University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai Japan

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1 AAC Accepts, published online ahead of print on 1 May 009 Antimicrob. Agents Chemother. doi:10.11/aac Copyright 009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. In vitro activity of garenoxacin against Streptococcus pneumoniae mutants with characterized resistance mechanisms Running title- The activity of garenoxacin against S. pneumoniae mutants Authors- Kazuko Yamamoto 1, Katsunori Yanagihara*, Kazuyuki Sugahara, Yoshifumi Imamura 1, Masafumi Seki 1, Koichi Izumikawa 1, Hiroshi Kakeya 1, Yoshihiro Yamamoto 1, Yoichi Hirakata 3, Shimeru Kamihira, and Shigeru Kohno 1 Institutions- 1 Department of Molecular Microbiology and Immunology, Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1--1 Sakamoto, Nagasaki City 5-501, Japan Downloaded from on July 1, 01 by guest 3 Department of Infection Control and Laboratory Diagnostics, Internal Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 90-5 Japan 1

2 Corresponding Author- Katsunori Yanagihara, M.D., Ph.D. Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1--1 Sakamoto, Nagasaki City 5-501, Japan Phone: FAX: Downloaded from on July 1, 01 by guest

3 1 ABSTRACT We evaluated the potency of garenoxacin in selecting resistant Streptococcus pneumoniae 3 mutants, by determining its mutant prevention concentration using strains with and without topoisomerase genes mutations, and compared its potency to that of other quinolones Garenoxacin had a significantly greater potency against pneumococci, including strains containing topoisomerase mutations-containing strains. Genetic analysis of the S. pneumoniae mutants created by garenoxacin revealed that the gyra gene was a primary target of garenoxacin. Downloaded from on July 1, 01 by guest 3

4 1 The emergence of Streptococcus pneumoniae strains with resistance to the β-lactams and macrolides has complicated the treatment of pneumococcal respiratory tract infections and 3 created a need for new antibiotic agents. Recently developed compounds within the quinolone group have demonstrated enhanced potency against S. pneumoniae. In particular, agents such as moxifloxacin, gatifloxacin, and levofloxacin have been recommended and used for therapy (1). However, S. pneumoniae strains with quinolone resistance have been observed in several countries (3,9,11,16,19). Furthermore, evidence suggests that increased usage of these compounds could lead to the development of further resistance and treatment failures (5,,0). Quinolone resistance in S. pneumoniae is mediated by amino acid substitutions within the quinolone resistance determining regions (QRDRs) of DNA gyrase (GyrA or GyrB) and/or topoisomerase IV (ParC or ParE), sometimes in combination with efflux (1,1,1). The mutant prevention concentration (MPC) of a drug is the concentration that prohibits the groh of mutants from a susceptible population of more than cells, and determining the MPC is a novel approach for evaluating quinolone potency (, 6). Additionally, the mutant selection window (MSW), which is defined as the range between the MIC and MPC, provides Downloaded from on July 1, 01 by guest 16 a means for defining the ability of an antibiotic to prevent the emergence of mutants (). 1 Garenoxacin (GAR) is a novel des-f(6) quinolone with a broad spectrum of activity against 1 respiratory tract pathogens with elevated or resistant-level fluoroquinolone MIC values, 19 including S. pneumoniae (,10,13,1). The aim of this study was to evaluate the potency of

5 1 GAR in selecting for resistant S. pneumoniae, and compare it to other quinolones, by determining the MPC and the MSW, using strains with and without mutations in the QRDRs 3 of topoisomerase genes. Additionally, to determine the targets of GAR, we examined the intrinsic development of resistant S. pneumoniae mutants that were created by GAR exposure, with detailed evaluation of additional QRDR mutations and efflux. A total of eight S. pneumoniae clinical isolates were used in this study. The QRDR genetic backgrounds and quinolone MICs of the isolates are summarized in Table 1. All strains were wild-type for GyrB and ParE. Isolates W001 and W00 were quinolone-susceptible isolates with wild-type ParC and GyrA. Isolate S001 had an Asp3Asn mutation in ParC with wild-type GyrA. Isolate S00 had a Ser9Phe mutation in ParC with wild-type GyrA. Isolates S003 and S00 had single GyrA mutations (Ser1Phe for S003 and Gly5Asn for S00). Isolates D001 and D00 had both GyrA and ParC mutations (D001, Ser9Phe in ParC and Gly5Lys in GyrA; D00, Ser9Phe in ParC and Ser1Phe in GyrA). All the strains were exposed to,,, 16, 3, and 6x the MICs for ciprofloxacin (CIP), levofloxacin (LVX), gatifloxacin (GAT), moxifloxacin (MXF), and GAR for - hr at Downloaded from on July 1, 01 by guest 16 3 C in 5% CO. The MPCs were measured using a procedure previously described (). 1 Briefly, 00µL of a culture containing 10 log 10 CFU/mL was applied to Mueller Hinton II 1 Agar plates containing 5% sheep blood and a drug, at various concentrations. MPCs were 19 recorded as the lowest concentration of the antibiotic that prevented bacterial colony 5

6 1 formation after hr. All determinations were done in duplicate, and the results were identical. Genomic DNA was extracted from mutants growing on the plates (a maximum of eight 3 mutants per plate were cultured individually, see Table ) using a QIAGEN Blood Mini Kit (QIAGEN, Hilden, Germany). All mutant DNA extracts were screened for QRDR mutations of the parc and gyra genes using a relatively new PCR-melting curve analysis (PCR-MCA) method, which we reported previously (). Briefly, probes labeled with LC-Red 60 and fluorescein were used with designated primers () that targeted four QRDR positions (Ser9 and Asp3 of the parc gene and Ser1 and Gly5 of the gyra gene). PCR was performed in a 0 µl volume containing 5 µl of DNA template, µl of LightCycler 0 Genotyping Master (Roche Diagnostics, Basel, Switzerland), 3 mm MgCl, 0. µm of each probe, and 0.5 µm of each primer. Thermal cycling was performed with an initial hold for 10 min at 95 C, followed by 35 cycles of 5 sec at 95 C, 10 sec at 55 C and 1 sec at C. A melting curve was generated by cooling to 0 C for 30 sec, followed by heating to 0 C at a rate of.0 C/s. The PCR-MCA assay was performed using LightCycler0 analysis software (Roche Diagnostics, Basel, Switzerland). The total assay time was approximately 1 hr. Nucleotide Downloaded from on July 1, 01 by guest 16 mismatches between the sequence and the hybridization probe resulted in a lower melting 1 temperature (T m ) for the mutant than for the wild-type. The assay made it possible to 1 quickly and easily differentiate a mutant strain from a wild-type strain. The QRDR sequences 19 for the topoisomerase genes (parc, pare, gyra, and gyrb) of the mutant strains were 6

7 1 confirmed at the nucleotide level by all directly sequenced, using the BigDye Terminator ver.3.1 Sequencing Standard Kit and an ABI PRISM TM 310 Genetic Analyzer (both by 3 Applied Biosystems, CA, USA) with published primers (1). The QRDR DNA sequencing results were compared with the R6 strain (GenBank accession no. NC_00309). MIC determination was done in parallel, both with and without an efflux inhibitor (10 µg of reserpine/ml). Efflux was considered to be present when a -fold reduction in the MIC was observed. The MPC and MSW ranges of the S. pneumoniae isolates are summarized in Figure 1. The MPCs that are shown were averaged between the W001 and W00, S001 and S00, S003 and S00, D001 and D00 strains. The MPC order in the S. pneumoniae wild-type strains was CIP ( µg/ml) > LVX ( µg/ml) > GAT (0.5 µg/ml) MXF (0.5 µg/ml) > GAR (0.1 µg/ml). GAR potency was 16- to 6-fold greater than LVX or CIP, and -fold greater than GAT or MXF. GAR also had a significantly narrower MSW than the other quinolones, even against strains with QRDR mutations. Between the strains, the MSW range (from lowest to highest) was the wild-type strains (W001 and W00) < strains with a single Downloaded from on July 1, 01 by guest 16 QRDR mutation (S001-00) < strains with two QRDR mutations (D001 and D00). LVX, 1 GAT, and GAR had narrower MSWs for strains with single parc mutations than for strains 1 with single gyra mutations, while CIP and MXF showed the opposite. The number of 19 pneumococcal mutants that were created by GAR exposure and cultured individually is

8 1 shown in Table. The QRDR wild-type strains generated mutants with GAR exposures of 1 to MIC, while strains with QRDR mutations generated mutants from 1 to 16-3 MIC. 3 The QRDR genetic changes in the S. pneumoniae mutants that were generated by GAR exposure are shown in Table 3. The PCR-MCA assays on the wild-type strain mutants (W001 and W00) created by GAR exposure revealed a high frequency of additional gyra mutations. In strains with single parc mutations (S001 and S00), a high percentage of additional gyra mutations in codon 1 was seen. On the other hand, in the strains with single gyra mutations (S003 and S00), additional parc mutations in codon 9 were seen at a high percentage. Strains with two QRDR mutations (D001 and D00) had additional gyra mutations resulting from GAR exposure. None of the mutants created by GAR exposure showed the existence of efflux, by MIC determination with reserpine. DNA sequence analysis of the mutants without gyra or parc mutations after GAR exposure, showed two W001 mutants and a W00 mutant each with an additional pare mutation (Asp35Asn). In addition, two W001 mutants, a W00 mutant, two S003 mutants, and a S00 mutant had an additional parc mutation (Lys13Asn). As for the gyrb gene, only one S001 mutant had an Asp35Asn Downloaded from on July 1, 01 by guest 16 mutation (data not shown). 1 In the present study, the MIC results showed that GAR was potently active against 1 pneumococci, including strains containing QRDR mutations, at a level significantly greater 19 than other quinolones. In addition, the significantly narrower MSW range and the low MPC

9 1 values, demonstrated that it was more difficult for pneumococci to acquire resistance to GAR compared to other quinolones. We used a relatively new PCR-MCA assay to make a detailed 3 genetic analysis of QRDR mutations from a vast numbers of mutants. Previous studies had difficulties analyzing large numbers of mutants, due to the time-consuming process of culturing individually and analyzing their DNA sequences. A high proportion of the mutants that were derived from isolates with single parc mutations acquired secondary gyra mutations and became highly resistant to all of the quinolones we used in our study. With the wild-type pneumococcal strains, a high percentage (50 % of mutants from strain W001 and 6% of mutants from strain W00) of additional GyrA mutations were seen in the mutants exposed to GAR. This may indicate that GAR has a more balanced affinity for the two target enzymes, with a slight initial preference for GyrA as an initial target. A total of out of 31 mutants (31%) did not have additional detectable gyra or parc mutations in this study. We expected that these remaining mutants would have acquired other additional QRDR mutations (pare or gyrb) or efflux, however, only ten mutants (1%) were detected by sequencing the topoisomerase genes. Therefore, these mutants may have acquired other resistance Downloaded from on July 1, 01 by guest 16 mechanisms, such as plasmid-based resistance. Further studies will be needed to clarify the 1 other resistance mechanisms of pneumococci to GAR. Although quinolone resistance in S. 1 pneumoniae isolates remains low with susceptibility based on MIC at or below the 19 susceptibility breakpoint, it may be of note the findings of Lim et al (15) which determined 9

10 1 the substantial percentage (60%) of S. pneumoniae isolates with MICs at µg/ml to LVX contained first step QRDR mutations. The opportunities to treat respiratory tract infections 3 with quinolone have increased, and the potential for forming resistance should thus be considered when specific quinolones are selected for treatment. Including MPCs as part of a dosing strategy may be one method for limiting the selection of quinolone-resistant mutants and preserving this class of antibiotics. In conclusion, the novel des-f(6) quinolone, garenoxacin, showed a low MPC value and a narrow MSW for QRDR mutation-containing pneumococcal strains, suggesting that garenoxacin will be useful for minimizing the selection of quinolone-resistant mutants with S. pneumoniae. Downloaded from on July 1, 01 by guest 10

11 References 1. Bast DJ, Low DE, Duncan CL, Kilburn L, Mandell LA, Davidson RJ, and Azavedo de JC.S Fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae: contributions of type II topoisomerase mutations and efflux to levels of resistance. Antimicrob Agents Chemother. : Blondeau JM, Zhao X, Hansen G, and K. Drlica Mutant prevention concentrations of fluoroquinolones for clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother. 5: Chen, D. K., A. McGeer, J. C. de Azavedo, and Low, D.E Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada. N. Engl. J. Med. 31: Christiansen KJ, Bell JM, Turnidge JD, and Jones RN. 00. Antimicrobial activities of garenoxacin (BMS 56) against Asia-Pacific region clinical isolates from the SENTRY program, 1999 to 001. Antimicrob Agents Chemother. : Davidson R., R. Cavalcanti, J. L. Brunton, D. J. Bast, J. C. Azavedo, P. Kibsey, C. Downloaded from on July 1, 01 by guest Fleming, and Low, D. E. 00. Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med. 36: Dong Y., X. Zhao, Kreiswirth, B. N., and Drlica K Mutant prevention concentration as a measure of antibiotic potency: studies with clinical isolates of 11

12 Mycobacterium tuberculosis. Antimicrob. Agents Chemother. : Endimiani A., G. Brigante, A. A. Bettaccini, F. Luzzaro, P. Grossi, and A. Q. Toniolo Failure of levofloxacin treatment in community-acquired pneumococcal pneumonia. BMC Infect Dis. 5:106.. Fukushima K. Y., Hirakata Y., Sugahara K, Yanagihara K, Kondo A, Kohno S, and Kamihira S Rapid screening of topoisomerase gene mutations using a novel melting curve analysis for early-warning of fluoroquinolone-reistant Streptococcus pnuemoniae emergence. J. Clin. Microbiol. : Goldsmith, C. E., J. E. Moore, P. G. Murphy, and J. E. Ambler Increased incidence of ciprofloxacin resistance in penicillin-resistant pneumococci in Northern Ireland. J. Antimicrob. Chemother. : Grohs P, Houssaye S, Aubert A, Gutmann L, and Varon E In vitro activities of garenoxacin (BMS 56) against Streptococcus pneumoniae, viridans group streptococci, and Enterococcus faecalis compared to those of six other quinolones. Antimicrob Agents Chemother. : Downloaded from on July 1, 01 by guest 11. Ho, P. L., R. W. Yung, D. N. Tsang, T. L. Que, M. Ho, W.H. Seto, T. K. Ng, W. C. Yam, and W. W. Ng Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong multicentre study in 000. J. Antimicrob. Chemother. :

13 1. Janoir C, Zeller V, Kitzis MD, Moreau NJ, and Gutmann L High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parc and gyra. Antimicrob Agents Chemother. 0: Jones RN, Fritsche TR, Sader HS, and Stilwell MG. 00. Activity of garenoxacin, an investigational des-f(6)-quinolone, tested against pathogens from community-acquired respiratory tract infections, including those with elevated or resistant-level fluoroquinolone MIC values. Diagn Microbiol Infect Dis. 5: Jorgensen JH, Weigel LM, Ferraro MJ, Swenson JM, and Tenover FC Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyra, parc, and pare loci. Antimicrob Agents Chemother.3: Lim S., D. Bast, A. McGeer, J. de Azavedo, and D. E. Low Antimicrobial susceptibility breakpoints and first-step parc mutations in Streptococcus pneumoniae: redefining fluoroquinolone resistance. Emerg Infect Dis. 9: Linares, J., A. G. de la Campa, and R. Pallares Fluoroquinolone resistance in Downloaded from on July 1, 01 by guest Streptococcus pneumoniae. N. Engl. J. Med. 31:156-15; author reply, Low, D. E. 00. Quinolone resistance among pneumococci: therapeutic and diagnostic implications. Clin. Infect. Dis. (Suppl. ):S Morrissey, I., and J. George Activities of fluoroquinolones against Streptococcus 13

14 pneumoniae type II topoisomerase purified as recombinant proteins. Antimicrob. Agents Chemother. 3: Pankuch, G. A., B. Bozdogan, K. Nagai, A. Tambic-Andrasevic, S. Schoenwald, T. Tambic, S. Kalenic, S. Plesko, N. K. Tepes, Z. Kotarski, M. Payerl-Pal, and P. C. Appelbaum. 00. Incidence, epidemiology, and characteristics of quinolone-nonsusceptible Streptococcus pneumoniae in Croatia. Antimicrob. Agents Chemother. 6: Trallero E. P., J. M. Marimon, L. Iglesias, and J. Larruskain Fluoroquinolone and macrolide treatment failure in pneumococcal pneumonia and selection of multidrug-resistant isolates. Emerg Infect Dis. 9: Zhanel GG, Palatnick L, Nichol KA, Bellyou T, Low DE, and Hoban DJ Antimicrobial resistance in respiratory tract Streptococcus pneumoniae isolates: results of the Canadian Respiratory Organism Susceptibility Study, 199 to 00. Antimicrob Agents Chemother. : Zhao X, and K. Drlica. 00. Restricting the selection of antibiotic-resistant mutant Downloaded from on July 1, 01 by guest bacteria: measurement and potential use of the mutant selection window. J Infect Dis. 15:

15 Table 1. S. pneumoniae strains W001 W00 S001 S00 S003 D001 QRDR genetic background a Asp3Asn Ser9Phe Gly5Lys MIC (µg/ml) parc gyra CIP LVX GAT MXF Ser9Phe Ser1Phe Abbreviation: a, wild type. CIP, ciprofloxacin; LVX, levofloxacin; GAT, gatifloxacin; MXF, moxifloxacin; GAR, garenoxacin S00 Gly5Asn D00 Ser9Phe Ser1Phe GAR Downloaded from on July 1, 01 by guest

16 Table. S. pneumoniae strains W001 W00 S001 S00 GAR MIC (µg/ml) No. of cultured mutants (n=31) GAR concentrations ( MICs) total Downloaded from S003 S00 D001 D Abbreviation: GAR, garenoxacin on July 1, 01 by guest

17 Table 3. S. pneumoniae strains W001 W00 S001 S00 S003 S00 D001 D00 QRDR genetic background parc a Asp3Asn Ser9Phe Ser9Phe gyra ParC9 0 / (0%) * 0 / (0%) 0 / 31 (0%) * no. of mutants with QRDR mutations / no. of isolated mutants (%) Ser1Phe Ser1Phe ** presented mutations with original isolates Abbreviations: a, wild type. QRDR mutations of mutants (n=31) ParC3 0 / (0%) 0 / (0%) ** 0 / 33 (0%) 1 / 3 (65%) 0 / 3 (0%) 5 / (1%) GyrA1 16 / (6%) 0/ (0%) / 31 (%) GyrA5 1 / (50%) 3 / (1%) 0 / 31 (0%) 16 / 33 (%) 3/ 33 (9%) 0 / 3 (0%) Ser1Phe 1 / 3 (56%) 0 / 3 (0%) 0 / 3 (0%) Ser9Phe Gly5Lys / 31 (3%) / 31 (0%) 1 / (50%) Downloaded from on July 1, 01 by guest

18 Figure 1. GAR MXF GAT LVX CIP ( ) (0.06 1) (0.06 ) (0.5 ) ( ) (0.1 ) (0.5 ) ( - 16) ( ) (0.5 ) (0.5 ) (0.5 - ) ( - 16) (1-16) (0.5 - ) W001 & W00 (1-3) 1 56 concentrations (µg/ml) (16-1) ( - 1) (1-6) (3-56) S001 & S00 S003 & S00 D001 & D00 Downloaded from on July 1, 01 by guest

19 Figure Legends Figure1. Mutant prevention concentrations and mutant selection windows of S. pneumoniae strains. Abbreviations: CIP, ciprofloxacin; LVX, levofloxacin; GAT, gatifloxacin; MXF, moxifloxacin; and GAR, garenoxacin. Horizontal axis represents concentrations of quinolones. White bar, striped bar, dotted bar, and black bar represent wild type strain (W001 and W00), parc single mutation-containing strain (S001 and S00), gyra single mutation-containing strain (S003 and S00), and strain with both parc and gyra mutations (D001 and D00), respectively. The left side of the bars, right end of the bars, and width of the bars represent minimal inhibitory concentration (MIC), mutant prevention concentration (MPC), and mutant selection window (MSW), respectively. The quinolone MIC (µg/ml)-mpc (µg/ml) values of each bar (white, striped, dotted, and black) are as follows: CIP (0.5-, -1, 1-6, and 3-56), LVX (0.5-,1-16,1-3,and 16-1), GAT ( , 0.5-, 0.5-, and -16), MXF ( , 0.1-, 0.5-, and -16), and GAR ( , , 0.06-, and 0.5-), as putted in the brackets beside the bars.. Downloaded from on July 1, 01 by guest

ACCEPTED. Goarant, 2 and S. Le Hello 1* Institut Pasteur de Nouvelle-Calédonie, Laboratoire d Epidémiologie Moléculaire, 1

ACCEPTED. Goarant, 2 and S. Le Hello 1* Institut Pasteur de Nouvelle-Calédonie, Laboratoire d Epidémiologie Moléculaire, 1 AAC Accepts, published online ahead of print on August 00 Antimicrob. Agents Chemother. doi:0./aac.000-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights