Assessing crAss-like phages as indicators of antibiotic resistance genes under environmental and disinfection conditions

Wastewater systems act as reservoirs for the dissemination of antibiotic resistance genes (ARGs), contributing to the global issue of antimicrobial resistance. CrAss-like phages, such as the prototypical p-crAssphage and the recently identified crAssBcn phages, exhibit high specificity for human fecal contamination, notable environmental persistence, and strong correlations with ARGs. This study assessed their potential as ARG indicators by comparing the persistence of naturally occurring crAss-like phages and three ARGs (tetW, sul1, and bla_TEM) in urban wastewaters under natural seasonal conditions (winter and summer) using a mesocosm approach. Additionally, inactivation rates were evaluated after disinfection treatments including UV irradiation (5.94-178.2 mJ/cm²), chlorination (10 ppm), and thermal inactivation (60°C and 80°C). Escherichia coli and somatic coliphages served as reference microbial indicators. CrAssBcn phages displayed greater environmental persistence than p-crAssphage in both seasons, showing stability comparable to or exceeding that of ARGs. Notably, crAssBcn remained stable throughout winter, whereas sul1 was the most persistent ARG, showing no detectable inactivation. Both crAss-like phages and ARGs were highly stable after chlorination and thermal treatments, while UV irradiation caused higher reductions in crAss-like phages than in ARGs. These findings highlight crAssBcn's potential as a reliable indicator for ARGs of anthropogenic origin under environmental conditions.