Prevalence Of Plasmid-mediated Quinolone Resistance Determinants In Association With β-lactamases,16S RRNA Methylase Genes And Integrons Amongst Clinical Isolates Of Shigella

Objective:To investigate the variation and distribution of the plasmid-mediated quinoloneresistance genes in clinical isolates of Shigella and their resistance to antimicrobialagents.To investigate the prevalence of plasmid-mediated quinolone resistance (PMQR)determinants in association with β-lactamases,16S rRNA methylase genes andintegronsMaterials and Methods:Isolates:From September2007to October2010，a total of137non-duplicate Shigella isolateswere collected from31hospitals in Anhui, China. Escherichia coli ATCC25922wasused as a quality control strain for antimicrobial susceptibility testing. Sodiumazide-resistant Escherichia coli J53AzRwas used as the recipient for conjugationexperiments.Method:Total DNA was extracted by suspending a few overnight colonies in0.5ml ofdouble-distilled water and heating the mixture at100°C for10min. Bacterial plasmidDNA was extracted from the clinical isolates by the rapid alkaline lysis protocol. qnr, aac(6)-Ib-cr and qepA genes were identified by polymerase chain reaction (PCR) in137clinical isolates of Shigella. All purified PCR products were directly sequenced bythe dideoxy chain termination method. Sequence alignment was compared with theGenBank nucleotide database using the Nucleotide BLAST program.Conjugation experiments for the PMQR-positive isolates were carried out inLuria-Bertani (LB) broth with Sodium azide-resistant Escherichia coli J53AzRas therecipient as previously described. The minimal inhibitory concentrations (MICs) ofwild-type isolates, recipient strains and transconjugants were tested by agar dilutionmethod for quinolones and other antimicrobial agents.The mutations in the quinolone resistance-determining regions (QRDRs), β-lactamasesgenes,16S rRNA methylase genes and integrons were determined by PCR with themethods described previously for the PMQR-positive isolates. All purified PCRproducts were directly sequenced and sequence alignment was compared with theGenBank nucleotide database using the Nucleotide BLAST program. β-lactamasesgenes,16S rRNA methylase genes and integrons were determined by PCR with themethods described previously for the transconjugants.The genetic relatedness of the PMQR-positive isolates was analysed by pulsed-field gelelectrophoresis (PFGE).Results:Amongst the137Shigella isolates,10isolates (7.3%) carried at least one PMQR gene.Four qnr-positive isolates (2.9%) were identified, and sequencing revealed one qnr B4allele and three qnrS2alleles (GenBank accession numbers of the complete sequenceHQ917003, and JF261185). Five isolates (3.6%) carrying aac(6)-Ib-cr were detected (GenBank accession number JF261186). Additionally, one isolate (0.7%) harbored qepA.The MICs of transconjugants for quinolones were increased differently compared torecipient strains.Amongst the10PMQR-positive isolates,7strains had a mutation at codon83(Ser83→Leu) of gyrA.2strains had a mutation at codon87(Asp→Tyr or Asp→Asn) ofgyrA,6strains had a mutation at codon80(Ser80→Ile) of parC. Nine strains of the10PMQR-positive isolates were detected to carry β-lactamases genes,7strains of whichharbouring blaCTX-M-14,7strains of which harbouring blaOXA-1and2strains of whichharbouring blaDHA-1. Three strains of the10PMQR-positive isolates were detected tocarry16S rRNA methylase genes,2strains of which harbouring RmtB and1strain ofwhich harbouring ArmA. Eight strains of the10PMQR-positive isolates were detectedto carry integrons,7strains of which harbouring intI1and6strains of which harbouringintI2. Additionally,2intI1-positive isolates carried dfrA17-aadA5gene cassettes, and allthe intI2-positive isolates carried dfrA1-sat1-aadA1gene cassettes. Close linkageamongst these resistance determinants localised on the same strain plays an importantrole in the prevalence of multidrug resistance (MDR) isolates. PMQR determinants withβ-lactamases,16S rRNA methylase genes and class1integrons were co-transferred.The analysis of PFGE of the10PMQR-positive isolates indicated that threeqnrS2-positive isolates and three aac(6)-Ib-cr-positive isolates belonged to the sameclone cluster, respectively. The other isolates were clonally unrelated.Conclusion:The plasmid-mediated quinolone resistance genes are lowly prevalent in clinical isolatesof Shigella.PMQR determinants along with β-lactamases,16S RNA methylase genes and class1 integrons on a transferable plasmid may spread to other bacterial species by horizontalexchange, which lead to the emergence and spread of multidrug-resistant pathogens.DNA fingerprinting profiles were analysed by PFGE, suggesting that part of the isolateswere clonally related.