Coevolutionary Mechanism Of Coresident Plasmids Bla_CTX-M-IncFⅡ And Mcr-1-IncⅠ2 In Escherichia Coli

The coexistence of plasmids in clinical strains can lead to interactions that affect the conjugation ability,stability,and fitness cost of plasmids.A chicken derived Escherichia coli(E.coli)strain was clinically isolated in our laboratory.And,this E.coli carried two conjugating plasmids: the bla CTX-M-bearing Inc FⅡ type plasmid p LWY24J-3(carrying rmt B,bla TEM-1b,and bla CTX-M-55 genes)and the mcr-1-bearing Inc Ⅰ2 type plasmid p LWY24J-mcr-1(carrying mcr-1 gene).Previous studies have confirmed that both plasmids can be transferred simultaneously or independently,and that the stability and adaptability of the two plasmids when coexisting are significantly enhanced.However,there is no relevant study on the interaction between the two plasmids.In this experiment,the interaction mechanism of bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids was studied,with a view to providing a basis for in-depth understanding of the interaction mechanism of plasmid coexistence and providing a reference for curbing the spread of bla CTX-M and mcr-1.The interaction between blaCTX-M-IncFⅡ and mcr-1-IncⅠ2 plasmids was detected by plasmid copy number assay,conjugation test,conjugation speed test,and motive ability test.The plasmid copy number results showed that both the bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids exhibited a significant decrease in copy number when the plasmids bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 coexisting.The plasmid conjugation test detected that the bla CTX-M-Inc FⅡ plasmid significantly reduced the conjugation rate of the mcr-1-Inc Ⅰ2 plasmid,while the mcr-1-Inc Ⅰ2 plasmid was more easily transferred to the recipient bacteria carrying the bla CTX-M-Inc FⅡ plasmid under the influence of the bla CTX-M-Inc FⅡ plasmid.The plasmid conjugation speed test showed that compared to the mcr-1-Inc Ⅰ2 plasmid,the bla CTX-MInc FⅡ plasmid was more easily conjugated transferred.When the bla CTX-M-Inc FⅡ and the mcr-1-Inc Ⅰ2 plasmid coexisted,the conjugation and transfer efficiency of the mcr-1-Inc Ⅰ2 plasmid was higher,and the speed and efficiency of co transfer of the two plasmids were significantly increased.In the motive ability test,we found that the motive ability of C600FⅡ+Ⅰ2 was significantly increased,which may be one of the reasons for the higher efficiency of co transfer of bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids.Morphological and physiological tests were conducted to detect the inner and outer membrane permeability,intracellular ROS levels,intracellular ATP levels,intracellular H+levels,and membrane potential levels of E.coli C600,C600 FⅡ,C600Ⅰ2 and C600FⅡ+Ⅰ2.The results showed that both the bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids could reduce the outer membrane permeability of host E.coli,while the decrease in outer membrane permeability was more significant when the two plasmids coexisted.And,the intracellularATP level was significantly increased of E.coli which carrying the bla CTX-M-Inc FⅡ plasmid alone,while the intracellular ATP level of host bacteria carrying both the bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids significantly decreased compared to C600 FⅡ,which means that the mcr-1-Inc Ⅰ2 plasmid can reduce the energy demand brought by the bla CTX-M-In FⅡ plasmid.The results of intracellular H+concentration detection showed that the mcr-1-Inc Ⅰ2 plasmid could increase the intracellular H+concentration of the host bacteria.In addition,in the membrane potential detection results,the bla CTX-M-Inc FⅡ plasmid caused some damage to the membrane potential of the host bacteria,while the mcr-1-Inc Ⅰ2 plasmid could inhibit the damage of the bla CTX-M-IncFⅡ plasmid to the membrane potential of the host E.coli.Through transcriptome studies,it was found that in the dual plasmid carrying strain C600FⅡ+Ⅰ2,compared to C600 FⅡ,the expression of E.coli type I flagellar system,biological adhesion,energy metabolism,toxin antitoxin system,and RNA chaperone protein encoding genes was increased,while the expression of outer membrane protein related genes was downregulated.Compared with C600Ⅰ2,the expression of metal ion transport systems and RNA chaperone related genes in C600FⅡ+Ⅰ2 is upregulated,while the expression of amino acid synthesis and metabolism related genes is downregulated.In summary,the coexistence of bla CTX-M-IncFⅡ and mcr-1-IncⅠ2 plasmids can improve the expression of genes related to the flagellar system of host bacteria,improve the motility of host bacteria,and thereby improve the rate and efficiency of cotransfer of bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids.Moreover,the host bacteria coexisting with the bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids exhibit higher environmental advantages by reducing amino acid synthesis and reducing their own energy requirements.At the same time,the bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids jointly regulate the metal ion transport channels of the host bacteria,restore the membrane potential damage caused by the bla CTX-M-Inc FⅡ plasmids,and enhance the environmental adaptability of the host bacteria.It also improves the transcriptional stability of the bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids by increasing the expression of the RNA chaperone csps family,and also improves the stability of vertical transmission of the two plasmids.These results indicate that when the bla CTX-M-Inc FⅡ and mcr-1-Inc Ⅰ2 plasmids coexist,they have more efficient horizontal transmission,more stable vertical transmission,and higher tolerance to harsh environments.