JAC Advance Access originally published online on October 25, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):28-34; doi:10.1093/jac/dkl428
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In vitro induction and selection of fluoroquinolone-resistant mutants of Streptococcus pyogenes strains with multiple emm types
1 Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University 811-1 Kimiidera, Wakayama, 641-8510, Japan 2 Clinical Center, National Institutes of Health, Department of Health and Human Services Bethesda, MD 20892, USA 3 Food and Drug Administration, Department of Health and Human Services Rockville, MD 20855, USA
*Corresponding author. Tel: +81-73-441-0650; Fax: +81-73-448-2434; E-mail: ynobi{at}wakayama-med.ac.jp
Received 10 August 2006; returned 12 September 2006; revised 25 September 2006; accepted 28 September 2006
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Abstract |
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Objectives: To perform a systematic analysis of point mutations in the quinolone resistance determining regions (QRDRs) of the DNA gyrase and topoisomerase genes of emm type 6 and other emm types of Streptococcus pyogenes strains after in vitro exposure to stepwise increasing concentrations of levofloxacin.
Methods: Twelve parent strains of S. pyogenes, each with a different emm type, were chosen for stepwise exposure to increasing levels of levofloxacin followed by selection of resistant mutants. The QRDRs of gyrA, gyrB, parC and parE correlating to mutants with increased MICs were analysed for point mutations.
Results: Multiple mutants with significantly increased MICs were generated from each strain. The amino acid substitutions identified were consistent regardless of emm type and were similar to the mechanisms of resistance reported in clinical isolates of S. pyogenes. The number of induction/selection cycles required for the emergence of key point mutations in gyrA and parC was variable among strains. For each parent-mutant set, when MIC increased, serine-81 of gyrA and serine-79 of parC were the primary targets for amino acid substitutions. No point mutations were found in the QRDRs of gyrB and parE in any of the resistant mutants sequenced.
Conclusions: Despite its intrinsic polymorphism in the QRDR of parC, emm type 6 is not more likely to develop high-level resistance to fluoroquinolones when compared with other emm types. All emm types seem equally inducible to high-level fluoroquinolone resistance.
Keywords: S. pyogenes , resistance , laboratory induction and selection , point mutations
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Introduction |
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Since the first report of multiple fluoroquinolone resistance in Streptococcus pyogenes,1 patient isolates with reduced susceptibility to fluoroquinolones have been reported by several investigators throughout the world.2–6 Published reports have indicated that the prevalence of S. pyogenes with reduced susceptibility to ciprofloxacin was 3.5% in 1998–99 in Spain, 5.4% in 1999–2002 in Belgium and 10.9% in 2002–03 in the United States.4,5,7 Orscheln et al.4 reported that all S. pyogenes emm type 6 isolates they investigated had intrinsic reduced susceptibility to fluoroquinolones due to a polymorphism in the quinolone resistance determining region (QRDR) of the parC gene, which codes for a change from serine-79 to alanine. Analysis of the gyrA and parC gene sequences of some of those fluoroquinolone-resistant isolates has demonstrated point mutations along the QRDRs analogous to previously reported mutations in S. pyogenes.1–4,6 The limited sequencing data from the wild-type clinical isolates shows a lack of a systematic correlation between fluoroquinolone resistance level and amino acid substitutions in the QRDRs of gyrA and parC, leaving uncertainty as to whether resistance to fluoroquinolones in S. pyogenes is a continuously evolving process or occurs as random events.
Stepwise acquisition of mutations in the QRDRs of the parC and gyrA genes of Streptococcus pneumoniae has been demonstrated in vitro after exposure to increasing concentrations of fluoroquinolones.8 Stepwise acquisition of point mutations in the QRDRs of S. pyogenes is implied because wild-type clinical isolates with higher MICs of fluoroquinolones have different amino acid substitutions at the same location as isolates with lower MICs.1–4,6 Laboratory-generated mutants of S. pyogenes strains through serial passages by an exposure and selection process may help investigators correlate the appearance of point mutations with increased MICs of fluoroquinolones.9–11 Previous reports of laboratory-induced mutants with reduced fluoroquinolone susceptibility did not include emm typing data, have incomplete analysis of sequencing information of the QRDRs of gyrA/parC genes, or did not evaluate mutations in the QRDRs in a stepwise fashion. A pioneer study of Schmitz et al.11 identified alterations in the gyrA and parC genes of resistant mutant isolates, but did not report the change in point mutations for each resistant mutant throughout the stepwise exposure to fluoroquinolones.
In this current project, 12 parent strains representing 12 different emm types were chosen for stepwise induction and selection by increasing levels of levofloxacin in order to determine whether isolates of various serotypes of S. pyogenes may be equally inducible to resistance and whether levels of resistance to fluoroquinolone correlate with particular substitutions of amino acid residues in the QRDRs. The findings of concomitant point mutations with specific amino acid substitutions correlated with increased resistance to fluoroquinolones in S. pyogenes of multiple emm types are essential for a better understanding of whether the emergence of fluoroquinolone resistance is more likely with certain emm types.
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Materials and methods |
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Bacterial strains and growth conditions
Twelve wild-type isolates of S. pyogenes of different emm types (1, 4-1, 6-1, 9, 11, 12, 28, 73-3, 75-5, 89, 94 and 103) with comparable initial susceptibilities (not more than 4-fold difference in MICs) to levofloxacin were selected for the induction assays. Ten isolates were recovered from swabs collected from patients with tonsillitis and one of each from patients with pharyngitis and rhinosinusitis. These 12 emm types are common both in Japan and in the USA. The strains were originally provided by Sugita ENT Clinic (Urayasu, Japan) and Tokyo Metropolitan Institute of Public Health (Tokyo, Japan). emm typing of the S. pyogenes strains was performed at the Tokyo Metropolitan Institute of Public Health by sequencing according to the recommendation of the Division of Bacterial and Mycotic Diseases, Center for Diseases Control and Prevention, and using the emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). S. pyogenes was identified using standard methods.4
Susceptibility testing
MICs of levofloxacin (Daiichi Pharmaceuticals, Tokyo, Japan) for parent and mutant strains were determined by a reference broth microdilution method in cation-adjusted Muller–Hinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with 5% lysed horse blood, as recommended by the Clinical Laboratory Standards Institute (CLSI, formerly NCCLS).12 Dilutions of levofloxacin ranged from 0.125 to 128 mg/L. S. pneumoniae ATCC 49619 was used for quality control with a QC range of 0.5–2 mg/L. In addition, susceptibility of the parent and mutant strains to other representative fluoroquinolones (ciprofloxacin, gatifloxacin, ofloxacin and norfloxacin) was determined using the Etest (AB Biodisk, Solna, Sweden) following the manufacturer's instructions.
Multicycle induction and selection of resistant mutants
Parent strains were grown in antibiotic-free Todd–Hewitt broth supplemented with 0.5% yeast extract (Difco Laboratories) at 37°C for 18 h, and 
1 x 108 cfu/mL (0.5 McFarland standard) of each strain was inoculated into 2 mL of Todd–Hewitt broth containing levofloxacin. The levofloxacin concentrations used for mutant induction ranged from 2x to 4x the MIC for the parent strain or sub-parent mutant strain resulting from the prior induction step. After an overnight incubation at 37°C, cells growing in the tubes with the highest levofloxacin exposure concentration were selected for mutants with increased MICs by plating onto levofloxacin embedded agar plates at concentrations of 0, 2, 4, 8 and 16 mg/L, or higher when necessary. Three colonies were randomly selected for MIC determination, and isolates with the highest MIC were subjected to sequence analysis and further induction. This was repeated when a higher exposure concentration was used for the next step of the induction/selection cycle until mutants with significantly high MICs were selected. The stability of the selected resistant mutants was confirmed by sub-culture onto antibiotic-free 5% sheep blood plates (Nippon Becton Dickinson Company Ltd, Tokyo, Japan) for 10 serial passages.
Amplification and DNA sequencing of the QRDRs
Mutational alterations in the QRDRs of all subunits for DNA gyrase and topoisomerase IV of both parent and fluoroquinolone-resistant mutants were investigated by DNA sequence analysis. Table 1 shows the primers used for amplification of fragments of gyrA/B and parC/E containing the QRDR. Multiple DNA sequencing reactions were performed for each QRDR of individual strains using the Applied Biosystems sequencing kit and ABI Prism 310 Genetic Analyzer (Perkin-Elmer, Applied Biosystems, Foster City, CA, USA).
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Fluoroquinolone-resistant mutants
Table 2 shows the MICs of levofloxacin for the parent strains and the mutants. MICs of the other fluoroquinolones tested for all mutants showed increases nearly identical with those observed for levofloxacin. All mutants reached MICs of ciprofloxacin >32 mg/L after four cycles of induction/selection except strains 6 and 1314 which took six and twelve cycles, respectively. MICs of gatifloxacin were >32 mg/L after three or more cycles of induction, and the mutants also had high MICs of ofloxacin and norfloxacin (data not shown).
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With each set of parent-mutants, the MIC of levofloxacin for mutants was increased following induction/selection cycles. However, the number of cycles required to generate mutants with significantly high MICs varied among the 12 strains studied, even in strains with the same pre-induction MICs. In nine parent-mutant sets, up to seven cycles of induction/selection cycles were required to yield resistant mutants with an MIC of levofloxacin of 128 mg/L. Mutants from strains 3 (emm 28) and 6 (emm 94) reached MICs of 64 and 32 mg/L, respectively, after seven and six cycles of induction/selection. Strain 30 (emm type 12) yielded mutants with MICs of 128 mg/L after only two steps of exposure, and strains 87 (emm type 73-3), 19 (emm type 75-5) and 79 (emm type 4-1) yielded numerous mutants with MICs of levofloxacin of 64 mg/L after 3–4 cycles of induction/selection and 128 mg/L following additional cycles. In contrast, following 11 induction/selection cycles, the MIC of levofloxacin for mutants derived from strain 1314 (emm type 6-1) only reached 8 mg/L and a maximum MIC of 64 mg/L was reached at cycle 14 (Table 2 and Figure 1).
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Point mutations in the QRDRs of gyrA/B and parC/E
No mutations were detected in the QRDRs of gyrB and parE in all mutants and parent strains; however, point mutations were found in gyrA and parC subunits. Individual mutations resulting in an amino acid substitution in the QRDRs of gyrA and parC corresponding to increased MICs for mutants are presented in Table 2.
As the MICs increased in mutants, except for strain 6 (emm type 94), residue 79 was changed from the initial residue to alanine, valine, tyrosine or phenylalanine dependent upon a particular parent-mutant set. The substitution of valine at position 79 of parC has not been observed from reported point mutations of clinical isolates of S. pyogenes. Strains 79 (emm type 4-1) and 6 (emm type 94) also developed a change in mutants from aspartic acid to glycine at position 83, which was not found in mutants derived from other strains. The remaining strains had no point mutation at this residue.
Point mutations were found in the QRDR of gyrA in mutants from each strain regardless of emm type and the point mutation only occurred in the serine residue at position 81, with two types of amino acid substitutions, either to Tyr-81 (in 8 of 12 strains) or Phe-81 (4/12). However, the occurrence of a point mutation at this residue did not consistently correlate to an increase in MICs. For example, it took as low as a 4-fold increase in MIC in strain 6 to reveal a substitution from Ser-81 to Phe-81, while for strain 13, the same residue replacement occurred after the MIC was increased more than 256-fold.
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Discussion |
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Although in vitro generation of fluoroquinolone-resistant mutants in S. pyogenes has been studied previously,10,11 our study has improvements over those studies for the purpose of further understanding the mechanism of fluoroquinolone resistance in S. pyogenes. Boos et al.10 induced S. pyogenes to become fluoroquinolone-resistant in vitro by exposure to fluoroquinolone drugs, but that study did not investigate the changes in the QRDRs of the DNA gyrase and topoisomerase IV genes and it did not determine the emm types of the parent strains studied. In addition, the highest MIC increase in their generated resistant mutants was only 9-fold. We show an increase in MICs up to 512-fold, suggesting that the appropriate combination of stepwise induction and selection cycles are an efficient way to produce resistant mutants in vitro. The availability of mutants with a wide MIC distribution reveals more variety of point mutations associated with an increase in resistance level. Schmitz et al.11 used ciprofloxacin, gatifloxacin, moxifloxacin and BMS-284756 (garenoxacin) to generate fluoroquinolone-resistant mutants and demonstrated that, despite the choice of fluoroquinolone used, the potential for induction of resistant mutants in S. pyogenes is comparable. In the current study, we used levofloxacin to generate fluoroquinolone-resistant laboratory mutants from 12 parent strains of different emm types. Instead of testing with different fluoroquinolone agents, we enhanced the efficiency by repeating the induction/selection process until significantly high-level resistant mutants were selected. For each set of parent-mutants, an array of mutants was produced with a stepwise increase in MICs of levofloxacin corresponding to point mutation information in the QRDRs of both gyrA and parC. This allows a systematic analysis of mutations through the study of the relationship between the changes of residues involved in the point mutations and increasing MICs.
In conjunction with previous studies, the findings from the current study provide the following conclusions. (i) Induction coupled with selection in a laboratory setting is a reliable method for yielding stable fluoroquinolone-resistant S. pyogenes mutants. (ii) Despite the fact that many mutants with a wide range of MICs were produced from a dozen parent strains with different baseline MICs, the number of residues affected and variety of amino acid substitutions found in gyrA and parC are limited (Table 2), which are comparable to those demonstrated in reported clinical resistant isolates.1–4,6,13–15 (iii) The concentration of a fluoroquinolone agent used and number of induction/selection cycles required to yield high MIC mutants vary with emm types/strains. (iv) Of the representative emm type strains, the majority of the non-emm 6 types were comparable in their ability to yield highly resistant mutants, suggesting that resistance to fluoroquinolones can develop independent of emm type.
Previous studies indicated that >90% of clinical isolates that are either resistant or have reduced susceptibility to fluoroquinolones are emm type 6.4,13 However, our emm type 6 isolate took 14 induction/selection cycles to produce resistant mutants with a maximum MIC of 64 mg/L (Table 2 and Figure 1), while 10 non-emm 6 types took fewer cycles (from 1 to 7 cycles) to reach the same level of resistance. We challenged six other emm 6 strains collected from different geographic regions in Japan to the same induction/selection experiments and found that the generation of highly resistant mutants in these emm 6 strains was also relatively difficult (data not shown).
The disparity of ease of yielding high-level fluoroquinolone-resistant mutants between emm 6 and other types was further analysed by comparison of amino acid substitutions. The emm 6 parent strain used in this study had an MIC of 2 mg/L of levofloxacin and ciprofloxacin and had alanine substituted for serine-79 in parC before induction. Orscheln et al.4 raised the concern that the intrinsic reduction in susceptibility to ciprofloxacin and the amino acid polymorphism at this residue of the parC gene in emm type 6 S. pyogenes might set the stage for the emergence of high-level resistance if a single point mutation were to occur in the gyrA gene. Our data seem to indicate that this may not be so. The mutation that leads to the substitution of tyrosine for serine-81 in gyrA took five steps to appear in the emm type 6 strain, but it took only one or two steps for a substitution of this residue with either tyrosine or phenylalanine in seven other emm type strains.
Point mutations in gyrA and parC appear to explain the primary mechanism of loss of susceptibility to fluoroquinolones in S. pyogenes. Stepwise acquisition of mutations in S. pneumoniae has been known for sometime.8,16,17 It has been suggested that S. pyogenes mutations are also acquired in a stepwise fashion.4,18 This study demonstrated stepwise acquisition of mutations in S. pyogenes through repeated in vitro exposure to increasing concentrations of a fluoroquinolone followed by a selection process. Based on comparison of our data and reports of point mutations in fluoroquinolone-resistant clinical isolates of S. pyogenes, amino acid substitutions associated with fluoroquinolone resistance in S. pyogenes seem far less complicated than that in S. pneumoniae.19–22 A limitation of our study was that we studied a single isolate of each emm type for all our 12 different types. Because isolates from different emm types demonstrate similar phenotypic and genotypic changes we would expect different isolates of the same emm type to respond similarly. We are now conducting experiments using multiple isolates of emm type 6 to determine whether different strains of the same emm type respond in the same way. Precise correlation between residue replacements and increased MIC in mutants may not be solely explained by the point mutations, because it is still difficult to explain why mutant strains with the same point mutations demonstrated various levels of MIC of levofloxacin. Therefore, exploration of other possible mechanisms, such as an activated efflux pump specific for fluoroquinolone drugs, among the resistant mutants may be necessary.
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No interest has influenced the conclusions drawn from the work.
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Acknowledgements |
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We would like to thank Dr Rinya Sugita (Sugita ENT Clinic, Urayasu, Japan) for providing us with strains. We highly acknowledge Dr Miyoko Endo (Tokyo Metropolitan Institute of Public Health, Tokyo, Japan) for emm typing of S. pyogenes. This work was not funded by any organization. Note: The opinions and information in this article are those of the authors, and do not represent the views and/or policies of S. S. Yan's affiliation.
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References |
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1 Yan SS, Fox ML, Holland SM, et al. (2000) Resistance to multiple fluoroquinolones in a clinical isolate of Streptococcus pyogenes: identification of gyrA and parC and specification of point mutations associated with resistance. Antimicrob Agents Chemother 44:3196–8.
2 Richter SS, Diekema DJ, Heilmann KP, et al. (2003) Fluoroquinolone resistance in Streptococcus pyogenes. Clin Infect Dis 36:380–3.[CrossRef][Web of Science][Medline]
3 Reinert RR, Lutticken R, Al-Lahham A. (2004) High-level fluoroquinolone resistance in a clinical Streptoccoccus pyogenes isolate in Germany. Clin Microbiol Infect 10:659–62.[CrossRef][Web of Science][Medline]
4 Orscheln RC, Johnson DR, Olson SM, et al. (2005) Intrinsic reduced susceptibility of serotype 6 Streptococcus pyogenes to fluoroquinolone antibiotics. J Infect Dis 191:1272–9.[CrossRef][Web of Science][Medline]
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Perez-Trallero E, Fernandez-Mazarrasa C, Garcia-Rey C, et al. (2001) Antimicrobial susceptibilities of 1,684 Streptococcus pneumoniae and 2,039 Streptococcus pyogenes isolates and their ecological relationships: results of a 1-year (1998–1999) multicenter surveillance study in Spain. Antimicrob Agents Chemother 45:3334–40.
6
Alonso R, Galimand M, Courvalin P. (2002) parC mutation conferring ciprofloxacin resistance in Streptococcus pyogenes BM4513. Antimicrob Agents Chemother 46:3686–7.
7
Malhotra-Kumar S, Lammens C, Chapelle S, et al. (2005) Clonal spread of fluoroquinolone non-susceptible Streptococcus pyogenes. J Antimicrob Chemother 55:320–5.
8 Gootz TD, Zaniewski R, Haskell S, et al. (1996) Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro. Antimicrob Agents Chemother 40:2691–7.[Abstract]
9
MacGowan AP, Bowker KE, Wootton M, et al. (1999) Exploration of the in-vitro pharmacodynamic activity of moxifloxacin for Staphylococcus aureus and streptococci of Lancefield Groups A and G. J Antimicrob Chemother 44:761–6.
10
Boos M, Mayer S, Fischer A, et al. (2001) In vitro development of resistance to six quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. Antimicrob Agents Chemother 45:938–42.
11
Schmitz FJ, Boos M, Mayer S, et al. (2002) In vitro activities of novel des-fluoro(6) quinolone BMS-284756 against mutants of Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus selected with different quinolones. Antimicrob Agents Chemother 46:934–5.
12 National Committee for Clinical Laboratory Standards. (2003) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6(PA, USA, NCCLS, Villanova).
13
Alonso R, Mateo E, Galimand M, et al. (2005) Clonal spread of pediatric isolates of ciprofloxacin-resistant, emm type 6 Streptococcus pyogenes. J Clin Microbiol 43:2492–3.
14
Powis J, McGeer A, Duncan C, et al. (2005) Prevalence and characterization of invasive isolates of Streptococcus pyogenes with reduced susceptibility to fluoroquinolones. Antimicrob Agents Chemother 49:2130–2.
15 Rivera A, Rebollo M, Sanchez F, et al. (2005) Characterization of fluoroquinolone-resistant clinical isolates of Streptococcus pyogenes in Barcelona, Spain. Clin Microbiol Infect 11:759–61.[CrossRef][Web of Science][Medline]
16 Tankovic J, Perichon B, Duval J, et al. (1996) Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro. Antimicrob Agents Chemother 40:2505–10.[Abstract]
17 Pan XS, Ambler J, Mehtar S, et al. (1996) Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob Agents Chemother 40:2321–6.[Abstract]
18
Alberti S, Garcia-Rey C, Dominguez MA, et al. (2003) Survey of emm gene sequences from pharyngeal Streptococcus pyogenes isolates collected in Spain and their relationship with erythromycin susceptibility. J Clin Microbiol 41:2385–90.
19 Zheng X, Johnson C, Lu Y, et al. (2001) Clinical isolates of Streptococcus pneumoniae resistant to levofloxacin contain mutations in both gyrA and parC genes. Int J Antimicrob Agents 18:373–8.[CrossRef][Web of Science][Medline]
20 Hooper DC. (2002) Fluoroquinolone resistance among Gram-positive cocci. Lancet Infect Dis 2:530–8.[CrossRef][Web of Science][Medline]
21
Weigel LM, Anderson GJ, Facklam RR, et al. (2001) Genetic analyses of mutations contributing to fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother 45:3517–23.
22 Montanari MP, Tili E, Cochetti I, et al. (2004) Molecular characterization of clinical Streptococcus pneumoniae isolates with reduced susceptibility to fluoroquinolones emerging in Italy. Microb Drug Resist 10:209–17.[Web of Science][Medline]
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