Temporal dynamics of class 1 and class 3 integron pools in treated wastewater

Wastewater is a hotspot for the spread and evolution of antimicrobial resistance (AMR), with integrons playing a key role in the dissemination of AMR. However, there is limited information available regarding the integron pool of wastewater environments. The present study aims to characterize the integron pool in outflow secondary-treated municipal wastewater samples collected weekly over the course of a year (n = 52 samples) and to identify the risks associated with the carriage of clinically relevant antimicrobial resistance gene cassettes (ARGCs). Integron arrays were amplified with the primer combinations intI-R/3'CS and intI3F/class3R. PacBio long-read sequencing was performed on 12 monthly pooled samples per primer pair. The analysis yielded 283,579 integron reads, capturing an estimated 10%-20% of the total integron pool, with the most abundant arrays successfully recovered. Most identified GCs had no known function (33.3% to 74.5% depending on integron class). Regarding GCs with known function, AMR was the most prevalent function in class 1 integrons. Class 3 integrons primarily encoded defence mechanisms and amino acid transport and metabolism, while cassettes associated with replication, recombination and repair were abundant in integrons of unknown class. The prevalence of ARGCs for class 1, 3 and integrons of unknown class was 61.7%, 19.2% and 9.6%, respectively. Most ARGCs (from the 146 identified) encoded resistance to aminoglycosides and beta-lactams, the most abundant being aadA10, aac(6')-IIc and bla_GES-5. Most beta-lactamase-encoding GCs (BLGCs) identified encoded enzymes with carbapenemase and/or extended-spectrum activity, with class 1 and class 3 integrons being relevant reservoirs of BLGCs. GCs encoding proteins involved in oxidative stress repair, as well as in other functions, were also identified and might promote co-selection of ARGCs. Large variation of the integron arrays and the GC pool was observed between seasons and pooled monthly samples, with resistance genotypes such as macrolide and phenicol resistance differentially abundant across seasons. Overall, these findings show that secondary-treated wastewater is a reservoir of highly diverse class 1 and class 3 resistance integrons, some encoding critically relevant resistance mechanisms (e.g., carbapenemases).