The Dissemination Mechanism Of PoxtA Gene In Enterococci Of Swine Origin

Enterococci is considered to be a repository of multiple drug resistance genes,which greatly accelerates the spread of drug resistance genes.After the emergence of poxtA gene,it can mediate the dual resistance of florfenicol and oxazolidinone at the same time.Therefore,there is an urgent need to study the diffusion mechanism of oxazolidinone cross resistance gene poxtA in En terococci in order to provide scientific basis for risk assessment of drug resistance gene transmission.poxtA gene is a new type of florfenicol-oxazolidinone-tetracycline resistance gene.The protein encoded by poxtA gene has 32%homology with optrA gene,and poxtA gene shows the typical characteristics of F family of ATP binding box(ABC)protein superfamily,which leads to antibiotic resistance through ribosome protection.poxtA gene can reduce the sensitivity of positive strains to amidols,oxazolidinones and tetracyclines.poxtA gene is usually located in a complex transposon-like structure containing two IS1216 elements,which can carry the horizontal transfer of drug resistance gene by forming a circular intermediate.The strains tested in this study are two strains of enterococci with positive poxtA gene preserved in the laboratory,namely Enterococcus faecalis Fac6 and Enterococcus hirae Fbc83.The experiment was carried out by means of PCR detection,Antimicrobial susceptibility test,MLST typing,conjugation test,electrotransformation test,plasmid stability test,S1-PFGE,Southern blot,whole genome sequencing and so on.The horizontal diffusion mechanism of poxtA gene within species and the cross-species transmission mechanism between species and genera were discussed.The experimental results of horizontal diffusion mechanism of poxtA gene within species showed that,The poxtA gene of Enterococcus faecalis Fac6 could be conjugated and transferred,and the conjugation efficiency was 1.87×10-3.By MLST typing,the ST typing of the transcongjugant was consistent with that of the recipient strain JH2-2,both of which were ST8.It was confirmed that the conjugation transfer of poxtA gene did occur.Antimicrobial susceptibility test showed that the MIC of T-Fac6 to linezolid was 8 times higher than that of florfenicol,and the MIC of florfenicol was 32 times higher than that of Linezolide.Sl-PFGE and Southern blot tests showed that the poxtA gene was located on the plasmid.After molecular blotting,the position of the plasmid was the same as that on the S1-PFGE map,and both were located in the plasmid.The results of three generations of whole genome sequencing showed that the poxtA gene was located on the 19.8 Kb plasmid(pFac6-1).It could be fused with the 101kb optrA positive conjugate plasmid(pFac6-2)to realize conjugation transfer.The size of the conjugated plasmid(pT-Fac6)was 121Kb.The mechanism of plasmid fusion is homologous recombination at bcrR gene site,which confirms that poxtA gene can spread horizontally within species.The results of the experiment on the mechanism of interspecies transmission of poxtA gene:The Fbc83 of porcine Enterococcus hirae was detected as poxtA gene positive strain by PCR.Two electrorotor TJ-Fbc83 strains(Enterococcus faecalis JH2-2 as receptor)and TR-Fbc83(Staphylococcus aureus RN4220 as receptor)were successfully obtained in electrotransformation test.The resistance of electrorotor TJ-Fbc83 to florfenicol was increased by 16 times and the MIC to linezolid was increased by 8 times.The resistance of electric rotor TR-Fbc83 to florfenicol was increased by 32 times and the MIC to linezolid was increased by 4 times.Plasmid stability test confirmed that poxtA gene could replicate in Enterococcus faecalis and Staphylococcus aureus.Staphylococcus aureus needs to be medicated in order to replicate stably.In the absence of drugs,the drug resistance genes were randomly lost from the passage to the fifth generation of poxtA-related drug resistance genes.The sequencing of the whole genome of the third generation of Fbc83 showed that the plasmid pT-Fbc83,containing 26.7 kb carried poxtA,fexB,tet(M)+tet(L).Among them,the poxtA gene is located in the flanking region of the plasmid pT-Fbc83,poxtA gene and contains a codirectional IS1216 sequence,which can form a multi-combinatorial circular intermediate,suggesting that the horizontal transfer of the drug resistance gene is associated with the transposable unit.To sum up,through the mapping,transferability and sequence analysis of poxtA gene,this study revealed the molecular mechanism of intraspecific and interspecific transmission of poxtA gene in porcine Enterococci,and provided scientific basis for risk assessment of poxtA gene transmission.