Antibiotic resistance genes are increased by combined exposure to sulfamethoxazole and naproxen but relieved by low-salinity

Combined pollution of antibiotic and non-antibiotic pharmaceutical residues is ubiquitous in realistic polluted environments, which is regarded as a complicated emerging pollution. Herein, high-throughput sequencing and high-throughput quantitative PCR were applied to profile the overall changes in microbial communities and antibiotic resistance genes (ARGs) of biofilms in response to a combination of naproxen and sulfamethoxazole pollution. After continuous operation for 120 days, naproxen or/and sulfamethoxazole were efficiently removed, and the salinity of 1.00% enhanced the removal rate of sulfamethoxazole. The high-throughput sequencing revealed that Eubacterium spp. with abundances of over 40.00% dominated in all samples, and combined pollution of naproxen and sulfamethoxazole more readily promoted the occurrence of multidrug-resistant microbes, including Pseudomonas and Methylophilus. The high-throughput quantitative PCR results showed that the combined pollution of naproxen and sulfamethoxazole increased the total abundance of ARGs to approximately 9 copies per cell. In contrast, increasing the salinity to 1.00% greatly reduced the overall abundance of ARGs to below 2 copies per bacterial cell. Mantel test and Procrustes analysis indicated that microbiomes from different treatments had tight links to their respective antibiotic resistomes. Furthermore, network analysis revealed that multidrug-resistant microbes were potential hosts for greatly enriched numbers of ARGs in the combined treatment. As increased salinity eliminated those multidrug-resistant but salt-sensitive microbes, the abundance of ARGs was significantly decreased. These results showed the high probability of the transmission of ARGs in biofilms exposed to combined pollution of naproxen and sulfamethoxazole, which could be relieved by increased salinity.