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Antimicrobial Agents and Chemotherapy, August 2005, p. 3325-3333, Vol. 49, No. 8
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.8.3325-3333.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania 17033
Received 2 March 2005/ Returned for modification 3 April 2005/ Accepted 10 April 2005
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0.002 to 2.0/0.06/0.5) and 128 coagulase-negative staphylococci (0.004 to 0.25/0.016/0.125). Among strains tested, 76 S. aureus strains and 51 coagulase-negative staphylococci were resistant to ciprofloxacin. DX-619 had the lowest MIC50/MIC90 values against 127 quinolone-resistant staphylococci (0.125/0.5), followed by sitafloxacin (0.5/4), moxifloxacin (2/8), gatifloxacin (4/16), levofloxacin (16/>32), and ciprofloxacin (>32/>32). Raised quinolone MICs were associated with mutations in GyrA (S84L) and single or double mutations in GrlA (S80F or Y; E84K, G, or V) in all S. aureus strains tested. A recent vancomycin-resistant S.
aureus (VRSA) strain (Hershey) was resistant to available quinolones and was inhibited by DX-619 at 0.25 µg/ml and
sitafloxacin at 1.0 µg/ml. Vancomycin (except VRSA), linezolid,
ranbezolid, tigecycline, and quinupristin-dalfopristin were active
against all strains, and teicoplanin was active against S.
aureus but less active against coagulase-negative staphylococci.
DX-619 produced resistant mutants with MICs of 1 to >32
µg/ml after <50 days of selection compared to 16 to
>32 µg/ml for ciprofloxacin, sitafloxacin,
moxifloxacin, and gatifloxacin. DX-619 and sitafloxacin were also more
active than other tested drugs against selected mutants and had the
lowest mutation frequencies in single-step resistance selection. DX-619
and sitafloxacin were bactericidal against six quinolone-resistant
(including the VRSA) and seven quinolone-susceptible strains tested,
whereas gatifloxacin, moxifloxacin, levofloxacin, and ciprofloxacin
were bactericidal against 11, 10, 7, and 5 strains at 4x MIC
after 24 h, respectively. DX-619 was also bactericidal
against one other VRSA strain, five vancomycin-intermediate S.
aureus strains, and four vancomycin-intermediate
coagulase-negative staphylococci. Linezolid, ranbezolid, and
tigecycline were bacteriostatic and quinupristin-dalfopristin,
teicoplanin, and vancomycin were bactericidal against two, eight, and
nine strains, and daptomycin and oritavancin were rapidly bactericidal
against all strains, including the VRSA. DX-619 has potent in vitro
activity against staphylococci, including methicillin-, ciprofloxacin-,
and vancomycin-resistant
strains. |
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Most methicillin-resistant staphylococci are also resistant to available quinolones, such as ciprofloxacin and levofloxacin (8, 23). Thus, the latter group of compounds may not be safely used in empirical therapy of patients with methicillin-resistant staphylococcal infections. DX-619 is a new des-F(6)-quinolone (Fig. 1) with excellent activity against gram-positive organisms. The current study examined (i) the activity of DX-619 against 259 methicillin- and quinolone-susceptible and -resistant staphylococci compared to those of sitafloxacin, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, vancomycin, teicoplanin, linezolid, ranbezolid, tigecycline, and quinupristin-dalfopristin by agar MIC testing and those of daptomycin and oritavancin by microdilution; (ii) the activities of the above-mentioned compounds against 13 staphylococci by time-kill analysis; and (iii) the capabilities of DX-619, sitafloxacin, moxifloxacin, gatifloxacin, and ciprofloxacin to select for resistance in 10 staphylococci.
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FIG. 1. Chemical structure of DX-619.
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Antibacterials and susceptibility testing. DX-619 susceptibility powder was obtained from Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan. Other antimicrobials were obtained from their respective manufacturers. For all drugs except daptomycin and oritavancin, agar dilution MICs using cation-adjusted Mueller-Hinton agar were used (18). For daptomycin (for which added calcium has been standardized only in broth) and oritavancin (which binds to agar and can be tested only in broth), microdilution was performed by standardized methodology, using cation-adjusted Mueller-Hinton broth (CAMHB; Difco) with added calcium for daptomycin testing (18). Vancomycin MICs were read after a full 24-h incubation. The antibacterial susceptibilities of VRSA strains were tested by the macrodilution method using cation-adjusted Mueller-Hinton broth (18).
Time-kill studies. Tubes containing 5 ml cation-adjusted Mueller-Hinton broth (Difco) with doubling antibiotic concentrations were inoculated with 5 x 105 to 5 x 106 CFU/ml and incubated at 35°C in a shaking water bath. The methods were those described previously by our group (21), and added calcium was used for daptomycin testing (18). The strains used for time-kill studies were tested by the double-disk test for macrolide resistance, using erythromycin (30 µg) and clindamycin (2 µg) disks.
Time-kill assays were analyzed by determining
the number of strains that yielded
log10 CFU/ml of
1, 2, and 3 at 0, 3, 6, 12, and 24
h compared to counts at time zero. Antimicrobials were considered
bactericidal at the lowest concentration that reduced the original
inoculum by
3 log10 CFU/ml (99.9%) at each of the
time periods and bacteriostatic if the inoculum was reduced by 0 to
<3 log10 CFU/ml. The problem of bacterial carryover
was addressed by dilution as described previously
(21). Six strains were
not tested with ciprofloxacin, four with levofloxacin, and one each
with gatifloxacin, moxifloxacin, and vancomycin due to resistance to
the individual antibiotics.
Multistep resistance selection. Ten staphylococcal strains, including three ciprofloxacin-susceptible (MIC < 2 µg/ml) and seven ciprofloxacin-resistant (MIC > 32 µg/ml) strains, were used in the study. Initial inocula (ca. 5 x 108 to 1 x 109 CFU/ml) were prepared by suspending growth from an overnight Trypticase soy blood agar plate (Difco) in CAMHB. Glass tubes containing 1 ml of antibiotic-free or antibiotic-supplemented CAMHB were inoculated; antibiotic concentrations in the tubes ranged from four doubling dilutions above to three doubling dilutions below the MIC of each drug for each strain. The tubes were incubated at 35°C for 24 h. Daily passages were then performed for up to 50 days by taking a 10-µl inoculum from the tube nearest the MIC (usually 1 to 2 dilutions below) which had the same turbidity as the antibiotic-free controls. From time to time, an aliquot from a tube used as an inoculum for the successive passage was frozen at 70°C in double-strength skim milk for subsequent analysis. When an MIC for a strain stabilized at >32 µg/ml (minimum amount, 14 passages) during four successive passages, serial transfer in the presence of subinhibitory concentrations of antibiotic was discontinued, and the strains were subjected to 10 passages in antibiotic-free medium to check the stability of acquired mutants. To determine whether resistant isolates obtained during and at the end of serial passages were indeed derived from the parental strains, the parental strains, intermediary clones with elevated MICs, and clones obtained after the final passage were examined by pulsed-field gel electrophoresis using a CHEF DR III apparatus (Bio-Rad, Hercules, CA) as previously described (1, 17).
Single-step resistance studies. The frequency of spontaneous single-step mutation was determined by spreading cultures (approximately 1010 CFU/ml) in a 100-µl volume of phosphate-buffered saline. Mueller-Hinton agar plates (100-mm diameter) contained each compound at 1x, 2x, 4x, and 8x MIC. The plates were incubated aerobically at 35°C for 24 h. Confluent growth at 1x MIC with initial inoculum dilutions of 101, 102, 103, and >300 colonies at 105 made it unfeasible to reliably interpret growth on plates. Additionally, quinolone MICs taken from growth on these plates were, in >90% of cases, equal to or 1 dilution above those of the parent strains. By contrast, single colonies were reproducibly found at drug concentrations above the MIC. We therefore elected to analyze results obtained at 2x, 4x, and 8x MIC only. Because colonies obtained at these higher MICs often occurred on a background of confluent growth (see above), retesting by agar dilution MIC was performed. To ensure the reproducibility and reliability of results, resistance in single-step studies was defined as a MIC >4-fold higher than that for the parent. The resistance frequency was calculated at each MIC as the number of resistant colonies per inoculum.
MICs of resistant clones. The MIC of each resistant clone to each of the quinolones in resistance selection studies was determined by broth macrodilution, as described above. The effect of reserpine (20 mg/liter dissolved in dimethyl sulfoxide) on MICs was also determined.
Determination of quinolone resistance mechanism. The PCR method was used to amplify quinolone resistance determinant regions (QRDR) in the gyrA, gyrB, grlA, and grlB genes using primers and cycling conditions described previously (7, 15). Template DNA for PCR was prepared using InstaGen Matrix, as recommended by the manufacturer (Bio-Rad Laboratories, Hercules, CA). After amplification, PCR products were purified from excess primers and nucleotides using a QIAquick PCR Purification kit (QIAGEN, Valencia, CA) and sequenced directly using the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA).
Determination of macrolide and methicillin resistance mechanisms. The presence of genes coding for macrolide resistance, ermA, ermB, ermC, and msrA, were studied by PCR as described by Sutcliffe and coworkers (27). The methicillin resistance determinant mecA gene was amplified by PCR from methicillin-resistant strains by using specific primers (20).
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TABLE 1. Summary of MICs (µg/ml) for Staphylococcus strains.
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TABLE 2. MIC
range, MIC50, and MIC90 of DX-619 compared to
those of other quinolones against quinolone-nonsusceptible
staphylococci
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The Hershey VRSA strain (VRS2) had the following MICs: DX-619, 0.25 µg/ml; sitafloxacin, 1.0 µg/ml; ciprofloxacin, >64.0 µg/ml; levofloxacin, 32.0 µg/ml; gatifloxacin, 8.0 µg/ml; moxifloxacin, 4.0 µg/ml; vancomycin, 32.0 µg/ml; teicoplanin, 4.0 µg/ml; linezolid, 1.0 µg/ml; ranbezolid, 1.0 µg/ml; tigecycline, 0.125 µg/ml; daptomycin, 0.5 µg/ml; oritavancin, 0.25 µg/ml; and quinupristin-dalfopristin, 1.0 µg/ml.
Mechanism of quinolone resistance. Seventy-six S. aureus strains resistant to ciprofloxacin were studied for the presence of mutations in quinolone resistance-determining regions in gyrA, gyrB, grlA, and grlB. The strains tested had at least one mutation in GyrA and one mutation in GrlA (Table 3). All quinolone-resistant S. aureus strains had the same mutation in GyrA: serine (S) at position 84 was replaced by leucine (L). Only four strains had a mutation in GyrB: arginine (R) at position 404 was replaced by leucine. All 76 strains had mutations in subunit A and only 18 in subunit B of topoisomerase IV. The modifications in GrlA were at position 80 by changing serine to phenylalanine (F) or tyrosine (Y) or at position 84 by changing glutamic acid (E) to lysine (K). Thirteen strains had double mutations; the mutations at position 80 were associated with a second mutation at position 84, by changing glutamic acid to glycine (G), lysine, or valine (V) (Table 3).
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TABLE 3. Mutations
detected in QRDR of 76 ciprofloxacin-resistant S. aureus
strains
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FIG. 2. (A) MIC distributions of S. aureus strains with single and double mutations in GrlA. Cipro, ciprofloxacin; Levo, levofloxacin; Moxi, moxifloxacin. (B) MIC distribution of S. aureus with double mutation in GrlA. x axis, MIC (µg/ml); y axis, no. of strains.
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Cross-resistance occurred in all clones with
acquired quinolone resistance (Table
4). The MICs of DX-619 were lowest against the resistant mutants selected
(with the exception of DX-619-resistant mutants originating from SA096
and SA077, whose DX-619 MICs were higher than those of sitafloxacin),
followed by those of sitafloxacin, moxifloxacin, gatifloxacin, and
ciprofloxacin. For moxifloxacin-, gatifloxacin-, and
ciprofloxacin-resistant clones, DX-619 MICs were
2 dilutions
lower than the corresponding sitafloxacin MICs (except for the
moxifloxacin-resistant clone of CN158 and the gatifloxacin-resistant
clone of CN111). All selected mutants had stable resistance phenotypes,
with no strains showing >1 dilution step decrease in MICs after
10 consecutive passages in antibiotic-free medium.
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TABLE 4. Results
of multistep resistance selection by DX-619 and other
comparatorsa
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We studied the QRDR nucleotide sequences of the gyrA, gyrB, grlA, and grlB genes of all mutant strains originating from S. aureus. The mutations identified are summarized in Table 4. Four out of six S. aureus parent strains were initially ciprofloxacin resistant; all had an S84L mutation in GyrA and either an S80F or an S80Y mutation in GrlA. Exposure to DX-619, sitafloxacin, moxifloxacin, and gatifloxacin in these strains resulted in various mutations in all four studied genes. In GyrA, three isolates had an E88K/V mutation, one had a G82C mutation, and one had an A119T mutation. In GrlA, the mutations were S81P (one strain) and E84V/A (two strains). Various mutations were found in GyrB: three mutants had a D437E mutation (in combination with V434S in one strain), and five mutants had a P456S mutation either alone (four of five) or with an L457F mutation (one of five). Several different mutations were found in GrlB: D432V (three mutants), S437T (one mutant), R444C (one mutant), E472V (one mutant), and E471K (two mutants).
All mutants (except a ciprofloxacin-resistant mutant of SA096) selected from two originally ciprofloxacin-susceptible strains acquired mutations in GyrA (S84L [except for the moxifloxacin-resistant mutant of strain SA505] and E88K [in the sitafloxacin-resistant mutant of SA505]) and GrlA (S80F/Y and/or E84K). The ciprofloxacin-resistant mutant of SA096 had no mutations in any of the QRDR; however, it had efflux for all studied quinolones. Additionally, three quinolone-resistant mutants of SA505 had L433I plus P456S mutations in GyrB (selected with moxifloxacin), a P451S mutation in GrlB (selected with sitafloxacin), and an S433T mutation in GrlB (selected with gatifloxacin). The DX-619-resistant mutant of SA096 had an E472G mutation in GrlB.
Single-step resistance selection studies. Frequencies of single-step mutations with DX-619, sitafloxacin, moxifloxacin, and gatifloxacin at 2x, 4x, and 8x MIC are summarized in Table 5. As can be seen, DX-619 and sitafloxacin had the lowest mutation frequencies of all quinolones at all MICs tested, with DX-619 selecting for fewer mutants than sitafloxacin.
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TABLE 5. Frequencies
of single-step resistance selection in staphylococci at different
quinolone concentrations
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TABLE 6. MICs
of strains tested by time-kills
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TABLE 7. Time-kill
analyses for 12 staphylococci and vancomycin-resistant S.
aureus (VRS2; Hershey)
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FIG. 3. Killing activities of DX-619 against vancomycin-nonsusceptible staphylococci. DX-619 was bactericidal at 2x MIC after 3 to 12 h regardless of the ciprofloxacin susceptibilities. The time-kill graphics of vancomycin-resistant S. aureus (VRS1), vancomycin-intermediate S. aureus (VISA; NRS1), and vancomycin-intermediate coagulase-negative staphylococcus (NRS8) are
shown.
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Linezolid, ranbezolid, tigecycline, and quinupristin-dalfopristin were mainly bacteriostatic after 24 h. Vancomycin, at 4x MIC, was bactericidal against nine strains after 24 h. Oritavancin and daptomycin had bactericidal effects for all strains after 12 h at 4x MIC. Daptomycin and oritavancin were both rapidly bactericidal, with activity demonstrable as early as 3 h. DX-619 and sitafloxacin were bactericidal at 2x MIC against the VRSA strain after 3 to 6 h.
The killing activities of DX-619 were also tested against the vancomycin-resistant S. aureus strain isolated in Michigan (VRS1), five vancomycin-intermediate S. aureus strains (NRS1, NRS12, NRS17, NRS56, and NRS63), and four vancomycin-intermediatecoagulase-negative staphylococci (NRS7, NRS8, NRS9, and NRS60). DX-619 had low MICs against vancomycin-nonsusceptible staphylococci regardless of their vancomycin and ciprofloxacin resistances and was bactericidal at 2x MIC after 3 to 12 h (Table 8).
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TABLE 8. Killing
activities of DX-619 against vancomycin-nonsusceptible staphylococci
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All 76 quinolone-resistant S. aureus strains had mutations in GyrA (S84L) and GrlA (S80F/Y). Thirteen strains had a second mutation in GrlA (E84G/K/V). These mutations have been shown to be the main mechanism of quinolone resistance in clinical strains of S. aureus (12, 13, 22). A few mutations affecting the gyrB and grlB genes have also been described in quinolone-resistant S. aureus (13, 25). Only 4 out of 76 ciprofloxacin-resistant strains had mutations in GyrB (R404L), and 18 strains had various mutations in GrlB; some of the latter mutations have been reported previously (25).
In the present study, we found that DX-619 produced resistant clones with MICs of 1 to >32 µg/ml after <50 days of antibiotic selection compared to 16 to >32 µg/ml with ciprofloxacin-, sitafloxacin-, moxifloxacin-, and gatifloxacin-resistant clones. Cross-resistance studies showed that DX-619 and sitafloxacin had lower MICs than ciprofloxacin, moxifloxacin, and gatifloxacin in the clones resistant to the latter group of quinolones. In addition, DX-619 and sitafloxacin also had the lowest mutation frequencies of all quinolones at all MICs tested in single-step tests. We have no explanation for the confluent growth (which on retesting did not represent resistant mutants) found at 1x MIC in single-step experiments.
All but one resistant clone obtained during multistep selection had mutations in GyrA (S84L and E88K) and GrlA (S80F/Y and E84K/V/A). Some of the strains had mutations in GyrB and GrlB; however, no correlation was observed between the presence of mutations in the last two, in addition to mutations in GyrA and GrlA, and an increase in quinolone MICs. Also, though testing with reserpine demonstrated the presence of an efflux pump in some resistant clones, it did not seem to contribute to quinolone resistance (MICs for the relevant drugs were not substantially higher than those in clones without efflux present). The only exception was the ciprofloxacin-resistant clone of SA096, which did not have any detectable mutations present in the QRDR but had efflux for all tested quinolones. The clinical significance of a strain with the latter genotype is unknown.
The MIC and time-kill results for other compounds tested against staphylococci are similar to those described previously (4-6, 8, 9, 11, 16, 24, 26): daptomycin and oritavancin had the most rapid bactericidal activities, followed by vancomycin, teicoplanin, and quinupristin-dalfopristin, with tigecycline, linezolid, and ranbezolid being mainly bacteriostatic. The bacteriostatic activity of quinupristin-dalfopristin resulted from the presence of erm genes in seven strains; in the remaining six strains, the lack of the drug's usual rapid bactericidal activity could not be explained.
The results of this study indicate a potential role for DX-619 in the treatment of staphylococcal infections. However, interpretation of these in vitro results must be complemented by toxicity and pharmacokinetic-pharmacodynamic studies before the drug can be recommended for clinical testing.
We thank NARSA and Focus Technologies, Herndon, Va., for provision of the Michigan VRSA and the vancomycin-intermediate S. aureus and VICoNS strains.
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