Epidemic Distribution Of Tet(X) Gene And Drug Resistance In Riemerella Anatipestifer

Riemerella anatipestifer is a gram-negative bacterium belonging to the family Flavobacteriaceae.The bacterium can cause severe acute or chronic sepsis in an array of avian animals,including ducks,geese,chickens,and turkeys.Tigecycline is the first antibacterial agent among glycylcyclines that is not cross-resistant with conventional tetracyclines.With the emergence of drug resistance genes blaNDM-1 and mcr-1 that mediate carbapenem and polymyxin,tigecycline is regarded as the ’last line of defense’ against infection by multi-drug resistant Gram-negative bacteria.The tet（X）gene,encoding the inactivating enzyme,is the first drug resistance gene capable of directly inactivating tigecycline.Furthermore,the identification of plasmid-mediated tet（X3）and tet（X4）in Acinetobacter baumannii and Escherichia coli（E.coli）has shown that these genes can be transferred through conjugation,promoting the spread of tigecycline resistance.Although many studies have investigated tet（X）gene,its origins are still unclear.In this study,we aim to explore the distribution and drug resistance mechanism of the tet（X）gene in Riemerella anatipestifer,including an analysis of a multi-drug resistance plasmid and a new trimethoprim resistance gene.First,we isolated and identified 139 Riemerella anatipestifer isolates from chickens,ducks,and geese raised in various farms located in Jiangsu Province and Guangdong Province from 2016 to 2020.The isolates had a 100%resistance rate to tetracycline and a 100%prevalence rate of the tet（X）gene.Among the 139 isolates,38 isolates were from Jiangsu,with 13.16%from chicken,23.68%from ducks and 63.16%from geese,while 101 isolates were from Guangdong and were exclusively from geese.In Jiangsu,the resistance rates of the isolates to doxycycline,ampicillin,kanamycin,florfenicol,tigecycline,and erythromycin were 86.8%,39.5%,65.8%,50%,55.3%,and 89.5%,respectively.In Guangdong,the resistance rates of the isolates to these antibiotics were 90%,63%,96%,27%,2%,and 91.8%,respectively.The tet（X）gene was prevalent,and so were the floR and ermF genes.The floR prevalence rates were 55.3%and 81%for Jiangsu and Guangdong isolates,respectively.The ermF prevalence rates,on the other hand,were 89.5%and 89.1%for Jiangsu and Guangdong isolates,respectively.Additionally,several ereD genes and insertion sequence ISCR2 were detected.The conjugation test showed that Riemerella anatipestifer,whether as donor or recipient,had host restriction.Under the current experimental conditions,tet（X）could not be transmitted across species by conjugation transfer.Then,we selected 38 isolates of Riemerella anatipestifer from Jiangsu with higher tigecycline resistance rate and analyzed the genomic characteristics of them positive for tet（X）.The results showed that the variable regions of tet（X）gene were located between the conservative regions rsmB-wzx and czcD-dam,and there were 12 kinds of genetic environment.Another kind of genetic environment is the classical structure of floR-ISCR2-tet（X）.Thirteen new variants of tet（X）were discovered on the chromosomes of Riemerella anatipestifer for the first time,including tet（X17）,tet（X16）,tet（X18）,tet（X19）,tet（X20）,tet（X21）,and others previously reported.These tet（X）variants can mediate the resistance of Escherichia coli BL21（DE3）to tigecycline.Additionally,they also influence the sensitivity to other tetracycline antibiotics（tetracycline,minocycline,oxytetracycline,doxycycline）,including the newly FDA-approved iracycline and oxocycline.The suicide plasmid pRE112 and the shuttle plasmid pTRA01 constructed in this study were used to knock out and supplement the tet（X）gene in Riemerella anatipestifer,respectively.The results showed that the tet（X）gene was indeed a gene regulating the resistance of the bacteria to tetracycline antibiotics.We also discovered a regulatory mechanism on the chromosome of Riemerella anatipestifer controlling the expression of the tet（X）gene.Increasing the copy number of the tet（X）gene on the chromosome raises the resistance level of Riemerella anatipestifer to tigecycline,resulting in an increase of over 16-fold.The tet（X）gene has a GC content similar to that of Riemerella anatipestifer,a member of Flavaceae.This suggests that Riemerella anatipestifer may be the origin of the tet（X）gene.Moreover,the tet（X）gene in sensitive or low-level resistance strains may provide an advantage in becoming a natural repository of tigecycline resistance genes,though more evidence is needed to support this claim.Finally,we analyzed the multi-drug resistance plasmid p20190305E2-2₂ in Riemerella anatipestifer 20190305E2-2,which carries several antibiotic resistance genes,including dfi A49,tet（X18）,catB,ermF,ereD,Δarr,and blaoXA,using Nanopore long-read sequencing technology and a bioinformatics platform.dfrA49 is a novel trimethoprim resistance gene identified from this plasmid,which can mediate the high level of trimethoprim resistance in E.coli BL21（DE3）and increase the MIC by at a minimum of 256-fold.To our knowledge,this is the first report of dfrA family genes in Riemerella anatipestifer.The analysis of genetic environment revealed that dfrA49 always coexists with the catB gene,and this structure was observed only in Flavaceae bacteria.We found nine different genetic environments of dfrA49-catB through linear comparison in this study.The presence of IS4351-tet（X18）suggests an interruption in the dfrA49-catB structure in p20190305E2-2₂,indicating that insertion hotspots may exist between dfrA49 and catB.Therefore,the host strain carrying dfrA49-catB structure has a higher risk of transferring and enriching ARGs.In summary,this study clarified the epidemic distribution of tigecycline resistance gene tet（X）and drug resistance in Riemerella anatipestifer,analyzed the genetic environment of tet（X）and dfra49-catB in Riemerella anatipestifer genome by Short-read Illumina sequencing and long-read Nanopore sequencing techniques,and verified the function of new variants of tet（X）and a new trimethoprim resistance gene dfrA49.This study introduces a novel approach to regulate the resistance of strains containing a single copy tet（X）gene to tigecycline.Moreover,our findings confirm that the prevalence of tet（X）is high in Riemerella anatipestifer isolates.Thus,it is crucial to investigate the transfer risks of tet（X）from Riemerella anatipestifer to other clinical pathogens.