Prevalence Of Plasmid-mediated Fosfomycin Resistance In Associatiott With β-lactamases Genes Amongst Clinical Isolates Of Shigella

Objectives To determine the the minimal inhibitory concentration(MIC) of shigella against 12 commonly used antibiotics and detect the plasmid-mediated fosfomycin resistant genes in fosfomycin resistance shigella and analyze its mode of transmission. To investigate the relation of plasmid-mediated fosfomycin resistance genes with β-lactamase genes.Materials and Methods Isolates Totally 263 non-duplicate clinical isolates of shigella was collected from antimicrobial resistance monitoring center of Anhui Province from 2011 to 2012.ATCC25922 which was used as quality control strain for antimicrobial susceptibility testing, sodium-resistant Escherichia coli J53 AZR, Klebsiella pneumoniae ATCC700603 which was ESBLs positive strains and fos A、fos A3、fos C positive Escherichia coli were saved by antimicrobial resistance monitoring center of Anhui Province.Methods 1.Antimicrobial susceptibility testing was performed by agar dilution method and ATCC25922 was used as quality control strain. 2.Using polymerase chain reaction(PCR) to detect the plasmid-mediated fosfomycin resistance genes in fosfomycin resistance strains,gene sequencing was performed to determine whether there are mutution in plasmid-mediated fosfomycin resistance genes. 3.conjugation experiment was performed in plasmid-mediated fosfomycin genespositive strains and J53 AZR, The MIC of recipient strains and transconjugants were tested by agar dilution method for fosfomycin and other antimicrobial agents,plasmid-mediated fosfomycin resistance genes in transconjugants were determined by PCR.4.enterobacterial intergenic consensus PCR(ERIC-PCR) was employed to understand the molecular homology of the fos A3-positive isolates. 5.using PCR to identify the genotypes of ESBLs-producing isolates.DNA sequencing of ESBLs gene-positive strains were analyzed and the conjugation experiment was performed.The MIC of recipient strains and transconjugants were tested by agar dilution method for fosfomycin and other antimicrobial agents. 6.performing PCR-based replicon typing in transconjuants.to determine the replicon type.Results 1.In 263 shigella strains fosfomycin resistance rate is 9.5% and Intermediate rate is 2.7%, Chloramphenicol resistance rate is the highest, reaching 87.8%, imipenem, piperacillin / tazobactam and cefoperazone / sulbactam are more sensitivity than hird-generation cephalosporin, levofloxacin and ciprofloxacin susceptibility rate was 60.5% and 64.3%, respectively.2.PCR results showed that 18 drug-resistant strains fos A3 positive, no fos A and fos C2 gene was detected, the Gen Bank accession numbers of fos A3 is KJ716852. The conjugation experiments were successfully carried out in 13 PCR-positive isolates.The MIC of transconjugants against fosfomycin increased differently compared to recipient strains.The homology of partly fos A3-positive isolates was comfirmed by ERIC-PCR. 3.All 18 fos A3-positive shigella was ESBLs-producing shigella and 1 isolate only take OXA genotype,3 isolates take CTX-M and OXA and TEM genotype, 14 isolates take CTX-M and OXA genotype.DNA sequencing reavaled that CTX-M gene isCTX-M-15、CTX-M-55、CTX-M-123 and Gen Bank accession numbers of CTX-M-123 is KJ871006,all TEM gene is TEM-1 and all OXA gene is OXA-30.The transconguant test of 13 ESBL-producing isolates were positive and these conjugations were resistant to fosfomycin and third generation cephalosporins and take the same resistance genes as donor.4.All 13 transconguants take Inc F type plasmid and of which 3 strains take Inc HI2 type plasmid,2 strains take Inc I1-Ir type plasmid and 1 strain take Inc N type plasmid.Conclusions: 1. The problem of antimicrobial resistance in Shigella in Anhui province is serious, but there is still a high sensitivity of fosfomycin, so it can be used for clinical treatment of bacillary dysentery. 2. Confirmed there are plasmid-mediated fosfomycin resistance of Shigella in Anhui province and for the first time detected plasmid-mediated fosfomycin resistance gene in shigella. 3. The plasmid-mediated fosfomycin gene fos A3 can lead to high level of fosfomycin resistance in shigella. 4. There are very high level success rate in transconguant test and homology in fos A3-positive shigella,so transmission for fos A3 gene is easy. 5. There may Presence plasmid-mediated fosfomycin resistance gene fos A3 and β-lactamase gene located on the same plasmid co-propagating, causing multi-drug resistant.