The Effect And Mechanism Of Zearalenone On Plasmid Conjugation Transfer

Irrational use of antibiotic accelerates the emergence and spread of antibiotic resistance.Plasmid-mediated conjugation transfer is the main mode of horizontal transmission of antibiotic resistance.Zearalenone(ZEA)and its derivatives are one of the most contaminated fungal toxins existed widely in animal feed and human food.Currently,most studies have focused on the toxicity of ZEA to humans and animals,but its effect on the horizontal transfer of plasmid containing antibiotic resistance gene(ARG)is unknown.Here,we explored the ability of ZEA to promote plasmidmediated horizontal transfer of ARG and its possible mechanism.The findings provide new insight into the role of ZEA in promoting horizontal transfer of ARG and may provide a basis for management program to mitigate the spread of antibiotic resistance by controlling this toxin in the context of actual condition.The main findings and results of this paper are as follows:1.First,we confirmed that ZEA of 3.125 ～ 50 μg/m L did not affect the growth of donor and recipient bacteria,which laid foundation for the subsequent conjugation transfer tests.The tests showed that 12.5 ～ 50μg/m L and 50 μg/m L ZEA significantly enhanced the transfer efficiency of plasmid pSD11(Inc X4 plasmid carrying cfr gene)in intra-genus(E.coli MG1655/pSD11 and E.coli C600)and inter-genus(E.coli MG1655/NDM and K.pneumoniae/kp85)bacteria,respectively.2.Secondly,we studied the mechanism of ZEA to promote the conjugation transfer efficiency of plasmid.ROS production in donor and recipient bacteria under ZEA exposure were measured by 2’,7’-dichlorofluorescence diacetate(DCFH-DA),and we found that both donor and recipient strains showed a increase of ROS production with the dose of ZEA exposure.In additon,the significant decreased membrane potential detected by JC-1 method confirmed the ROS overproduction in the recipient bacteria exposed to ZEA of high concentration.Cell morphology observation by transmission electron microscopy showed damaged cell membrane,blurred cell boundaries and more intercellular contacts of the donor and recipient bacteria under 50 μg/m L ZEA exposure.Cell membrane permeability was determined by flow cytometry after propidium iodide(PI)staining,and it was found that the cell membrane permeability of the donor and recipient bacteria increased with the ZEA concentration.Meanwhile,detection of cell wall integrity by measuring the extracellular alkaline phosphatase(AKP)content showed that ZEA could disrupt the integrity of bacterial cell wall in a dose-dependent manner.In summary,ZEA promoted the conjugation transfer of plasmid through the co-regulation of ROS production,increased membrane permeability,decreased membrane potential,and cell wall damage.The expression levels of conjugation transfer-related genes virB4,virB6 and virB9 on the plasmid were significantly up-regulated under the maximum concentration of ZEA(50 μg/m L),and that of virD2 and virD4 decreased with the ZEA dose.ZEA significantly inhibited the expressions of conjugation-related genes h-ns and cpxR on chromosome,and significantly improved the expression of pilT which however was gradually weakened with the treatment dose.These results indicated that these genes were involved in promoting the plasmid conjugation-transfer by ZEA.3.Finally,transcriptome change in strain MG1655/pSD11 under ZEA exposure(12.5 μg/m L)was analyzed,and it was found that 73 and 71 genes were significantly up-regulated and down-regulated,respectively.KEGG enrichment analysis of the differentially expressed genes revealed that ZEA mainly affected plasmid conjugation transfer through flagellar assembly,ABC transporter protein and tryptophan biosynthesis.