Emergence And Transmission Mechanisms Of Plasmid-Mediated High-Level Tigecycline Resistance Gene Tet(X4)in Escherichia Coli From Animals

With the global emergence and spread of antimicrobial resistance to colistin and carbapenems,tigecycline has acted as one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria.However,the tigecycline resistance also sporadically occurs in recent years,primarily due to overexpression of chromosome-encoding efflux pumps and ribosome protection proteins.On the other hand,tigecycline has been confirmed to be one of substrates for the flavin-dependent monooxygenase gene tet（X）,which needs FAD,Mg²⁺,O₂ and NADPH for the degradation activity,but its clinical significance remains questionable.Therefore,we conducted a further study and reported the emergence of one novel plasmid-mediated mobile tigecycline resistance mechanism in Escherichia coli strains from China.The index isolate E.coli LHM10-1 of pig origin,resistant to both tigecycline and colistin,was initially characterized by conjugation and genomic DNA sequencing.Insterestingly,the tigecycline resistance could be successfully transferred and a potential novel tigecycline resistance gene tet（X4）,located on an Inc Q1 plasmid（p LHM10-1-p6）,was identified.The similarities of Tet（X4）with previously reported Tet（X）～Tet（X3）subtypes were between 68.7%and 95.1%.Subsequently,it was subcloned and its function was further confirmed by microbiological degradation and liquid chromatography-mass spectrometry experiments.It could inactivate and confer resistance to all of the tetracyclines,even for descreased susceptibilities against the newly FDA-approved eravecycline（4μg/m L）and omadacycline（8μg/m L）.In an in vivo murine model,the presence of tet（X4）also led to the failure of tigecycline treatment.Epidemiologically,45 tet（X4）-positive E.coli strains were identified at a low detection rate between 2017 and 2018 from pigs（1.3%）,chickens（0.8%）,migratory birds（0.2%）,dusts（0.9%）and soils（0.6%）.They belonged to 13 pulsotypes and 11 sequence types（STs）that similarly consistent with the core-genome evolutional analysis,of which 24（53.3%）strains carried the same p LHM10-1-p6-like plasmid.Geographically,these isolates were distributed in five provinces in eastern and southern China,including Fujian（3.2%）,Jiangsu（2.4%）,Guangdong（1.3%）,Guangxi（1.1%）and Jiangxi（0.9%）.It should be noted that the tet（X4）-positive E.coli strains belonging to ST8302,ST1196,ST515,ST761 and ST10exhibited the clonal relationship among different provinces and sources,suggesting the potential risk for further spread of tet（X4）-mediated tigecycline resistance.Then the minimum inhibitory concentrations（MICs）of tet（X4）-positive E.coli isolates mentioned above were determined.The result showed that all of them were resistant to tigecycline,tetracycline,florfenicol and compound sulfamethoxazole,with additional high resistance rates to ciprofloxacin（35.6%）,cefotaxime（28.9%）,gentamicin（24.4%）and colistin（20.0%）,while they were susceptible to meropenem,amikacin and fosfomycin.Notablely,they also conferred high MICs to eravacycline（≥4μg/m L）and omadacycline（≥8μg/m L）.Worrisomely,besides the virulence genes che Y,csg D and others,100.0%of them were also multidrug-resistant,and the proportions of strains resistant to 3～7 classes of antibiotics ranged from 4.4%to 55.6%.Among them,all of the 13 cefotaxime-resistant E.coli isolates carried the bla_CTX-M-14（n=8）,bla_CTX-M-65（n=4）and bla_CTX-M-27（n=1）genes,respectively,even the nine colistin-resistant isolates of them also harbored the mcr-1 gene.Besides E.coli LHM10-1 carrying the Inc Q1 plasmid p LHM10-1-p6,Nanopore sequencing indicated that the tet（X4）genes were also located on the Inc HI1/F-:A8:B-hybrid plasmid（p2FT38-2-1）in E.coli 2FT38-2,the F-:A18:B-plasmid（p2FT39-3）in E.coli2FT39,the novel plasmid（p STB20-1）in E.coli STB20-1 and the chromosome（c2ZN37-2）in E.coli 2ZN37-2.Conjugation experiments by filter mating revealed that both the tet（X4）-harboring plasmids p LHM10-1-p6 and p2FT38-2-1 could be successfully transferred into the recipient clinical and laboratory E.coli,Klebsiella pneumoniae and Salmonella Typhimurium strains at high frequencies of 10^-2 to 10^-6,while the plasmids p2FT39-3 and p STB20-1 were further transferred by transformation into the competent E.coli DH5α.In addition,we also confirmed that the Inc X4,Inc I2 and Inc FII helper plasmids carrying mcr-1could contribute to the transfer process of the Inc Q1 plasmid p LHM10-1-p6(10^-2 to 10^-5).Furthermore,a phylogenetic analysis of tet（X4）and other tet（X）homologous genes was conducted.The result indicated that a total of five tet（X）subgroups have been generated until now.The cloning expression and microbiological degradation assays confirmed that tet（X3）,tet（X4）,tet（X5）and their variants could confer the similar strong activity against tetracyclines,which were also always associated with the mobile element ISCR2.By species,they were widely distributed in various bacterial hosts,especially in Acinetobacter spp.and Enterobacteriaceae strains,and the environmental Flavobacteriaceae bacteria might act as their ancestor.In the NCBI database,a matching distribution of ISCR2 was also found out from Acinetobacter spp.（54.5%）,E.coli（17.7%）and Flavobacteriaceae（0.6%）strains,further confirming the potential ISCR2-mediated evolutionary route of tet（X）genes.In this study,the detection of tet（X4）-positive E.coli strains in food-producing animal,migratory bird and their surrounding environment is a warn for public health and dramatically threatens the clinical efficacy of tigecycline as one last-resort treatment option.This emphasizes the seriousness of current bacterial resistance once again and the necessity for rational usage of antibiotics in human,animal and agriculture.In order to avoid the situation that no antibiotics are available in the future,we need to start from human,animal and environment to jointly fight against the problem of antibiotic resistance under the concept of“One Health”.