Prevalence Of Chromosome-and Plasmid-mediated Quinolone Resistance In Association With β-lactamases And16s RRNA Methylase Genes Amongst Clinical Isolates Of Serratia Marcescens

ObjectiveTo investigate the prevalence of plasmid-mediated quinolone resistance (PMQR)determinants (qnr, aac(6′)-Ib-cr and qepA) and mutations in quinolone resistance-determining regions (QRDRs) of Serratia marcescens and their association withfluoroquinolone susceptibility in Anhui.To investigate the prevalence of chromosome-plasmid-mediated quinolone resistance(PMQR) determinants in association with β-lactamases, and16S rRNA methylase genes.Materials and MethodsIsolatesA total of non-duplicate104strains of S. marcescens were collected from various clinicalspecimens from34hospitals in Anhui, China from2005to2010were analysed. Escherichia coliATCC25922was used as a quality control strain for antimicrobial susceptibility testing. Sodiumazide-resistant Escherichia coli J53AzRwas used as the recipient for conjugation experiments.Method1. The MICs of S. marcescens were determined by broth microdilution method. The resultswere judged according to the criteria recommended by CLSI2010.2. 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. gyrA,parC, qnr, aac(6′)-Ib-cr and qepA genes were identified by polymerase chain reaction(PCR) in104clinical isolates of S. marcescens. All purified PCR products were directlysequenced by the dideoxy chain termination method. Sequence alignment was comparedwith the GenBank nucleotide database using the Nucleotide BLAST program.3. 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) of 4. wild-type isolates, recipient strains and transconjugants were tested by agar dilutionmethod for quinolones and other antimicrobial agents.5. The β-lactamases genes, and16S rRNA methylase genes were determined by PCR withthe methods 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.Results1. The majority of S. marcescens were isolated from the specimen of sputum, accounting for59.6%. The bacteria was the frequently distribution in Respiratory Department, followed byIntensive Care Unit, Gerontology Department. The results of antimicrobial susceptibility testshowed that S. marcescens was highly resistant to the1st and2nd generation cephalosporins(79.8%-91.3%), but sensitive to levofloxacin, gatifloxacin, and the4th generationcephalosporins. Carbapenems were the most active antimicrobial agents against S.marcescens. Cefoxitin-resistant strains were highly resistant to most antimicrobial agents incomparison with non-cefoxitin-resistant strains. There was significantly statistical deferenceof antimicrobial agents (P<0.05).2.2(6.5%) strains of the104S. marcescens isolates harboured a qnrB6gene，and one (3.2%)harboured a qnrS2gene, whereas no qnrA-, qnrC-or qnrD-positive isolate was detected. Theaac(6’)-Ib-cr gene was detected in four isolates.(GenBank accession numbers are JQ034317,JQ041635and JQ034318). None of the31isolates carried the qepA gene. Mutations in theQRDR of gyrA and parC genes were detected in9and7isolates, respectively.3. The conjugation experiments were successfully carried out in5isolates of6PMQRdeterminants-postive strains. The MICs of conjugants were increased evidently compared torecipient strains.4.7strains of the11gyrA or parC mutants and qnr, aac(6’)-Ib-cr-positive strains were detectedto carry β-lactamases genes,3strains of which harbouring blaSHV-5,2strains of whichharbouring blaCTX-M-14,4strains of which harbouring blaOXA-1,2strains of which harbouringblaTEM-1and1strains of which harbouring blaDHA-1.2strains of the11isolates weredetected to carry16S rRNA methylase genes,2strains of which harbouring armA. Closelinkage amongst these resistance determinants localised on the same strain plays animportant role in the prevalence of multidrug resistance (MDR) isolates. PMQRdeterminants with β-lactamases, and16S rRNA methylase genes were co-transferred. Conclusion1. S. marcescens is one of the major conditional pathogenic bacteria coursing hospitalinfections, and resistant to many kinds of antimicrobial agents in Anhui. The surveillanceof antimicrobial resistance in S. marcescens should be strengthened for purpose ofpreventing the transmission of multidrug resistant strains.2. To our knowledge, we report the first detection of qnr in S. marcescens isolates in China.Mutations in QRDRs are also important for resistance in S. marcescens.3. The plasmid-mediated AmpC β-lactamases genes were also detected in S. marcescensisolates, and co-exist with PMQR genes.4. gyrA or parC mutants and PMQR determinants along with β-lactamases, and16S RNAmethylase genes could co-exist in a single isolate, which may spread to other bacterialspecies by horizontal exchange, leading to the emergence and spread of multidrug-resistant pathogens.