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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 Pheretimaguillelmi 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 Clostridiumsensu 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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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