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_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). Among the 146 _S. marcescens_ isolates, the total resistance rates to
ciprofloxacin, levofloxacin and gatifloxacin were 27.4% (40/146), 17.1% (25/146), and 15.8% (23/146), respectively. The quinolone resistance rates for all isolates per year were shown in
Table 1. The quinolone resistance rates in our study were higher than the reports of Yang _et al._,1 which shown that the resistance rate of ciprofloxacin and levofloxacin in _S. marcescens_
collected from 15 teaching hospitals in China in 2005 was 15.0% and 7.5%, respectively. The differences may be due to the extensive use of quinolone in clinic condition in recent years in
China. PMQR determinants were present in seven (4.8%) isolates with _qnr_ and _aac(6′)-Ib-cr_ detected alone or in combination. Mutations in _gyrA_ and/or _parC_ were identified in those
seven PMQR-positive isolates simultaneously (Table 2). Those seven PMQR-positive isolates were all resistant to fluoroquinolones according to CLSI 2012. The most frequently found gene was
_aac(6′)-Ib-cr_, which was identified in 5 (3.4%) isolates. Two _aac(6′)-Ib-cr_-positive isolates carried _qnrS2_ and _qnrB6_, respectively. This agrees with the previous reports that _qnr_
alleles were coexpressed with _aac(6′)-Ib-cr_ on the same plasmid.18 _qnrS_ and _qnrB_ were identified in two isolates, respectively. However, other PMQR genes (_qnrA_, _qnrC_, _qnrD_,
_qepA_ and _oqxA_) were not found in these isolates. Kim SY _et al._19 observed significant association of _aac(6′)-Ib-cr_ with _qnrA_ and _qnrS_ in a Korean study. And most isolates with
both _aac(6′)-Ib-cr_ and _qnr_ genes showed higher levels of quinolone resistance than those with _aac(6′)-Ib-cr_ alone. Mutations in the quinolone resistance-determining regions of _gyrA_
and/or _parC_ were identified amongst the seven PMQR-positive isolates, whereas no _gyrB_ or _parE_ mutations were found. The most common mutation found in PMQR-positive isolates was at
codon 83 of _gyrA_ (TCG→TTG transition), resulting in the replacement of serine by leucine. Four isolates were found with a combination of a mutation in _parC_ at codon 80 and a mutation in
the _gyrA_ at codon 83 or codon 87. The mutations of _parC_, plus other _gyrA_ mutations, conferred a higher level of resistance to fluoroquinolones than the mutation at codon 80 of _parC_
in the study. Earlier studies have described alterations in DNA gyrase in quinolone-resistant _S. marcescens_ isolates but did not examine the topoisomerase IV genes.20, 21 Weigel LM _et
al._21 reported that the fluoroquinolone-resistant clinical isolates of _S. marcescens_ displayed the greatest diversity in mutations, including Gly-81 to Cys, Ser-83 to Ile or Arg and
Asp-87 to Asn. Interestingly, in contrast with other enterobacterial species, mutation of Ser-83 was not required for high-level fluoroquinolone resistance in _S. marcescens._ Kim JH _et
al._20 reported that the Ser-83-to-Arg substitution in GyrA protein might lead to high-level quinolone resistance in _S. marcescens_ by introducing a bulky amino-acid residue into the
protein and also by decreasing the hydrogen-bonding capacity between amino-acid residues. In our study, the mutation at codon 83 (C→T transition) in _gyrA_ has also been detected, resulting
in the replacement of serine (TCG) by leucine (TTG). Among the seven PMQR-positive isolates, plasmids from six of the isolates were successfully transferred to the recipients, suggesting
that the dissemination of the PMQR determinants is mostly due to the transmission of plasmids by horizontal exchange. An increase in the MICs of quinolones was detected in the
transconjugants compared with the recipients (Table 2). Our study described clinical isolates of _S. marcescens_ in China carrying plasmid-mediated quinolone-resistant genes (_qnrB6_,
_qnrS2_ and/or _aac(6′)-Ib-cr_) together with mutations in _gyrA_ and _parC_ genes. These mechanisms were likely to have contributed individually to the high level of ciprofloxacin,
levofloxacin and gatifloxacin resistance in _S. marcescens_. NUCLEOTIDE SEQUENCE ACCESSION NUMBERS The sequences of the _qnrB6_, _qnrS2_ and _aac(6′)-Ib-cr_ reported in this article have
been deposited in the GenBank database and assigned accession numbers JQ034317, JQ041635 and JQ034318, respectively. _gyrA_ and _parC_ genes have also been deposited in GenBank under
accession number JQ034320 and JQ235843 for _gyrA_, JQ235844 for _parC_. ACCESSION CODES ACCESSIONS GENBANK/EMBL/DDBJ * JQ034317 * JQ034318 * JQ034320 * JQ041635 * JQ235843 * JQ235844
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Enterobacteriaceae. _Antimicrob. Agents Chemother._ 42, 2661–2667 (1998). Article CAS PubMed PubMed Central Google Scholar Download references ACKNOWLEDGEMENTS We thank all the
contributing hospitals that provided isolates for this study. This work was supported by grants from the National Natural Science Foundation of China (no. 30972631and no. 81101288) and the
Natural Science Foundation of Anhui Province, China (no. 11040606Q23). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Infectious Disease, the First Affiliated Hospital of Anhui
Medical University, Hefei, China Hai-Fei Yang, Jun Cheng, Li-Fen Hu, Ying Ye & Jia-Bin Li * Institute of Bacterium Resistance, Anhui Medical University, Hefei, China Ying Ye &
Jia-Bin Li * Anhui Center for Surveillance of Bacterial Resistance, Hefei, China Ying Ye & Jia-Bin Li Authors * Hai-Fei Yang View author publications You can also search for this author
inPubMed Google Scholar * Jun Cheng View author publications You can also search for this author inPubMed Google Scholar * Li-Fen Hu View author publications You can also search for this
author inPubMed Google Scholar * Ying Ye View author publications You can also search for this author inPubMed Google Scholar * Jia-Bin Li View author publications You can also search for
this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Jia-Bin Li. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. RIGHTS AND
PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Yang, HF., Cheng, J., Hu, LF. _et al._ Detection of the plasmid-mediated quinolone resistance determinants in
clinical isolates of _Serratia marcescens_ in China. _J Antibiot_ 65, 531–533 (2012). https://doi.org/10.1038/ja.2012.63 Download citation * Received: 15 March 2012 * Revised: 24 May 2012 *
Accepted: 26 June 2012 * Published: 01 August 2012 * Issue Date: October 2012 * DOI: https://doi.org/10.1038/ja.2012.63 SHARE THIS ARTICLE Anyone you share the following link with will be
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initiative KEYWORDS * fluoroquinolones * Qnr * resistance * _Serratia marcescens_