Emergence Of Plasmid-Mediated Fosfomycin Resistant Gene FosA10 Among ESBL-producing Escherichia Coli

Fosfomycin is a phosphoenolpyruvic acid analog and has a broad-spectrum bactericidal activity against a variety of gram-positive and gram-negative bacteria.It is used to treat simple urinary tract infection caused by extended-spectrum β-lactamases(ESBL)producing and carbapenm resistant Enterobacteriaceae(CRE).The fosA genes are the most common resistant gene of Enterobacteriaceae to fosfomycin.The fosA genes can locate on plasmids or chromosomes,while most of plasmid-mediated fosfomycin resistance genes in E.coli originate from other Enterobacteriaceae,such as Kluyvera spp.(fosA3,fosA4),Klebsiella spp.(fosA5,fosA6 and fosA9),and Leclercia spp.(fosA8).This study discovered a novel fosfomycin resistance gene fosA10 from a ESBL-producing E.coli,and found it originated from Klebsiella pneumoniae.This study analyzed 983 ESBL-producing Enterobacter(393 human-derived,362animal-derived and 228 food-derived strains)in Guangzhou from 2011 to 2014,and 9food-derived E.coli were from Pakistan in 2018.Agar dilution method was used to detect their antibiotc susceptibility to fosfomycin.The result showed that 182 ESBL-producing E.coli from Guangzhou and a chicken meat-derived ESBL-producing E.coli(PK9)from Pakistan were resistant to fosfomycin.PCR detection of the known fosfomycin resistance genes,such as fosA1～fosA7 and fosC2,was conducted for all fosfomycin-resistant strains.The result showed that 182ESBL-producing E.coli from Guangzhou were positive to fosA3 gene,while fosA and fosC2 were not detected in E.coli PK9 from Pakistan.The conjugation experiment of PK9 and E.coli C600 was carried out,and a transconjugatant with fosfomycin resistance was obtained,which proved that the fosfomycin resistant gene was located on a conjugative plasmid.Performed the whole genome sequencing(WGS)analysis on strain PK9.The result showed that the PK9 strain had a genome of 5.4 Mb in length and belonged to ST38.The PK9 Strain carried three plasmids.A 420 bp gene that encoded a FosA-like protein was located on a 53 kb Inc FII(F35:A-:B-)plasmid(p HNPK9-FOS).The amino acid sequence of this FosA-like protein had 56.1% to 98.6% amino acid sequence homology with reported FosA proteins(FosA1～FosA9).The fosA-like gene amplification product was cloned into the p MD~TM19-T vector and transferred into competent cells E.coli DH5α.And the recombinant strain showed high resistance to fosfomycin(MIC>128 μg/m L),which proved that the gene mediates fosfomycin resistance.Thus,the fosfomycin resistance gene was designated as fosA10.The gene environment analysis of fosA10 showed that there were two reversed IS10 s on both sides of it,and they formed a transposon(IS10-fosA10-IS10).Besides,there were 9bp reverse repeats at both ends of the insertion position,which proved that the fosA10 was transferred into p HNPK9-FOS by forming a transposon with IS10 s.Analyzing by BLAST,the 1670 bp of fosA10 gene and its upstream and downstream sequences had 100%nucleotide identity with the chromosome sequences of 18 Klebsiella pneumoniae in the NCBI database.Thus,it was presumably originated from Klebsiella pneumoniae.The prevalence of fosA10 in 182 ESBL-producing E.coli with fosfomycin resistance was investigated.The result showed that none of them carried fosA10 gene.Although Klebsiella pneumoniae chromosomes all carry fosA genes inherently,the levels of fosfomycin resistance in different Klebsiella pneumoyniae strains are quite different.In order to confirm whether the various kinds of fosA genes on Klebsiella pneumoniae chromosomes could mediated different fosfomycin-resistant phenotypes in E.coli.12vegetable-derived Klebsiella pneumoniae with different fosfomycin resistance levels were ligated to the p MD~TM19-T vector,transferred into competent cells E.coli DH5α,and tested for the antibiotics susceptibility.The results showed that some of the fosA genes on Klebsiella pneumoniae chromosome showed similar fosfomycin resistance level to that on E.coli plasmids,but some of them showed significant different resistance levels between two different species.In summary,this study discovered a novel resistant gene fosA10 in E.coli.The fosA10 gene formed a transposon with IS10 s and transferred from Klebsiella pneumoniae chromosome to E.coli via Inc FII plasmid.As fosA genes derived from Klebsiella pneumoniae are likely to propagate fosfomycin resistance genes in E.coli and other Enterobacteria through horizontal transfer of mobile elements,and the fosA genes might further spead by plasmids.Thus,we should pay attention to the emergence of novel plasmid-mediated fosA genes,and strengthen the monitoring of fosfomycin application in humans and animals,in order to prevent the further spread of fosfomycin resistance.