Study On The Fitness Cost Of Tet(X4)-positive Plasmid In Escherichia Coli

In the past few decades,the massive use of antibiotics in the healthcare system and livestock and poultry breeding industry has led to the emergency and rapid spread of bacterical resistance.Tigecycline is considerded to be one of the important antibacterial agents for the treatment of resistant Gram-negative infections.However,the emergence of plasmid-mediated tigecycline resistance gene tet(X4)has seriously affected the effectiveness of tigecycline and posed a potential to public health.Therefore,there is an urgent need to establish feasible strategies to prevent the spread of multidrug-resistant bacteria.Studies have found that the fitness cost caused by drug resistance plasmids is a key factor affecting the maintenance and spread of antibiotic resistance genes within the host bacteria.The fitness cost refers to that the resistant strain is more competitive than susceptible strain in the presence of antibiotics,while in the absence of antibiotics,the resistant strains will be at a disadvantage compared with the susceptible strains.It has been found that tet(X4)resistance genes can be located in a variety of plasmid types and have a wide host range.However,it is not clear whether the fitness cost of tet(X4)in different plasmids is different or whether there are scientific questions such as a dominant plasmid.Based on this,this study will evaluate the differences of fitness cost of tet(X4)-positive plasmids in E.coli TOP10,so as to understand the mechanism of persistence and spread of resistant clonal strains.First,we selected strains carrying different tet(X4)positive plasmids,the tet(X4)positive plasmids were extracted and transferred into electroporate into the competent E.coli TOP 10 to obtain transformants,the successful transformants was varified by screeening the single colony on tigecycline plates and by polymerase chain reaction(PCR).Drug sensitivity test was used to analyze the sensitivity of the transformants with eight antibiotics in clinical,it is helpful to better understand the resistance of tet(X4)-positive plasmids in E.coli.It was found that most strains showed high resistance to tetracycline,oxytetracycline,chlortetracycline,aureomycin,and tigecycline,and most strains were sensitive to enrofloxacin.These data tables indicated that strains showed a trend of multi-drug resistance to most tetracyclines.Then,20 tet(X4)positive transformants with different plasmid types,including IncX1,IncFIB,IncA,IncQ1,IncFIA and IncF?,were selected to evaluate their fitness costs by measuring their growth rate,biofilm formation,virulence,plasmid stability and in vitro competitive ability.The results showed that most tet(X4)positive plasmid showed that the fitness of different tet(X4)-positive plasmid in E.coil TOP 10 were significantly different.Compared with TOP 10,most tet(X4)transformants showed reduced growth rate of resistant strains and lower competitive index.The introduction of tet(X4)-positive plasmid can reduce the ability of biofilm formation and improve the survival ability of G.mellonella larvae,indicating that the virulence of host bacteria is significantly reduced.Compared with other plasmid types,the IncFII plasmid carrying tet(X4)gene caused the lowest fitness cost and could be stable in TOP 10,which may imply that the IncFII plasmid may become the dominant plasmid for epidemic transmission in the future.Finally,the transformants carrying tet(X4)resistance gene were concinuously cultured for 100 generations under different drug pressure,the fitness cost changes of evolutionary transformants were evaluated by in vivo and in vitro competition,drug sensitivity test of the passaged strains,plasmid stability and detection of drug resistance gene expression level.The results indicated that most tet(X4)positive plasmids showed modest fitness cost on host bacteria,but bacterial evolution under antibiotic pressure could significantly improve their fitness.After continuous culture of transformants,it was found that tigecycline selection pressure was not necessary for the evolution of fitness in tet(X4)-positive strains.Further studies showed that tetracyclines selective pressure is contributed to the fitness evolution of tet(X4)-positive strains.This result provides a posssible explanation for the fact that tigecycline is not currently approved for veterinary clinical use,but the tet(X4)resistance gene is widely spread in livestock and poultry.To conclude,the study indicates that the introduction of most of tet(X4)-harboring plasmids can impose varying degrees of fitness cost on host bacteria,but the fitness cost can be quickly alleviated during the evolution,which may be responsible for the epidemic spread of dominant plasmids in the clinical setting.In addition,the specific genes located in every plasmid may also affect the traits of host bacteria,which required a more comprehensive investigation.