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Antimicrobial Agents and Chemotherapy, March 2005, p. 1190-1193, Vol. 49, No. 3
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.3.1190-1193.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Laboratoire de Bactériologie, Université Victor Segalen Bordeaux 2, Bordeaux, France1
Received 30 July 2004/ Returned for modification 13 October 2004/ Accepted 9 November 2004
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The purpose of this study was to determine and compare the abilities of six older and newer fluoroquinolones to select for fluoroquinolone-resistant mutants of M. pneumoniae. Furthermore, our previous data on fluoroquinolone resistance in M. hominis were completed by selecting for mutants of this mycoplasma that were resistant to the newer compounds levofloxacin, moxifloxacin, gemifloxacin, and gatifloxacin.
Growth conditions and antibiotic susceptibility testing of the mycoplasma strains have been previously described (12). MICs of fluoroquinolones were determined in the presence and absence of reserpine (20 µg/ml) as described previously (11). Two selection methods, with either broth or agar medium, were used for M. pneumoniae FH (ATCC 15531), while only the agar-based selection was done for M. hominis PG21 (ATCC 23114). Broth-selected mutants were obtained by serial transfers of M. pneumoniae FH in Hayflick modified broth medium containing subinhibitory concentrations of each fluoroquinolone, as previously described (10). For the first passage, the reference strain M. pneumoniae FH was inoculated in Hayflick modified medium with increasing twofold dilutions of each antibiotic. The MIC was determined as the lowest concentration of antimicrobial agent that prevented a color change in the medium at the time when the drug-free growth control first showed a color change (after about 5 days of incubation at 37°C). The culture containing the highest antibiotic concentration with visible growth (subinhibitory concentration) was used to inoculate another antibiotic dilution panel for the following passage. Fifteen passages were performed for each selector antibiotic except for gatifloxacin, and two of the five clones subcultured from passages 5, 7, 10, and 15 were studied. With gatifloxacin, the characterization of passage 2 was added and the selection was conducted up to 10 passages. Subinhibitory concentrations ranged from 0.06 to 16 µg/ml, depending on the selector fluoroquinolone and the selected passage. For the stepwise selection on agar, M. pneumoniae FH cells and M. hominis PG21 cells were concentrated 100-fold by centrifugation and directly filtered through a 0.45-µm-pore size filter (Millipore) to reach an inoculum titer of approximately 109 and 1010 color changing units/ml, respectively. Then, selection of fluoroquinolone-resistant mutants was performed as previously described by plating a 100-µl inoculum onto Hayflick modified agar medium containing increasing inhibitory concentrations of the selector antibiotic (2). For each selection experiment, three steps were performed with fluoroquinolone concentrations at 2, 4, 8, and 10 times the MIC for the respective parent strain. The mutation frequency was determined as the number of colonies appearing on the plate with antibiotic divided by the number of colonies in the inoculum.
Amplification of the gyrA, gyrB, parC, and parE QRDRs was carried out with 2 µl of a broth culture for the resistant mutants and 1 µM (each) primer, as described elsewhere (2). For M. pneumoniae, primer sets were chosen from the complete genome sequence (6) to amplify a 550-bp gyrA fragment (nucleotides [nt] 28 to 577), a 297-bp gyrB fragment (nt 1258 to 1554), a 588-bp parC fragment (nt 22 to 609), and two parE fragments of 269 bp (nt 5 to 273) and 300 bp (nt 1219 to 1518). For M. hominis, gyrA and parC fragments were obtained with primer set MHA1 (5'-ATGAGTGTCATAGTTTCTCG-3') and MH4 (2) and primer set MHC1 (5'-GCCGATATAATGTCTGATAG-3') and MHC2 (5'-TGTTGCATCAATAACTTCGC-3'), respectively. gyrB and parE QRDRs were amplified with primers MH6-7 and MH28-29, respectively (2). A 5' parE fragment was also amplified with primers MHE1 (5'-AAATAATTACGAAGCTAGCG-3') and MHE2 (5'-ACTCGTGTTTATTGACAGG-3'). PCR products were directly sequenced by using an ABI PRISM dRhodamine terminator cycle sequencing ready reaction kit (Applied Biosystems).
We were able to select quinolone-resistant M. pneumoniae mutants after serial passages in subinhibitory concentrations of all the six fluoroquinolones used in this study. Two fluoroquinolones, ciprofloxacin and gatifloxacin, selected for M. pneumoniae mutants only with the broth method, using subinhibitory antibiotic concentrations. In contrast, resistant M. hominis mutants were obtained in the presence of inhibitory concentrations of the four fluoroquinolones studied. For M. pneumoniae, mutation frequencies ranged from 1.3 x 10–6 to 2.9 x 10–7 with sparfloxacin, while they ranged from 3 x 10–8 to 7 x 10–9 with levofloxacin, moxifloxacin, and gemifloxacin. For M. hominis, overall, mutation rates were lower with moxifloxacin, gemifloxacin, and gatifloxacin (about 10–8) than with levofloxacin (about 10–7). The susceptibility profiles of the mutants according to their gyrA, gyrB, parC, and parE QRDR status are shown Table 1 for M. pneumoniae and in Table 2 for M. hominis. These mutants exhibited cross-resistance for fluoroquinolones, with the MICs depending on the number of mutations and on the altered positions in the QRDRs. Moxifloxacin, gemifloxacin, and gatifloxacin but not ciprofloxacin and levofloxacin stayed active against M. pneumoniae or M. hominis mutants with one mutation. No efflux mechanism was detected for M. pneumoniae and M. hominis mutants in the presence of reserpine.
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As in M. hominis, distinct fluoroquinolones seem to have different primary targets in M. pneumoniae. Thus, according to our genetic studies with M. pneumoniae, sparfloxacin and ciprofloxacin selected mutants with a single modification in parC alone, while levofloxacin and moxifloxacin selected single mutants with a mutation in gyrA or gyrB alone. Gemifloxacin selected single mutants with a modification in either gyrA or parC, in support of dual activity on both DNA gyrase and topoisomerase IV in M. pneumoniae, as in Streptococcus pneumoniae (9). It should be noted that the same fluoroquinolone did not have the same preferential target in both mycoplasmas species. For instance, sparfloxacin targeted primarily topoisomerase IV in M. pneumoniae (Table 1) but DNA gyrase in M. hominis (2, 8). For M. hominis, first-step mutants that were selected with newer fluoroquinolones, levofloxacin and gatifloxacin, had mutations in parC or parE. Here again, first-step mutants of M. hominis selected with gemifloxacin harbored mutations in either gyrA or parC. The preferential targets of moxifloxacin with M. hominis and gatifloxacin with M. pneumoniae were not elucidated in our study, with no single mutants selected with these compounds.
To our knowledge, this is the first description of fluoroquinolone resistance acquired in M. pneumoniae. Even though lower mutation rates were obtained with broader-spectrum quinolones, such as moxifloxacin, gemifloxacin, and gatifloxacin, than with ciprofloxacin for both mycoplasma species, all fluoroquinolones tested selected for resistance, confirming the necessity of a judicious use of these compounds in order to limit development of resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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