Occurrence of antibiotic resistance genes and multidrug-resistant bacteria during wastewater treatment processes

Wastewater treatment plants (WWTPs) constantly receive a wide variety of contaminants, including pharmaceuticals, and are potential reservoirs of antibiotic resistance genes (ARGs). This favors the development of multidrug-resistant bacteria (MRB) through horizontal gene transfer. Samples from five different WWTP processes were collected in September 2020 and January 2021 to monitor ARG resistomes and culturable MRB in the presence of eight different antibiotics. Nanopore-based ARG abundance and bacterial community analyses suggested that ARG accumulation favors the generation of MRB. Activated and mixed sludges tended to have lower bacterial diversity and ARG abundance because of selective forces that favored the growth of specific microorganisms during aeration processes. Escherichia strains enriched in WWTPs (up to 71%) were dominant in all the samples, whereas Cloacamonas species were highly abundant only in anaerobically digested sludge samples (60%-79%). Two ARG types [sulfonamide resistance genes (sul1) and aminoglycoside resistance genes (aadA1, aadA13, and aadA2)] were prevalent in all the processes. The total counts of culturable MRB, such as Niabella, Enterococcus, Bacillus, and Chryseobacterium species, gradually increased during aerobic WWTP processes. Genomic analyses of all MRB isolated from the samples revealed that the resistome of Enterococcus species harbored the highest number of ARGs (7-18 ARGs), commonly encoding ant(6)-la, lnu(B), erm(B), and tet(S/M). On the other hand, Niablella strains possibly had intrinsic resistant phenotypes without ARGs. All MRB possessed ARGs originating from the same mobile genetic elements, suggesting that WWTPs are hotspots for the migration of ARGs and emergence of MRB.