Serratia marcescens is a prominent opportunistic pathogen responsible for serious infections in immunocompromised individuals, due primarily to its high intrinsic antibiotic resistance. Quinolones are commonly prescribed antimicrobial agents in China, and quinolone-resistant S. marcescens isolates have emerged. Yang et al.1 reported that the resistance rate of ciprofloxacin and levofloxacin in S. marcescens collected from 15 teaching hospitals located in different areas in China in 2005 was 15.0% and 7.5%, respectively. Three kinds of plasmid-mediated quinolone resistance (PMQR) determinants have been detected, conferring low-level resistance to quinolones by different mechanisms: quinolone-resistance proteins (Qnr), AAC(6′)-Ib-cr, and QepA efflux.2 Recently, oqxAB has been identified as another mechanism of PMQR.3 As the discovery of Qnr, the presence of different qnr (A, B, S, C and D) in plasmids has been found worldwide in different bacterial pathogens.4, 5 Resistance to quinolones can also be caused by mutations in the chromosomal genes that code for DNA gyrase and/or DNA topoisomerase IV.6 Fujimaki K et al.7 reported that DNA gyrase alterations are the basis of quinolone resistance in clinical isolates of S. marcescens. Watanabe M et al.8 showed that the mutation in DNA gyrase and mutations that appear to decrease drug permeation occurred frequently in quinolone-resistant isolates of S. marcescens. It has been found that the amino-acid residues most frequently mutated in GyrA from S. marcescens occur at Gly-81, Ser-83 and Asp-87. Park et al.9 examined 166 isolates of S. marcescens and reported one positive for qnrA and three for qnrB. We conducted this study to estimate the prevalence of PMQR determinants and the role of mutations in DNA gyrase and/or DNA topoisomerase IV in S. marcescens in Anhui, China.

A total of 146 nonduplicate S. marcescens isolates were collected in 34 hospitals from 2005 to 2011 in Anhui, China. Species identification was performed with the Vitek 2 system (bioMérieux, Marcy l’ Étoile, France) and confirmed with API 20E (bioMérieux). The minimum inhibitory concentrations (MICs) for ciprofloxacin, levofloxacin and gatifloxacin (Oxoid) were further determined by the agar dilution method in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI, 2012).10 All isolates were screened for the presence of qnrA, qnrB, qnrS by multiplex PCR using the primers as Robicsek et al.11 as described. Amplification products were provisionally identified from their sizes in ethidium bromide-stained agarose gels. Positive results were confirmed by amplification with primers 5′-ATGACGCCATTACTGTATAA-3′ and 5′-GATCGCAATGTGTGAAGTTT-3′ for qnrB.12 aac(6′)-Ib was amplified by PCR as Park et al.12 described. All positives were further analyzed by direct sequencing of the PCR products with primer 5′-CGTCACTCCATACATTGCAA-3′ to identify aac(6′)-Ib-cr. qnrC, qnrD, qepA, and oqxA genes were screened by using the primers as described previously.3, 13, 14, 15 The gyrA and gyrB genes of DNA gyrase and the parC and parE genes of topoisomerase IV were amplified by PCR using the primers, as previously described.16 All the purified PCR products were sequenced on an ABI PRISM3730 sequencer analyzer (Applied Biosystems, Foster City, CA, USA). The nucleotide and deduced protein sequences were analyzed with software available from the National Center for Biotechnology Information. Conjugation experiments were carried out in Luria–Bertani (LB) broth with sodium azide-resistant Escherichia coli J53 as the recipient, as previously described.17 Transconjugants were selected on LB agar plates supplemented with sodium azide (100 μg ml−1) (Sigma Chemical Co., St Louis, MO, USA) and ciprofloxacin (0.25 μg ml−1).

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