Résumé
Objective To investigate the prevalence and plasmid size of qnrD determinant in Morganella morganii (M.morganii) isolates.Methods A total of 100 non-duplicated M.morganii clinical isolates were collected from inpatients.Standard ager dilution method was used to determine the minimum inhibitory concentrations (MICs) of fluoroquinolones against M.morganii isolates.PCR were performed to detect plasmid-mediated quinolone resistance determinants (PMQRs) in M.morganii isolates and the prevalence of extended-spectrum β-lactamase (ESBL) genes and AmpC β-lactamase genes in PMQRs-positive M.morganii strains.The homology analysis among qnrD-positive M.morganii strains were conducted by using pulsed-field gel electrophoresis (PFGE).The location of qnrD gene and the size of plasmid carrying it were determined by southern hybridization.The transferability of qnrD gene was determined by conjugation experiment.Results Thirty out of 100 M.morganii isolates (30％) were found carrying PMQRs including 17 qnrD-positive strains,14 aac (6')-Ib-cr-positive strains and 5 qepA-positive strains.PCR and sequencing confirmed that thirty PMQRs-positive isolates carried blaDHA-1.Among them,six isolates were positive for ESBLs genes (four for blaCTX-M-14,one for blaCTX-M-3 and one for blaCTX-M-24) and four isolates were positive for blaTEM-1.Almost all PMQRs-positive M.morganii isolates showed reduced susceptibility to fluoroquinolones.Moreover,seventeen qnrD-positive M.morganii isolates harbored blaDHA-1 including five (29.4％) harboring aac(6')-Ib-cr gene,four (23.5％) harboring blaCTX-M-14,two (11.8％) harboring blaTEM-1 and one harboring aac(6')-Ib-cr gene,blaCTX-M-14 and blaDHA-1.PFGE analysis showed that the 17 qnrD-positive M.morganii isolates were divergent from each other and not clone-related.Southern hybridization analysis showed that qnrD genes of all M.morganiiis isolates were mainly located in a 2.7 kb plasmid,but only a few of them were located in a size of 5.1 kb plasmid.M.morganiiis isolates failed to transfer qnrD gene to E.coli EC600 through conjugation.Conclusion PMQRs were widely distributed in M.morganiiis isolates.qnrD gene was the predominant determinants with a high prevalence rate of 17.0％,followed by aac(6')-Ib-cr gene.qnrD gene was located on a non-conjugative plasmid of approximately 2.7 kb or 5.1 kb.One qnrD-positive M.morganii isolate carrying aac(6')-Ib-cr gene,blaCTX-M-14 and blaDHA-1 was detected.
Résumé
Objective To investigate the prevalence and molecular characteristics of the extended -spectrum β-lactamase ( ESBL) and AmpC enzyme-producing Proteus mirabilis ( P.mirabilis) strains isola-ted in Shenzhen People′s Hospital.Methods The production of ESBLs and AmpC enzymes by P.mirabilis isolates were detected by a screening and confirmatory test for ESBLs and AmpC disk test , respectively .The PCR assays followed by DNA sequencing of the products were employed to analyze the multiple genes inclu -ding the ESBLs genes, AmpC genes, insertion sequences (ISs) upstream of the ESBLs or AmpC genes, plasmid -mediated quinolone resistance ( PMQR ) determinants , quinolone resistance-determining region (QRDR) genes , the integrase genes, and class1 integron cassette.The epidemiological analysis of the iso-lates was performed by pulsed field gel electrophoresis .Results There were 130 P.mirabilis clinical iso-lates collected from Shenzhen People′s Hospital in China during the year 2004 to 2010.Among them, 13 isolates (10%) produced ESBLs, that accounted for 0%-9.1%in the year 2004-2009 and up to 29.4%in 2010, and 3 isolates (2.3%) produced AmpC enzymes.The predominant genotype of ESBLs -producing isolateswas b al CTX-M-14(n=7), followed by blaCTX-M-65(n=3), blaCTX-M-55(n=1), blaCTX-M-24(n=1) and blaPER-1 (n =1).The clinical isolate of PER-1-producing P.mirabilis was reported for the first time in China.Twoisolates carried an AmpC β-lactamase gene of blaCMY-2 and one isolate carried an unidentified AmpC gene .ISEcp1 located upstream of blaCTX-M and blaCMY-2 were detected in 91.7% (11/12) of CTX-M-producing isolatesand one CMY-2-producing isolate, respectively.ISPa12 was present upstream of blaPER-1 in one studiedisolate.Approximately 66.7% (10/15) of ESBL and /or AmpC-producing isolates harbored PMQR genes including2 carrying qnrD, 5 carrying aac-Ib-cr and 3 carrying both qnrD and aac-Ib-cr.Twelve ESBL and /orAmpC-producers with high level of resistance to ciprofloxacin carried the similar mutation profiles of S 83I inGyrA, S80I or S80R in ParC and among them, six strains showed E466D mutation in GyrB.Approximately86.7% (13/15) of ESBL and/or AmpC-producing isolates carried class 1 integron.Fourteen PFGE typeswere observed among 15 ESBL and/or AmpC-producers.Conclusion The prevalence of CTX-M β-lactamasesin P.mirabilis isolates contributed to the increased resistance to extended -spectrum cephalosporins.The qnrD and/or aac-Ib-cr genes were detected among the most of ESBL and /or AmpC-producing P.mirabilis clinical isolates.