Prevalence And Transmission Characteristic Of Tigecycline Resistance Gene Tet(X4) In Different Animal Farms

In recent years,the abuse of antibiotics has led to the emergence of antibiotic resistance and its widespread spread in bacterial populations.Multidrug resistant bacteria are increasing,and bacterial antibiotic resistance has become a serious threat to human and animal health.Tegacycline,a tetracycline antibiotic,is used as a last resort to treat multidrug resistant bacteria(especially carbapenem resistant bacteria).However,plasmid mediated horizontal transfer makes the tet(X4)gene of Tegacycline resistance spread among different bacteria,especially in Enterobacteriaceae and Acinetobacter baumannii,which affects the efficacy of Tegacycline.At present,most of the tet(X4)-positive bacteria isolated are related to livestock.Therefore,the monitoring of tegacyclin resistance genes in animal strains can provide a theoretical basis for guiding rational drug use and provide a new strategy for controlling the transmission of tegacyclin resistance genes.In 2021,289 samples of chicken,cattle and pig origin were collected from different farms in Jiangxi Province and Henan Province,and 222 strains of Escherichia coli were isolated and identified.The sensitivity of the isolates to ampicillin,ceftazidime,cefotaxime,cefquinome,meropenem,tigecycline,tetracycline,doxycycline,amikacin,streptomycin,ciprofloxacin,fosfomycin,colistin and florfenicol were detected by agar microdilution method.The results showed that the resistance rate of E.coli to florfenicol(87.84%)and tetracycline(78.83%)was the highest,followed by doxycycline(72.07%)and ampicillin(55.41%);The resistance rate to fosfomycin(9.46%),tegacyclin(5.86%),amikacin(1.80%),and myxin(0.45%)was low,and they were all sensitive to meropenem.Among them,the resistance of chicken Escherichia coli was the most serious,and the resistance trend of pig and cattle sources was similar.The isolated Escherichia coli strains resistant to four drugs at the same time were the most,accounting for 18.02%(n=40),and the strains resistant to 12 drugs(0.90%)were the least.PCR results showed that 22.97%(n=51)of the strains detected the tested genes,and the majority of them were chicken derived Escherichia coli.29 strains detected blaCTX-M gene,13 strains detected tet(X4)gene,8 strains detected blaCTX-M and fosA3 genes at the same time,1 strain detected tet(X4)and blaCTX-M genes at the same time,9 strains detected tet(X4),blaCTX-M and fosA3 genes at the same time,1 strain detected blaCTX-M,fosA3 and mcr-1 genes at the same time.44 strains carrying tet(X4)were screened with tegacyclin resistant plate,including 43 Escherichia coli and 1 Citrobacter freundii.Drug resistance characteristics were analyzed together with 13 tet(X4)-positive Escherichia coli in Test 1.The drug sensitivity results showed that tet(X4)-positive strains had the highest resistance rate to tetracyclines and amide alcohol drugs florfenicol(100%),followed by streptomycin and ampicillin(more than 90.00%),cefotaxime,cefquinoxime,ciprofloxacin and ceftazidime(59.65.80%～68.42%),and fosfomycin(47.37%),The resistance rate to amikacin and colistin was below 10.00%,and the resistance rate to meropenem(1.75%)was the lowest.The resistance of chicken strains was the most serious,followed by pig strains,and the resistance rate of bovine strains was the lowest among the three animals.The analysis of multiple drug resistance of tet(X4)-positive strains showed that they were at least resistant to 5 drugs at the same time,and at most resistant to 12 drugs at the same time.The number of strains resistant to 11 drugs was the largest,accounting for 30.51%(n=18),followed by 18.64%of strains resistant to 6 drugs,and the number of strains resistant to 10 drugs was the smallest,accounting for 3.39%.Most of these strains came from chickens.Among 57 tet(X4)-positive strains,12 strains carried blaCTX-M gene,1 strain carried blaNDM gene,24 strains carried blaCTX-M and fosA3 genes,and 2 strains carried blaCTX-M and mcr-1 genes at the same time.30 of 57 tet(X4)-positive strains were successfully conjugated.The results of replicon typing showed that there were many plasmid types,among which IncHI1 and IncFII had the highest detection rate.The transmission characteristics of the above 57 tet(X4)positive strains were analyzed.Through PFGE,tet(X4)-positive Escherichia coli could be classified into 15 pulse types,and 30 tet(X4)-positive strains were successfully conjugated.The results of replicon typing showed that the tet(X4)-positive E.coli plasmid was IncHI1-IncFII type(n=16),IncHI1 type(n=11)and IncHI1-IncFII-IncFIB type(n=2),respectively.The plasmid of Citrobacter freundii was IncX1 type.The whole genome of R121 carrying both tet(X4)and blaNDM genes and Citrobacter freundii W1 carrying tet(X4)genes were sequenced.Sequence analysis showed that strain R121 carried a chromosome and six plasmids of different sizes,namely,tet(X4)-positive multidrug resistant IncHI1 plasmid,multidrug resistant IncFIB plasmid,Non-resistant IncY plasmid,blaNDM-5positive IncX3 plasmid RmtB positive multidrug resistant IncN plasmid and blaCMY-2 positive IncX4 plasmid.The chromosome of multidrug resistant strain R121 carries the mdf gene and the virulence genes gad and astA,and its plasmid carries the drug resistant genes tet(X4),blaTEM-1,aph(3’)-la,qnrS2,floR,blaNDM-5,tet(M)and tet(A).Strain W1 has one chromosome and three plasmids,which are non anti plasmid,multi drug resistant IncR plasmid and tet(X4)-positive multi drug resistant IncX1 plasmid,respectively.In addition to tet(X4),strain W1 also carries a variety of resistance genes,including blaCMY-41,blaSHV-12,qnrB,aac(3’)-Ⅱb,sul2,sul3,aadA1,aadA2,cmlA1,dfrA12,lnu(F),sul2,floR,and tet(A).The tet(X4)-positive IncX1 plasmid has a high homology with similar plasmids reported,which is the first time that an IncX1 plasmid containing tet(X4)has been detected in Citrobacter freundii.To sum up,the drug resistance of animal derived Escherichia coli from different farms in Jiangxi Province and Henan Province is relatively serious,and different regions,animal species,and drug habits may lead to different drug resistance.The tegacyclin resistance genes in the detected farms were all tet(X4),and no other tet(X)variants were detected.Tet(X4)can be mediated by plasmid and spread among different bacteria through horizontal transfer.This suggests that we should take measures to control the rapid spread of drug-resistant bacteria.