抗生素耐药性（AMR）已成为全球性环境污染问题，生物膜作为细菌在环境中最常见的聚集生活方式，在抗生素抗性基因（ARGs）的环境传播中扮演着重要角色。然而，生物膜形成是一个动态发育的过程，不同阶段的生物膜在物理化学性质和代谢活性等方面都有显著差异，因而可能对ARGs的水平基因转移（HGT）产生不同的影响。目前对生物膜形成过程的HGT动态变化规律缺乏系统性的研究。本研究旨在建立可控、重复的生物膜形成模型，探究生物膜发育过程对ARGs的HGT（特别是接合转移）的影响并解析其分子机制。通过优化并确定菌株培养条件和添加不同浓度（0~1000 μg/L）的信号分子（C4-HSL、3-oxo-C12-HSL和AI-2），模拟不同发育阶段的生物膜。发现生物膜的发育过程对ARGs的接合转移具有双重调控作用：在生物膜形成的初期阶段，ARGs的接合转移频率显著提高（属内：最高达1.64×10-6，是对照组的2.81倍；属间：最高达1.31×10-6，是对照组的6.93倍）；而生物膜发育成熟后，ARGs的接合转移频率大幅下降（属内：最低为4.07×10-7，仅为对照组的69.7%；属间：最低为1.26×10-7，仅为对照组的66.7%）。该发现揭示了生物膜动态变化与ARGs传播风险紧密关联。进一步通过转录组学分析，系统比较了生物膜不同发育阶段的基因表达谱。结果表明，在生物膜形成的初期阶段，供体和受体菌中与质粒复制、接合桥形成等接合转移关键基因普遍表达上调，为质粒在细菌间的高效的HGT提供了分子基础。而当生物膜发育进入成熟期后，接合相关基因的表达水平则显著下调，使得接合转移发生受限。值得注意的是，Ⅵ型分泌系统（T6SS）作为一种普遍存在于革兰氏阴性菌中的蛋白分泌装置，可介导细菌间的竞争或拮抗作用，在成熟期大幅激活（如clpV3、icmF3等基因），意味着T6SS可能通过调节生物膜内部细菌的相互作用，间接影响ARGs在菌株间的传播。最后，我们通过构建T6SS关键基因clpV3的缺失突变株，验证了T6SS对生物膜内接合转移的抑制作用，以及对游离ARGs释放和摄取的影响。总之，本文从宏观表型到微观分子机制，系统揭示了细菌生物膜发育过程对ARGs传播的调控规律，为深入理解ARGs在环境中的传播机制提供了新的视角。上述发现有助于开发针对性的干预策略，遏制耐药细菌的环境扩散，对于应对日益严峻的全球性AMR危机具有重要意义。
Antimicrobial resistance （AMR） has become a global environmental pollution problem, and biofilms, as the most common aggregated lifestyle of bacteria in the environment, play a crucial role in the environmental dissemination of antibiotic resistance genes （ARGs）. However, biofilm formation is a dynamic developmental process, and biofilms at different stages exhibit significant differences in physicochemical properties and metabolic activities, which may exert distinct influences on the horizontal gene transfer （HGT） of ARGs. Currently, there is a lack of systematic research on the dynamic changes of HGT during the biofilm formation process.This study aims to establish a controllable and reproducible biofilm formation model to investigate the impact of biofilm development on the HGT of ARGs （especially conjugative transfer） and elucidate its molecular mechanisms. By optimizing and determining the bacterial culture conditions and adding different concentrations （0~1000 μg/L） of signaling molecules （C4-HSL, 3-oxo-C12-HSL, and AI-2）, biofilms at different developmental stages were simulated. It was found that the biofilm development process exerts a dual regulatory effect on the conjugative transfer of ARGs: in the early stage of biofilm formation, the conjugative transfer frequency of ARGs significantly increased （within bacterial genera: up to 1.64×10-6, 2.81 times of the control group; across bacterial genera: up to 1.31×10-6, 6.93 times of the control group）; however, as the biofilm matured, the conjugative transfer frequency of ARGs decreased substantially （within bacterial genera: down to 4.07×10-7, only 69.7% of the control group; across bacterial genera: down to 1.26×10-7, only 66.7% of the control group）. This finding reveals a close association between the dynamic changes of biofilms and the dissemination risk of ARGs.Furthermore, through further transcriptomic analysis, gene expression profiles at different developmental stages of the biofilm were systematically compared. The results indicated that in the initial stage of biofilm formation, the expression of key genes related to plasmid replication, conjugative bridge formation, and other conjugative transfer processes was generally upregulated in both donor and recipient bacteria, providing a molecular basis for the efficient HGT of plasmids between bacteria. However, when the biofilm development entered the mature stage, the expression levels of conjugation-related genes were significantly downregulated, limiting the occurrence of conjugative transfer. Besides, it is noteworthy that the type VI secretion system （T6SS）, a protein secretion apparatus widely present in Gram-negative bacteria that can mediate competition or antagonism between bacteria, was substantially activated in the mature stage （e.g., genes such as clpV3 and icmF3）. This suggests that T6SS may indirectly influence the spread of ARGs among strains by regulating the interactions between bacteria within the biofilm.Finally, we constructed a deletion mutant of the key T6SS gene clpV3 to validate the inhibitory effect of T6SS on conjugative transfer within biofilms and its impact on the release and uptake of extracellular ARGs. In conclusion, this study systematically elucidates the regulatory patterns of bacterial biofilm development on ARG dissemination, from macroscopic phenotypes to microscopic molecular mechanisms, providing new perspectives for in-depth understanding of the dissemination mechanisms of ARGs in the environment. The aforementioned findings contribute to the development of targeted intervention strategies to curb the environmental spread of resistant bacteria, which is of great significance for addressing the increasingly severe global AMR crisis.
