Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hong, S. G. Articles by Thomson, K. S. Search for Related Content PubMed PubMed Citation Articles by Hong, S. G. Articles by Thomson, K. S.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, April 2007, p. 1512-1514, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.00959-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Vitro Studies with DQ-113 and Comparison Fluoroquinolones To Determine Propensities To Select Resistance in Gram-Positive Cocci

Seong Geun Hong, Ellen Smith Moland, Paul A. Wickman, Jennifer A. Black, Ashfaque Hossain, Nancy D. Hanson, and Kenneth S. Thomson^*

Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, 2500 California Plaza, Omaha, Nebraska 68178

Received 2 August 2006/ Returned for modification 7 September 2006/ Accepted 27 December 2006

Top Abstract Text References

 
DQ-113 was compared in vitro to sitafloxacin, moxifloxacin, levofloxacin, and ciprofloxacin for potential to select mutational resistance in multiresistant staphylococci, pneumococci, and enterococci. Its ability to select less-susceptible mutants varied according to species, being lowest with staphylococci, intermediate with pneumococci, and greatest with enterococci.

Top Abstract Text References

 
DQ-113 is an investigational fluoroquinolone with enhanced anti- gram-positive activity (Fig. 1) (6, 8, 10). We report investigations into its potential to select less-susceptible mutants from clinical isolates of Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus spp.

View larger version (8K): [in this window] [in a new window]

FIG. 1. Structure of DQ-113. (Reprinted from reference 10 with permission.)

Single-step mutational frequencies were determined after exposure of approximately 10⁷ to 10⁹ CFU of five clinical isolates each of methicillin-susceptible S. aureus (MSSA), methicillin- resistant S. aureus (MRSA), penicillin-resistant S. pneumoniae (PRSP), and vancomycin-resistant Enterococcus spp. (VRE) (one E. faecalis and four E. faecium strains) to DQ-113, sitafloxacin, moxifloxacin, ciprofloxacin, and levofloxacin (powders provided by their manufacturers) at the MIC, and two, four, and eight times the MIC in appropriate media for 48 to 72 h at 35°C in air (5% CO₂ for S. pneumoniae) as previously described (1). Mutants were also selected at subinhibitory fluoroquinolone concentrations by the agar gradient plate method of Bryson and Szybalski (2) after five passages on drug-containing media. The MRSA strains were relatively susceptible to fluoroquinolones and not representative of frequently encountered nosocomial strains. Antibiotic susceptibilities were determined by the Clinical and Laboratory Standards Institute (CLSI) agar dilution methodology using CLSI-recommended quality control strains (3). Both fluoroquinolones and the nonfluoroquinolone agents vancomycin, oxacillin, erythromycin, gentamicin, chloramphenicol, tetracycline, and penicillin were tested. Known quinolone resistance determining regions (QRDRs) for the DNA gyrase subunit genes gyrA and gyrB, the topoisomerase IV subunit genes grlA and grlB (S. aureus) and parC and parE (S. pneumoniae), and the promoter regions of the norA and pmrA efflux pump genes were amplified by PCR and sequenced directly. The primer sequences used for amplicon generation and sequencing are shown in Table 1.

View this table: [in this window] [in a new window]

TABLE 1. S. aureus and S. pneumoniae primers for amplicon generation and sequencing

DQ-113 was less effective than sitafloxacin, moxifloxacin, and levofloxacin in preventing the emergence of single step mutants from the PRSP strains. Sitafloxacin selected single-step mutants from only two of the five strains, moxifloxacin and levofloxacin selected single-step mutants from three strains, DQ-113 selected single-step mutants from four strains, and ciprofloxacin selected single-step mutants from all five strains. All mutants were selected at mutational frequencies of 10^–5 to 10^–7. DQ-113 was the most potent agent, with all mutants being susceptible to 0.007 µg/ml, compared to 0.06 µg of sitafloxacin/ml, 0.25 µg of moxifloxacin/ml, 2 µg of levofloxacin/ml, and 8 µg of ciprofloxacin/ml (data not shown). Mutants were selected from all strains after five exposures to subinhibitory drug concentrations and were more resistant. All were inhibited by 0.12 µg of DQ-113/ml, 4 µg of sitafloxacin/ml, 8 µg of moxifloxacin/ml, 64 µg of levofloxacin/ml, and 128 µg of ciprofloxacin/ml (Table 2).

View this table: [in this window] [in a new window]

TABLE 2. Influence of exposure of S. pneumoniae to subinhibitory fluoroquinolone concentrations on activity of DQ-113 and comparator fluoroquinolones

The QRDR sequences of gyrA, gyrB, parC, and parE revealed mutations only in the QRDR of parC and only in 2 of the 20 single-step mutants. No mutations were detected in the promoter of the pmrA gene. Both mutants with QRDR changes had the mutation Ser(79)Tyr. One of these was a double mutant with Glu(120)Asp as its second mutation (data not shown).

DQ-113 was the most effective agent for preventing the emergence of single-step mutants of MSSA and MRSA, with only one mutant selected from the 10 strains compared to mutants being selected from 4 strains by moxifloxacin, 5 strains by sitafloxacin, 7 strains by levofloxacin, and 8 strains by ciprofloxacin. All single-step mutants were susceptible to 0.03 µg of DQ-113/ml, 0.25 µg of sitafloxacin/ml, 0.5 µg of moxifloxacin/ml, 2 µg of levofloxacin/ml, and 8 µg of ciprofloxacin/ml, with frequencies of 10^–6 and 10^–10, with no apparent differences between the fluoroquinolones (data not shown).

Mutants were selected by subinhibitory drug concentrations from all parents with all mutants inhibited by 2 µg of DQ-113/ml, 16 µg of sitafloxacin/ml, 32 µg of moxifloxacin/ml, 256 µg of levofloxacin/ml, and >128 µg of ciprofloxacin/ml. Reduced susceptibility to gentamicin (four mutants with MICs increasing from 0.12 to 1 µg/ml [one mutant], 0.25 to 1 µg/ml [two mutants], and 0.25 to 2 µg/ml [one mutant]) and chloramphenicol (MIC increased from 4 to 16 µg/ml [one mutant]) was detected, and increased susceptibility to erythromycin (0.5 to 0.12 µg/ml [two mutants]) and tetracycline (MIC decreased from 1 to 0.25 µg/ml [1 mutant]) was detected (data not shown).

Mutations in the single-step mutants occurred in the QRDR of grlA in 17 of the 28 mutants, with the most frequent mutation being Ser(80)Phe. Other mutations were Ser(80)Tyr, Glu(84)Lys, and Pro(144)Ser. A Pro(144)Ser mutation occurred outside the defined QRDR of grlA of a ciprofloxacin-selected mutant, which also had a Ser(80)Phe mutation and three point mutations in the 5'untranslated region of the norA gene (data not shown).

Single-step mutants of VRE were selected at frequencies of 10^–5 and 10^–8 from each strain with each fluoroquinolone except sitafloxacin, which selected mutants from four strains. All mutants were susceptible to 2 µg of DQ-113/ml, 4 µg of sitafloxacin/ml, 8 µg of moxifloxacin/ml, and 16 µg of levofloxacin and ciprofloxacin/ml. The mutants selected by subinhibitory concentrations were more resistant than the single-step mutants, being inhibited by 8 µg of DQ-113 and sitafloxacin/ml, 64 µg of moxifloxacin/ml, 128 µg of ciprofloxacin/ml, and 256 µg of levofloxacin/ml. Vancomycin resistance was lost from a ciprofloxacin-selected mutant of E. faecium (MIC decreased from >128 to 2 µg/ml; data not shown).

Overall, DQ-113 was approximately 10-fold or more potent than the comparator fluoroquinolones. Its capability to select less-susceptible mutants varied according to species, being lowest with staphylococci, intermediate with pneumococci, and greatest with enterococci. The clinical relevance of this will be determined by the concentrations that can be safely achieved in serum and tissues. Its propensity to select less-susceptible mutants of PRSP in vitro may possibly be offset clinically if its high potency inhibits and prevents mutants from emerging during therapy. The absence of target mutations in many pneumococcal mutants suggested the involvement of another resistance mechanism. Although no mutations were detected in the promoter of the PmrA efflux pump (4), other types of mutations may have been responsible for increased PmrA activity, or other resistance mechanisms may have been involved, e.g., other efflux pumps (5) or ABC transporters such PatA and PatB (7, 9).

None of the study fluoroquinolones was highly active against the mutants of vancomycin-resistant E. faecium, suggesting that they would be unsuitable for monotherapy of serious infections caused by this pathogen.

 
We thank Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan, for the grant supporting this research.

We thank Daiichi Pharmaceutical Co. for supplying DQ-113 and sitafloxacin powders and P. Appelbaum (Pennsylvania State University, Milton S. Hershey Medical Center, Hershey, PA) for two strains of PRSP with wild-type QRDRs. We also thank Thomas J. Lockhart for excellent technical assistance.

 
* Corresponding author. Mailing address: Creighton University School of Medicine, Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-4096. Fax: (402) 280-1875. E-mail: kstaac{at}creighton.edu

Published ahead of print on 12 January 2007.

Top Abstract Text References

 

1
1. Brenwald, N. P., M. J. Gill, and R. Wise. 1998. Prevalence of a putative efflux mechanism among fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42:2032-2035.[Abstract/Free Full Text]
2
2. Bryson, V., and W. Szybalski. 1952. Microbial Selection. I. Science 116: 45-48.[Free Full Text]
3
3. Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing; 15th informational supplement M100-S15. Clinical and Laboratory Standards Institute, Wayne, PA.
4
4. Gill, M. J., N. P. Brenwald, and R. Wise. 1999. Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 43:187-189.[Abstract/Free Full Text]
5
5. Hoskins, J., W. E. Alborn, Jr., J. Arnold, L. C. Blaszczak, S. Burgett, B. S. DeHoff, S. T. Estrem, L. Fritz, D. J. Fu, W. Fuller, C. Geringer, R. Gilmour, J. S. Glass, H. Khoja, A. R. Kraft, R. E. Lagace, D. J. LeBlanc, L. N. Lee, E. J. Lefkowitz, J. Lu, P. Matsushima, S. M. McAhren, M. McHenney, K. McLeaster, C. W. Mundy, T. I. Nicas, F. H. Norris, M. O'Gara, R. B. Peery, G. T. Robertson, P. Rockey, P. M. Sun, M. E. Winkler, Y. Yang, M. Young-Bellido, G. Zhao, C. A. Zook, R. H. Baltz, S. R. Jaskunas, P. R. Rosteck, Jr., P. L. Skatrud, and J. I. Glass. 2001. Genome of the bacterium Streptococcus pneumoniae strain R6. J. Bacteriol. 183:5709-5717.[Abstract/Free Full Text]
6
6. Kaneko, Y., K. Yanagihara, Y. Miyazaki, K. Tsukamoto, Y. Hirakata, K. Tomono, J. Kadota, T. Tashiro, I. Murata, and S. Kohno. 2003. Effects of DQ-113, a new quinolone, against methicillin- and vancomycin-resistant Staphylococcus aureus-caused hematogenous pulmonary infections in mice. Antimicrob. Agents Chemother. 47:3694-3698.[Abstract/Free Full Text]
7
7. Marrer, E., K. Schad, A. T. Satoh, M. G. Page, M. M. Johnson, and L. J. Piddock. 2006. Involvement of the putative ATP-dependent efflux proteins PatA and PatB in fluoroquinolone resistance of a multidrug-resistant mutant of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 50:685-693.[Abstract/Free Full Text]
8
8. Otsu, Y., K. Yanagihara, Y. Fukuda, Y. Miyazaki, K. Tsukamoto, Y. Hirakata, K. Tomono, J. I. Kadota, T. Tashiro, I. Murata, and S. Kohno. 2003. In vivo efficacy of a new quinolone, DQ-113, against Streptococcus pneumoniae in a mouse model. Antimicrob. Agents Chemother. 47:3699-3703.[Abstract/Free Full Text]
9
9. Robertson, G. T., T. B. Doyle, and A. S. Lynch. 2005. Use of an efflux-deficient Streptococcus pneumoniae strain panel to identify ABC-class multidrug transporters involved in intrinsic resistance to antimicrobial agents. Antimicrob. Agents Chemother. 49:4781-4783.[Abstract/Free Full Text]
10
10. Tanaka, M., E. Yamazaki, M. Chiba, K. Yoshihara, T. Akasaka, M. Takemura, and K. Sato. 2002. In vitro antibacterial activities of DQ-113, a potent quinolone, against clinical isolates. Antimicrob. Agents Chemother. 46:904-908.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, April 2007, p. 1512-1514, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.00959-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hong, S. G. Articles by Thomson, K. S. Search for Related Content PubMed PubMed Citation Articles by Hong, S. G. Articles by Thomson, K. S.