Metagenomic analysis reveals wastewater treatment plants as hotspots of antibiotic- and biocide-metal resistome and mobilome

Abstract

The intensive use of antibiotics due to their broad applications and incessant release into the environment has led to the widespread emergence of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Antibiotic resistance has turned into a genuine threat to human health. Wastewater treatment plants (WWTPs) have been identified as one of the major sources of ARGs and biocide-metal resistance genes (BMRGs), known to provide numerous environmental conditions potentially supporting the selection of ARGs and their dissemination into the environment mostly via horizontal gene transfer (HGT). Even though the distribution of antibiotic- and biocide-metal resistome (the ensemble of resistance genes) in WWTPs has been profoundly investigated, in-depth knowledge of the prevalence and abundance of mobilome (transposable elements and integrative and conjugative elements) and their association to ARGs is still lacking. Moreover, little is known regarding the pool of antibiotic- and biocide-metal resistome along with the mobilome in the chromosomal and plasmid metagenome. In this study, using Illumina high-throughput sequencing based metagenomic approach, we investigated the prevalence and abundance of antibiotic- and biocide-metal resistome and mobilome in the influent, effluent, upstream and downstream samples of three different WWTPs located at Seoul, Gwangju, and Jangseong city of South Korea. Our results showed that 21 out of 24 ARG types were detected in all the WWTPs. Moreover, BMRG abundance was found to be approximately 2 times higher than that of ARGs. Genes conferring resistance to multi-drug and rifampicin were detected high in all the samples. Additionally, our results suggested that mobile MCR-5 colistin resistance gene may be present on a plasmid in the influent sample of Gwangju WWTP. Metal resistance gene abundance was found to be higher than that of the biocide resistance gene, while genes conferring resistance to both biocides and metals were a few. The influent samples showed higher antibiotic and biocide-metal resistome abundance than those of the effluent (10 times and 5-7 times, respectively), suggesting the successful elimination of antibiotic- and biocide-metal resistome by the WWTPs. Similar trends were observed for the mobilome abundance in all the WWTPs. Nevertheless, the downstream showed a higher abundance of antibiotic- and biocide-metal resistome and mobilome than the upstream in the two WWTPs. On the other hand, we detected a higher abundance of integrative and conjugative elements (ICE) than that of the transposable elements in all the samples. Furthermore, a large number of mobile ARGs were associated with ICE and located on the chromosomes, while several mobilizable and conjugative plasmids were found to carry mobile ARGs along with BMRGs and virulence genes. It has been reported that plasmids carrying mobile ARGs were designated to various human pathogenic bacteria such as Vibrio cholerae, Salmonella enterica subsp. enterica serovar Heidelberg, Klebsiella pneumoniae, Salmonella enterica subsp. enterica serovar Corvallis, and Pseudomonas syringae. Therefore, regardless the abundance, those plasmids should be monitored with caution. Our results showed that the plasmids can persist the treatment processes in the WWTPs, depending on the total abundance of plasmids. On analyzing the microbial community, the phylum Proteobacteria dominated all the WWTP samples. At the genus level, there was a substantial difference between the influent and effluent samples although the downstream and upstream samples did not show any significant difference in microbial community composition. Moreover, various potentially pathogenic species belonging to genera Pseudomonas and Acinetobacter were detected in all the WWTPs. Overall, our results suggest that the WWTPs may serve as a potential hotspot for antibiotic- and biocides-metal resistome and mobilome, although a substantial amount of resistome was successfully removed. Yet, WWTPs likely contribute to the dissemination of these genes into the surrounding environment, thus resistome near WWTPs should be monitored continuously and intensively.