Pollutants discharged from wastewater are the main cause of the spread of antibiotic resistance in river biofilms. There is controversy regarding the primary contribution of environmental selectors such as antibiotics and heavy metals to the development of antibiotic resistance in bacterial communities. Here, this study compared the effect of environmental safety concentration Cu²⁺ and enrofloxacin (ENR) on the evolution of antibiotic resistance by examining phenotypic characteristics and genotypic profiles of bacterial communities in a river biofilm, and then distinguished the major determinants from a comprehensive perspective. The pollution induced community tolerance in ENR-treated group was significantly higher than that in Cu²⁺-treated group (at concentration levels of 100 and 1000 μg/L). Metagenomic sequencing results showed that ENR significantly increased the number and total abundance of antibiotic resistance genes (ARGs), but there was no significant change in the Cu²⁺- treated group. Compared with Cu²⁺, ENR was the major selective agent in driving the change of taxonomic composition because the taxonomic composition in ENR was the most different from the original biofilm. Comparing and analyzing the prokaryotic composition, the phylum of Proteobacteria was enriched in both ENR and Cu²⁺ treated groups. Among them, Acidovorax and Bosea showed resistance to both pollutants. Linking taxonomic composition to ARGs revealed that the main potential hosts of fluoroquinolone resistance genes were Comamonas, Sphingopyxis, Bradyrhizobium, Afipia, Rhodopseudomonas, Luteimonas and Hoeflea. The co-occurrence of ARGs and metal resistance genes (MRGs) showed that the multidrug efflux pump was the key mechanism connecting MRGs and ARGs. Network analysis also revealed that the reason of Cu²⁺ selected for fluoroquinolones resistant bacterial communities was the coexistence of multidrug efflux gene and MRGs. Our research emphasizes the importance of antibiotics in promoting the development of antibiotic resistant bacterial communities from the perspective of changes in community structure and resistome in river biofilms.

废水排放的污染物是河流生物膜中抗生素耐药性传播的主要原因。关于抗生素和重金属等环境选择剂对细菌群落抗生素耐药性发展的主要贡献存在争议。在这里，本研究通过检查河流生物膜中细菌群落的表型特征和基因型谱，比较了环境安全浓度 Cu ²⁺和恩诺沙星 (ENR) 对抗生素耐药性演变的影响，然后从综合角度区分了主要决定因素. ENR处理组的污染诱导群落耐受性显着高于Cu ²⁺-处理组（浓度水平为 100 和 1000 μg/L）。宏基因组测序结果显示，ENR显着增加了抗生素抗性基因（ARGs）的数量和总丰度，但Cu ²⁺ - 处理组没有显着变化。与Cu ²⁺相比，ENR是驱动生物分类组成变化的主要选择剂，因为ENR中的分类组成与原始生物膜的差异最大。比较和分析原核组成，变形菌门在ENR和Cu ²⁺处理组中均富集。其中，西瓜果和氧化包西氏对两种污染物均表现出抵抗力。联的分类学组合物的ARG透露，氟喹诺酮的抗性基因的主要潜在主机名丛毛单胞，鞘脂单胞，慢生根瘤菌，Afipia，红假单胞菌属，Luteimonas和Hoeflea。ARGs 和金属抗性基因 (MRGs) 的共存表明，多药外排泵是连接 MRGs 和 ARGs 的关键机制。网络分析还揭示了Cu ²⁺选择氟喹诺酮类耐药细菌群落的原因是多药外排基因和 MRGs 的共存。我们的研究从河流生物膜群落结构和耐药组变化的角度强调了抗生素在促进抗生素耐药细菌群落发展中的重要性。