活性氧对蚯蚓肠道中抗生素抗性基因扩散的影响

Impact of reactive oxygen species on the dissemination of antibiotic resistance genes in earthworm intestines

摘要:

蚯蚓肠道富含糖类与有机酸，被视为抗生素抗性基因(antibiotic resistance genes, ARGs)水平转移的潜在热点。然而，在厌氧条件下蚯蚓肠道是否产生活性氧(reactive oxygen species, ROS)以及活性氧如何调控质粒接合转移仍缺乏直接证据。目的 探究蚯蚓肠道内有机物代谢对ROS生成的贡献以及活性氧如何影响质粒接合转移。方法 以威廉环毛蚓(Pheretima guillelmi)为模式生物，构建模拟蚯蚓肠道原位底物浓度的厌氧微宇宙体系，设置葡萄糖、乳酸盐、乙酸盐和氨基酸4种唯一碳源处理；通过添加活性氧清除剂验证活性氧的作用。采用荧光探针技术、离子色谱和实时荧光定量聚合酶链式反应(quantitative real-time PCR, qPCR)等方法，分别测定?OH、O₂^?-和H₂O₂的生成水平、有机底物消耗情况以及接合转移相关基因(gfp、mCherry、trfA和trbB)的丰度变化。结果 所有处理组均检测到活性氧，葡萄糖组在第2天?OH、O₂^?-和H₂O₂产量最高，分别为0.684、0.988、6.371 μmol/L，显著高于其他处理组，乙酸盐组最低。底物消耗速率与活性氧生成趋势一致：葡萄糖组>乳酸盐组>氨基酸组>乙酸盐组。葡萄糖组中gfp、trfA、trbB基因丰度及接合频率均最高，分别为3.47×10⁶、6.73×10⁶、7.86×10⁶ copies/μg DNA和8.9×10^-4，乙酸盐组最低。清除活性氧后各处理组的接合频率均显著下降73%?92%。Mantel分析显示，羟基自由基与接合频率、trfA和trbB的相关性最为显著，表明羟基自由基是驱动接合转移的核心活性氧物种；肠杆菌科未分类属(unclassified Enterobacteriaceae)与狭义梭菌属10 (Clostridium sensu stricto 10)是活性氧耦合接合过程的核心菌群。结论 蚯蚓厌氧肠道中有机物代谢过程可显著促进活性氧的生成，活性氧通过改变与接合相关基因的丰度，进一步调控抗生素抗性基因在微生物间的接合转移。

Abstract:

Objective Earthworm intestines, rich in carbohydrates and organic acids, are considered potential hotspots for the horizontal transfer of antibiotic resistance genes (ARGs). However, direct evidence is lacking regarding whether reactive oxygen species (ROS) are produced under anaerobic conditions in earthworm intestines and how ROS regulate plasmid conjugation. This study aimed to investigate the contribution of organic matter metabolism to ROS generation in earthworm intestines and how ROS affected the conjugative transfer of plasmids.Methods Pheretima guillelmi was used as a model organism to establish the anaerobic microcosm systems simulating in-situ substrate concentrations of earthworm intestines. Four treatments with glucose, lactate, acetate, and amino acids as sole carbon sources were set up. The role of ROS was verified by adding ROS scavengers. Using the fluorescent probe technology, ion chromatography, and qPCR, we determined the production levels of ?OH, O₂^?-, and H₂O₂, the consumption of organic substrates, and the abundance changes of the conjugation-related genes gfp, mCherry, trfA, and trbB, respectively.Results ROS was detected in all the treatments. The glucose group showed the highest ?OH, O₂^?-, and H₂O₂ yields (0.684, 0.988, and 6.371 μmol/L, respectively) on day 2, which were significantly higher than those in other groups, while the acetate group showed the lowest yields. The substrate consumption rate followed the trend of glucose>lactate>amino acids>acetate, which was consistent with the ROS generation trend. Correspondingly, the glucose group exhibited the highest abundance of gfp, trfA, and trbB (3.47×10⁶, 6.73×10⁶, and 7.86×10⁶ copies/μg DNA) and conjugation frequency (8.9×10^-4), which were the lowest in the acetate group. After ROS scavenging, the conjugation frequencies in all the treatments significantly decreased by 73%?92%. Mantel analysis revealed that hydroxyl radical showed the most significant correlation with conjugation frequency and abundance of trfA and trbB, indicating that ?OH was the core ROS driving conjugative transfer. Unclassified Enterobacteriaceae and Clostridium sensu stricto 10 were identified as the core microbial taxa coupling ROS generation and conjugation.Conclusion Organic matter metabolism in the anaerobic earthworm intestine can significantly promote ROS generation. ROS further regulates the conjugative transfer of ARGs among microbial strains by altering the abundance of conjugation-related genes.