a Department of Pathology (Clinical Microbiology), Hershey Medical Center, 500 University Drive, Hershey, PA 17033; b Department of Pathology (Clinical Microbiology), Case Western Reserve University, Cleveland, OH 44106, USA
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Abstract |
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Introduction |
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Fluoroquinolones present potential for empirical treatment of adult respiratory infections, in part due to the increase in the incidence of penicillin-resistant pneumococci and also because of their activity against Haemophilus influenzae, Moraxella catarrhalis, Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila.3,4 However, the abuse of this class of antimicrobial agent could lead to the emergence of resistant mutants. There were no pneumococci with raised ciprofloxacin MICs in a 1997 US surveillance study,5 and worldwide incidence of quinolone resistance in pneumococci is less than 1%.5 However, resistance to levofloxacin (5.5%) and trovafloxacin (2.2%) was recently reported from Hong Kong.6 In Canada, the prevalence of pneumococci with reduced susceptibilities to fluoroquinolones in adults increased from 0% in 1993 to 1.7% in 1997 and 1998.7 The prevalence of pneumococci with higher ciprofloxacin MIC (4 mg/L) also increased from 0.9% in 19911992 to 3.0% in 19971998 in Spain.8 It is of concern that these resistant strains may spread to other parts of the world and the overuse of this class of antimicrobial agents could lead to the emergence of resistant mutants.
The primary targets of fluoroquinolone are type II topoisomerases: topoisomerase IV and DNA gyrase, which alter DNA topology through a transient double-stranded DNA break.9 Topoisomerase IV is composed of ParC and ParE subunits, which are encoded by parC and parE genes, respectively. DNA gyrase is composed of GyrA and GyrB, which are encoded by gyrA and gyrB genes, respectively. Point mutations in the quinolone resistance-determining regions (QRDRs) of topoisomerase IV (primarily parC) and DNA gyrase (primarily gyrA) genes are associated with quinolone resistance. Mutations in the QRDRs of parE and gyrB are also believed to play a role in fluoroquinolone resistance, albeit to a lesser extent.1012
There are three types of fluoroquinolones: (i) those that have topoisomerase IV as their primary target; (ii) those that have DNA gyrase as their primary target; and (iii) those that target both topoisomerase IV and DNA gyrase. Among pneumococci, topoisomerase IV is thought to be the primary target for ciprofloxacin and trovafloxacin,4,10,13 whereas DNA gyrase is the primary target of gatifloxacin and moxifloxacin.14,15 Fluoroquinolones that target both enzymes include clinafloxacin and gemifloxacin.16,17
The purpose of this study was to determine and compare the ability of gemifloxacin, trovafloxacin, ciprofloxacin, gatifloxacin and moxifloxacin to select quinolone-resistant mutants of S. pneumoniae.
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Materials and methods |
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Sixteen clinical isolates of S. pneumoniae isolated during the past 5 years were studied. MICs of quinolones for these strains are shown in Table 1. Eight of 16 strains chosen for this study were susceptible to ciprofloxacin (based on a FDA breakpoint of
1 mg/L) and the other eight strains were resistant to ciprofloxacin (MICs
8 mg/L). Gemifloxacin powder for susceptibility testing was obtained from SmithKline Beecham Laboratories, Harlow, UK. The other drugs were obtained from their respective manufacturers.
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MIC testing for all parent strains and derived mutants was carried out by the broth dilution method with incubation in air at 35°C for 2024 h according to current NCCLS guidelines.18 Susceptible breakpoints for trovafloxacin, gatifloxacin and moxifloxacin were MICs 1 mg/L according to the NCCLS.18 A gemifloxacin MIC of
1 mg/L was considered susceptible based on pharmacokinetic/pharmacodynamic (PK/PD) data.5,19,20
Multi-step resistance selection
Multi-step resistance selection for each quinolone was performed as described previously.2123 Briefly, glass tubes, each containing 1 mL of cation-adjusted MuellerHinton broth (BBL Microbiology Systems, Cockeysville, MD, USA) with 5% lysed horse blood and doubling antibiotics, were initially inoculated with approximately 5 x 105 cfu/mL at antibiotic concentrations three doubling dilutions above and three doubling dilutions below the MIC. Inocula were diluted to achieve a final concentration of 5 x 105 cfu/mL in each tube. The tubes were incubated at 35°C for 24 h. For each subsequent daily passage, 10 µL inocula were taken from the first tube containing a subinhibitory drug concentration and subcultured into the next passage tubes containing each diluted drug. Daily subculturing was done 50 times, or until mutants with elevated MICs (4 x) to the selecting drug emerged. Mutants were inoculated from the last subculturing tube onto drug-free media using a cotton swab. Fifteen to 20 colonies of resistant clones were subcultured for 10 days on drug-free media then frozen in double-strength skimmed milk (Difco Laboratories, Detroit, MI, USA) at 70°C until further testing. One purified colony from frozen culture was used for MIC testing, DNA sequencing and pulsed-field gel electrophoresis (PFGE).
Single-step resistance selection
The frequency of spontaneous single-step mutation was determined by spreading cultures (c. 1010 cfu/mL) in a 100 µL volume of saline onto brainheart infusion agar (BBL) supplemented with 10% lysed horse blood plates (100 mm diameter) containing 1 x, 2 x, 4 x, 8 x and 16 x MICs of each compound.16,23 Plates were incubated aerobically at 35°C for 4872 h. The mutation frequency was calculated as the number of resistant colonies per inoculum.16,23 MICs of mutant strains were determined by agar dilution method on MuellerHinton agar (BBL) with 5% lysed sheep blood.1,24,25 Gene sequencing and the presence of an efflux mechanism were determined for some single-step mutant strains (see below).
Serotyping
Serotyping of parent and mutant strains was performed by the standard Quellung method with sera from Statens Seruminstitut (Copenhagen, Denmark).
PFGE
To determine whether resistant isolates obtained at the end of serial passages were derived from those used at the beginning of the study, the parent strains and the mutants with increased MICs obtained after the last passage were tested by PFGE with a CHEF DR III apparatus (Bio-Rad, Hercules, CA, USA) as described previously.21,26
PCR of quinolone resistance determinants and DNA sequence analysis
To determine whether mutants that developed resistance to quinolones had alterations in topoisomerase IV or DNA gyrase compared with the parent strains, parC, parE, gyrA and gyrB were amplified by PCR and sequenced as described previously.1,21,23 Mutants with mutations widely described in the literature (e.g. Ser-79Tyr or Phe in ParC and Ser-81
Tyr or Phe in GyrA) were sequenced once in the forward direction. Mutants with no mutations in a particular gene, or with a previously undescribed mutation, were sequenced twice in the forward and once in the reverse direction on products of independent PCR experiments.1,21,23 Only clones obtained at the end of subculturing were examined by DNA sequencing and for the presence of an efflux mechanism (see below).
Determination of efflux mechanism
MICs were determined in the presence and absence of 10 mg/L of reserpine (Sigma Chemicals, St Louis, MO, USA) as described previously.21,23,27 Sixteen parent strains and all 80 quinolone mutants selected by repeated exposure to subinhibitory concentrations of fluoroquinolones were tested. An efflux mechanism was believed to be present when the MIC in the presence of reserpine was at least four-fold less (two doubling dilutions) than the MIC in the absence of reserpine (tests were repeated in triplicate).21,23
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Results |
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The frequencies of single-step mutations with gemifloxacin (2.0 x 104 to <1.0 x 1010) and gatifloxacin (3.0 x 104 to <1.0 x 1010) were lower than those with trovafloxacin (1.9 x 102 to 2.4 x 107), ciprofloxacin (>2.3 x 102 to <5.0 x 109) or moxifloxacin (3.0 x 104 to <2.0 x 109) (Table I). Single-step mutation frequencies of trovafloxacin and ciprofloxacin for some isolates appeared higher than the frequencies reported previously.11,13,15,28 It is thought that this was owing to isolate variation.
Gene sequencing of the QRDRs of ParC, GyrA, ParE and GyrB was performed for single-step mutants of strains 1 and 14. Mutant isolate 1 selected at 4 x MIC of trovafloxacin had a mutation in ParC (Ser-79Tyr), but no mutations in GyrA; mutant isolate 1 selected at the moxifloxacin MIC had a mutation in GyrA (Glu-85
Gln), but no mutations in ParC. The mutants selected at 2 x MIC of trovafloxacin or 2 x MIC of gatifloxacin from isolate 14 had mutations in GyrA (Cys-81
Trp) compared with their parent, but no mutations in ParC; mutants of isolate 14 selected at 1 x and 2 x MIC of moxifloxacin MIC had mutations in GyrB (1 x MIC of moxifloxacin: Leu-412
Arg; 2 x MIC of moxifloxacin: Pro-454
Ser). There were no mutations in ParE. MICs of all isolate 1 and 14 single-step mutants were not changed in the presence of reserpine.
Multi-step resistance selection by subculturing in subinhibitory concentrations
Initial MICs of parents and resistant mutants resulting from serial daily subculturing in subinhibitory concentrations of antimicrobials are summarized in Table 2. Subculturing in the presence of gemifloxacin selected six resistant mutants, trovafloxacin selected nine, ciprofloxacin selected 11, gatifloxacin selected 13 and moxifloxacin selected 12.
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The distribution of MICs for each quinolone among 80 multi-step mutant strains is shown in Table 3. Using the breakpoints described earlier, all mutants were resistant to ciprofloxacin (80/80), followed by gatifloxacin (66/80), moxifloxacin (57/80), trovafloxacin (50/80) and gemifloxacin (13/80) in descending order.
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The 16 pneumococcal isolates tested comprised serotypes, type 1 (strains 5 and 7), type 4 (strain 14), type 6b (strain 1), group 7 (strain 4), group 9 (strain 12), type 14 (strains 2, 8, 9 and 10), group 19 (strains 3, 6, 11 and 16) and group 23 (strains 13 and 15). All parent strains had unique PFGE patterns. All mutants had serotypes identical to those of the parent strains, and all had PFGE patterns identical to those of the parent strains.
Mutations in topoisomerase IV and DNA gyrase
DNA sequencing of the QRDRs of parC, parE, gyrA and gyrB of resistant mutants is shown in Table II. Most of the mutations associated with quinolone resistance were found in ParC at Ser-79
Phe or Tyr and Asp-83
Asn, and in GyrA at Ser-81
Ala, Cys, Phe or Tyr and Glu-85
Lys. Mutations not previously described for S. pneumoniae that were associated with quinolone resistance were Glu-85
Ala and Ala-123
Ser in GyrA. The Glu-85
Ala mutation in GyrA was selected by gemifloxacin and was associated with a two-fold increase in trovafloxacin, gatifloxacin and moxifloxacin MICs and an eight-fold increase in gemifloxacin MIC. The Ala-123
Ser mutation in GyrA was selected by gatifloxacin and was associated with a four-fold increase in the trovafloxacin and gatifloxacin MICs, and an eight-fold increase in the moxifloxacin and gemifloxacin MICs. The mutation of Ala-115
Val in ParC has also not been reported previously in S. pneumoniae, and was associated with quinolone resistance in the isolate 7 mutant selected by trovafloxacin. The mutation of Val-71
Ile in GyrA was found in one mutant (isolate 14) selected by trovafloxacin; however, it was not associated with quinolone resistance. A mutant selected by repeated subculturing of isolate 9 in subinhibitory concentrations of moxifloxacin had the highest gemifloxacin MIC (4 mg/L) and had both Ser-81
Phe and Glu-85
Tyr mutations in GyrA.
Three mutants had ParE mutations at Asp-435Asn or Val that were associated with quinolone resistance. Mutations in ParE, which have not been described previously, were found in six resistant mutants at the following positions: Leu-431
Ile, Arg-447
Cys, Asn-473
Ile and Ile-476
Phe. The significance of these mutations was not clear since other ParC and GyrA mutations were also found in these mutants. Arg-447
Ser mutation was selected by gemifloxacin from parent strain 4 and was associated with increased MICs (eight- to 32-fold) to all the fluoroquinolones tested.
In two resistant mutants the previously described Glu-474Lys mutation in GyrB16 was found (strains 1 and 13). Mutations in GyrB that have not been described previously were Lys-404
Ile, Ser-405
Phe, Gly-406
Asp, Cys or Ser, Leu-412
Arg, Ser-438
Tyr, Ser-440
Tyr, Arg-445
Gly, Asp-453
Val, Pro-454
Ser and Glu-475
Val. It is unclear what role these GyrB mutations have in quinolone resistance. However, Pro-454 mutation in GyrB was found in both single- and multi-step mutant strains selected by moxifloxacin. The strain 12-derived mutant selected by moxifloxacin had a four amino acid deletion in GyrB (Asp-411LeuProGly-414; C1684CTTCCAGGGAA1695) compared with the parent strain and the MIC for moxifloxacin was increased eight times after selection.
Efflux mechanism
MICs determined in the presence and absence of reserpine are shown in Table 2. In the presence of reserpine, 53 of the 80 mutants had ciprofloxacin MICs that were 432 x lower; 39 mutants had lower gemifloxacin MICs (432 x), 13 mutants had lower gatifloxacin MICs (48 x), whereas none had lower trovafloxacin or moxifloxacin MICs.
Reserpine lowered the ciprofloxacin MICs in seven of the 11 resistant mutants with no mutations in ParC, GyrA, ParE and GyrB (mutants of isolates 4, 5, 7, 8, 12, 13 and 16 selected by ciprofloxacin), and the gemifloxacin MICs in two of the six resistant mutants (of isolates 15 and 16 selected by gemifloxacin) in which no mutations in the QRDRs were found. Resistant mutants selected by trovafloxacin (from isolates 6 and 12) and selected by moxifloxacin (from isolates 13 and 15) had no mutations in any of the QRDRs or lower MICs in the presence of reserpine.
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Discussion |
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The known types of resistance mechanisms for quinolones are target modification and active efflux. Mutations in the DNA gyrase and topoisomerase IV that confer resistance to quinolones usually occur in the QRDRs.
Topoisomerase IV is thought to be the primary target for trovafloxacin and ciprofloxacin.4,10,13 In our study trovafloxacin-selected mutants were found to be modified in their parC gene alone except for one derived mutant selected by trovafloxacin, which had a single modification in Ser-81Phe in gyrA or with additional mutations in gyrA, parE or gyrB. Higher trovafloxacin MICs were associated with more than one QRDR mutation.
The only mutation selected by ciprofloxacin among the ciprofloxacin-susceptible strains was Ser-79Tyr in parC with additional mutations in gyrA at Ser-81
Phe or Tyr. Resistance by efflux mechanism was found in all of the mutant strains selected by ciprofloxacin. Activation or induction of this efflux mechanism by ciprofloxacin was the most common mechanism of resistance.27
DNA gyrase is the primary target of gatifloxacin and moxifloxacin.14,15 Gatifloxacin-selected mutants were found to be modified in their gyrA alone or with additional mutations in parC, parE and gyrB, except for one strain which had two mutations in parC and one mutation in gyrB and efflux mechanisms. Moxifloxacin selected mutations in gyrB similar to gyrA. Eight mutants selected by moxifloxacin had modification in gyrB gene alone or associated with parC, parE and/or gyrA.
Gemifloxacin is thought to have activity on both DNA gyrase and topoisomerase IV.17 In support of this activity, gemifloxacin selected mutants with single modifications in gyrA, parC or parE alone. Recently, Boos and co-workers29 presented multi-step resistance selection of S. pneumoniae with subinhibitory concentration of gemifloxacin with five other quinolones. Derived clones had single mutations of gyrA or parC alone or double mutations both in gyrA and parC. It is possible that repeated exposure with subinhibitory concentrations of gemifloxacin might target all QRDRs with the exception of gyrB.
Most mutants with raised quinolone MICs had mutations in ParC at Ser-79 or Asp-83 and GyrA at Ser-81 or Glu-85 as previously reported.1,10,11,1316,28,30 Three mutants had a mutation in ParE at Asp-435Asn, which has been demonstrated to confer low-level quinolone resistance and one mutant strain had mutation of Arg-447
Ser, as previously reported.17,21 Mutation of Glu-474
Lys in GyrB previously reported by Pan & Fisher,16 was found in two quinolone-resistant mutants.
Several unique mutations in the QRDRs that have not been reported previously were found among some of the derived clones. While many of these mutations were associated with increased fluoroquinolone MICs, it was unclear whether the mutations contributed to the raised MICs, because often the mutants contained other QRDR mutations that have been shown to contribute to increased MICs. Experiments involving transformation of wild-type strains with the above mutations are ongoing to determine whether these previously undescribed mutations contributed to increased fluoroquinolone MICs.
Twenty-eight mutants did not have any detectable mutations in the QRDRs compared with parent strains, but most had lower MIC in the presence of reserpine, suggesting that an efflux mechanism might play a role in their quinolone resistance.27,31 However, the increased fluoroquinolone MICs compared with parent strains in some mutants could not be explained by QRDR mutations or an efflux mechanism. Mutants of this type were very common among those derived from ciprofloxacin-resistant parent strains 1416. These parent strains already had parC and gyrA mutations know to cause fluoroquinolone resistance. These observations indicate that other resistance mechanisms are involved. It is possible that mutations outside the QRDR or in other genes were responsible for the increased fluoroquinolone MICs. Recently, Janoir et al.32 described a parE mutation outside the QRDR that contributed to raised fluoroquinolone MICs. Additionally, an efflux mechanism unaffected by reserpine could be involved. Others have found that trovafloxacin and moxifloxacin are poor substrates for PmrA.33 Efflux mechanisms other than PmrA may play a role in these strains (M. J. Gill & N. P. Brenwald, personal communication). Studies looking for other mechanisms in these mutants are currently underway.
The clinical efficacy of fluoroquinolones has been described to be associated with free AUC24/MIC ratios exceeding 25 in immunocompetent patients.5,19 The AUC of gemifloxacin at the standard oral dose used during development of 320 mg/day is 8.4 mgh/L and the AUC24/MIC ratio exceeds 25 for isolates with MICs of <0.25 mg/L. However, the AUC24 of gemifloxacin 800 mg once daily by oral administration is 31.4 ± 7.6 mg/L,34 and AUC24/ MIC ratios exceed 25 for isolates with MIC of 1 mg/L. As MICs of gemifloxacin were
1 mg/L for 84% of mutants (67/80) in this study, gemifloxacin at 800 mg/day has potential for treatment of many pneumococcal infections due to isolates with raised quinolone MICs. Single- and multi-step studies showed that all quinolones tested selected for resistance. To avoid resistance selection with broader spectrum quinolones such as levofloxacin, gatifloxacin, moxifloxacin and gemifloxacin, judicious use and proper dosing will be necessary in order to limit development of resistance.
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Acknowledgements |
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Notes |
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References |
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34
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Received 22 November 2000; returned 23 April 2001; revised 29 May 2001; accepted 1 June 2001