| Carbapenems are considered one of the most effective antimicrobial agents for the treatment of multidrug-resistant Gram-negative bacteria.However,with the widespread use of carbapenems,carbapenem-resistant bacteria have emerged and rapid spread worldwide,which has become an important issue threatening public health.Carbapenems is not approved for veterinary.Previous studies showed that the detection rate of carbapenem-resistant bacteria from animal origin was increasing rapidly,and it is possible for carbapenem-resistant bacteria to become fully prevalent in animal origin.Therefore,to exploring the relation between important carbapenem resistance genes and their plasmids in Enterobacter from different origin,a systematically study of the prevalence distribution and molecular transmission mechanism of carbapenem-resistant bacteria from animal origin in China was performed.From 2015 to 2017,6302 samples were collected from 21 provinces or municipality,including 2,771 samples from food animals,3,188 samples from migratory birds and 343samples from companion animals.A total of 2255 carbapenem non-susceptible bacteria were screened from 1791 samples by medium containing meropenem,and the samples positive rate was 28.4%(1791/6302),including 654 positive samples from food animals,1067samples from migratory birds and 70 samples from companion animals.The modified Carba NP test was used to detect the carbapenemase-producing bacteria.The results showed that1002 strains capable of producing carbapenemases,which were isolated from 850 samples and 208 samples from food animals,619 from migratory birds and 23 from companion animals.Carbapenemase-producing bacteria were test for carbapenems resistant genes and the identification of species was conduct in gene positive strain.The result indicated that 593NDM-positive E.coli,10 VIM-positive P.putida,4 VIM-positive S.maltophilia and 2 NDM-positive E.coli.In addition,the predominant strains from food animals and companion animals were E.coli and in migratory birds and their environment were Klebsiella,E.cloacae and Proteus.The MIC in 609 strains revealed that most tested strains were multi-drug resistant,and more than 80%of them were resistant to tetracycline,sulfonamide/trimethoprim,florfenicol and gentamicin.Moreover,the resistant rate of amikacin,aztreonam,fosfomycin,colistin and ciprofloxacin in food animals was higher than in migratory birds.Pulsed field gel electrophoresis(PFGE)was carried out to analyze the relationship of330 bla NDM-carrying Enterobacteriaceae.The result showed that the relationship of Enterobacteriaceae carrying bla NDMgene was diversity.There were 4 group of E.coli carrying bla NDMgene were isolated from food animal,among which two strains were clone related in each group.In addition,69 bla NDM positive E.coli strains belonged to different phylogenetic groups,and 6 strains isolated from companion animals were divided into five different phylogenetic groups.The genotyping results of migratory bird origin strains demonstrated that 230 positive Klebsiella strains were divided into 5 phylogenetic groups,of which 202 strains belonged to group B,indicating that there was a dominant clonal group in the migratory bird,and bla NDM gene may be transmitted through the dominant clonal group in the bird population.The horizontal transfer ability of 330 bla NDM-positive Enterobacteriaceae were conducted by conjugation experiments.The results showed that the success rate of conjugation in the strain carring bla NDM gene from animal was higher,and from food animal,migratory bird and pet was more than 85%,indicating that Enterobacteriaceae carring bla NDM gene had a good transferability.The plasmid characteristics of bla NDM gene were further analyzed by plasmid replicon type,S1-PFGE and Southern blot.The result showed that the Inc X3 plasmid was the main vehicle to mediate bla NDM gene transmission in animals.This indicates that although the clone relationship between bla NDM positive bacteria from different sources was far,bla NDM gene can be transmitted between different strains through the Inc X3 plasmid,which is an important reason for the wide distribution of this gene in animal origin in China.A total of 130 bla NDM positive strains from 78 food animal origin,46 migratory bird origin and 2 companion animal origin were selected for whole genome sequencing,the results showed that the high detection rate of resistant genes in bla NDM positive strains from food animal were class of tetracycline,sulfonamide,aminoglycoside and amide alcohols,and from migratory birds were the class of?-lactamase,aminoglycoside,fluoroquinolone,tetracyclines and sulfonamides.The results of MLST typing showed that the most prevalence clonal groups in bla NDM positive E.coli from food animain were ST48,ST165,ST405 and ST34,and in bla NDM positive Klebsiella from migratory birds was ST1697.This dominant clonal group was the hybrid strain formed by recombination of K.pneumoniae subsp.pneumoniae and K.quasipneumoniae subsp.similipneumoniae.There are 4 types environments mediating the spread of bla NDM gene in China,and the dominant structure is IS26-tat-trp F-ble MBL-bla NDM-ISAba125-IS3000-?Tn2.In addition,it was found that an Inc X3 and Inc X4 hybrid plasmid maintained in original E.coli isolate and its transconjugant,and this hybrid plasmid harbouring both bla NDM-5 and mcr-1 gene,which could mediate resistance to carbapenems and colistin simultaneously.In conclusion,the carbapenem non-susceptible strains and carbapenemases producing strains from animal are widely spread in China.The bla NDM gene is the main reason of strain resistance to carbapenem.The Inc X3 plasmid is the important carrier mediated the spread of bla NDM genes in animal.The current study reports the identification of a transferable Inc X3–X4 hybrid plasmid harbouring both bla NDM-5 and mcr-1 gene,and first reported for the first time that NDM-5 is carried in migratory birds,and its dominant clone is formed by hybrid formation between two subspecies of Klebsiella.This implied that horizontal transfer of resistant genes,recombination between plasmids and strains plays an important role in the spread of antibiotic resistant genes and the evolution of bacteria. |
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