JAC Advance Access originally published online on October 20, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1279-1282; doi:10.1093/jac/dkl427
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In vitro activity of sitafloxacin against clinical strains of Streptococcus pneumoniae with defined amino acid substitutions in QRDRs of gyrase A and topoisomerase IV
1 Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases, Graduate School of Medicine, University of the Ryukyus 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan 2 Yonabaru Central Hospital Okinawa, Japan 3 Nakagami General Hospital Okinawa, Japan 4 Department of Laboratory Medicine, Graduate School of Medicine, University of the Ryukyus Okinawa, Japan
*Corresponding author. Tel: +81-98-895-1144; Fax: +81-98-895-141; E-mail: fhiga{at}med.u-ryukyu.ac.jp
Received 10 June 2006; returned 17 August 2006; revised 22 September 2006; accepted 28 September 2006
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Abstract |
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Objectives: Fluoroquinolone-resistant Streptococcus pneumoniae are increasing worldwide rapidly. In vitro activities of sitafloxacin were evaluated against clinical isolates of S. pneumoniae resistant to levofloxacin (MIC of levofloxacin
4 mg/L), which were characterized genetically. Methods: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE of these strains were analysed by PCR-based sequencing. MICs of sitafloxacin and other quinolones were determined by a microdilution broth method.
Results: All 18 strains had at least one amino acid substitution in the QRDRs of GyrA and ParC, which included Ser-81
Tyr/Phe and Glu-85Lys in GyrA and Ser-79Phe/Ile/Tyr, Asp-83Tyr, Asn-91Asp, Ser-107Phe, Lys-137Asn and Ala-142Ser in ParC. Most isolates had Asp-435Asn/Ile-460Val/Ala-596Thr substitutions in ParE, while no amino acid substitution in GyrB was noted in all isolates. Ten isolates for which levofloxacin MICs were 16 or 32 mg/L had multiple mutations in both GyrA and ParC. The MIC80 value of sitafloxacin for levofloxacin-resistant isolates was 0.25 mg/L. The range of MICs of sitafloxacin for isolates resistant to levofloxacin (MIC 4–32 mg/L) was 0.016–0.5 mg/L.
Conclusions: These findings warrant further studies to evaluate the usefulness of sitafloxacin in the treatment of levofloxacin-resistant S. pneumoniae infection.
Keywords: levofloxacin-resistant S. pneumoniae , drug resistance , sitafloxacin , target alteration , efflux pump
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Introduction |
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Streptococcus pneumoniae is the major cause of respiratory tract infections, bacteraemia and bacterial meningitis. For a long time, penicillin was the most effective drug against such infections. However, the incidence of multidrug-resistant S. pneumoniae is currently increasing throughout the world.1 The rapid spread of pneumococcal clones resistant to ß-lactams and macrolides has promoted the use of selected fluoroquinolones for the treatment of pneumococcal infections. Therefore, fluoroquinolones with antipneumococcal activity, such as levofloxacin, moxifloxacin, gatifloxacin and gemifloxacin, may play an important role in the management of pneumococcal disease.2
Accordingly, the increase in S. pneumoniae resistance to fluoroquinolones that has been reported recently is of great concern. So far, two mechanisms responsible for the reduced susceptibility to fluoroquinolones have been identified in clinical isolates: target alteration and/or reduced drug accumulation due to drug efflux.3 The targets of fluoroquinolones are DNA gyrase and topoisomerase IV, which are encoded by gyrA, gyrB, parC and parE. Fluoroquinolone-resistant strains show amino acid substitutions in quinolone resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV. Multiple mutations within QRDRs of both gyrA and parC result in high-level resistance to levofloxacin.4 In this study, the in vitro activities of sitafloxacin,5 a newer quinolone, were evaluated against clinical isolates of S. pneumoniae resistant to levofloxacin (MICs of levofloxacin 4 mg/L), which were characterized genetically.
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Materials and methods |
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Bacterial strains
Eighteen clinical isolates of S. pneumoniae with resistance to fluoroquinolones (MICs of levofloxacin 4 mg/L) were used in this study. The strains were collected from isolates from various specimens submitted to the clinical laboratory of Ryukyu University Hospital from January 1994 through December 2004. The isolates were confirmed to be S. pneumoniae by colony morphology, optochin susceptibility and bile solubility, and the presence of the autolysin gene lytA was confirmed by PCR (Wakunaga Pharmaceuticals, Co., Hiroshima, Japan). The bacteria were grown on 5% sheep blood agar (Kyokuto Co., Tokyo, Japan) at 37°C in an atmosphere enriched with 5% CO2. A levofloxacin-susceptible clinical strain, S. pneumoniae WP90, was used for sequencing analysis to compare its amino acid sequence with those of the other strains. The quality control strain S. pneumoniae ATCC 49619 was also used for MIC determination.
Antimicrobial susceptibility testing
Antimicrobial susceptibility was determined by the 2-fold broth microdilution method according to the guidelines of the CLSI (formerly the NCCLS).6 Cation-adjusted Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA) was supplemented with 3% lysed horse blood. Microdilution trays (final volume, 100 µL/well) were inoculated with an automatic MIC-2000 inoculator (Dynatech Laboratories, Inc., Alexandria, VA, USA). Final inocula contained 
5x104 cfu/well. The MIC of each drug was defined as the lowest concentration resulting in the complete inhibition of visible growth after 18 h of incubation. MICs were also determined in the presence and absence of 10 mg/L reserpine (Sigma Aldrich Japan, K. K., Tokyo) to evaluate the presence of an efflux mechanism. A change in MIC of more than 4 times was assumed significant. The following quinolone antimicrobial agents, obtained as laboratory-grade powders from their respective manufacturers, were tested: levofloxacin (Daiichi Pharmaceutical Co., Tokyo, Japan), sparfloxacin (Dainippon Pharmaceutical Co., Osaka, Japan), gatifloxacin (Kyorin Pharmaceutical Co., Tokyo) and sitafloxacin (Daiichi Pharmaceutical Co.).
DNA sequencing and analysis
Mutations in the QRDRs of the gyrA, gyrB, parC and parE genes of fluoroquinolone-resistant strains were investigated by the PCR method. The primer sequences used in this study were described previously.7 Bacterial genomic DNA was prepared from several colonies of S. pneumoniae grown on a blood agar plate by boiling with Chelex-100 (Bio-Rad, Hercules, CA, USA). Subsequently, 5 µL of the extract was added to 50 µL of a PCR solution [1x PCR buffer, 200 µM of each dNTP, 2.5 U of Taq polymerase (Takara Biomedical, Kyoto, Japan) and 0.5 µM of sense and reverse primers]. PCR conditions were as follows: 35 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. The PCR products were electrophoresed on an agarose gel to confirm the presence of the product, and were then purified with a PCR purification kit (Qiagen Sciences Inc., Germantown, MD, USA) to prepare a sequencing template. The sequencing reaction was conducted with a Rhodamine Terminator Cycle Sequencing FS Ready Reaction kit (PE Biosystems, Foster city, CA, USA). The reaction mixtures were placed in a thermal cycler and denatured at 94°C for 2 min. They were then subjected to 25 PCR cycles (94°C for 10 s, 50°C for 5 s and 60°C for 4 min). The nucleotide sequences were determined with an ABI PRISM3100 DNA sequencer.
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Results |
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Sequencing of the region encoding the QRDRs of gyrA, gyrB, parC and parE was carried out to investigate the involvement of gene mutations in fluoroquinolone-resistant clinical isolates. The results of sequencing analysis were reproducible. Table 1 summarizes the substitutions of deduced amino acid sequences within QRDRs of GyrA, ParC and ParE of the 18 levofloxacin-resistant strains. All 18 strains had at least one amino acid substitution in the QRDRs of GyrA and ParC, which included Ser-81
Tyr/Phe and Glu-85Lys in GyrA and Ser-79Phe/Ile/Tyr, Asp-83Tyr, Asn-91Asp, Ser-107Phe, Lys-137Asn and Ala-142Ser in ParC. Among them, 10 strains had amino acid substitutions in both QRDRs of GyrA and ParC. Most isolates had Asp-435Asn/Ile-460Val/Ala-596Thr substitutions in ParE, while no amino acid substitution in GyrB was noted in all isolates. Multiple amino acid substitutions in both GyrA and ParC were detected in 10 strains with levofloxacin MICs of 16 or 32 mg/L.
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The MIC80 of sitafloxacin for levofloxacin-resistant isolates was 0.25 mg/L. The range of MICs of sitafloxacin for isolates highly resistant to levofloxacin (MIC 16–32 mg/L) was 0.125–0.5 mg/L. The addition of reserpine did not change the susceptibility of the strains to quinolones (data not shown). MICs of levofloxacin, sitafloxacin, sparfloxacin and gatifloxacin against the control strain ATCC 49619 were 0.5, 0.032, 0.25 and 0.125 mg/L, respectively.
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Discussion |
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Predominantly, the fluoroquinolone resistance of S. pneumoniae clinical isolates is attributed to amino acid substitutions at positions Ser-81 in GyrA and Ser-79 in ParC to either Phe or Tyr.1,4 The present study showed that the same amino acid substitutions were detected in Japanese levofloxacin-resistant strains, confirming the significant role of these substitutions within QRDRs of GyrA and ParC. The Glu-85
Lys substitution in GyrA was also detected in high-level levofloxacin-resistant strains, suggesting this mutation is associated with the quinolone resistance as well as Ser-81Phe/Tyr. The Ser-79Ile substitution in ParC (strains V7 and HC37) was first detected in this study, and this substitution seemed to be associated with the quinolone resistance. The Lys-137Asn substitution in ParC is detected frequently in levofloxacin-susceptible strains;4 therefore, this mutation may be unrelated to the resistance. Other mutations in ParC (Asn-91Asp, Ser-107Phe and Ala-142Ser) were detected in strains with high resistance to levofloxacin (MICs 16–32 mg/L). The significance of these substitutions is still unclear. Multiple substitutions in ParC may increase the resistance, even when each single substitution would cause no effect. Further studies are required to clarify the significance of these mutations. The Ile-460Val substitution in ParE is prevalent in Japanese isolates less susceptible to fluoroquinolones.8 Amino acid substitutions in ParE detected in the present study (Asp-435Asn, Ile-460Val and Ala-596Thr) may be related to low-level quinolone resistance.
Sitafloxacin, a newer quinolone, is potent against Gram-positive cocci as well as Gram-negative bacilli, and excellent activity against S. pneumoniae has been reported.5 In the present study, this drug had significantly lower MICs for S. pneumoniae with resistance to levofloxacin, compared with the other quinolones tested, and these findings support previous reports showing a similar potency of this drug against levofloxacin-resistant S. pneumoniae with defined multiple mutations within both gyrase A and topoisomerase IV.9,10 Gemifloxacin is also potent against such strains with double mutations both in gyrase A and topoisomerase IV.11 MICs of sitafloxacin were higher for strains with a single Ser-81 mutation in GyrA (strains TZ2–11 and 3568) than those with a single Ser-79 mutation in ParC (strains 2E19, 4426, DS-1, 4511 and V91), suggesting that the primary target of sitafloxacin may be GyrA rather than ParC. The MIC of sitafloxacin for strain VY5 with a Ser-81Tyr substitution in GyrA was lower than those for strains with Ser-81Phe substitutions. Ser-81Tyr may not affect the affinity of GyrA for sitafloxacin. In contrast, Glu-85Lys substitutions in GyrA resulted in higher MICs of sitafloxacin.
In summary, levofloxacin-resistant S. pneumoniae isolated in Japan had multiple amino acid substitutions in QRDRs of GyrA, ParC and ParE, as described previously in other countries. Sitafloxacin was potent against levofloxacin-resistant S. pneumoniae with multiple mutations in QRDRs of gyrase A and topoisomerase IV. Further studies are warranted to evaluate the usefulness of sitafloxacin against the infections caused by levofloxacin-resistant S. pneumoniae.
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Conflict of interest. None to declare.
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References |
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Jolley A, Andrews JM, Brenwald N, et al. (1993) The in-vitro activity of a new highly active quinolone, DU-6859a. J Antimicrob Chemother 32:757–63.
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7
Pestova E, Beyer R, Cianciotto NP, et al. (1999) Contribution of topoisomerase IV and DNA gyrase mutations in Streptococcus pneumoniae to resistance to novel fluoroquinolones. Antimicrob Agents Chemother 43:2000–4.
8 Kawamura-Sato K, Hasegawa T, Torii K, et al. (2005) Prevalence of Ile-460-Val/ParE substitution in clinical Streptococcus pneumoniae isolates that were less susceptible to fluoroquinolones. Curr Microbiol 51:27–30.[CrossRef][ISI][Medline]
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Jones ME, Sahm DF, Martin N, et al. (2000) Prevalence of gyrA, gyrB, parC, and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from worldwide surveillance studies during 1997–1998 respiratory season. Antimicrob Agents Chemother 44:462–6.
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11
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