Transmission Mechanism And Epidemic Of Tet(X4) Gene In Escherichia Coli Strains Form Companion Animals

In recent years,the massive use and abuse of antimicrobials has resulted in the increasing rates of resistance,and the emergence and spread of drug-resistant bacteria in the Enterobacteriaceae pose a serious threat to the health of humans and animals.Tigecycline with broad-spectrum antibacterial activity,has become one of the last resorts for the treatment of multi-drug resistant Gram-negative bacterial infections.The emergence of plasmid-mediated high-level tigecycline resistance gene tet(X)variants,especially the widespread prevalence of tigecycline resistance gene tet(X4)in different bacteria,seriously threatened the clinical use of tigecycline,which has a great impact on public health.In this study,the epidemic situation and transmission mechanism of tet(X4)-positive Escherichia coli strains from companion animals in Zhengzhou,Henan Province,were analyzed to evaluate the risk of transmission of tet(X4),so as to provide basic theoretical basis for traceability of human drug resistant strains and the intelligent use of antimicrobials.From July to September 2019,a total of 202 fecal samples of pet cats and dogs were collected from different pet hospitals in Zhengzhou,and a total of 186 Escherichia coli strains were isolated and identified with an isolation rate of 92.08%.These strains were highly resistant to ampicillin,tetracycline,doxycycline and florfenicol,and the resistance rate ranged between 74.66% and 87.30%;Followed by cefotaxime,cefquinome,streptomycin,ceftazidime,fosfomycin and ciprofloxacin,with resistance rates ranging from 22.04% to 54.30%;and the lowest drug resistance rates of 7.53%,4.84% and 3.23% to amikacin,colistin and tigecycline,respectively.No meropenem-resistant strains weren detected.Six tet(X4)-positive Escherichia coli were detected by PCR,and the detection rate was 3.23%.The tet(X4)-positive strains were resistant to ampicillin,tetracycline,doxycycline,streptomycin and florfenicol.Among them,five of the tet(X4)-positive strains carried tet(A)gene,four of the strains carried bla CTX-M gene,two of strains carried fos A3 and mcr-1 gene,and bla NDM gene was not detected in all strains.Two pan-drug-resistant strains carried four drug resistant genes including tet(X4),bla CTX-M,fos A3 and mcr-1.m LST typing results showed that six tet(X4)-positive strains harbored five ST types including ST10(n=2),ST2178,ST48,ST642 and ST7366,a novel ST type.Six tet(X4)-positive strains were divided into A,B,C,D,E and F pulsed field patterns by PFGE.The tet(X4)gene in tet(X4)-positive isolates could be successfully transferred from the donor strains to the recipient strain E.coli C600 by conjugation assay.All tet(X4)-positive plasmids belonged to Inc HI1 type plasmid,and the conjugation frequency of Inc HI1 plasmid was 5.5×10-7.The tet(X4)-positive Inc HI1 plasmid remained stable for 15 days of passage in an antibiotic-free environment,and presented a competitive disadvantage against the host bacteria.The tet(X4)-positive Inc HI1 plasmids p T16R-1 and p T28R-1 shared 99.83% homology with 83%nucleotide sequence of plasmid R27 from Salmonella.The backbone sequences in tet(X4)-positive plasmids and R27 were highly conserved,and the most visible difference lay in the multidrug resistance region.The tet(X4)in Inc HI1 plasmids located in the △ISCR2-hp-ISCR2-tet(X4)-abh-IS1 region.In summary,the tet(X4)-positive E.coli strains from companion animals in Zhengzhou showed a severe drug resistance.The genetic relationship between tet(X4)-positive strains was far away,and there was no obvious sign of clone spread.The high stability and competitive advantage of tet(X4)-positive plasmids facilitated the transmission of tet(X4)gene.Meanwhile the presence of ISCR2 upstream of the tet(X4)gene was closely related to the transfer of tet(X4).The multiple transfer mode may accelerate the transmission of the resistance gene,which bringed a great challenge to the prevention and control of tigecycline-resistant strains as well as clinical treatment.The results of this study may provide a theoretical basis for controlling the spread and evolution of tigecycline-resistant pet E.coli strains.