Fluoroquinolone Resistance Mechanisms in an Escherichia coli Isolate, HUE1, Without Quinolone Resistance-Determining Region Mutations

ORIGINAL RESEARCH ARTICLE published: 24 May 2013 doi: 10.3389/fmicb.2013.00125 Fluoroquinolone resistance mechanisms in an Escherichia coli isolate, HUE1, without quinolone resistance-determining region mutations Toyotaka Sato 1 , Shin-ichi Yokota 2 , Ikuo Uchida 3 , Torahiko Okubo 1 , Masaru Usui 1, Masahiro Kusumoto 4 , Masato Akiba 4 , Nobuhiro Fujii 2 and Yutaka Tamura 1* 1 Laboratory of Food Microbiology and Food Safety, Department of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Japan 2 Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan 3 Dairy Hygiene Research Division, Hokkaido Research Station, National Institute of Animal Health, Sapporo, Japan 4 Bacterial and Parasitic Disease Research Division, Safety Research Team, National Institute of Animal Health, Ibaraki, Japan Edited by: Kunihiko Nishino, Osaka University, Japan Reviewed by: Axel Cloeckaert, Institut National de la Recherche Agronomique, France Junichi Yamagishi, Nihon Pharmaceutical University, Japan *Correspondence: Yutaka Tamura, Laboratory of Food Microbiology and Food Safety, Department of Health and Environmental Sciences, School of Veterinary Medicine, Rakuno Gakuen University, 582 Midorimachi-Bunkyoudai, Ebetsu 069-8501, Japan. e-mail: Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB–TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli. Keywords: AcrAB, efflux pump, Escherichia coli, fluoroquinolone resistance, oqxAB, qnrS INTRODUCTION Fluoroquinolones are widely used in the clinical treatment of various bacterial infections, such as urinary tract and blood stream infections caused by Escherichia coli. Many studies have reported the isolation of fluoroquinolone-resistant strains (Peña et al., 1995; Cizman et al., 2001; Sanchez et al., 2012). Fluoroquinolone resistance is mainly caused by point mutations in the quinolone resistance-determining regions (QRDRs) of the DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE) subunits (Yoshida et al., 1991; Conrad et al., 1996; Heisig, 1996; Breines et al., 1997). A slight decrease in susceptibility to fluoroquinolones is attributed to a single mutation in gyrA. Secondary mutations in gyrA and additional mutations in parC and/or parE are required to exceed the breakpoint of the fluoroquinolone MIC (Conrad et al., 1996; Heisig, 1996; Breines et al., 1997). Recently, we reported a fluoroquinolone-resistant E. coli isolate without QRDR mutations, named HUE1 (Sato et al., 2011). Its MICs for fluoroquinolones, such as ciprofloxacin (CIP) and levofloxacin (LVX), exceeded the breakpoints established by the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2011). HUE1 possesses two www.frontiersin.org plasmid-mediated quinolone-resistant determinants (PMQRs), viz., oqxAB and qnrS. In this bacterium, OqxAB is a plasmidencoded efflux pump; however, the gene is present on the chromosomal DNA in most Klebsiella pneumoniae and Enterobacter cloacae strains (Bin Kim et al., 2009). The presence of this pump confers resistance to several antimicrobial agents, such as olaquindox (OLA), trimethoprim (TMP), and chloramphenicol (CHL), and decreases bacterial susceptibility to fluoroquinolones (Hansen et al., 2007). QnrS, on the other hand, is a member of the pentapeptide-repeat protein family that protects DNA gyrase (and probably also topoisomerase IV) from binding to fluoroquinolones, thereby decreasing fluoroquinolone susceptibility (Jacoby, 2005). However, acquisition of these PMQRs alone results in only a low level of fluoroquinolone resistance, with MICs that do not exceed the breakpoints for fluoroquinolones (Jacoby, 2005; Hansen et al., 2007). E. coli isolates lacking QRDR mutations in gyrA and parC and showing concomitant acquisition of oqxAB and qnrS have previously been reported in China; however, these isolates did not exceed the breakpoint for CIP (Zhao et al., 2010). Our previous findings suggested that the fluoroquinolone resistance of HUE1, which lacks QRDR mutations, is associated May 2013 | Volume 4 | Article 125 | 1 Sato et al. Fluoroquinolone resistance in non-QRDR-mutated E. coli with not only with the presence of oqxAB and qnrS but also with other fluoroquinolone-resistance mechanism(s) (Sato et al., 2011). In the current study, we investigated the fluoroquinoloneresistance mechanisms of the HUE1 strain. METHODS BACTERIAL ISOLATES E. coli HUE1 had been isolated from the urinary catheter of a 77-year-old female patient at Hokkaido University Hospital (Sapporo, Japan) in 2007 (Sato et al., 2011). The somatic (O) serotype was determined by the slide agglutination test by using Escherichia coli O antisera (Denka Seiken, Tokyo, Japan), and the flagellar (H) serotype was determined using reference sera obtained from the Statens Serum Institut (Hillerød, Denmark). SUSCEPTIBILITY TESTING AND GENETIC ANALYSIS Norfloxacin (NOR) was purchased from Sigma-Aldrich (St Louis, MO). Other antibiotics were obtained as described previously (Sato et al., 2011). Susceptibility to fluoroquinolones [CIP, LVX, urifloxacin (URX), sitafloxacin (STX), and NOR], nalidixic acid (NAL), CHL, and TMP was determined by the agar plate dilution method, according to CLSI guidelines (Clinical and Laboratory Standards Institute, 2011). Phe-Arg-β-naphthylamide (PAβN; final concentration, 20 mg/L), which is an inhibitor of the resistance-nodulation-division (RND)-type efflux pump, was purchased from Sigma-Aldrich. The presence of oqxA, oqxB, and qnrS was determined by PCR (Sorensen et al., 20 (...truncated)