| Recently,the increasing clinical application of antibiotics has led to a growing number of multidrug-resistant gram-negative bacterial infections.Although fosfomycin has been used for 50 years since its discovery,it still has antibacterial activity against gram-negative bacteria as well as some gram-positive bacteria due to its unique antibacterial mechanism and chemical structure,and it has good synergistic effect with other drugs.As a clinical drug for human medicine,fosfomycin-resistant E.coli and Klebsiella pneumoniae isolated from animals and environment are increasing in recent years.Fosfomycin resistance is mainly mediated by fos A3.The fos A3 gene is often detected in different strains and plasmids,and can be co-transmitted with a variety of drug-resistant genes.However,there are few reports on fosfomycin resistance strains of vegetable sources.In order to prevent the spread of drug resistance and provide a basis for the rational use of antibiotics in veterinary clinics,the prevalence of the fosfomycin resistance gene fos A3 in Enterobacteriaceae from vegetable was studied.In this study,233 strains of ESBLs-producing Enterobacteria isolated from the vegetable samples purchased from supermarkets in Guangzhou from 2015 to 2016 were tested for fosfomycin resistance genes fos A,fos A3,fos C2,and other common drug resistance genes.Seventeen fos A3 positive strains carrying the bla CTX-M gene were detected.Antimicrobial susceptibility test of the fos A3-carrying isolates was performed by the agar dilution method.The results showed that the MIC values of fosfomycin of 18fos A3 positive strains were over 2048μg/m L.All strains were resistant to at least 5 drugs,showing multiple drug resistance.All strains were sensitive to imipenem.Fifteen fos A3-positive strains were subjected to pulsed-field gel electrophoresis.All strains were successfully identified.The 7 pairs of housekeeping genes of Escherichia coli and Klebsiella pneumoniae were amplified by PCR to obtain the MLST number.The results showed that LC3 and LDH4-2 belonged to the new MLST classification.The results of PFGE typing and MLST typing were consistent,indicating that there was no clonal transmission relationship between fos A3 positive bacteria.Transconjugants and transformants with single plasmid were successfully obtained by conjugation assay and transformation assay to study the transferability of fos A3-bearing plasmids.Southern hybridization results identified that fos A3 of LDL3-3 was located on chromosome.Part of the fos A3-carring transconjugants and transformants extracte plasmids were selected for sequencing and analyzing the plasmid characteristics as well as fos A3 genetic environment.Except LDL3-3,which fos A3 was located on chromosome,fos A3 gene of other bacteria were located on plasmids.Most plasmids carrying fos A3 were Inc HI2,Inc FII and its hybrid or hypotype plasmids.Both Inc HI2 and Inc FII plasmids have a typical skeletal structure,but their variable regions differ greatly.The Inc FII heterozygous plasmids were consist of the common Inc FII-N and the uncommon Inc FII-R.The latter was formed by fusing a partial framework region of the Inc R-type plasmid near the variable region of the Inc FII type plasmid.Furthermore,fos A3 was found to be located on an untyped plasmid of about 50 kb in two strains and the structure was dissimilar with other published plasmids.As the genetic environments were divided into five different types by cluster analysis,which showed that fos A3 can locate on different plasmids through IS26.Other insertion sequences,such as IS903 and ISEcp1,carring bla CTX-M genes,often co-transfered with fos A3.In summary,the fos A3 gene from vegetable has a broad range of hosts and is carried by different plasmid types.Besides,fos A3 is often co-transfer with the bla CTX-M gene horizontally,mainly through the Inc HI2 type plasmid,Inc FII type plasmid and its hybrid plasmid or hypotype plasmid.In addition,IS26 plays an important role in the horizontal transfer of plasmids.Owing to the particularity that vegetable is edible directly,the drug resistant genes can be transmitted to the human body indirectly through the food chain and cause certain harm to public health problems,and therefore it is necessary to carry out further monitoring of fosfomycin resistance. |
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