Dissemination mechanisms of unique antibiotic resistance genes from flowback water to soil revealed by combined Illumina and Nanopore sequencing

Abstract

As a byproduct of shale gas extraction, flowback water (FW)is produced in large quantities globally. Due to the unique interactions between pollutants and microorganisms, FW always harbor multiple antibiotic resistance genes (ARGs) that have been confirmed in our previous findings, potentially serving as a point source for ARGs released into the environment. However, whether ARGs in FW can disseminate or integrate into the environmental resistome remains unclear. In this study, unique ARGs from FW were identified, and the ARG profiles in soil and FW-spiked soil were compared using a combination of Illumina and Nanopore sequencing. The results indicated that the total abundance of the soil resistome increased by 30.8% in soil contaminated with FW. Of this increase, 11.1% was attributable to the integration of exogenous ARGs from FW into the soil resistome. Sequence alignment at the gene level further confirmed the successful integration of 20 unique ARG sequences classified as multidrug and vancomycin resistance genes into the soil resistome. These 20 ARG sequences were detected only in the FW. Multiple lines of evidence indicated that horizontal gene transfer dominated ARG dissemination in soil contaminated by FW. This conclusion is supported by the discrepancy between changes in mobile ARGs and host abundance, the upregulation of oxidative stress-related genes (SOD1 and SOD2) and the SOS response (lexA and recA), as well as the upregulation of genes related to quorum sensing (virD4, virB9, and virB3) and naked DNA uptake (pilD, pilT, and pilQ).