| Fosfomycin has a good antibacterial activity against multi-drug resistant Gram-negative bacteria and a low resistance rate to Gram-negative bacteria and Gram-positive bacteria,thus has attracted people’s attention.Although fosfomycin has not been approved for use in animals in China,fosfomycin-resistant bacteria have been found in Enterobacteriaceae,fos A3 has become the most popular plasmid-mediated fosfomycin resistance gene among animal Enterobacteriaceae in China,especially in E.coli.Pigeons,as food animals,although is limited in the use of antibacterial drugs,are still considered to be a potential reservoir of bacteria and may pose a threat to human public health.72 pigeon and environmental samples were collected from a pigeon farm in Foshan,Guangdong Province,and the bacteria from samples were isolated and identified using selective medium and MALDI-TOF-MS.PCR were used for detecting plasmid-mediated fosfomycin resistance genes fos A3,fos C2 and fos A.The results showed that the 143 isolates identified as E.coli（108）,P.mirabilis（22）,K.pneumoniae（2）,P.vulgaris（5）,C.freundii（1）,S.provencal（1）,P.putida（1）,A.aeruginosa（1）.The results showed that 49 E.coli,16 P.mirabilis and 1 C.freundii were positive for fos A3,and no fos C2 and fos A genes were detected.The resistant phenotype of fos A3-positive E.coli and its conjugations and P.mirabilis were determined by agar dilution method and micro-broth dilution method respectively.The results showed that the 49 fos A3 positive E.coli were resistant to fosfomycin（MIC>256μg/m L）,ampicillin,cefotaxime and ciprofloxacin.The rates of resistance to tetracycline,sulfonamides,fluorobenzene,gentamicin,colistin,amikacin were 97%,73.47%,38.78%,16.33%,8.20%and 2%respectively;but all the isolates were sensitive to chloramphenicol,meropenem and tigecycline.All of the 16 fos A3-positive P.mirabilis isolates were resistant to fosfomycin（MIC>256μg/m L）,ampicillin,cefotaxime,tetracycline,tigecycline,colistin,ciprofloxacin.In addition,the resistance rate to gentamicin was 18.75%.The phylogenetic relationship of the strains was analyzed by pulsed-field gel electrophoresis（PFGE）,whole genome sequencing,construction of core genome evolutionary tree and multi-locus sequence typing（MLST）.The PFGE results found that the fos A3-positive E.coli and P.mirabilis were clone spread in the pigeon farm;nine ST types of E.coli were present in this pigeon farm,three of the which were detected in human samples and the predominant ST type of the pigeon farm is ST6856（n=12）.Different PFGE isolates were selected for genome-wide sequencing and phylogenetic tree analysis.It was found that between isolates from human samples and E.coli and P.mirabilis isolated in this study all had less SNP,suggesting that fos A3-positive E.coli and P.mirabilis could be transmitted between human and animals.The horizontal transmission of fos A3 gene was performed by conjugation experiment.The results indicated that fos A3 in31 E.coli could transferred into E.coli,but the 16 fos A3-positive P.mirabilis failed.S1-PFGE and Southern-blot showed that the fos A3 gene was located on plasmids of different sizes ranging from 70 to 380 kb.I-Ceu I PFGE and Southern-blot were carried out on the fos A3 gene-positive P.mirabilis,and found that fos A3 was located on the chromosome of P.mirabilis.fos A3 positive isolates differed in PFGE patterns were selected for whole genome sequencing,and PCR-mapping method was used to analyze the genomic context flanking fos A3 gene.The results showed that the genetic environment gene was 100%similar to the previous reported sequences,and fos A3 was located in three different types of genomic context.11 E.coli strains carried the genomic context IS26-?orf2-orf1-fos A3-?IS903D-bla CTX-M-14-IS26.Three strains including 1 E.coli and 2 P.mirabilis harbored the genetic structure IS26-orf3-?orf2-orf1-fos A3-IS26.Four E.coli comprised the gene cassette array IS26-orf3-?orf2-orf1-fos A3-IS26-?IS903D-blaCTX-M-65-?ISEcp1-IS26.This study investigated the prevalence and spread of the fosfomycin resistance gene fos A3 in Enterobacteriaceae from a pigeon farm.The results showed a high prevalence of fosfomycin resistance gene fos A3 in E.coli and P.mirabilis in the pigeon farm.The fos A3gene may transfer via the spread of plasmid and could be clone spread among samples from various sources in E.coli.In P.mirabilis,the fos A3 gene was mainly located on the chromosome and transfer via clone spread.In addition,the complex transposon structure IS26-fos A3-IS26 is likely to mediate the transfer of the fos A3 gene between plasmids and chromosomes among Enterobacteriaceae,and facilitate the dissemination of fos A3.In this research,we studied the prevalence and the epidemiological features of fosfomycin resistance genes in Enterobacteriaceae from a pigeon farm.This study will provide important scientific evidence for controlling the transmission of multi-drug resistant bacteria in poultry. |
