Activity of clinafloxacin, compared with six other quinolones, against Acinetobacter baumannii clinical isolates

Jordi Vilaa,*, Anna Riberaa, Francesc Marcoa, Joaquim Ruiza, Josep Mensaa, Josep Chavesb, Gonzalo Hernandezb and M. Teresa Jimenez De Antaa

a Institut Clinic d'Infeccions i Immunologia, IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Villarroel, 170, 08036 Barcelona; b Parke-Davis, S.L., Poligono Industrial Manso Mateu, s/n, 08820 El Prat de Llobregat, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The in vitro activity of clinafloxacin was studied in comparison with ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, sparfloxacin and trovafloxacin against Acinetobacter baumannii clinical isolates. Clinafloxacin showed a MIC90 of 4 mg/L, whereas the remaining quinolones showed a MIC90 equal to or higher than 16 mg/L. MIC50 determination in the presence of reserpine resulted in a two-fold decrease, except for trovafloxacin, which decreased four-fold, and for moxifloxacin and nalidixic acid, which did not change. The effect of reserpine was most pronounced among strains with a low level of resistance to quinolones. The MIC of clinafloxacin for strains with no mutation in either gyrA or parC genes ranged from 0.008 to 0.25 mg/L. In strains with a single mutation at amino acid codon Ser83 of the gyrA gene, the MIC of clinafloxacin ranged from 0.12 to 1 mg/L, whereas strains with a double mutation, one in the gyrA gene and another in the parC gene, showed a range of MIC of clinafloxacin from 1 to 8 mg/L. Therefore, clinafloxacin shows good activity against strains carrying a single mutation in the gyrA gene, and hence a second mutation is required for the microorganism to express resistance.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the last few years the importance of Acinetobacter baumannii in nosocomial infections has been steadily rising.1–3 Several outbreaks, many of them in intensive care units, caused by multiply resistant strains of Acinetobacter spp. have been documented.4–10

Previously, quinolones had good activity against Acinetobacter strains11–14 even compared with expanded-spectrum cephalosporins and aminoglycosides. However, resistance to these antibiotics has rapidly emerged in clinical isolates.15–18 Multiply resistant A. baumannii infections are usually treated with imipenem or sulbactam.19 However, recent data show an emergence of resistance even to these antimicrobial agents.20 Colistin is among the few antimicrobial agents that can still be used to treat infections caused by multiresistant strains, but this antibiotic can cause nephrotoxicity, neuromuscular blockade and neurotoxicity.20 Thus, there is a need to find treatment alternatives and the new fluoroquinolones may be a therapeutic option in the treatment of severe infections caused by multidrug-resistant A. baumannii.

New fluoroquinolones are broad-spectrum antibacterial agents that present an enhanced activity in comparison with old quinolones.21 The protein targets for quinolones are type II topoisomerases (DNA gyrase and topoisomerase IV).22 Both are tetrameric enzymes with two A subunits and two B subunits, encoded by the gyrA and gyrB genes, respectively, in the case of DNA gyrase, and by the parC and parE genes in the case of topoisomerase IV.22,23 There is a region in these genes that is known as the quinolone resistance determining region (QRDR), where mutations associated with the acquisition of quinolone resistance have been located.22 The mutations that play the most important role in the acquisition of resistance are located in the QRDR of the gyrA and parC genes.22,24 Mutations affecting the QRDR of the gyrB gene seem to be more frequent in quinolone-resistant strains obtained in vitro25 than in clinical isolates.26,27

Mutations affecting the parE gene are extremely unusual among clinical isolates of Gram-negative microorganisms,28,29 although mutations in in vitro quinolone-resistant strains of Escherichia coli have been described by Breines et al.30

Another mechanism of resistance to quinolones, different from alterations in target proteins, is a decrease in the accumulation of the quinolone, both by decrease in permeability and/or by an increase in the active efflux of the antibiotic. These mechanisms have not been studied in depth in Acinetobacter.24

Reserpine is a well established inhibitor of efflux pumps among Gram-positive microorganisms,31 and has recently shown its ability to inhibit an active efflux system in Bacteroides fragilis.32,33 Moreover, previous results obtained in our laboratory show its potential to act concomitantly with quinolones against other non-fermenting Gram-negative microorganisms such as Stenotrophomonas maltophilia.34

The main aim of the present study was to compare the activity of clinafloxacin, a novel fluoroquinolone, against clinical isolates of A. baumannii with other quinolones both in the presence and absence of reserpine, and to correlate their activity with mechanisms of resistance.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial isolates

A total of 42 epidemiologically unrelated isolates of A. baumannii were recovered from different biological samples, mainly respiratory secretions, submitted to the Clinical Laboratory of Microbiology of the Hospital Clinic of Barcelona, Spain and to other hospitals in Spain (La Princesa, Madrid; Doce de Octubre, Madrid; Ramon y Cajal, Madrid; San Pablo, Barcelona; Vall D'Hebron, Barcelona; Virgen del Rocío, Sevilla; Virgen de la Macarena, Sevilla; San Joan, Reus and Virgen del Pino, Canary Islands). Isolates were identified as A. baumannii using standard biochemical procedures following the criteria of Bouvet & Grimont.35

Antimicrobial susceptibility testing

Susceptibility testing was carried out using a broth microdilution assay, in either the absence or the presence of reserpine at a concentration of 25 mg/L, in accordance with the guidelines established by the National Committee for Clinical Laboratory Standards.36 An inoculum of 5 x 10 5 cfu/mL of each isolate was inoculated on to freshly prepared medium containing serial dilutions of ciprofloxacin and moxifloxacin (Bayer, Leverkusen, Germany), nalidixic acid (Sigma, St Louis, MO, USA), levofloxacin (Hoechst Marion Roussell, Romainville, France), sparfloxacin, (Rhone-Poulenc, Vitry, France), trovafloxacin (Pfizer Ltd, Sandwich, UK) and clinafloxacin (Parke-Davis, Ann Arbor, MI, USA). The quality control strains used were: E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212.

The breakpoints proposed by the NCCLS36 were used for ciprofloxacin (S <= 1 mg/L; R >= 4 mg/L); levofloxacin (S <= 2 mg/L; R >= 8 mg/L); and nalidixic acid (S <= 16 mg/L; R >= 32 mg/L), whereas the breakpoints proposed for MENSURA37 (Mesa Española de Normalización de la Sensibilidad y Resistencia a los Antimicrobianos) were used for the quinolones that do not possess a breakpoint established by the NCCLS,36 as was the case for sparfloxacin (S <= 1 mg/L; R >= 4 mg/L); clinafloxacin (S <= 1 mg/L; R >= 4 mg/L); moxifloxacin (S <= 2 mg/L; R >= 4 mg/L); and trovafloxacin (S <= 1 mg/L; R >= 4 mg/L).

To evaluate the intrinsic activity of reserpine, the MIC for this agent was determined following the procedure mentioned above.

Amplification and DNA sequencing of the QRDR of the gyrA and parC genes

The PCR amplification of the QRDR of the gyrA and parC genes was carried out using the primers and following the conditions previously described.38,39 PCR was carried out using a DNA thermal cycler 480 (Perkin-Elmer Cetus, Emeryville, CA, USA). Amplified DNA products were resolved by electrophoresis in agarose gels containing 0.5 mg of ethidium bromide per litre. The PCR product was recovered from the agarose gel and purified with the Concert Rapid Purification System according to the manufacturer's instructions (Gibco-BRL, Life Technologies Inc., Gaithersburg, MD, USA). The sample was then directly processed for DNA sequencing using the dRhodamine Terminator Cycle Sequencing kit and was analysed in an automatic DNA sequencer (Abi Prism 377; Perkin-Elmer).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The susceptibility of 42 clinical isolates of A. baumanni was determined either in the presence or in the absence of reserpine and the calculated MIC50 and MIC90 of different quinolones are shown in Table 1Go. Resistant and intermediate strains were classified together as resistant. Clinafloxacin showed better activity than ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, sparfloxacin and trovafloxacin, with a MIC90 of 4 mg/L of clinafloxacin versus a MIC90 equal to or higher than 16 mg/L of the remaining quinolones. Overall, 55% of the analysed clinical isolates were resistant to clinafloxacin, whereas 57% were resistant to sparfloxacin and trovafloxacin and 60, 62, 81 and 81% were resistant to moxifloxacin, levofloxacin, ciprofloxacin and nalidixic acid, respectively (Table 1Go). When reserpine was added, the percentage of resistance decreased in all cases, except for levofloxacin. Clinafloxacin remained the most active, with 43% resistance, whereas 55% of the strains were resistant to sparfloxacin, trovafloxacin and moxifloxacin. The resistance levels to ciprofloxacin and nalidixic acid decreased to 76 and 79%, respectively (Table 1Go).


View this table:
[in this window]
[in a new window]
 
Table 1. In vitro activity of seven quinolones against 42 A. baumannii clinical isolates in the presence and absence of reserpine
 
A two-fold decrease in the MIC50 of ciprofloxacin, levofloxacin, sparfloxacin and clinafloxacin was observed when it was determined in the presence of reserpine, whereas the MIC50 of trovafloxacin decreased four-fold, and of nalidixic acid and moxifloxacin the MIC50 did not change (Table 1Go). Nineteen (45.2%) out of 42 clinical isolates of A. baumannii showed a decrease in the MIC of at least one of the quinolones when the MIC was calculated in the presence of reserpine. The effect of the reserpine on the MIC of quinolones was most pronounced among the strains with lower MIC values, including eight out of nine strains (88.9%) with a ciprofloxacin MIC of <4 mg/L. However, in only two strains (10.5%) of the 19 strains affected by reserpine was the MIC of nalidixic acid decreased in the presence of this compound. The 19 strains varied depending on the quinolone(s) that was affected by this inhibitor (Table 2Go). Only a decrease of at least four-fold was considered to be a real effect of reserpine. The maximal decrease in the MIC, 16-fold for clinafloxacin, was observed in a strain having a MIC of clinafloxacin of 0.5 mg/L in the absence and 0.03 mg/L in the presence of reserpine. In this strain, the MIC of ciprofloxacin, levofloxacin and sparfloxacin also decreased four-fold.


View this table:
[in this window]
[in a new window]
 
Table 2. Fold reduction in quinolone MICs for clinical isolates of A. baumannii in the presence of reserpine
 
To measure any possible intrinsic antibacterial activity of reserpine, the MIC of this compound was determined. The MIC of reserpine was >256 mg/L for all the strains included in this study.

The correlation between the mutations in the gyrA and parC genes and the MIC of ciprofloxacin and clinafloxacin are presented in Table 3Go. Twenty-two strains had a double mutation, a mutation at the amino acid codon Ser83 in the gyrA gene, which generated a change from Ser to Leu, plus a mutation at the amino acid codon Ser80 of the parC gene, which also produced a substitution to Leu in all of these strains. The range of MICs of ciprofloxacin for these strains was from 32 to 256 mg/L and of clinafloxacin was from 1 to 8 mg/L.


View this table:
[in this window]
[in a new window]
 
Table 3. Association between mutations in the gyrA and parC genes of clinical isolates of A. baumannii and MICs of ciprofloxacin and clinafloxacin
 
One strain had a double mutation at amino acid codon Ser83 of the gyrA gene, and at the amino acid codon Glu84 of the parC gene; in the latter a change of Glu to Lys was produced. Eleven strains had only a single mutation in the amino acid codon Ser83 of the gyrA gene and the remaining eight strains did not show any mutation either in the QRDR of the gyrA or in the parC genes.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A. baumannii is an opportunistic nosocomial pathogen that often shows susceptibility to colistin, imipenem and sulbactam, although a trend towards decreased susceptibility to the last two antibiotics has been observed in most nosocomial strains.20 This scenario makes it necessary to search for therapeutic alternatives other than colistin to treat nosocomial infections caused by A. baumannii. We have compared the in vitro activity of clinafloxacin with other quinolones, showing that this fluoroquinolone, among the tested quinolones, has better activity against A. baumannii. In fact, the MIC90 of clinafloxacin was at least four-fold lower than that seen for other quinolones.

The analysis of the mutations in the QRDR of the gyrA and parC genes showed that alterations in Ser83 of GyrA were most frequently found among the strains with a MIC of ciprofloxacin >=2 mg/L, whereas a double mutation at the amino acid codon Ser83 of the gyrA gene plus a mutation at the amino acid codon Ser80 of the parC gene was found in strains with a MIC of ciprofloxacin >=32 mg/L. These amino acid substitutions were also identified in our previous studies38,39 and by other authors.40 The substitution at amino acid Ser83 of GyrA to Leu was found in strains with a range of MICs of clinafloxacin from 0.12 to 4 mg/L, although the strain having a MIC of 4 mg/L may possess other mechanisms of resistance to clinafloxacin, which may result in this level of MIC. The strains with a double mutation, one in the gyrA gene and another in the parC gene, showed a range of MICs of clinafloxacin from 1 to 8 mg/L. Therefore, clinafloxacin may retain potentially useful clinical activity against strains carrying a single mutation in the gyrA gene, and a second mutation is required for the microorganism to express higher levels of resistance.

All the strains of A. baumannii analysed in this study were able to grow in the presence of 256 mg/L reserpine. Therefore, the effect observed on the MIC of quinolones in the presence of 25 mg/L of this compound was not due to the antibacterial activity of the reserpine.

The effect of reserpine on the MICs of different quinolones is heterogeneous, affecting from only one quinolone (ciprofloxacin or clinafloxacin) to several quinolones at the same time. It is worth mentioning that the majority of reserpine inhibition is observed in clinical isolates having a MIC of ciprofloxacin equal to or lower than 4 mg/L. Therefore, in a strain with a MIC of ciprofloxacin within the range 0.25–4 mg/L, a decrease in the MIC due to reserpine can clearly be observed. This effect is more difficult to appreciate in a strain already having a MIC of 128 mg/L, since it is not sufficient to decrease the MIC below the previous dilution (64 mg/L). However, the effect of reserpine was also detected among some strains with high levels of resistance to quinolones.

The exact mechanisms of action of reserpine in A. baumannii remain unknown. It could be possible that reserpine affects some hypothetical efflux pumps, as is well established among Gram-positive microorganisms.31 Previous reports have suggested the ability of reserpine to inhibit an efflux pump of Bacteroides fragilis32,33 and Stenotrophomonas maltophilia.34 However, no direct evidence is available to attribute, unequivocally, the effect of reserpine over A. baumannii to reserpine-inhibited efflux pumps. Therefore, further studies to elucidate this question will be carried out.

In summary, clinafloxacin shows a good activity against A. baumannii, being a potential alternative to conventional treatments. Moreover, in combination with reserpine its activity is clearly enhanced, thus further investigations are required to establish the mechanisms of action of reserpine in A. baumannii.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by grants from Parke-Davis, Barcelona, Spain and Fondo de Investigaciones Sanitarias (FIS 00/0623 and 00/0997), Spain. A.R. has a fellowship from the Ministerio de Educación y Ciencia, Spain.


    Notes
 
* Corresponding author. Tel: 34-93-2275522; Fax: 34-93-2275454; E-mail: vila{at}medizina.ub.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Bergogne-Berezin, E., Joly-Guillou, M. L. & Vieu, J. F. (1987). Epidemiology of nosocomial infections due to Acinetobacter calcoaceticus. Journal of Hospital Infection 10, 105–13.[ISI][Medline]

2 . Siegman-Igra, Y., Bar-Yosef, S., Gorea, A. & Avram, J. (1993). Nosocomial Acinetobacter meningitis secondary to invasive procedures: report of 25 cases and review. Clinical Infectious Diseases 17, 843–9.[ISI][Medline]

3 . Tilley, P. A. G. & Roberts, F. J. (1994). Bacteremia with Acinetobacter species: risk factors and prognosis in different clinical settings. Clinical Infectious Diseases 18, 896–900.[ISI][Medline]

4 . Beck-Sagué, C. M., Jarvis, W. R., Brook, J. H., Culver, D. M., Polts, A. & Gay, E. (1990). Epidemic bacteremia due to Acinetobacter baumannii in five intensive care units. American Journal of Epidemiology 132, 723–33.[Abstract]

5 . Cefai, C., Richards, J., Gould, F. K. & McPeake, P. (1990). An outbreak of Acinetobacter respiratory tract infection resulting from imcomplete disinfection of ventilatory equipment. Journal of Hospital Infection 15, 177–82.[ISI][Medline]

6 . French, G. L., Casewell, M. W., Roncoroni, A. J., Knight, S. & Phillips, I. (1980). A hospital outbreak of antibiotic resistant Acinetobacter anitratus. Epidemiology and control. Journal of Hospital Infection 1, 125–31.[Medline]

7 . Harstein, A. I., Rashad, A. L., Liebler, J. M., Luis, A. A., Freeman, J. R. & Rourve, J. W. (1988). Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspecies anitratus respiratory infection and colonization associated with contaminated, reusable ventilator circuits and resuscitation bags. American Journal of Medicine 85, 624–31.[ISI][Medline]

8 . Holton, J. (1982). A report of further hospital outbreak caused by a multiresistant Acinetobacter anitratus. Journal of Hospital Infection 3, 305–9.[Medline]

9 . Marcos, M. A., Abdalla, S., Pedraza, F., Andreu, A., Fernandez, F., Gomez-Lus, R. et al. (1994). Epidemiological markers of Acinetobacter baumannii clinical isolates from a spinal cord injury unit. Journal of Hospital Infection 28, 39–48.[ISI][Medline]

10 . Vila, J., Almela, M. & Jimenez de Anta, M. T. (1989). Laboratory investigation of hospital outbreak caused by two different multiresistant Acinetobacter calcoaceticus subsp. anitratus strains. Journal of Clinical Microbiology 27, 1086–9.[ISI][Medline]

11 . Bergogne-Berezin, E. & Joly-Guillou, M. L. (1985). An underestimated nosocomial pathogen, Acinetobacter calcoaceticus. Journal of Antimicrobial Chemotherapy 16, 535–8.[ISI][Medline]

12 . Higgins, P. G., Coleman, K. & Amyes, S. G. (2000). Bactericidal and bacteriostatic activity of gemifloxacin against Acinetobacter spp. in vitro. Journal of Antimicrobial Chemotherapy 45, Suppl. 1, 71–7.[Abstract/Free Full Text]

13 . King, A. & Phillips, I. (1986). The comparative in vitro activity of eight newer quinolones and nalidixic acid. Journal of Antimicrobial Chemotherapy18, Suppl. D, 1–20.[ISI][Medline]

14 . Rolston, K. V. I., Ho, D. H., Leblanc, B., Gooch, G. & Bodey G. P. (1988). Comparative in vitro activity of the new difluoroquinolone temafloxacin (A-62254) against bacterial isolates from cancer patients. European Journal of Clinical Microbiology and Infectious Diseases 7, 684–6.[ISI][Medline]

15 . Acar, J. F., O'Brien, T. F., Goldstein, F. W. & Jones, R. N. (1993). The epidemiology of bacterial resistance to quinolones. Drugs 45, Suppl. 3, 24–8.

16 . Seifert H., Baginski, R., Schulze, A. & Pulverer, G. (1993). Antimicrobial susceptibility of Acinetobacter species. Antimicrobial Agents and Chemotherapy 37, 750–3.[Abstract]

17 . Traub, W. H. & Spohr, M. (1989). Antimicrobial drug susceptibility of clinical isolates of Acinetobacter species (A. baumannii, A. haemolyticus, genospecies 3 and genospecies 6). Antimicrobial Agents and Chemotherapy 33, 1617–9.[ISI][Medline]

18 . Vila, J., Marcos, M. A., Marco, F., Abdalla, S., Vergara, Y., Reig, R. et al. (1993). In vitro antimicrobial production of ß-lactamases, aminoglycoside-modifying enzymes, and chloramphenicol acetyltransferase by and susceptibility of clinical isolates of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 37, 2477–9.

19 . Villar, H. E., Laurino, G. & Hoffman, M. (1996). Bactericide activity of sulbactam before bacteria belonging to the Acinetobacter calcoaceticus–Acinetobacter baumannii complex. Enfermedades Infecciosas y Microbiologia Clinica 14, 524–7.[Medline]

20 . Levin, A. S., Barone, A. A., Penco, J., Santos, M. V., Marinho, I. S., Arruda, E. A. et al. (1999). Intravenous colistin as therapy for nosocomial infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. Clinical Infectious Diseases 28, 1008–11.[ISI][Medline]

21 . Pascual, A., Lopez-Hernandez, I., Martinez-Martinez, L. & Perea, E. J. (1997). In vitro susceptibilities of multiresistant strains of Acinetobacter baumannii to eight quinolones. Journal of Antimicrobial Chemotherapy 40, 140–2.[Free Full Text]

22 . Hooper, D. C. & Wolfson, J. S. (1993). Quinolone Antibacterial Agents, 2nd edn. American Society for Microbiology, Washington, DC.

23 . Fuchs, L. Y., Reyna, F., Chihu, L. & Carrillo, B. (1996). Molecular aspects of fluoroquinolone resistance. In Antibiotic Resistance: From Molecular Basics to Therapeutic Options, (Amábile-Cuevas, C. F., Ed.), Springer, Berlin, Germany.

24 . Vila, J. (1998). Mechanisms of antimicrobial resistance in Acinetobacter baumannii. Reviews of Medical Microbiology 9, 87–97.

25 . Nakamura, S., Nakamura, M., Kojima, T. & Yoshida, H. (1989). gyrA and gyrB mutations in quinolone-resistant strains of Escherichia coli. Antimicrobial Agents and Chemotherapy 33, 254–5.[ISI][Medline]

26 . Ouabdesselam, S., Hooper, D. C., Tankovic, J. & Soussy, C. J. (1995). Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations. Antimicrobial Agents and Chemotherapy 39, 1667–70.[Abstract]

27 . Vila, J., Ruiz, J., Marco, F., Barceló, A., Goñi, P., Giralt, E. et al. (1994). Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrobial Agents and Chemotherapy 38, 2477–9.[Abstract]

28 . Everett, M. J., Fang, J. Y., Ricci, V. & Piddock, L. J. (1996). Contribution of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrobial Agents and Chemotherapy 40, 2380–6.[Abstract]

29 . Ruiz, J., Casellas, S., Jimenez de Anta, M. T. & Vila, J. (1997). The region of the parE gene, homologous to the quinolone-resistant determining region of the gyrB gene, is not linked with the acquisition of quinolone resistance in Escherichia coli clinical isolates. Journal of Antimicrobial Chemotherapy 39, 839–40.[Free Full Text]

30 . Breines, D. M., Ouabdesselam, S., Ng, E. V., Tankovic, J., Shah, S., Soussy, C. J. et al. (1997). Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV. Antimicrobial Agents and Chemotherapy 41, 175–9.[Abstract]

31 . Kaatz, G. W. & Seo, S. M. (1997). Mechanisms of fluoroquinolone resistance in genetically related strains of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 41, 2733–7.[Abstract]

32 . Miyamae, S., Nikaido, H., Tanaka, Y. & Yoshimura, F. (1998). Active efflux of norfloxacin by Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 42, 2119–21.[Abstract/Free Full Text]

33 . Peterson M. L., Hovde, L. B., Wright, D. H., Hoang, A. D., Raddatz, J. K., Boysen, P. J. et al. (1999). Fluoroquinolone resistance in Bacteroides fragilis following sparfloxacin exposure. Antimicrobial Agents and Chemotherapy 43, 2251–5.[Abstract/Free Full Text]

34 . Ribera, A., Jurado, A., Ruiz, J., Marco, F., Valle, O., Mensa, J. et al. (2002). In vitro activity of clinafloxacin with other quinolones against Stenotrophomonas maltophilia clinical isolates in the presence and absence of reserpine. Diagnostic Microbiology and Infectious Diseases, in press.

35 . Bouvet, P. J. M. & Grimont, P. A. (1986). Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., Acinetobacter junii sp. nov., and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. International Journal of Systematic Bacteriology 36, 228–40.

36 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing: Approved Standard M100-S11. NCCLS, Wayne, PA.

37 . Mesa Española de Normalización de la Sensibilidad y Resistencia a los Antimicrobianos. (2000). Recomendaciones del grupo MENSURA para la selección de antimicrobianos en el estudio de la sensibilidad y criterios para la interpretación del antibiograma. Revista Española de Quimioterapia 13, 73–86.

38 . Vila, J., Ruiz, J., Goñi, P. & Jimenez de Anta, M. T. (1997). Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy 39, 757–62.[Abstract]

39 . Vila, J., Ruiz, J., Goñi, P., Marcos, A. & Jimenez de Anta, M. T. (1995). Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 39, 1201–3.[Abstract]

40 . Seward, R. J. & Towner, K. J. (1998). Molecular epidemiology of quinolone resistance in Acinetobacter sp. Clinical Microbiology and Infection 4, 248–54.[Medline]

Received 25 June 2001; returned 16 November 2001; accepted 26 November 2001