Table 1_The ecological security risks of bronopol: a focus on antibiotic resistance gene dissemination.docx

Disinfectants are commonly utilized by humans to combat microorganisms. However, residual disinfectants may promote environmental antimicrobial resistance by facilitating horizontal gene transfer (HGT) of antibiotic resistance genes. Bronopol is a routinely used disinfectant that persists in the environment, and previous studies have concentrated on its ecotoxicity rather than its implications on the propagation of resistance genes. This study aimed to establish an in vitro conjugation model to investigate whether bronopol promotes the transfer of antibiotic resistance genes (ARGs) via plasmid conjugation. Using Escherichia coli DH5α and DC8855 as donors harboring RP4-7 and blaNDM-4-positive IncFII(K) plasmids, respectively, and J53 as the recipient strain, we found that sub-inhibitory concentrations of bronopol (2 μg/L and 20 μg/L) significantly increased the conjugative transfer frequency (CTF) of both plasmids. Mechanistic analysis revealed that bronopol enhanced bacterial membrane permeability, as demonstrated by propidium iodide (PI) staining, 1-N-phenylnaphthylamine (NPN) fluorescent probes, transmission electron microscopy (TEM), and upregulation of the outer membrane protein gene ompC. Additionally, bronopol treatment upregulated RP4 plasmid-encoded genes involved in DNA transfer/replication (trfAp) and the global regulator of HGT (kilA/kilB). These findings highlight a previously unrecognized role of bronopol in facilitating the dissemination of antibiotic resistance genes, particularly those of clinical significance.

消毒剂是人类对抗微生物的常用手段。然而，残留消毒剂可通过促进抗生素抗性基因的水平基因转移（horizontal gene transfer, HGT），推动环境抗菌耐药性的传播。溴硝醇是一种在环境中持久残留的常用消毒剂，此前的研究多聚焦于其生态毒性，而非其对抗生素抗性基因传播的潜在影响。本研究旨在构建体外接合模型，探究溴硝醇是否可通过质粒接合过程促进抗生素抗性基因（antibiotic resistance genes, ARGs）的转移。本研究分别以携带RP4-7质粒与携带blaNDM-4阳性IncFII(K)质粒的大肠杆菌DH5α和DC8855作为供体菌，以大肠杆菌J53作为受体菌。实验结果显示，亚抑菌浓度（2 μg/L与20 μg/L）的溴硝醇可显著提升两种质粒的接合转移频率（conjugative transfer frequency, CTF）。机制分析表明，溴硝醇可增强细菌细胞膜通透性，这一结果通过碘化丙啶（propidium iodide, PI）染色、1-N-苯基萘胺（1-N-phenylnaphthylamine, NPN）荧光探针实验、透射电子显微镜（transmission electron microscopy, TEM）以及外膜蛋白基因ompC的表达上调得以证实。此外，溴硝醇处理可上调RP4质粒编码的参与DNA转移与复制的基因（trfAp），以及水平基因转移的全局调控基因（kilA/kilB）的表达。本研究结果揭示了溴硝醇此前未被认知的一项功能——促进抗生素抗性基因的传播，尤其是具有临床重要性的抗性基因。