominated upstream and in the effluent, occurring approximately twofold more frequently than E. faecalis. Downstream, E. faecalis increased in relative abundance, shifting the species ratio of E. faecium/E. faecalis from 2.3–3.2 to 0.89. E. casseliflavus was detected at low frequency, and E. gallinarum was not observed. Virulence-associated genes (esp, gelE) were identified in ~75% of E. faecalis isolates, consistent with enhanced environmental persistence. Although upstream and effluent patterns reflected a strong human fecal signal, the downstream enrichment of E. faecalis suggests additional secondary inputs and/or naturalization. This study provides empirical evidence of species shifts in a tropical stream, with an increase in E. faecalis downstream of a WWTP despite E. faecium dominance in the effluent highlighting the likely influence of other nonpoint fecal sources within the watershed. Overall, these results suggest that the WWTP effluent did not contribute substantially to enterococci concentrations nor significantly influence the species composition of enterococci downstream in Quebrada Mondongo, highlighting the likely influence of other nonpoint fecal sources within the watershed.