Study On Antimicrobial Resistance And Transmission Characteristics Of The Bacteria Carrying Tet(X3)/tet(X4) Gene From Animal Sources

The plasmid carried tet(X)variant genes as a threat to the treatment of Carbapenem-Resistant-Enterobacteriaceae(CRE).Moreover,the tet(X)variant genes makes it difficult to choose the drug therapy for livestock and poultry breeding.Plasmid carrying tet(X3)and tet(X4)genes could generate high resistance to tigecycline in bacteria was discovered in recent years.Meanwhile,it also can exhibit the resistance to tetracyclines.Although tigecycline has not been applied for the treatment of animals,a wide variety of bacteria carrying tet(X)variant genes identified from various animal origin bacteria,such as pigs,cows and chickens from different geographical divisions of China.There is limited information on the mechanisms of occurrence and prevalence of tet(X)variant genes in bacteria from animal sources.It is important to survey and analyse the epidemic trend and transmission mechanism for preventing the transmission of resistance genes and clinical medication.In this study,we collected 1362 feces of pigs,goats and crested lbis from eight provinces including Shannxi,Ningxia,Shanxi,Sichuan,Qinghai,Guangxi,Xingjiang and Zhejiang.We investigated the distribution about the tet(3)/tet(X4)gene in those regions.At the same time,the minimum inhibitory concentration(MIC)was determined by standard broth microdilution tests to understand the resistance situation.Then we analysed the whole genome sequence(WGS)by complete genome sequencing and compared the genetic environments for the tet(X3)/tet(X4).The conjugation assay,fitness cost and genetic stability were tested in order to study the transmission characteristics of tet(X3)and tet(X4)genes.Firstly,we screened for resistance bacteria in the way of agar medium with tigecycline(2 mg/L)in 1362 samples.Building the Multiplex-PCR test method to detect tet(X3)and tet(X4)genes in the resistance bacteria.We analysed the risk factors and epidemic regularity of tet(X3)/tet(X4)gene in different animals and regions.The result showed the isolation rate of tet(X3)and tet(X4)positive isolates in feces from pigs is 56%(14/25)in Ningxia,54.9%(112/204)in Guangxi,51.33%(58/113)in Shannxi,26.67%(4/15)in Shanxi and 8.73%(11/126)in Sichuan that included Acinetobacter spp.,Escherichia coli(E.coli),Kleber pneumoniae(K.pneumoniae)and Enterobacter cloacae(E.cloacae).The risk factors analysis indicated that the prevalence of resistance bacteria was significant differences in regions and the size of hoggery.In this study,we found that the tet(X3)/tet(X4)gene was widely present in pig farms in Ningxia,Guangxi and Shaanxi.Secondly,the susceptibility of the 203 tet(X3)/tet(X4)positive isolates to 14 antibiotics were detected by standard broth microdilution method.The results indicated that the isolates carrying tet(X3)or tet(X4)gene exhibited high resistance to tetracycline,doxycycline,tigecycline.The multi-resistant was serious that the common resistance of florfenicol and sulfasomizole were most serious.Most of the isolates were resistant to ampicillin,amoxicillin-clavulanic acid and enrofloxacin.All of the isolates were sensitive to meropenem and polymyxin.The different resistance rates were observed in different regions about ceftiofur,ceftazidime,imipenem,gentamicin and enrofloxacin.The situation of multi-resistant is serious,with flufenicol,sulfamethoxazole and ampicillin being the most servere resistant antibiotics.Thirdly,in order to obtain the genomic information and plasmids information of the strains carrying tet(X3)/tet(X4)gene,we analyzed the genetic characteristics of some strains by second-generation whole gene sequencing,Pulsed field gel electrophoresis(PFGE)and Southern blot.The results showed that the MLST types of E.coli were diversified and had no obvious clone spread.And the ST48 and ST10 types of E.coli were more than those of other types.The tet(X4)gene was mainly located on the plasmids of E.coli(91.02%),and most of these strains carried Inc X1 replicon.K.pneumoniae had clonal spread(ST727)from the faces of pigs in Guangxi,but there was also plasmid spread of drug-resistant genes.All of the tet(X4)were located on the plasmids of K.pneumoniae.Most of the tet(X3)genes located on the plasmids in various Acinetobacter species,and the resistant genes were mainly spread by the plasmids.We found that plasmid is an important factor in the transmission of tet(X3)/tet(X4)gene,and the Inc X1 plasmid is the main type for transmission of tet(X4)gene in E.coli.Fourth,to further obtain the genetic contexts of tet(X3)/tet(X4)gene in different strains,we conducted the whole genome sequencing of different strains carrying tet(X3)/tet(X4)gene through nanopore sequencing.Then we compared the plasmids information and core genetic structure of tet(X3)/tet(X4)gene.The results showed that the plasmids in K.pneumoniae and E.cloacae were similar to E.coli that were isolated from different regions.The plasmids carrying tet(X3)in A.towneri were not correlated with the plasmids carrying tet(X4)in other bacteria.Unexpectedly,seventeen copies of tet(X4)genes were found to be arranged in a tandem array in A.indicus.ISCR2 might transpose tet(X4)via a circular,the core tet(X3)-bearing genetic contexts in this study was ?ISCR2-IS26-Lys R-aph(3')-Ia-IS26-xer D-tet(X3)-resolvase-hp-ISCR2 and the core tet(X4)-bearing genetic contexts were abh-tet(X4)-ISCR2 and ISCR2-abhtet(X4)-ISCR2.Finaly,the strains carrying tet(X3)/tet(X4)gene were conjugated with E.coli 26R793 to study the plasmid trasferability in the wild strain.The previous studies explored that the tet(X3)/tet(X4)gene may be existing host options,which may due to fitness cost in bacteria.We cloned the strains E.coli DH10?-tet(X3)and E.coli DH10?-tet(X4)to compare the growth curve with E.coli DH10?.Then,we tested the stability of the tet(X4)in the bacteria by continuous passage culture in the Brain Heart Infusion Broth(BHI)with or without tigecycline(4 ?g/m L).The result showed that the resistant plasmids could be transferred to E.coli 26 R 793 which in a part of strains E.coli(57.14%)and E.cloacae(50%).The strains E.coli DH10?,E.coli DH10?-tet(X3)and E.coli DH10?-tet(X4)were not showed a significant different of growing rate.Then the tigecycline is important to generate and maintenance the multiple copies of tet(X4).The multiple copies of tet(X4)could enhance the MIC of tigecycline in bacteria.This study confirmed that the tet(X4)gene in E.coli is mainly transmitted by plasmid,and the insertion sequence ISCR2 gene can mediate the production of tandem repeat tet(X4)gene.Although the strains carrying tet(X)variant genes has been detected in hospitals is still limited.There are various bacteria carrying tet(X)variant genes in kinds of animals that they are collected from number of provinces and cities.Most of those strains could transfer the tet(X)variant genes horizontally by plasmids.Moreover,the genes between the insertion element(IS)have a potential spread risk.Not only does it cause tet(X)variant genes to spread widely among animals that would limit the effect of tetracycline antibiotics.It may have the risk of spread from animal bacteria to human at some time.It is a threat to treat the patients infected by CRE.In this study,we investigated the prevalence of tet(X3)/tet(X4)gene in bacteria,and analyzed the genetic environment of tet(X3)/tet(X4)gene.The results illustrate the the epidemic regular of bacteria and the transmission characteristics of the tet(X3)/tet(X4)gene.The results can provide data support for effective prevention and control of ticacycline resistant bacteria.