摘要
      由于在温室农业生产中大量使用农药和有机肥料，土壤中农药残留和抗生素抗性基因的共同污染正在增加。非抗生素胁迫，包括来自农业杀菌剂的胁迫，是ARGs水平转移的潜在共同选择因素，但其潜在机制尚不清楚。建立了抗生素抗性质粒RP4的属内和属间偶联转移系统，以检测四种广泛使用的杀菌剂：三唑酮、百菌清、嘧菌酯和多菌灵在胁迫下的偶联转移频率。使用透射电子显微镜、流式细胞术、RT-qPCR和RNA-seq技术在细胞和分子水平上阐明了其机制。质粒RP4在大肠杆菌菌株之间的结合转移频率随着百菌清、嘧菌酯和多菌灵暴露浓度的增加而增加，但在大肠杆菌和恶臭假单胞菌之间被高杀菌剂浓度（10µg/mL）抑制。三唑酮对偶联转移频率没有显著影响。对潜在机制的探索表明：（i）百菌清暴露主要促进细胞内活性氧的产生，刺激SOS反应，并增加细胞膜通透性，而（ii）嘧菌酯和多菌灵主要增强质粒上结合相关基因的表达。这些发现揭示了与质粒结合相关的杀菌剂触发机制，并强调了非杀菌农药在ARGs传播中的潜在作用。
Abstract
Co-pollution of soil with pesticide residues and antibiotic resistance genes (ARGs) is increasing due to the substantial usage of pesticides and organic fertilizers in greenhouse-based agricultural production. Non-antibiotic stresses, including those from agricultural fungicides, are potential co-selectors for the horizontal transfer of ARGs, but the underlying mechanism remains unclear. Intragenus and intergenus conjugative transfer systems of the antibiotic resistant plasmid RP4 were established to examine conjugative transfer frequency under stress from four widely used fungicides: triadimefon, chlorothalonil, azoxystrobin, and carbendazim. The mechanisms were elucidated at the cellular and molecular levels using transmission electron microscopy, flow cytometry, RT-qPCR, and RNA-seq techniques. The conjugative transfer frequency of plasmid RP4 between Escherichia coli strains increased with the rising exposure concentrations of chlorothalonil, azoxystrobin, and carbendazim, but was suppressed between E. coli and Pseudomonas putida by a high fungicide concentration (10 µg/mL). Triadimefon did not significantly affect conjugative transfer frequency. Exploration of the underlying mechanisms revealed that: (i) chlorothalonil exposure mainly promoted generation of intracellular reactive oxygen species, stimulated the SOS response, and increased cell membrane permeability, while (ii) azoxystrobin and carbendazim primarily enhanced expression of conjugation-related genes on the plasmid. These findings reveal the fungicide-triggered mechanisms associated with plasmid conjugation and highlight the potential role of non-bactericidal pesticides on the dissemination of ARGs.

https://www.sciencedirect.com/science/article/abs/pii/S0043135423002245