Abstract
We analyzed the frequencies of selection, the order of acquisition, and the mutations selected on moxifloxacin in two wild-type pneumococcal strains, R6 and 5714. The first selection step showed either a single GyrA mutation or no mutation in any of the quinolone resistance-determining regions. Second-step mutants selected had either a second mutation in ParC or in ParE. Moxifloxacin could belong to these fluoroquinolones, which preferentially target GyrA though probably acting equally through both gyrase and topoisomerase IV.
Moxifloxacin (MXF) is an 8-methoxyquinolone very active against penicillin-susceptible as well as penicillin-resistant Streptococcus pneumoniae (2, 14), with MICs at which 90% of the isolates tested are inhibited ranging from 0.12 to 0.25 μg/ml. If resistance of S. pneumoniae to fluoroquinolones (FQs) appears still very low (3, 4, 14), in the clinical setting resistant mutants have nevertheless been described that harbor different mutations in topoisomerase IV and gyrase either alone or associated (3, 12-15). Several studies have established that FQs can be grouped in at least two main classes (7, 9,10). The first class, including levofloxacin, pefloxacin, ciprofloxacin, and trovafloxacin, will select quinolone resistance-determining region (QRDR) mutations first in topoisomerase IV and then in gyrase. The second group, including sparfloxacin, gemifloxacin, gatifloxacin, clinafloxacin, and BMS-284756, will select mutations first in gyrase and then in topoisomerase IV. No FQ able to target either topoisomerase IV or gyrase at the first selection step in S. pneumoniae has been described so far, but that fact does not exclude that both enzymes may simultaneously contribute to the activity of a defined FQ (9, 10, 18). In a previous study (22) it was shown, using isogenic transformants harboring different mutations in the QRDR of the ParC subunit of the topoisomerase IV or of the GyrA subunit of the gyrase, that a similar low level of resistance was obtained for MXF, although this did not indicate if a preferential target existed. In this study, using a stepwise selection method, we demonstrated in the presence of MXF the order of acquisition and the type of mutations in the QRDR of the topoisomerase genes for two strains of S. pneumoniae.
S. pneumoniae R6 is a susceptible derivative of the encapsulated Rockefeller University strain R36A; 5714 is an FQ-susceptible clinical isolate of S. pneumoniae (11). For the first selection step, 109 to 1010 cells were plated onto Mueller-Hinton agar supplemented with 5% horse blood containing various concentrations of MXF or levofloxacin and were incubated aerobically for 48 to 72 h. Colonies were restreaked on plates containing the same drug concentration. The next selection step was done in the same manner using one of the mutants selected during the first step. Frequencies of resistant mutants were calculated from the ratio of the count of CFU obtained on drug-containing plates to the count of CFU obtained on drug-free plates. MICs were determined in triplicate on Mueller-Hinton agar plates supplemented with 5% horse blood using a Steers replicator device with an inoculum of 104 to 105 CFU per spot. MICs were read after 18 h at 37°C. MICs were also determined in the presence of reserpine (10 μg/ml) (Sigma, St. Quentin-Fallavier, France) in order to detect the increased active efflux mechanism of resistance (8). Chromosomal DNA extractions and PCR experiments were done as previously described for the amplification of the entire genes of GyrA, GyrB, ParC, and ParE and of the regions encompassing their QRDRs (11, 12), except for the QRDR of ParE, where the primers PNC15 (5′-CCAATCTAAGAATCCTGCTA-3′) and PNC16 (11) were used. Direct sequencing was performed using the dRhodamine BigDye Terminator sequencing kit (Perkin-Elmer, Applied Biosystems Division) with the oligonucleotides used for amplification.
Two strategies were used for the selection of stepwise resistant mutants. Two stepwise resistant mutants were selected first by two rounds of selection on MXF. Since only GyrA but no ParC mutants were selected at the first step (Table 1), we were interested in knowing what would be the frequency of selection by MXF of the second-step mutants if ParC mutants were selected first. Thus, in a second set of experiments we successively used levofloxacin known to select ParC mutants (6, 20) and then MXF as selectors. Frequencies of selection of first-step and second-step mutants did not differ significantly, whatever the FQ used as first selector, ranging from 10−7 to 10−9 (Table 2). Selection occurred only at two or four times the MIC for the wild-type strain considered. Interestingly, frequencies of selection of first- or second-step mutants were always higher (4- to 20-fold) for R6 than for 5714. This apparent strain-to-strain dependence was observed with six other clinical strains (Table 2). A 24-fold difference in the range of selection frequencies was observed (from 1.3 × 10−7 to 5.5 × 10−9), demonstrating a strain-to-strain dependence. Compared to frequencies for gatifloxacin, another 8-methoxy-FQ for which frequencies of selection of first-step mutants were reported to be not less than 3.7 × 10−9 (6, 7), frequencies of selection by MXF appeared generally higher. Since only one strain (6, 7) was tested with gatifloxacin, we cannot exclude that this favorable number might relate to the one strain studied.
TABLE 1.
MICs and amino acid substitutions detected in the QRDRs of the different first (Mx)- and second (MxMx)-step mutants selected on MXF
Strain (n)a | MIC (μg/ml) of:
|
Mutation(s) in QRDR ofc
|
||||||||
---|---|---|---|---|---|---|---|---|---|---|
MXF | SPXb | OFXb | NORb | LVXb | CIPb | PEFb | GyrA | ParC | ParE | |
R6 | 0.125 | 0.25 | 1 | 4 | 0.5 | 1 | 8 | —d | — | — |
R6-derived 1st-step mutants | ||||||||||
R6Mx1 to −7 (7) | 0.25 | 0.5 | 1-2 | 4-8 | 0.5-1 | 1-2 | 8-16 | — | — | — |
R6Mx8 (1) | 0.5 | 0.5 | 2 | 4 | 1 | 1 | 8 | S81A | — | — |
R6Mx9 (1) | 0.5 | 1 | 2 | 4 | 1 | 2 | 8 | S81F | — | — |
R6Mx10 to −12 (3) | 0.5 | 1 | 2 | 4-8 | 1 | 2 | 8-16 | S81F | — | — |
R6Mx9-derived 2nd-step mutants | ||||||||||
R6Mx9Mx1 (1) | 4 | 8 | 32 | 128 | 16 | 32 | 128 | S81F | S79Y | — |
R6Mx9Mx2 (1) | 2 | 8 | 16 | 32 | 8 | 16 | 64 | S81F | S79A | — |
R6Mx9Mx3 (1) | 4 | 16 | 32 | 64 | 16 | 32 | 128 | S81F | S79F | — |
R6Mx9Mx4 (1) | 4 | 8 | 16 | 64 | 8 | 16 | 128 | S81F | D83N | — |
R6Mx9Mx5 to −7 (3) | 2 | 4 | 16 | 32 | 8 | 16 | 64 | S81F | — | D435N |
5714 | 0.125 | 0.25 | 1 | 4 | 0.5 | 1 | 4 | — | — | — |
5714-derived 1st-step mutants | ||||||||||
5714Mx1 to −3 (3) | 0.25 | 0.5 | 1-2 | 4-8 | 0.5-1 | 1-2 | 8 | — | — | — |
5714Mx4 (1) | 0.5 | 0.5 | 2 | 8 | 1 | 1 | 8 | S81F | — | — |
5714Mx5 to −7 (3) | 0.5 | 1 | 2 | 4 | 1 | 1 | 8 | S81Y | — | — |
5714Mx4-derived 2nd-step mutants | ||||||||||
5714Mx4Mx1 to −5 (5) | 4 | 8 | 32 | 128 | 16 | 32 | 128 | S81F | S79Y | — |
5714Mx4Mx6 (1) | 2 | 2 | 8 | 32 | 4 | 4 | 32 | S81F | — | — |
n, number of mutants analyzed.
SPX, sparfloxacin; OFX, ofloxacin; NOR, norfloxacin; LVX, levofloxacin; CIP, ciprofloxacin; and PEF, pefloxacin.
No mutation was found in the GyrB QRDR.
—, no change in the amino acid sequence.
TABLE 2.
Frequencies of apparition of mutant strains selected with levofloxacin or MXF
Mutant | MXF MIC (μg/ml) | Selecting agent at following multiple of MIC
|
|||
---|---|---|---|---|---|
2 × MICb | 4 × MICb | 2 × MICc | 4 × MICc | ||
1st-step mutantsa | |||||
R6-derived | 2.0 × 10−7d | <1.0 × 10−9 | 1.3 × 10−7 | <1.0 × 10−9 | |
5714-derived | 5 × 10−8 | <1.0 × 10−9 | 6.5 × 10−9 | <1.0 × 10−9 | |
2nd-step mutants | |||||
R6-derived | 7.2 × 10−8 | 2.1 × 10−8 | 2.0 × 10−7 | 1.6 × 10−8 | |
5714-derived | 1.1 × 10−8 | 6 × 10−9 | 4.1 × 10−8 | <1.0 × 10−9 | |
Clinical strain 1st-step mutants | |||||
1 | 0.125 | —e | — | 1.4 × 10−7 | — |
2 | 0.125 | — | — | 6.2 × 10−8 | — |
3 | 0.25 | — | — | 3.5 × 10−8 | — |
4 | 0.125 | — | — | 2.1 × 10−8 | — |
5 | 0.25 | — | — | 8.0 × 10−9 | — |
6 | 0.25 | — | — | 5.5 × 10−9 | — |
R6 wild-type laboratory strain; 5714, wild-type clinical strain.
First and second-step mutants were selected on levofloxacin and MXF, respectively.
First and second-step mutants were selected on MXF.
Reproducibility of the mutant frequencies was confirmed in repeated experiments.
—, not done.
First-step mutants.
The impact on susceptibility, the order and the type of altered targets, and the identification of QRDR changes were studied after stepwise selection. After a first selection step on twice the MIC of MXF, analysis of 19 R6- or 5714-derived mutants revealed that two main types of mutants coexisted (Table 1). One type was represented by 10 derivatives from R6 (R6Mx1 to -7) or 5714 (5714Mx1 to -3), for which only a twofold but repeatable increase in the MICs of MXF and sparfloxacin was recorded, while, for those of the other compounds tested, MICs were unchanged or did not exceed twice the initial MIC. No mutations were found in the QRDRs of GyrA, GyrB, ParC, and ParE. Using total DNA from R6Mx1 and R6Mx5, we could transform R6 to the same phenotype with a frequency of 2 × 10−4, suggesting that a single recombination event occurred. However, no transformant could be selected using PCR fragments containing the entire gene of GyrA, GyrB, ParC, or ParE. Such first-step mutants selected on ciprofloxacin, gemifloxacin, or trovafloxacin harboring no mutation in their QRDRs were previously reported (6, 10, 17) and were explained by the presence of an efflux mechanism. For our mutants, the addition of reserpine (10 μg/ml) neither reduced the MICs of MXF nor those of ciprofloxacin or norfloxacin (data not shown), suggesting that, if an efflux mechanism was involved, it had to be different from that involving pmrA (8, 24). All other eight mutants (R6Mx9 to -12 and 5714Mx4 to -7) showed a single GyrA mutation at position 81 (Table 1). It was associated with only a two- to fourfold increase in the MICs of MXF and sparfloxacin and at most a twofold increase in the MICs of ofloxacin, levofloxacin, norfloxacin, ciprofloxacin, and pefloxacin consistent with the GyrA phenotype previously described (22). It proves that MXF, like gatifloxacin, gemifloxacin, and sparfloxacin, belongs to the subclass of FQs for which GyrA of S. pneumoniae is the preferential target. As expected (6, 20), when levofloxacin was used as selecting agent at twice the MIC in the first selection step, the only mutants selected were those for which was found a more pronounced increase (four- to eightfold) in MICs of pefloxacin, norfloxacin, and ciprofloxacin than in the MICs of sparfloxacin and MXF (two- to fourfold) corresponding to the ParC phenotype (22). MICs and single-amino-acid substitutions of some mutants (R6Lv1 and -2 and 5714Lv1 and -2) are presented in Table 3. To ensure that only one event had occurred, amplified fragments of parC encompassing the QRDR from R6Lv1 and R6Lv2 were used to transform R6. This was indeed the case, since transformants with the same phenotype as R6Lv1 and R6Lv2 were selected at a frequency of ca. 10−3 (control, 10−7) (data not shown). As observed for the first-step GyrA mutants selected on MXF, for the first-step ParC mutants selected on MXF, at least for R6, a fourfold increase in the MXF MIC was found. It suggested that, although GyrA could be the preferential target as far as selection by MXF is concerned, at a certain extent this compound would act as previously suggested through both gyrase and topoisomerase IV (22).
TABLE 3.
MICs and amino acid substitutions detected in the QRDRs of the different first-step mutants (Lv) selected on levofloxacin and second-step mutants (LvMx) selected on MXF
Strain (n)a | MIC (μg/ml) of:
|
Mutation(s) in the QRDR ofc
|
||||||||
---|---|---|---|---|---|---|---|---|---|---|
MXF | SPXb | OFXb | NORb | LVXb | CIPb | PEFb | GyrA | ParC | ParE | |
R6 | 0.125 | 0.25 | 1 | 4 | 0.5 | 1 | 8 | —d | — | — |
R6-derived 1st-step mutants | ||||||||||
R6Lv1 (1) | 0.5 | 0.5 | 4 | 32 | 2 | 4 | 64 | — | S79Y | — |
R6Lv2 (1) | 0.5 | 1 | 4 | 32 | 2 | 4 | 32 | — | D83N | — |
R6Lv2-derived 2nd-step mutants | ||||||||||
R6Lv2Mx1 to −2 (2) | 4 | 8-16 | 16-32 | 32 | 8-16 | 16 | 32-64 | S81Y | D83N | — |
R6Lv2Mx3 to −4 (2) | 4 | 16 | 16-32 | 32 | 8-16 | 16 | 64 | S81F | D83N | — |
R6Lv2Mx5 to −6 (2) | 4 | 16 | 8-16 | 32 | 8-16 | 8 | 32 | S85G | D83N | — |
5714 | 0.125 | 0.25 | 1 | 4 | 0.5 | 1 | 4 | — | — | — |
5714-derived 1st-step mutants | ||||||||||
5714Lv1 to −2 (2) | 0.25 | 0.5 | 4 | 32 | 2 | 4 | 32 | — | S79Y | — |
5714Lv1-derived 2nd-step mutants | ||||||||||
5714Lv1Mx1 to −3 (3) | 0.5-1 | 2 | 4-8 | 64 | 8-16 | 8 | 64 | — | S79Y | — |
5714Lv1Mx4 to −6 (3) | 4 | 8 | 32 | 128 | 16 | 32 | 128 | S81F | S79Y | — |
5714Lv1Mx7 (1) | 4 | 8 | 32 | 128 | 16 | 32 | 128 | S81Y | S79Y | — |
n, number of mutants analyzed.
SPX, sparfloxacin; OFX, ofloxacin; NOR, norfloxacin; LVX, levofloxacin; CIP, ciprofloxacin; and PEF, pefloxacin.
No mutation was found in GyrB QRDR.
—, no change in amino acid sequence.
Second-step mutants.
Second-step mutants selected on twice or four times the MIC of MXF were obtained from the first-step mutants R6Mx9 (GyrA S81F) and 5714Mx4 (GyrA S81F) selected on MXF (Table 1). Compared to what was found for the wild-type strains R6 and 5714, for the 13 second-step R6Mx9- and 5714Mx4-derived mutants, a 16- to 32-fold increase in MICs of MXF and an 8- to 64-fold increase in MICs of the other compounds were recorded. Due to its intrinsic better activity, MXF had the lowest MICs, not exceeding 4 μg/ml. All mutants had a second mutation either in ParC at position 79 or 83 or in ParE at position 435. Interestingly among the seven R6Mx9-derived second-step mutants, five different substitutions were found: four in ParC (S79Y, S79A, S79F, and D83N) and one in ParE (D435N). For this latter mutation located in the EGDSA motif of the ParE QRDR (18, 23), at most a twofold increase in sparfloxacin MIC and an 8- to 16-fold increase in ciprofloxacin MIC (19) were reported. If it was associated to an additional mutation in the GyrA QRDR, at least an eightfold increase in the MIC of sparfloxacin was observed (19) (this study). The presence of the ParE D435N mutation has also been described in some clinical isolates along with other mutations in ParE or GyrA (1, 16), although the selecting agents were not mentioned. In contrast to the R6Mx9-derived mutants, five out of six 5714Mx4-derived mutants showed the same S79Y ParC substitution. Thirteen second-step mutants selected on MXF and derived from R6Lv2 (ParC D83N) and 5714Lv2 (ParC S79Y) selected on levofloxacin were studied (Table 3). Three of them (5714Lv1Mx1 to -3) showed no additional mutations in any QRDRs. Compared to the results for their parental strain 5714Lv1, for these strains only two- to eightfold-increased MICs of the different FQs were found, and again no obvious decrease in MICs was observed in the presence of 10 μg of reserpine/ml. The same unknown mutation suspected in some first-step mutants selected on MXF could therefore be present associated to ParC mutations in these mutants. The other 10 mutants showed, associated to the ParC mutation, a mutation in GyrA at position 81 (S81Y, S81F) or 85 (E85G). The MICs were very similar to that of the second-step mutants selected after two rounds of selection on MXF.
This study indicates GyrA to be the preferential target of MXF, since mutations in the GyrA QRDR lead to a fourfold increase in the MICs of this compound. Since single mutations in ParC also affect the MICs of MXF at levels that are almost equal or equal to those of the mutants with an altered GyrA subunit (22) (this work), this compound, as well as clinafloxacin (18), gemifloxacin (10), or BMS-284756 (9), could belong to this particular class of FQs that target preferentially GyrA, though probably acting through both gyrase and topoisomerase IV.
An interesting finding was the easy selection of first-step mutants on MXF for which only a twofold increase in the MICs of the different compounds tested was observed but for which no mutation in DNA gyrase or topoisomerase IV was found. Since no decrease in MICs was obtained in the presence of reserpine, the origin of the resistance has still to be determined. Interestingly in a previous in vitro study (21) where it was shown that MXF was a poor substrate for active efflux in S. pneumoniae, neither second-step ParE mutants nor this type of mutants was selected on MXF.
In this work, we showed that a two-step mutation process was necessary to lead to MXF resistance, involving in all cases GyrA mutations and various topoisomerase IV mutations located in the QRDR of either ParC or ParE. Finally, MXF would still be considered active against mutants harboring ParC mutations, as was recently demonstrated in an endocarditis model (5). However, as shown with levofloxacin, any other FQ that selects for ParC mutants could alter the MXF efficacy, since it would facilitate the selection of second-step GyrA mutants exhibiting high-level resistance.
Acknowledgments
This work was supported by a grant from Bayer-Pharma, Puteaux, France.
We thank Erika Marie-Joseph for secretarial assistance.
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