Bacteriostatic and bactericidal activities of eight fluoroquinolones against MexAB-OprM-overproducing clinical strains of Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy (2005) 55, 518 522 doi:10.1093/jac/dki030 Advance Access publication 18 February 2005 JAC Bacteriostatic and bactericidal activities of eight fluoroquinolones against MexAB-OprM-overproducing clinical strains of Pseudomonas aeruginosa Philippe Dupont 1, Didier Hocquet 1, Katy Jeannot 1, Pascal Chavanet 2 and Patrick Plésiat 1 * 1 Laboratoire de Bactériologie, Centre Hospitalier Universitaire J. Minjoz, 25030 Besançon; 2 Service des Maladies Infectieuses, Centre Hospitalier Universitaire du Bocage, 21034 Dijon, France Received 17 August 2004; returned 29 September 2004; revised 18 December 2004; accepted 22 December 2004 Objectives: To assess the impact of stable overproduction of efflux system MexAB-OprM on the bacteriostatic and bactericidal activities of fluoroquinolones against clinical Pseudomonas aeruginosa strains. Methods: The minimal inhibitory concentrations (MICs) and the minimal bactericidal concentrations (MBCs) of eight fluoroquinolones (pefloxacin, norfloxacin, ofloxacin, moxifloxacin, levofloxacin, ciprofloxacin, trovafloxacin and grepafloxacin) were determined for nine post-therapy resistant isolates of P. aeruginosa overexpressing MexAB-OprM. Clinical significance of low-level resistance conferred by the efflux mechanism was evaluated with a Monte Carlo simulation. Results: Compared with their pre-therapy susceptible counterparts, seven out of the nine post-therapy efflux mutants exhibited a modest two- to eight-fold increase in resistance to all the fluoroquinolones tested. Interestingly, stronger variations in resistance (up to 64-fold) were observed in two other mutants, one of which had acquired a GyrB target mutation in addition to efflux under chemotherapy. Time kill experiments showed that MexAB-OprM up-regulation did not confer tolerance to fluoroquinolones as the ratio of MBC to MIC was less than 4 for most of the strains. To gain an insight into the clinical significance of resistance conferred by MexAB-OprM, a Monte Carlo simulation was conducted with various fluoroquinolone regimens. With this model, low levels of resistance to ciprofloxacin (MIC >_ 0.25 mg/l) or levofloxacin (MIC >_ 1 mg/l), such as those due to overproduced MexAB-OprM, were predicted to result in poor clinical outcomes. Conclusions: Altogether, these data strongly suggest that when derepressed, MexAB-OprM provides P. aeruginosa with a resistance that may be sufficient to impair the efficacy of single therapy with highly potent fluoroquinolones, such as ciprofloxacin and ofloxacin. Keywords: resistance, Monte Carlo simulation, efflux Introduction The active efflux system MexAB-OprM is known to play a major role in the natural resistance of Pseudomonas aeruginosa to antibiotics. Constitutively produced in wild-type bacteria, this polyspecific pump tends to prevent the intracellular accumulation of a wide range of antimicrobial agents including b-lactams (except imipenem), quinolones, chloramphenicol and tetracyclines. 1 Alterations of regulator genes controlling the expression of operon mexab-oprm, such as mexr (nalb mutants), 2 PA3721 (nalc mutants) 3 or as yet uncharacterized loci (nald mutants), 4 have indeed been shown to reduce the susceptibility of P. aeruginosa to the MexAB-OprM pump substrates, in laboratory or clinical isolates. The therapeutic impact of the moderate resistance resulting from MexAB-OprM up-regulation (MIC increased four- to eight-fold) remains unclear, but could be important in those situations where antibiotics only reach low concentrations at the infection site (e.g. poor local penetration, insufficient dosage, unfavourable pharmacokinetics). Since antipseudomonal therapy usually aims at the rapid eradication of inocula, especially in immunocompromised patients, 5 the question arises whether the... *Corresponding author. Tel: +33-3-81-66-82-86; Fax: +33-3-81-66-89-14; E-mail: patrick.plesiat@univ-fcomte.fr... 518 q The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org

Activity of fluoroquinolones against P. aeruginosa resistance provided by MexAB-OprM might impair the concentration-dependent bactericidal activity of antibiotics such as fluoroquinolones. This study thus examines the bacteriostatic (MICs) and bactericidal (MBCs) activities of eight fluoroquinolones against a series of well characterized MexAB-OprM overproducing mutants of P. aeruginosa recovered from infected patients. 6 The clinical significance of fluoroquinolone resistance due to efflux is also analysed by using a Monte Carlo simulation of various fluoroquinolone regimens. Pharmacokinetic/pharmacodynamic determination and Monte Carlo simulations Unbound, steady-state plasma concentration profiles for antibiotic drugs were simulated. Concentrations (C) of unbound drug versus time (t) were calculated according to published pharmacokinetic models for ciprofloxacin 10 14 and levofloxacin. 15 20 The following formulae were used for one- and two-compartment models, respectively: C ¼ðRinf=½k el 1 V1ŠÞ ð1 2 e 2kel1 Tinf Þ e 2kel1 t Materials and methods Bacterial strains Nine pairs of clonally related isolates of P. aeruginosa were recovered from patients undergoing chemotherapy at the University Hospital of Besançon, France. 6 Each of these pairs includes a baseline (pre-therapy) strain and its resistant (post-therapy) MexAB-OprMoverproducing mutant. The post-therapy mutants were initially recognized in individual patients for their resistance to ticarcillin, compared with previously isolated susceptible strains by RAPD (randomly amplified polymorphic DNA) and shown to overproduce protein OprM by western blotting experiments. 6 Strain 4098 (a derivative of wild-type reference strain PAO1 producing very low, non-inducible amounts of AmpC b-lactamase) and its MexAB- OprM up-regulated mutant 4098E were used as controls. 7 PCR amplification and DNA sequencing The quinolone resistance determining regions (QRDRs) of gyra, gyrb, parc and pare in three bacterial pairs exhibiting either high fluoroquinolone MICs (strains 284/283) or a strong increase in resistance during therapy (couples 18/17 and 141/128) were amplified and sequenced as described previously. 8 Drug susceptibility testing MICs were determined by the microbroth dilution assay in cationadjusted Mueller Hinton liquid medium (MHB; BBL, Cockeysville, MD, USA) with final inocula of 5 10 5 cfu/ml, following standard protocols. 9 Strains 4098 and 4098E were used as controls. Bactericidal activities of fluoroquinolones (MBCs) were assessed with the killing curve method. Briefly, exponentially growing bacteria were adjusted to 5 10 5 cfu/ml in 50 ml of pre-warmed MHB containing the test antibiotic at concentrations equal to multiples of the MIC, and incubated in a shaker (200 rpm) at 378C. Independent samples (3 50 ml) were taken immediately, and 1, 2, 4, 6 and 24 h after drug exposure. Each sample was diluted appropriately in MHB and inoculated at the surface of an antibiotic-free trypticase soy agar plate (Bio-Rad, Yvry-sur-Seine, France) by the means of a Spiral Plater apparatus (AES Laboratoire, Combourg, France). The plates were then incubated at 378C for 24 h, and killing curves were plotted using the mean colony counts at each time point. Preliminary experiments demonstrated no significant drug carry over with this method (data not shown). The MBC was defined as the lowest antibiotic concentration able to kill at least 99.9% (reduction of 3 log 10 ) of the initial inoculum. The fluoroquinolones tested were kindly supplied as titrated powders by Bayer Pharma (ciprofloxacin, moxifloxacin), GlaxoSmithKline (grepafloxacin), Pfizer (trovafloxacin), Aventis (ofloxacin, levofloxacin, pefloxacin) and Merck (norfloxacin). C ¼½ðRinf=½k el 1 V1ŠÞ ð1 2 e 2kel1 Tinf Þ e 2kel1 t Š þ½ðrinf=½k el 2 V2ŠÞ ð1 2 e 2kel2 Tinf Þ e2 kel2 t Š where Rinf is the infusion rate, k el 1andk el 2 are the constants of elimination of compartments one and two, respectively [k el is calculated as ln2 divided by the product of the half-life of elimination (t 1/2 ) and the volume of distribution of this compartment], V1 and V2 are the volumes of distribution and Tinf is the infusion duration. Based on population pharmacokinetic data, estimates for ciprofloxacin were protein-bound fraction = 40%, V1 =1.8L/kgandt 1/2 =4h. The estimates for levofloxacin were protein-bound fraction = 35%, V1 = 0.82 L/kg and t 1/2 (a) = 0.14 h, V2 = 1.05 L/kg and t 1/2 (b) = 9.16 h. t 1/2 was adjusted for renal function. 21 The patient population studied included age, sex, weight and creatinine clearance of 350 patients hospitalized in medical wards at the University Hospital of Dijon. For each simulation, peak/mic and AUC 0 24 /MIC were calculated. After several trials (not shown), we found that with 1000 simulations the asymptotic situation was reached. Based on results from various clinical studies 22,23 including bacteraemic patients with P. aeruginosa, 14 we chose an AUC 0 24 /MIC ratio >_ 125 and a C max /MIC ratio >_ 10 as the pharmacodynamic targets predicting clinical outcome. Results and discussion Bacteriostatic activities of fluoroquinolones against MexAB-OprM-overproducing strains Nine baseline/resistant paired isolates of P. aeruginosa illustrating the emergence of MexAB-OprM-mediated resistance in vivo 6 were assessed for their susceptibility to eight fluoroquinolones. As shown in Table 1, the fluoroquinolones exhibited very different intrinsic activities (MICs) against the post-therapy efflux mutants (multiple target mutant 283 excepted): ciprofloxacin (0.5 4 mg/l), grepafloxacin (1 8 mg/l), trovafloxacin (2 8 mg/l), levofloxacin and norfloxacin (2 16 mg/l), moxifloxacin (4 16 mg/l), ofloxacin (4 32 mg/l) and pefloxacin (8 64 mg/l). Compared with the pre-therapy strains, overexpression of MexAB-OprM in the post-therapy isolates 12, 92, 109, A2, C2 and M2 resulted in a modest increase in the MICs (from two- to eight-fold) of all the fluoroquinolones tested, including lastgeneration compounds such as moxifloxacin and grepafloxacin. In vitro nalb mutant 4098E used as a control also turned out to be two- to eight-fold more resistant than its wild-type parent 4098 (PAO1 derivative lacking an inducible AmpC b-lactamase) to the antibiotics, suggesting the absence of additional resistance mechanisms to fluoroquinolones in the clinical strains. Comparable results were reported by Zhang et al. 24 with another nalb mutant of PAO1 (OCR1). Altogether, these data indicated that MexAB- OprM overproduction impacts uniformly on the bacteriostatic activities of these drugs whatever the origin of the strain and 519

P. Dupont et al. Table 1. Bacteriostatic and bactericidal activities of various fluoroquinolones against clinical MexAB-OprM efflux mutants of Pseudomonas aeruginosa Strains CIP PEF NOR OFL MOX LVX TVA GRX Pre-/post-therapy 14/12 0.25 a /1 b (2 c ) 2/8 (32) 1/8 (8) 2/8 (16) 2/8 (16) 1/4 (8) 1/4 (8) 1/2 (4) 18/17 0.12/4 (8) 1/64 (128) 0.5/16 (32) 1/32 (128) 2/16 (32) 0.5/16 (16) 0.5/8 (16) 0.5/8 (32) 70/92 0.25/1 (2) 2/8 (16) 1/8 (8) 2/4 (4) 2/8 (8) 1/4 (4) 0.5/4 (4) 0.5/2 (2) 96/109 0.25/1 (2) 2/8 (32) 1/8 (8) 2/8 (16) 4/8 (32) 1/4 (8) 1/4 (8) 1/2 (8) 141/128 0.12/1 (8) 2/64 (256) 1/16 (16) 1/32 (64) 2/8 (64) 1/4 (32) 0.5/8 (32) 0.5/4 (32) 284/283 64/128 512/ > 1024 128/256 256/512 128/256 128/256 128/128 256/512 A1/A2 0.25/0.5 (1) 2/8 (16) 1/8 (8) 2/4 (16) 2/4 (16) 1/2 (4) 1/2 (4) 0.5/1 (4) C1/C2 0.12/0.5 (2) 1/8 (16) 1/2 (8) 1/4 (16) 2/8 (16) 0.5/2 (8) 0.5/2 (8) 0.5/2 (8) M1/M2 0.12/0.5 (2) 1/8 (16) 0.5/4 (16) 1/4 (16) 2/8 (32) 0.5/2 (4) 1/4 (8) 0.5/2 (8) 4098/4098E 0.25/0.5 (1) 1/8 (16) 0.5/2 (8) 1/4 (8) 1/4 (16) 0.5/2 (4) 1/2 (16) 0.5/2 (8) CIP, ciprofloxacin; PEF, pefloxacin; NOR, norfloxacin; OFL, ofloxacin; MOX, moxifloxacin; LVX, levofloxacin; TVA, trovafloxacin; GRX, grepafloxacin. a MIC (mg/l) for the pre-therapy strain. b MIC (mg/l) for the post-therapy strain. c MBC (mg/l) for the post-therapy strain. the nature of the fluoroquinolone. However, because of different intrinsic potencies of fluoroquinolones against wild-type P. aeruginosa, the six post-therapy mutants remained S to ciprofloxacin (MIC <_ 1 mg/l), whereas 3/6 were S (MIC <_ 2 mg/l) or R (MIC > 2 mg/l) to levofloxacin, 6/6 were I (MIC, 4 8 mg/l) to ofloxacin, and 1/6 and 5/6 were I (MIC, 2 4 mg/l) or R (MIC > 4 mg/l) to moxifloxacin, respectively, according to the BSAC breakpoints. 25 Target mutations DNA sequencing of the QRDRs of gyra, gyrb, parc and pare was performed in the bacterial pairs exhibiting high levels of fluoroquinolone resistance (284/283) or a strong increase in resistance during therapy (18/17 and 141/128). A single mutation in gyrb leading to a Ser-464! Phe substitution in target GyrB, already described by Akasaka et al., 26 was identified in isolate 17 ( I to ciprofloxacin) compared with the pre-therapy strain 18. Reminiscent of this observation, we recently described the emergence under ciprofloxacin therapy of a GyrB/MexAB-OprM double mutant of P. aeruginosa strongly resistant to fluoroquinolones. 27 Our present data thus confirm that clinical strains may evade fluoroquinolone therapy by combining two low-level resistance mechanisms. Isolates 284 and 283 displayed identical alterations in GyrA (Thr-83! Ile, Asp-87! Tyr) and in ParC (Ser-80! Leu) consistent with the high MICs of fluoroquinolones found for these bacteria. 26,28 Interestingly, MexAB-OprM overexpression in 283 was still able to add some resistance (MICs enhanced two-fold) to that conferred by the multiple target mutations in gyra and parc, thus contributing to the development of very recalcitrant strains, as suggested previously with heavily mutagenized PAO1 mutants. 29 In contrast, the QRDRs of the 141/128 pair turned out to be identical to that of PAO1, indicating the probable involvement of additional fluoroquinolone resistance mechanisms in post-therapy mutant 128. However, reverse-transcriptase real-time PCR experiments 4 demonstrated that no other efflux pump (MexCD-OprJ, MexEF-OprN or MexXY) was overproduced in strains 141 and 128 (data not shown). Bactericidal activities of fluoroquinolones In Escherichia coli, Goldman et al. 30 found that overexpression of the multiple antibiotic resistance locus mar had a protective effect against cell killing by fluoroquinolones. The mar locus is known to control a regulon of at least 40 genes, including those encoding the efflux system AcrAB-TolC. 31 To see whether MexAB-OprM might be involved in tolerance to fluoroquinolones in P. aeruginosa, we determined the MBCs of the fluoroquinolones for eight of the nine post-therapy strains overexpressing MexAB-OprM (Table 1). The MBCs were in general two- to four-fold higher than the MICs (extreme values from one- to eight-fold). This clearly shows that MexAB-OprM up-regulation is insufficient by itself to abolish the bactericidal activity of fluoroquinolones in vitro. On the other hand, such an increase in MBCs might reduce the clinical efficacy of fluoroquinolones in severely ill patients. Pharmacodynamic evaluation of fluoroquinolone efficacy Animal and in vitro studies have found a correlation between the efficacy of chemotherapy (i.e. bacterial eradication, resistant mutant prevention) and pharmacodynamic indices, such as AUC 0 24 /MIC (area under the concentration time curve for 24 h divided by the MIC) or C max /MIC (peak concentration divided by the MIC) for antibiotics like fluoroquinolones that exhibit concentration-dependent activity. 32 These notions were also supported by extensive clinical investigations showing that patient outcomes were significantly improved when AUC 0 24 /MIC >_ 125 and/or C max /MIC >_ 8 12 for fluoroquinolones. 14,22,23 To gain an insight into the clinical significance of MexAB-OprM up-regulation in P. aeruginosa, a 1000-patient Monte Carlo simulation (Crystal Ball 2000 software; Decisioneering, Inc.) was conducted to calculate estimates of AUC 0 24 /MIC and C max /MIC ratios for ciprofloxacin and levofloxacin administered at various dosing regimens, in relation to bacterial resistance levels conferred by efflux. As shown in Table 2, the likelihood of these regimens attaining the appropriate pharmacodynamic targets was low when MICs of ciprofloxacin and levofloxacin were >_ 0.25 mg/l and >_ 1 mg/l, 520

Activity of fluoroquinolones against P. aeruginosa Table 2. Target attainment rates of various ciprofloxacin and levofloxacin regimens (Monte Carlo simulation, see Methods) versus various MICs of fluoroquinolones (the most important results are in bold type) Treatment Target attainment rate (%) Drug total daily dosage (mg) unitary dose interval (h) MIC (mg/l) C max /MIC > 10 AUC/MIC > 125 Ciprofloxacin 1200 8 0.12 66 87 0.25 6 7 1600 6 0.12 66 90 0.25 5 12 2400 8 0.12 98 100 0.25 60 85 0.5 4.2 3.7 Levofloxacin 500 24 0.5 70 40 1 4 3 1000 12 0.5 72 72 1 4 5 750 24 0.5 97 79 1 32 14 1000 24 0.5 100 91 1 41 72 respectively, even with simulation of aggressive treatments [intravenous (iv) ciprofloxacin 800 mg three times a day, iv levofloxacin 1000 mg once a day]. Predicted rates of favourable outcomes were < 5% with the other fluoroquinolones including moxifloxacin (not presented), a result that confirms that these molecules are not appropriate for the treatment of P. aeruginosa infections. Conclusions In agreement with previous findings, 32 this work thus strongly suggests that MexAB-OprM overproduction may have a significant impact on clinical outcomes, and that increasing fluoroquinolone doses is insufficient by itself to eradicate efflux mutants in patients under single therapy. Because fluoroquinolones are widely prescribed in P. aeruginosa infections, it would be highly desirable to inform clinicians of strains exhibiting low-level resistance (MIC >_ 0.25 mg/l) to these compounds in order to choose another class of antibiotics (i.e. b-lactams) or to use combination therapy if fluoroquinolones are an option. It should be stressed that an S designation on a strain does not necessarily mean therapeutic success of a drug; this is mainly indicated by MICs. Making the link between clinical trials, animal models and in vitro pharmacodynamic studies, Schentag et al. 33 suggested that a AUC/MIC ratio >_ 250 would be a better index for predicting clinical outcomes and bacterial eradication. According to this proposal, increase in MBCs caused by MexAB-OprM efflux might be sufficient to impair the bactericidal activity of fluoroquinolones in vivo, thereby rendering bacterial eradication more difficult especially in infections implying heavy inocula. Whether fluoroquinolones are essentially bacteriostatic in vivo against MexAB-OprM-overproducing mutants of P. aeruginosa requires further support from clinical experience. Acknowledgements This work was supported by a grant from the French association against cystic fibrosis Vaincre la Mucoviscidose. We are grateful to Véronique Dupont and Christiane Bailly for their excellent technical contributions. References 1. Li, X. Z., Nikaido, H. & Poole, K. (1995). Role of MexA-MexB- OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 1948 53. 2. Poole, K., Tetro, K., Zhao, Q. X. et al. (1996). Expression of the multidrug resistance operon mexa-mexb-oprm in Pseudomonas aeruginosa: mexr encodes a regulator of operon expression. Antimicrobial Agents and Chemotherapy 40, 2021 8. 3. Cao, L., Srikumar, R. & Poole, K. (2004). MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalc gene encoding a repressor of PA3720-PA3719. Molecular Microbioliology 53, 1423 36. 4. Llanes, C., Hocquet, D., Vogne, C. et al. (2004). Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrobial Agents and Chemotherapy 48, 1797 802. 5. Craig, W. A. & Ebert, S. C. (1994). Antimicrobial therapy in Pseudomonas aeruginosa infections. In Pseudomonas aeruginosa Infections and Treatment (Baltch, A. L. & Smith, R. P., Eds), pp. 441 517. Marcel Dekker, Inc., New York, NY, USA. 6. Ziha-Zarifi, I., Llanes, C., Köhler, T. et al. (1999). In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM. Antimicrobial Agents and Chemotherapy 43, 287 91. 7. Li, X. Z., Ma, D., Livermore, D. M. et al. (1994). Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to b-lactam resistance. Antimicrobial Agents and Chemotherapy 38, 1742 52. 521

P. Dupont et al. 8. Hocquet, D., Bertrand, X., Köhler, T. et al. (2003). Genetic and phenotypic variations of a resistant Pseudomonas aeruginosa epidemic clone. Antimicrobial Agents and Chemotherapy 47, 1887 94. 9. Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5 16. 10. Birmingham, M. C., Guarino, R., Heller, A. et al. (1999). Ciprofloxacin concentrations in lung tissue following a single 400 mg intravenous dose. Journal of Antimicrobial Chemotherapy 43, Suppl. A, 43 8. 11. Forrest, A., Ballow, C. H., Nix, D. E. et al. (1993). Development of a population pharmacokinetic model and optimal sampling strategies for intravenous ciprofloxacin. Antimicrobial Agents and Chemotherapy 37, 1065 72. 12. Montgomery, M. J., Beringer, P. M., Aminimanizani, A. et al. (2001). Population pharmacokinetics and use of Monte Carlo simulation to evaluate currently recommended dosing regimens of ciprofloxacin in adult patients with cystic fibrosis. Antimicrobial Agents and Chemotherapy 45, 3468 73. 13. Shah, A., Lettieri, J., Kaiser, L. et al. (1994). Comparative pharmacokinetics and safety of ciprofloxacin 400 mg i.v. thrice daily versus 750 mg po twice daily. Journal of Antimicrobial Chemotherapy 33, 795 801. 14. Zelenitsky, S. A., Harding, G. K. M., Sun, S. et al. (2003). Treatment and outcome of Pseudomonas aeruginosa bacteraemia: an antibiotic pharmacodynamic analysis. Journal of Antimicrobial Chemotherapy 52, 668 74. 15. Chien, S. C., Rogge, M. C., Gisclon, L. G. et al. (1997). Pharmacokinetic profile of levofloxacin following once-daily 500- milligram oral or intravenous doses. Antimicrobial Agents and Chemotherapy 41, 2256 60. 16. Chow, A. T., Fowler, C., Williams, R. R. et al. (2001). Safety and pharmacokinetics of multiple 750-milligram doses of intravenous levofloxacin in healthy volunteers. Antimicrobial Agents and Chemotherapy 45, 2122 5. 17. Furlanut, M., Brollo, L., Lugatti, E. et al. (2003). Pharmacokinetic aspects of levofloxacin 500 mg once daily during sequential intravenous/oral therapy in patients with lower respiratory tract infections. Journal of Antimicrobial Chemotherapy 51, 101 6. 18. Geerdes-Fenge, H. F., Wiedersich, A., Wagner, S. et al. (2000). Levofloxacin pharmacokinetics and serum bactericidal activities against five enterobacterial species. Antimicrobial Agents and Chemotherapy 44, 3478 80. 19. Lubasch, A., Keller, I., Borner, K. et al. (2000). Comparative pharmacokinetics of ciprofloxacin, gatifloxacin, grepafloxacin, levofloxacin, trovafloxacin, and moxifloxacin after single oral administration in healthy volunteers. Antimicrobial Agents and Chemotherapy 44, 2600 3. 20. Preston, S. L., Drusano, G. L., Berman, A. L. et al. (1998). Levofloxacin population pharmacokinetics and creation of a demographic model for prediction of individual drug clearance in patients with serious community-acquired infection. Antimicrobial Agents and Chemotherapy 42, 1098 104. 21. Levey, A. S., Bosch, J. P., Lewis, J. B. et al. (1999). A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Annals of Internal Medecine 130, 461 70. 22. Forrest, A., Nix, D. E., Ballow, C. H. et al. (1993). Pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrobial Agents and Chemotherapy 37, 1073 81. 23. Preston, S. L., Drusano, G. L., Berman, A. L. et al. (1998). Pharmacodynamics of levofloxacin: a new paradigm for early clinical trials. Journal of the American Medical Association 279, 125 9. 24. Zhang, L., Li, X.-Z. & Poole, K. (2001). Fluoroquinolone susceptibilities of efflux-mediated multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia. Journal of Antimicrobial Chemotherapy 48, 549 52. 25. Andrews, J. M. (2004). BSAC standardized disc susceptibility testing method (version 3). Journal of Antimicrobial Chemotherapy 53, 713 28. 26. Akasaka, T., Tanaka, M., Yamaguchi, A. et al. (2001). Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrobial Agents and Chemotherapy 45, 2263 8. 27. Le Thomas, I., Couetdic, G., Clermont, O. et al. (2001). In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy. Journal of Antimicrobial Chemotherapy 48, 553 5. 28. Mouneimné, H., Robert, J., Jarlier, V. et al. (1999). Type II topoisomerase mutations in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 62 6. 29. Lomovskaya, O., Lee, A., Hoshino, K. et al. (1999). Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 1340 6. 30. Goldman, J. D., White, D. G. & Levy, S. B. (1996). Multiple antibiotic resistance (mar) locus protects Escherichia coli from rapid cell kiling by fluoroquinolones. Antimicrobial Agents and Chemotherapy 40, 1266 9. 31. Martin, R. G. & Rosner, J. L. (2002). Genomics of the mara/soxs/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data. Molecular Microbiology 44, 1611 24. 32. Wright, D. H., Brown, G. H., Peterson, M. L. et al. (2000). Application of fluoroquinolone pharmacodynamics. Journal of Antimicrobial Chemotherapy 46, 669 83. 33. Schentag, J. J., Meagher, A. K. & Forrest, A. (2003). Fluoroquinolone AUIC break points and the link to bacterial killing rates. Part 2: human trials. Annals of Pharmacotherapy 37, 1478 88. 522