Study On QnrB-mediated Quinolone Resistance Among Enterobacteriaceae Species

Backgroup and Objective:Quinolone resistance is traditionally mediated by chromosomal mutations. First discovery of plasmid-mediated quinolone resistance (PMQR) in Klebsiella pneumoniae UAB 1 was made inadvertently by Martinez- Martinez and colleagues in 1998. Plasmids harbouring qnr gene will result in an increase of MIC of Ciprofloxacin against E. coli J53 transjugants, for that Qnr protein could block the action of Ciprofloxacin against both DNA Gyrase and TopoisomeraseⅣ. Many findings revealed the worldwide distribution of qnr. Many subtype of qnr were reported, such as qnrA, qnrB, qnrS and qnrC. Especially, a new qnr-like gene, qnrB described in 2006 displays low identity to qnrA, only 49.5% for nucleotide and 39.5% for amino acid. However, little is known about the epidemiology and origin of this gene variant. Until now, the gene has only been detected in India, North America, Taiwan and Korea. The linkage between qnrB and bla gene encoding extended-spectrum(ESBLs) or plasmid-mediated AmpCβ-lactamase has been noted in lots of research, such as bla gene for SHV-12, CTX-M-15, IMP-8, DHA, etc. To investigate the prevalence of qnrB in West China, 120 unique strains of Enterobacteriaceae species isolated from October 2005 to Apirl 2006 in our Hospital were studied. The detection of qnrA and qnrB was performed by PCR using specific primers, and conjugation experiment was done to determine whether the qnr determinant was transferable to E. coli J53 Azide^R. The presence of bla for ESBLs or plasmid-mediated AmpCβ-lactamase was investigated by Multiplex PCR as previously described.Method:1. Identification and susceptability of Enterobacteriaceae species were performed by Microscan Walkaway 40 Automatic System.2. Plasmid DNA were extracted with E.Z.N.A. Plasmid Mini kit according to the manufacture's recommendations. Screening of qnrA and qnrB was carried out by PCR amplification with specific primers to give a 413bp or 469bp products, respectively. Positive and negative controls were included in each run. And products were provisionally identified from their sizes in ethidium bromide-stained 1% agarose gels. Reference stains, K. pneumoniae UABl(for qnrA) and E. coli J53 plus pMG298(for qnrB) were kindly provided by George A. Jacoby (Infectious Disease Department, Lahey Clinic, Burlington) and Wang Minggui (Institute of Antibiotics, Huashan Hospital, Shanghai).3. Tranfer of qnr in vitro was determined by conjugation experiments with Azide resistant E. coli J53 as receipt. And co-transcojugants were selected in Tryptic Soy Agar plates containing 100μg/mL sodium azide for counterselection and 100μg/mL Ampicillin, 8μg/mL Ceftaxime for selection. Colonies were plated onto TSA with or without 0.06μg/mL Ciprofloxacin to determine the cotransfer of quinolone resistance.4. MICs of donors, cotransconjugants and recipient were measured by agar dilution method in accordance with the guidelines of CLSI. The antibiotics tested were Ampicillin, Cefotaxime, Cefoxitin, Atrezonam, Ceftazidime, Ciproxacin, Levofloxacin, Gatifloxacin, Enixacin, Sparfloxacin, Lomenfloxacin. 5. The presence of orf513 and orf1005 was evaluated by PCR in strains with qnrA or qnrB. And sequencing of qnrB was performed by PCR using primers qnrB1/B2-CDS. Then cycle sequencing was carried out with an ABI PRISMTM 3730XL DNA Analyzer by Invitrogen Biotechnology. The NCBI BLAST program was utilized for sequence comparisons.6. Different species or strains collected from the same patient were detected to determine whether there was horizontal transfer of qnr in vivo. A strain of Acinetobacter baumannii was found to harbour qnrA and qnrB, then the purified qnr gene was ligated to pMD 18-T vector, and introduced into E. coli DH5 a with selection on TSA plates containing Ampicillin. Plasmid DNA of positive clones were sequenced.7. The existence ofβ-lactamase in qnr-bearing strains was evaluated by Nitrocefin Test.β-lactamase was extracted by freezing-thawing method, then to detect the ESBLs, plasmid-mediated AmpCβ-lactamase and metallo-β-lactamase using improved three-dimension test. Identification of the bla_SHV, bla_CTX-M-3-like, bla_{CTX-M-14-like} and bla gene for plasmid-mediated AmpCβ-lactamase was done by multiplex PCR.Results:1. Distribution ofqnr: qnrA and qnrB were detected in 37 and 33 isolates, respectively. And 13 strains containing both two genes. The prevelance of qnr in Enterobacter sp., Klebsiella sp. and E. coli was 52.9%,57.1% and 50.0%, respectively.2. Conjugation experiments. All qnr-borne isolates were transferable except a strain of K. pneumoniae. However, a isolate of transconjugants could not grow in TSA containing Ciprofloxacin.3. Susceptability. The MIC oftransconjugants increased many times with comparison to that of E. coli J53. The prevalence of resistance to Gatifloxacin and Cefpime in qnr-positive bacteria was relatively low.4. Detection of orf513 and orf1005 : The prevalence of orf513 and orf1005 was 18.9%, 21.2% among qnrA-bearing and qnrB-bearing strains, respectively.5. Sequencing of qnrB: Eighteen qnrB-borne isolates were amplified to give 681 bp or 645bp products. The sequence of qnrB from some strains was perfectly identity to that of qnrB1, while 4 isolates had almost identical sequences which differed from that of qnrB3 by three nucleotides (201 A→T, 271T→G, 498G→A) resulting in changes of two amino acids(Lys67Asn, Ser91Ala).6. Transfer of qnr in vivo: During 7 different strains from 5 patients with qnr-bearing pathogens, all contained qnr gene except one isolate of E. coli. And the latter isolate may harbour the same qnr gene as the former, or contain both two types of gene. The incomplete sequence(469bp) of qnrB in A. baumannii was identical to qnrA1, but that of the qnrA(413bp) has 99% identity of nucleotide acid to qnrA1 (C431T) and results in a change of amino acid.7. Study ofβ-lactamase in qnr-bome strains: All qnr-positve strains could turn Nitrocefin Disks to red. And improved three-dimension tests showed thirty-three strains produce ESBLs or AmpCβ-lactamase. bla_SHV or bla_CTX-M were present in 49 strains of qnr-bearing Enterobacteriaceae, and 49.0% of these isolates carried both two bla genes. Almost 70 percent of isolats carrying qnr were detected to harbour bla gene for AmpCβ-lactamase, and bla_EBC, bla_DHA were in the majority (31strains, 54.4%).Conelusion:The results suggested emergence of qnr-borne Enterobacteriaceae species in our hospital from October 2005 to Apirl 2006. It is the first report of qnrB-mediated quinolone resistance in China, and a new qnrB-like gene was found. We also firstly report the presence of PMQR among A. baumannii. The linkage between qnr and ESBLs or plasmid-mediated AmpCβ-lactamase was confirmed, and qnrB-borne strains may carry bla genes for SHV, CTX-M, DHA, et al.