Fitness Effect And Evolution Mechanism Of The Tet(x4)-positive Plasmid On The Multi-drug Resistant Escherichia Coli Carrying Bla_NDM-5 And Mcr-1

The spread of bacterial antibiotic resistance is a serious threat to human and animal health,especially the horizontal transfer of plasmid-mediated resistance genes.In recent years,the emergence and wide dissemination of blaNMD meditating carbapenem resistance,mcr meditating polymyxin resistance,and the high-level tigecycline resistance gene tet(X4),have seriously undermined the effectiveness of "the last resort antibiotics" in clinical.The findings of bacteria including two of these three genes are frequently reported.According to its dissemination pattern,the superbug carrying all the three important resistance genes may occur in the future,which will lead to a no-cure situation.The fitness of the resistance gene or plasmid in the host is the key figure to accessing its dissemination rate.A multidrug-resistant E.coli N31 carrying blaNDM-5 and mcr-1 gene was isolated from swine feces in our lab.To access the possibility and the evolution rules of the superbug with three resistance genes,we transfer a conjugative tet(X4)-positive plasmid into E.coli N31.A comprehensive evaluation of the fitness of the conjugon CN31-pF65-tet(X4)under tigecycline pressure or without antibiotic pressure was conducted by analyzing the growth rate,competition index,and stability of plasmid.Also,the evolution patterns and mechanisms of evolved strains in the absence of antibiotics as well as in the presence of antibiotics were explored by evaluating the fitness,plasmid stability,gene expression,and whole-genome sequencing.The conjugon CN31-pF65-tet(X4)has evolved under antibiotics(tigecycline)pressure and without antibiotics pressure for 100 generations.The plasmid stability and tet(X4)loss rate,competition index,and generation time of the conjugon in different generations were tested.The tet(X4)-positive plasmid is stable under antibiotics pressure and unstable without antibiotics pressure.The generation time of conjugon CN31-pF65-tet(X4)was prolonged compared to N31,and the generation time was shorter through serial transfer evolution,But without significant difference.The competition index(W)of the conjugon was greater with the increasing of passage,but the W for all the evolution strains in different generations was less than 1.These results demonstrated that the tet(X4)-positive plasmid imposed a fitness cost on the multidrug-resistant E.coli N31,while the plasmid fitness improved by passaging evolution both under the antibiotics pressure or without antibiotics pressure.To explore the evolution rules,five evolved strains carrying the tet(X4)-positive plasmid were randomly selected from the 100 generation populations passaged with or without antibiotics respectively.They were used in the follow-up studies.The loss rate of plasmid and tet(X4),conjugation frequency,the minimum inhibitory concentration(MIC),and time-killing curve of the ancestor conjugon and evolved strains were measured.The plasmid loss rate was below 25%for the antibiotic-free evolved strain(5/5)and antibiotic evolved strains(3/5),indicating the improved plasmid persistence of evolved strains.The tet(X4)in the antibiotic-free evolved strains was stable,while the tet(X4)in the antibiotic evolved strains was unstable.The conjugation frequency of the tet(X4)positive plasmid in the evolved strains was improved significantly compared to that of the ancestor,and so was the blaNDM-5 positive plasmid.The expression levels of conjugation transfer-related genes and tet(X4)in the evolved strains were determined.The expression levels of conjugative regulators such as traJ/traY/traM and tral were upregulated.These regulators promote the transcription of the downstream tra genes thus elevating the conjugation frequency.The MICs of tetracyclines(including tigecycline)of antibiotic-free evolved strains were 2-4 fold lower than that of the ancestor strain.However,the MIC values of antibiotic evolved strains were 2 fold higher than that of the ancestral strain.What's more,the killing curve shows that tigecycline had a bactericidal effect at 16 ?g/mL against the antibiotic-free evolved strains,while a slight inhibitory effect against the antibiotic evolved strain.In addition,the tet(X4)expression level of the antibiotic evolved strains was increased more than 20-fold compared to the ancestral strain,while the value of antibiotic-free evolved strains was decreased significantly compared to the ancestral strain(P<0.0001).the results indicated that the increased expression level of tet(X4)enhanced the tigecycline resistance level,but increased the fitness cost of the plasmid,while,the antibiotic-free evolved strains improved its fitness by inhibiting its expression of tet(X4)gene.Eventually,the antibiotic evolved plasmid maintains stability through the loss of its tet(X4).Whole-genome sequencing analysis of the ancestral strain and the evolved strains(with high stability of tet(X4)positive plasmid)revealed a large number of mutations in genes encoding conjugative transfer-related genes in the evolved plasmids.At the same time,the evolved strains had klcA mutations in the chromosomal genome.The expression of klcA gene facilitates DNA transfer.These mutations may be associated with the elevated conjugative frequency of plasmids.In addition,a MarR mutation was observed in the chromosomal genome of the antibiotic evolved strains.the MarR was an efflux pump and such mutation may be associated with elevated levels of resistance.Nanopore sequencing analysis of the ancestral and evolved plasmids without antibiotics shows that the evolved plasmids lost 43 kb of the multi-drug resistance region compared to the ancestral plasmids,while identified changes were observed in the stable evolved plasmids with antibiotics.In contrast,the antibiotic evolved plasmids with low stability are formed by fusion with another plasmid.these results illustrated that the loss of the multi-drug resistance region ameliorated the cost of plasmid and increased the stability of the plasmids.The plasmid-host co-evolution promotes phenotypic changes in evolved strains.To further verify the co-evolution pattern of plasmid stability enhancement,the ancestral and evolved plasmids were transferred into TOP 10 engineered bacteria,and the ancestral plasmid was transferred to the evolved strains without tet(X4)positive plasmid.The stability,conjugation frequency,and loss rate of tet(X4)were significantly higher in TOP 10 with antibiotic-free and antibiotic evolved plasmids than that in TOP 10 with the ancestral plasmid.The fitness cost of the evolved plasmid with antibiotics was higher than that of ancestral plasmids.However,opposite results were observed in evolved plasmid without antibiotics,demonstrating the highly expressed tet(X4)enhanced the fitness cost of the plasmid.On the other hand,the plasmid stability and conjugation frequency of the ancestral plasmids were significantly higher in the antibiotic evolved strains compared to the ancestral bacteria.These results validated a plasmid-host co-evolution pattern of tet(X4)positive plasmid and blaNDM5,mcr-1 bearing strain.In this study,we found that antibiotic-free evolution can improve the stability of tet(X4)positive plasmid in blaNDMs,mcr-1 bearing strain by increasing their conjugation frequency,decreasing the expression level of resistance genes,and losing multidrug resistance fragments.The results of the study provide a theoretical basis for further exploration of the formation of multidrug resistant bacteria and the dissemination of antibiotic resistance.