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Antimicrobial Agents and Chemotherapy, February 2006, p. 796-798, Vol. 50, No. 2
0066-4804/06/$08.00+0 doi:10.1128/AAC.50.2.796-798.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Omaha, Nebraska
Received 22 September 2005/ Returned for modification 22 November 2005/ Accepted 29 November 2005
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ABSTRACT |
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Quinolone resistance in S. pneumoniae is mediated by amino acid substitutions within the quinolone resistance determining regions (QRDRs) of DNA gyrase (GyrA or GyrB) and/or topoisomerase IV (ParC or ParE), sometimes in combination with efflux (2, 3). In general, the selection of high-level resistance involves a stepwise process with a mutational event in the primary target (either gyrA or parC), followed by additional mutations in secondary targets (6, 11). In vitro evidence suggests that the selection of a GyrA mutation coupled with a ParC change is often sufficient to increase the MICs of newer quinolones beyond the susceptible breakpoint. Bast et al. (1) observed that among 26 clinical isolates harboring a GyrA mutation and a ParC mutation, MICs of ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin were elevated and ranged from 4 to 64 µg/ml, 4 to 32 µg/ml, 2 to 8 µg/ml, and 1 to 8 µg/ml, respectively. Therefore, the introduction of more potent agents into clinical practice may be necessary if S. pneumoniae strains with multiple QRDR mutations begin to emerge.
DX-619 is a novel des-fluoro(6) quinolone with potent anti-gram-positive activity (5). To evaluate the potential of DX-619 as an antipneumococcal agent, a study was designed to compare the in vitro activities of DX-619, ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin against a panel of characterized mutants possessing single and multiple amino acid substitutions within the QRDRs. This panel consisted of selected mutants from a laboratory collection and was designed to provide detailed information about how the activities of DX-619 and the comparator quinolones are affected by a combination of various QRDR changes in S. pneumoniae.
The panel was selected from four parental strains of S. pneumoniae: two penicillin- and quinolone-susceptible strains, R6 and CP1000 (CP), and two penicillin-resistant, quinolone-susceptible clinical isolates, SP334 and SP335. The latter strains possess wild-type QRDRs and were kindly donated by P. Appelbaum, Pennsylvania State University, Milton S. Hershey Medical Center, Hershey, PA. Mutants were selected as described elsewhere (submitted for publication). Briefly, to select mutants, the parental strains were exposed to inhibitory concentrations of various quinolones in brain heart infusion agar (Becton Dickinson, Sparks, MD) supplemented with 10% sheep blood (Colorado Serum Company, Denver, CO). After 48 to 72 h of incubation at 37°C in 5% CO2, mutational decreases in susceptibility to the quinolones were determined. Representative mutants generated after one exposure to the quinolones were chosen, and the selection procedure was repeated to produce second- and third-step mutants.
To characterize the QRDRs of panel mutants, the nucleotide sequences of gyrA, gyrB, parC, and parE were amplified by PCR as described elsewhere (submitted for publication) and compared to the corresponding sequences of the parental strains. PCR amplifications were conducted in a total volume of 50 µl containing 0.25 mM of each deoxynucleoside triphosphate, 2 mM MgCl2, 0.5 µM of each primer (see the supplemental material), 2 units of a Taq Plus Precision polymerase mixture (Stratagene, La Jolla, CA) and 2 µl of template. PCRs were performed for 25 cycles with the following parameters: 95°C for 30 s, 50°C for 15 s, and 72°C for 2 min. PCR products were generated at least two separate times and sequenced directly by automated PCR cycle sequencing with dye-terminator chemistry using a DNA stretch sequencer (Applied Biosystems, Foster City, CA).
The panel comprised 20 mutants: three with a single QRDR change, seven with both a GyrA and a ParC change, one with a GyrB and a ParC change, and five with a total of three QRDR changes in GyrA and ParC. Four mutants did not have QRDR mutations despite reductions in susceptibility to some quinolones of 4- to 16-fold. To further characterize these mutants, semiquantitative reverse transcription-PCR was used to investigate the potential involvement of the quinolone efflux pump, PmrA. However, no differences in the expression of pmrA were observed between these mutants and their respective parental strains (data not shown). This suggests that a novel mechanism of resistance is responsible for reducing the susceptibility to quinolones in these strains.
MICs were determined with the agar dilution method according to CLSI (formerly NCCLS) (12) methodology, using 104 CFU per spot, on Mueller-Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, England) plates supplemented with 5% sheep blood and doubling dilutions of DX-619, moxifloxacin, ciprofloxacin, levofloxacin, and gatifloxacin. Plates were inoculated with a Steers replicator and incubated in 5% CO2 at 37°C overnight.
As shown in Table 1, DX-619 was the most potent quinolone against the mutants, inhibiting all mutants at 0.5 µg/ml. In comparison, 32 µg/ml of gatifloxacin and moxifloxacin, 128 µg/ml of levofloxacin, and 512 µg/ml of ciprofloxacin were required to inhibit all mutants. Using concentrations at which 90% of the bacteria tested were inhibited as the basis for comparison, DX-619 was 64- to 1,024-fold more potent than the comparator quinolones.
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In 15 of the 20 mutants, the smallest quinolone MIC increases occurred with DX-619, indicating that it was the quinolone least affected by the variety of mutations and combinations of mutations of the panel. In contrast, the largest MIC increases occurred most frequently with ciprofloxacin (12 of the 20 mutants).
The enhanced potency of DX-619 in comparison to the other quinolones was particularly noticeable against mutants with three QRDR alterations. While DX-619 was able to inhibit all mutants within this group at a concentration of 0.5 µg/ml, concentrations of 4 to 32 µg/ml, 8 to 32 µg/ml, 32 to 128 µg/ml, and 32 to 128 µg/ml for moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin were required for inhibition with these comparator agents. Therefore, these results indicated that DX-619 retained activity against strains that have already accumulated multiple QRDR alterations. These data suggest that it may have considerable potential for the treatment of pneumococcal respiratory tract infections, including those with reduced susceptibility to the comparator quinolones.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Supplemental material for this article may be found at http://aac.asm.org/.
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