The Prevalence And Transmission Mechanism Of Tigecycline Resistance Genes TmexCD-toprJ And Tet（a）Variant In Porcine Bacteria

As a semisynthetic parenteral glycylcycline with a broad spectrum of antimicrobial activity,tigecycline is regarded as one of the last-resort antimicrobials for the treatment of serious infections caused by Gram-negative bacteria.However,the emergence and transmission of novel tigecycline resistance genes seriously threaten the efficacy of tigecycline and pose a serious challenge to global public health security.Recently,one plasmid-mediated RND efflux pump gene cluster named tmexCD1-toprJ1,which can reduce the sensitivity of multiple antimicrobials,including tigecycline,was identified in Klebsiella pneumoniae of animal and human origins.But the epidemiological and genomic characteristics of this novel resistance determinant have not been systematically studied.Furthermore,the mutated efflux pumps of Tet（A）was the most common mechanism that contribute to tigecycline resistance.Therefore,this paper mainly explores the potential effect of the RND efflux pump gene cluster tmexCD-toprJ and MFS family efflux pump gene tet（A）variant on the formation of tigecycline resistance in the pork production chain,and clarifies the prevalence and transmission mechanisms.First of all,we isolated 25 tmexCD-toprJ-positive strains from 682 samples collected along the pork production chain,including swine farms,slaughterhouses,and retail pork.Then we found that tmexCD-toprJ-positive strains were most prevalent in slaughterhouses（7.32%）.Most of the positive strains were K.pneumoniae（23/25）,followed by Proteus mirabilis（2/25）.Then we isolated 45 tet（X）-and tmexCD-toprJ-negative tigecycline resistant strains from 382 samples in the above slaughterhouses,with MICs from 16 to 32 mg/liter.This may be the result of a combination of tet（A）mutations and inactivation of ramR and oqxR.3 tet（A）variants,type 1 and type 3 and tet（A）-v,were identified,type 3 tet（A）was novel.Subsequently,we focused on analyzing the genomic characteristics of 25 tmexCD-toprJpositive strains.The tmexCD1-toprJ1 gene cluster carried by 23 K pneumoniae was found on plasmids,while we characterized a novel tmexCD3-toprJ3 gene cluster,located on an SXT/R391 integrative and conjugative element（ICE）from other 2 P.mirabilis for the first time.We obtained 5 detailed maps of tmexCD1-toprJ1 positive plasmids based on the Nanopore MinION sequencing technology.Significant differences were found in their multidrug resistance regions.And multiple copies of tmexCD1-toprJ1 were present in multiple plasmids.These phenomenon illustrated that the multidrug resistance region was variable and that the copy number could change,then leading to the structural diversity of tmexCD1-toprJ1 positive plasmids.Then all tmexCD1-toprJ1 positive plasmids were divided into two types:IncFIB（Mar）/IncHI1B hybrid plasmids（20/23）and IncR plasmids（3/23）.IncFIB（Mar）/IncHI1B hybrid plasmids were mainly vectors for tmexCD1-toprJ1 and dominated the horizontal dissemination of tmexCD1-toprJ1 among pathogens,especially K.pneumoniae isolates.In addition,we detected translocatable units containing tmexCD1-toprJ1 gene cluster in multiple tmexCD1-toprJ1 positive plasmids,which has been reported to promote the mobilization of resistance genes.Genetic analysis showed the core genetic environment of tmexCD1-toprJ1 was conserved and the umuC gene was the potential insertion site of this novel resistance determinant.The evolution assays elucidated that tigecycline resistance levels,the expression of tmexCD1-toprJ1 gene cluster and the copy numbers of tmexCD1-toprJ1 can be elevated with tigecycline selection pressure.Finally,in order to explore the reason for the high prevalence of tmexCD-toprJ in K pneumoniae,we evaluated the fitness of the recombinant plasmid pUC19-tmexCD-toprJ in different bacteria host by monitoring their growth curve,relative fitness and the ability of bio film formation.The results showed that the expression of tmexCD-toprJ gene cluster would result in significant fitness cost in Escherichia coli rather than in K.pneumoniae,which may explain that the high prevalence of this gene cluster in K.pneumoniae was associated with its lower fitness cost.In conclusion,this study elucidated the epidemiological characteristics and transmission mechanisms of tigecycline resistance genes tmexCD-toprJ and tet（A）variant in porcine K.pneumoniae,revealed the prevalence characteristics of tmexCD-toprJ gene cluster from the perspective of fitness cost,then enriched the epidemiological and genomic characterization of tmexCD-toprJ gene cluster.The promoting role of traditional tetracyclines in the formation of tmexCD-toprJ gene cluster and tet（A）variants needs to be further explored due to the prohibition of tigecycline in veterinary clinic.