J Med Microbiol 55 (2006), 1395-1401; DOI: 10.1099/jmm.0.46636-0
© 2006 Society for General Microbiology
ISSN 1473-5644
Topoisomerase mutations and efflux are associated with fluoroquinolone resistance in Enterococcus faecalis
Yoshihiro Oyamada1,
Hideaki Ito1,
Matsuhisa Inoue2 and
Jun-ichi Yamagishi3
1 Pharmacology Research Laboratories, Dainippon Sumitomo Pharma Co. Ltd, Enoki 33-94, Osaka 564-0053, Japan
2 Department of Environmental Infectious Disease, Graduate School of Medical Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan
3 Technology Research and Development Center, Dainippon Sumitomo Pharma Co. Ltd, Ebie 1-5-51, Fukushima-ku, Osaka 553-0001, Japan
Correspondence
Hideaki Ito
hideaki-ito{at}ds-pharma.co.jp
Received 20 March 2006
Accepted 29 June 2006
To understand better the mechanisms of fluoroquinolone resistance in Enterococcus faecalis, fluoroquinolone-resistant mutants isolated from Ent. faecalis ATCC 29212 by stepwise selection with sparfloxacin (SPX) and norfloxacin (NOR) were analysed. The results showed the following. (i) In general, fluoroquinolone-resistance mechanisms in Ent. faecalis are similar to those in other Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae, namely, mutants with amino acid changes in both GyrA and ParC exhibited high fluoroquinolone resistance, and single GyrA mutants and a single ParC mutant were more resistant to SPX and NOR, respectively, than the parent strain, indicating that the primary targets of SPX and NOR in Ent. faecalis are DNA gyrase and topoisomerase IV, respectively. (ii) Alterations in GyrB (
KGA, residues 395–397) and ParE (Glu-459 to Lys) were associated with fluoroquinolone resistance in some mutants. Moreover, the facts that the NOR MIC, but not the SPX MIC, decreased in the presence of multidrug efflux pump inhibitors, that NOR accumulation decreased in the cells, and that the EmeA mRNA expression level did not change, strongly suggested that a NorA-like efflux pump, rather than EmeA, was involved in resistance to NOR.
Abbreviations: ABC, ATP-binding cassette; Acr, acriflavine; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; MDR, multidrug resistance efflux pump; MF, major facilitator; NOR, norfloxacin; QRDR, quinolone resistance-determining region; SPX, sparfloxacin.
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INTRODUCTION
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Enterococcus faecalis is one of the nosocomial pathogens capable of causing infections in various body systems, such as the gastrointestinal tract, the skin and skin structures, the urinary tract, the blood stream, and the heart. As multiresistance to antibacterial agents commonly used to treat enterococcal infections, including aminoglycosides, penicillins and glycopeptides, has increased, the choice of effective therapies for these infections is limited. In addition, the number of fluoroquinolone-resistant clinical isolates of Ent. faecalis has been increasing with the increased use of fluoroquinolones.
In Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae, fluoroquinolone resistance is essentially mediated by alteration of target enzymes such as DNA gyrase (a heterotetramer in the form A2B2 encoded by the gyrA and gyrB genes) and topoisomerase IV (a heterotetramer in the form C2E2 encoded by the parC and parE genes), and/or decreased intracellular accumulation (Piddock, 1999; Ruiz, 2003). Point mutations involved in fluoroquinolone resistance have been shown to occur in defined regions in the gyrA and gyrB genes, termed quinolone resistance-determining regions (QRDRs), and those in the parC and parE genes of topoisomerase IV have been reported to occur in similar regions (Piddock, 1999). Interestingly, the more sensitive enzyme to fluoroquinolones (namely the primary target) determines the susceptibility of Gram-positive organisms (Yamagishi et al., 1996), and the primary target differs depending on the structure of fluoroquinolones (Pan & Fisher, 1997; Yamagishi et al., 1996). On the other hand, the major cause of decreased accumulation of fluoroquinolones in bacterial cells is efflux of these agents. In the case of Staph. aureus, the efflux pumps involved in fluoroquinolone resistance are NorA, NorB and NorC, which belong to the major facilitator (MF) superfamily, and MepA, a member of the multidrug and toxin extrusion (MATE) family (Kaatz et al., 2005; Truong-Bolduc et al., 2005, 2006; Yoshida et al., 1990b). However, in the case of Strep. pneumoniae, the pump proposed to be involved in fluoroquinolone resistance is PmrA, a NorA homologue (Gill et al., 1999), although other transporters have been implicated (Piddock et al., 2002). Besides, it has recently been reported that an ATP-binding cassette (ABC) transporter is associated with ciprofloxacin (CIP) and norfloxacin (NOR) resistance in Strep. pneumoniae (Marrer et al., 2006; Robertson et al., 2005).
It has already been demonstrated that fluoroquinolone resistance in Ent. faecalis, as well as in other Gram-positive organisms, is associated with alterations of GyrA and/or ParC (Kanematsu et al., 1998; Korten et al., 1994; Onodera et al., 2002; Tankovic et al., 1996, 1999). However, no report has investigated the association between point mutations in the other subunit genes of DNA gyrase and topoisomerase IV (i.e. gyrB and parE) and fluoroquinolone resistance in Ent. faecalis. Although the efflux pump EmeA, which is a NorA homologue, has been shown to be associated with Ent. faecalis resistance to NOR, a hydrophilic fluoroquinolone (Jonas et al., 2001), there has been no direct evidence that EmeA contributes to fluoroquinolone resistance in Ent. faecalis resistant strains.
In order to understand better the mechanisms of fluoroquinolone resistance in Ent. faecalis, we isolated, in this study, spontaneous fluoroquinolone-resistant mutants from Ent. faecalis ATCC 29212 by stepwise selection with sparfloxacin (SPX), a hydrophobic fluoroquinolone, and NOR, a hydrophilic fluoroquinolone, and characterized the mutants by analysing point mutations in QRDRs of gyrB and parE, in addition to those of gyrA and parC. We also examined whether EmeA was involved in the fluoroquinolone resistance of the isolated mutants.
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METHODS
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Bacterial strains and culture conditions.
Ent. faecalis ATCC 29212 was obtained from the American Type Culture Collection (ATCC). All Ent. faecalis strains were grown aerobically at 37 °C in brain heart infusion broth (Difco Laboratories).
Chemicals and antibacterial agents.
SPX and NOR were synthesized at our Chemistry Research Laboratories. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), acriflavine (Acr), verapamil, lansoprazole and reserpine were purchased from Sigma-Aldrich. Other reagents were purchased from Nacalai Tesque, unless otherwise indicated.
Determination of MIC.
MICs were determined by the two-fold agar dilution method with brain heart infusion agar, as recommended by Clinical Laboratory Standards Institute, formerly National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, 2003).
Selection of fluoroquinolone-resistant mutants.
Approximately 1010 c.f.u. of Ent. faecalis ATCC 29212 was spread onto brain heart infusion agar containing SPX at a concentration two or four times MIC, or NOR at a concentration two or four times MIC, and incubated aerobically at 37 °C for 48 h. The second- and third-step mutants were selected in a similar manner to the first-step mutants. All mutants were derived by selection using a single bacterial culture. As mutants were isolated by stepwise selection, resistance was defined when a mutant had twofold or greater decreased susceptibility to the quinolone tested compared with the parent strain, Ent. faecalis ATCC 29212. In addition, high-level resistance was defined as MIC for SPX 
8 µg ml–1 and MIC for NOR 64 µg ml–1, and intermediate resistance was defined as 1 µg ml–1
MIC for SPX<8 µg ml–1, and 8 µg ml–1MIC for NOR<64 µg ml–1.
PCR amplification and DNA sequence analysis.
Ent. faecalis total DNA was prepared as described by Hudson & Curtiss (1990). DNA fragments containing the regions around the QRDRs of the gyrA, gyrB, parC and parE genes were amplified by PCR with the primers shown in Table 1
. PCR was performed with a GeneAmp PCR System 9600 using Takara Ex Taq polymerase (Takara Shuzo). The reactions were repeated for 30 cycles (15 s at 94 °C for denaturation, 30 s at 55 °C for annealing, and 1 min at 72 °C for polymerization). PCR-amplified fragments were sequenced by the cycle sequencing method using an ABI Prism Big Dye terminator version 3.1 cycle sequencing kit, and then applied to a Genetic analyser 3100 (Applied Biosystems).
Accumulation assays.
Accumulation assays were carried out essentially as described by Celesk & Robillard (1989). The amount of accumulated fluoroquinolones was measured by an agar well diffusion bioassay with Escherichia coli KP as assay organism (Gonzalez et al., 1985).
Quantification of EmeA mRNA.
Total RNA was isolated from Ent. faecalis cells in exponential phase using the RNeasy Mini kit (Qiagen) according to the manufacturer's recommended protocol. cDNA was synthesized from total RNA and random hexamers using the SuperScript III first-strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer's instructions. Real-time RT-PCR was carried out in an ABI Prism 7900HT sequence detection system with SYBR Green PCR Master Mix (Applied Biosystems) (2 min at 50 °C, 10 min at 90 °C, and 40 cycles for 15 s at 95 °C and 1 min at 60 °C). The sequences of primers were as follows: for EmeA, 5'-AGCCCAAGCGAAAAGCGGTTT-3' and 5'-CCATCGCTTTCGGACGTTCA-3'; for GyrB as an internal standard, 5'-TAGCAACTTGCCAGGGAAGC-3' and 5'-TGGAATTCACGGCTACGTCC-3'. Final quantification was performed by comparison with the internal standard.
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RESULTS AND DISCUSSION
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Isolation of fluoroquinolone-resistant mutants of Ent. faecalis
In order to examine the mechanisms of fluoroquinolone resistance in Ent. faecalis in detail, a variety of spontaneous fluoroquinolone-resistant mutants were isolated from Ent. faecalis ATCC 29212 by stepwise selection with SPX, a hydrophobic fluoroquinolone, and NOR, a hydrophilic fluoroquinolone (Fig. 1). When 2x MIC of SPX and NOR was used to isolate the first-step mutants from Ent. faecalis ATCC 29212, a large number of colonies were obtained, and some did not become resistant, indicating that 2xMIC was too low to isolate the mutants. Hence, 4x MIC of the fluoroquinolones was used for isolation of the first-step mutants. As MICs of SPX, NOR and Acr, a major substrate of NorA, for the first-step mutant 1NOR1 were the same as those for the first-step mutant 1SPX1, and both strains had no amino acid change in either GyrA or ParC, 1SPX1 was used for isolation of the second-step mutants. Selection of the second-step mutants from the first-step mutant 1SPX1 with 4x MIC of the fluoroquinolones failed. Consequently, the second-step mutants were isolated by selection with 2xMIC of SPX and NOR. It is unclear why the second-step mutants could be selected with 2xMIC of the fluoroquinolones, while 4x MIC was needed in the first-step selection. Compared with the frequency of the first-step mutants (2.7x10–7 with SPX and 6.5x10–7 with NOR), the frequency of the second-step mutants was low (6.4x10–9 with SPX and 3.2x10–8 with NOR), regardless of the agent used for isolation. From these findings, it is believed that the gene mutation rate in the first-step mutant 1SPX1 might have slackened. Since, by selection with SPX, the second-step mutant 2S-SPX2 was much more frequently isolated than the second-step mutant 2S-SPX1, 2S-SPX2 was used for isolation of the third-step mutants. At the third selection step, 2xMIC was generally too low to isolate the mutants, and the mutant frequency was higher than at the second selection step.
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Fig. 1. Relationships among Ent. faecalis ATCC 29212 and its fluoroquinolone-resistant mutants. First-, second- and third-step mutants (within boxes) are designated by the prefixes 1, 2 and 3, respectively. The numbers and powers above the boxes show the concentrations of quinolones (µg ml–1) used in each selection and the mutant frequency, respectively.
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Mechanism of fluoroquinolone resistance in Ent. faecalis
At each selection step, MICs of SPX and NOR for a randomly selected 30–100 isolates were determined, and five mutants selected from the isolates that showed a similar MIC pattern were subjected to QRDR analysis. As the selected five mutants had the same amino acid change(s) in QRDR(s) of GyrA and/or ParC in all cases, one of the mutants was selected as a representative strain.
The fluoroquinolone-resistance mechanisms involving GyrA and ParC in Ent. faecalis were generally similar to those described in previous reports and those in other Gram-positive bacteria, such as Staph. aureus and Strep. pneumoniae, in the following respects. (i) The amino acid changes (Ser-83 to Arg and Glu-87 to Gly in GyrA, and Ser-80 to Ile and Ser-80 to Arg in ParC) detected in this study, except for the amino acid change Gly-105 to Asp in GyrA, have already been found in fluoroquinolone-resistant Ent. faecalis and Enterococcus faecium (Brisse et al., 1999; Kanematsu et al., 1998; Korten et al., 1994; Leavis et al., 2006; Onodera et al., 2002; Oyamada et al., 2006; Tankovic et al., 1996, 1999). In addition, equivalent amino acid changes have been detected in fluoroquinolone-resistant strains of Staph. aureus and Strep. pneumoniae (Piddock, 1999). (ii) The single amino acid change detected in 2S-NOR1, 2S-SPX1 and 2S-SPX2 conferred low or intermediate fluoroquinolone resistance, and the mutants 3SN-SPX2, 3SN-SPX3, 3SS-NOR1 and 3SS-SPX1, which had amino acid changes in both GyrA and ParC, exhibited high-level fluoroquinolone resistance (Brisse et al., 1999; Kanematsu et al., 1998; Onodera et al., 2002; Oyamada et al., 2006; Piddock, 1999). (iii) Mutants with an amino acid change in GyrA (2S-SPX1 and 2S-SPX2) and those with an amino acid change in ParC (2S-NOR1) were more resistant to SPX and NOR, respectively, than their parent strain 1SPX1, indicating that the primary targets of SPX and NOR are DNA gyrase and topoisomerase IV, respectively, that the primary target in Ent. faecalis differs depending on the structure of the fluoroquinolone (Pan & Fisher, 1997; Yamagishi et al., 1996), and that the more sensitive type II topoisomerase (i.e. DNA gyrase or topoisomerase IV) determines the susceptibility of micro-organisms to fluoroquinolones (Yamagishi et al., 1996). Hence, GyrA and ParC double mutants (3SN-SPX2, 3SN-SPX3, 3SS-NOR1 and 3SS-SPX1) were resistant to both SPX and NOR.
The amino acid change Gly-105 to Asp in GyrA detected in this study is novel. Although no equivalent amino acid change has been reported, Yoshida et al. (1990a) have shown that the amino acid change Gln-106 to His in GyrA of E. coli confers low-level fluoroquinolone resistance, and that Gly-105 in Ent. faecalis GyrA is equivalent to Gly-105, which is located next to Gln-106 in E. coli GyrA. These findings suggest that the amino acid change Gly-105 to Asp in GyrA is involved in fluoroquinolone resistance in strain 2S-SPX1 (namely Ent. faecalis).
Among the mutants isolated in this study, first-step mutant 1SPX1 had fourfold decreased susceptibility to both SPX and NOR compared with its parent strain, Ent. faecalis ATCC 29212; however, 1SPX1 had no amino acid substitution in GyrA or ParC. Third-step mutants 3SN-NOR1, 3SN-SPX1 and 3SS-NOR2, which had an amino acid change in either GyrA or ParC, exhibited high-level fluoroquinolone resistance to SPX and/or NOR. Moreover, the strains 3SN-NOR1 and 3SS-NOR2, which had the same amino acid substitution in the QRDR of GyrA or ParC as their parent strains 2S-NOR1 and 2S-SPX2, respectively, showed greater than fourfold and eightfold decreased susceptibility to NOR compared with their parent strains, respectively. In addition, the strain 3SN-NOR1 exhibited fourfold decreased susceptibility to Acr, a substrate of a multidrug resistance efflux pump (MDR). The strain 3SN-SPX1, which also had no additional amino acid change, was four times more resistant to SPX, and tended to be more susceptible to NOR and Acr than its parent strain 2S-NOR1. Hence, the strains described above were further examined.
Alterations in GyrB and ParE and their association with fluoroquinolone resistance in Ent. faecalis
It has already been demonstrated that alterations in GyrB and ParE are associated with fluoroquinolone resistance in Gram-positive bacteria such as Staph. aureus, Strep. pneumoniae and Ent. faecium (Oyamada et al., 2006), but not in Ent. faecalis. Hence, we sequenced the regions around the QRDR of the gyrB and parE genes in order to examine the mechanisms of fluoroquinolone resistance in 1SPX1, 3SN-NOR1, 3SN-SPX1 and 3SS-NOR2 (Table 2). Strain 3SS-NOR2 had an additional amino acid substitution (Glu-459 to Lys) in ParE, which did not occur in the EGDSA and PLRGK motifs of ParE, commonly implicated in resistance and identified as the ParE QRDR. The mutation occurred in the C terminal of the PLRGK motif. Previous studies of fluoroquinolone-resistance mechanisms have identified a Ser-463 to Lys substitution in GyrB in Salmonella (Gensberg et al., 1995), and a Glu-474 to Lys substitution in GyrB in Strep. pneumoniae (Pan & Fisher, 1998), located at positions corresponding to the position which was altered in ParE of strain 3SS-NOR2. Moreover, an Asn-470 to Asp mutation in the C terminal of the PLRGK motif has been described for Staph. aureus ParE (Fournier & Hooper, 1998). Taken together, these findings strongly suggest that the amino acid change Glu-459 to Lys in ParE is involved in fluoroquinolone resistance in Ent. faecalis. Furthermore, strain 3SS-NOR2, having an additional amino acid change in ParE, was eight times more resistant to NOR than its parent strain 2S-SPX2, with no effect of SPX MIC. These results support the idea that the primary target of NOR is topoisomerase IV. Surprisingly, strain 3SN-SPX1 acquired a 9 bp in-frame deletion in gyrB, resulting in loss of residues KGA at positions 395–397 in GyrB. Pan et al. (2002) have reported that a fluoroquinolone-resistant mutant of Staph. aureus selected with SPX has a gyrB deletion (RKSAL, residues 405–409) that affects a trypsin-sensitive region connecting ATPase- and GyrA-interacting domains of Staph. aureus GyrB. As the alignment of Staph. aureus GyrB and Ent. faecalis GyrB showed that the deleted region of Staph. aureus GyrB was equivalent to Ent. faecalis GyrB residues 394–398 (data not shown), the loss of residues KGA at positions 395–397 in GyrB was considered to be associated with fluoroquinolone resistance in Ent. faecalis. It is interesting that the deletions found in fluoroquinolone-resistant Staph. aureus and Ent. faecalis occurred when selection of mutants was achieved with SPX, and that the mutants carrying the deletions exhibited a fourfold increased resistance to SPX at its MIC, with little effect of NOR at its MIC compared with their parent strains. These findings demonstrate that the molecular interaction between GyrB and a given fluoroquinolone differs depending on the physico-chemical properties and chemical structure of the compound. Unfortunately, no mutation in QRDRs of the gyrB and parE genes from strains 1SPX1 and 3SN-NOR1 was detected.
Involvement of an efflux pump in the development of resistance of Ent. faecalis to fluoroquinolones
Thus far, the mechanisms of acquisition of fluoroquinolone resistance in Ent. faecalis cannot be explained by QRDR mutations only. It is well known that MDRs such as NorA and PmrA pump out the hydrophilic fluoroquinolones CIP and NOR from bacterial cells, and are involved in the development of resistance to fluoroquinolones in Staph. aureus and Strep. pneumoniae (Gill et al., 1999; Yoshida et al., 1990b). In addition, the possibility that two multidrug efflux pumps contribute to fluoroquinolone resistance in Ent. faecalis has been reported. One of these pumps is EmeA, a NorA homologue and a member of the MF superfamily (Jonas et al., 2001), and the other is EfrAB, an ABC transporter (Lee et al., 2003). Therefore, we examined the susceptibility of 1SPX1, 3SN-NOR1, 3SN-SPX1 and 3SS-NOR2 to SPX, NOR and Acr, with or without MDR inhibitors. Three MDR inhibitors were used as follows: reserpine (a competitive pump blocker), verapamil (a calcium-channel blocker and a potent inhibitor of ATP-dependent transporters; Endicott & Ling, 1989), and lansoprazole (a H+ and K+ ATPase pump inhibitor). The susceptibility of 1SPX1, 3SN-SPX1 and 3SS-NOR2 to SPX, NOR and Acr did not change, or was only marginally changed, by MDR inhibitors (Table 2
, or data not shown), strongly suggesting that no efflux pump is involved in fluoroquinolone resistance in these strains. Although strain 1SPX1 was four times more resistant to SPX and NOR than its parent ATCC 29212, it had no mutation in the regions around the QRDRs of the gyrA, gyrB, parC and parE genes, and an efflux pump did not seem to be involved in its resistance. In addition, the parent ATCC 29212 did not have any plasmids. Therefore, it is suggested that another mechanism(s), rather than qnr (Tran & Jacoby, 2002) or modified acetyltransferase (Robicsek et al., 2006), contributes to fluoroquinolone resistance in 1SPX1. In the case of strain 3SN-NOR1, which compared to its parent 2S-NOR1 exhibited a fourfold or more increase in resistance to NOR and Acr at their MICs and was not affected by SPX at its MIC, the susceptibility to NOR and Acr increased twofold or more in the presence of reserpine, verapamil and lansoprazole (Table 2, data not shown). However, the susceptibility of strain 3SN-NOR1 to SPX did not change. The susceptibility changes observed for 3SN-NOR1 in the presence of MDR inhibitors were very similar to those observed for Staph. aureus RN4220 and its NorA overexpressed derivative in the presence of the same inhibitors (Table 2, data not shown). Indeed, NOR accumulation in 3SN-NOR1 was about 30 % that in its parent 2S-NOR1 or in the wild-type strain ATCC 29212. When the protonophore CCCP was added, the intracellular concentration of NOR in 3SN-NOR1 became almost the same as that in 2S-NOR1 or ATCC 29212 (Fig. 2). In contrast, SPX accumulation was similar in 3SN-NOR1 and its parent 2S-NOR1 (data not shown). These results are highly consistent with those of previous reports showing that NorA mediates fluoroquinolone efflux in Staph. aureus (Ng et al., 1994; Yoshida et al., 1990b). Taken together, these results strongly suggest that a NorA-like efflux pump is involved in NOR resistance in strain 3SN-NOR1. Hence, EmeA mRNAs from the strains ATCC 29212, 2S-NOR1 and 3SN-NOR1 were measured by real-time RT-PCR and compared. Unexpectedly, expression levels of mRNAs in the three strains were almost identical (data not shown), meaning that EmeA was not overexpressed in 3SN-NOR1. It did not seem that EmeA in 3SN-NOR1 was expressed inducibly, because non-treated 3SN-NOR1 was used in the accumulation assay and decreased NOR in 3SN-NOR1 was detected. Using a bioinformatic approach, Davis et al. (2001) have demonstrated that Ent. faecalis has 34 potential mulitdrug efflux pump genes, and that nine of them, including emeA and a bmr homologue, belong to the MF superfamily. Thus, it is suggested that another pump of the MF superfamily, rather than EmeA, is involved in fluoroquinolone resistance in 3SN-NOR1. However, to determine which pump in the MF superfamily is associated with fluoroquinolone resistance in Ent. faecalis, further studies are needed.
We have recently reported the mechanisms of resistance to fluoroquinolones in Ent. faecium (Oyamada et al., 2006). Comparing the results of that study to those found here, Ent. faecalis and Ent. faecium appear to resist fluoroquinolones via similar mechanisms. However, there are some differences, including that no ParE mutants are found in Ent. faecium and that an unidentified efflux pump, which pumps out both NOR and SPX, contributes to fluoroquinolone resistance in Ent. faecium. It has been reported that an ABC transporter(s) is involved in fluoroquinolone resistance in Strep. pneumoniae, which is phylogenetically close to the enterococci (Marrer et al., 2006; Robertson et al., 2005). In addition, Davis et al. (2001) have shown that Ent. faecalis possesses 23 ABC transporter homologues, and that one of these transporters confers intrinsic resistance to ofloxacin. It may be that an ABC transporter plays an important role in the development of fluoroquinolone resistance in enterococci.
In summary, we examined in this study fluoroquinolone-resistant Ent. faecalis strains with mechanisms of resistance that cannot be explained by alterations in GyrA and/or ParC. Our results demonstrate that alterations in GyrB (KGA, residues 395–397), ParE (an amino acid substitution, Glu-459 to Lys), and a NorA-like efflux pump which belongs to the MF superfamily, rather than EmeA, are involved in fluoroquinolone resistance in Ent. faecalis.
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ACKNOWLEDGEMENTS
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We thank Hiroaki Yoshida for his critical reading of the manuscript and useful discussion.
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TOP
INTRODUCTION
METHODS
, Strain 3SN-NOR1; 
, the parent strain 2SN-NOR1; 
, wild-type strain ATCC 29212. CCCP was added to a final concentration of 0.1 mM at the time indicated by the arrow. Solid lines, without CCCP; dotted lines, with CCCP.