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

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1 Journal of Antimicrobial Chemotherapy (2005) 55, doi: /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, Besançon; 2 Service des Maladies Infectieuses, Centre Hospitalier Universitaire du Bocage, 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: ; Fax: ; patrick.plesiat@univ-fcomte.fr q The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oupjournals.org

2 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 and levofloxacin 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 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 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

3 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/ 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/ /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/ /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/ /1 (2) 2/8 (32) 1/8 (8) 2/8 (16) 4/8 (32) 1/4 (8) 1/4 (8) 1/2 (8) 141/ /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/ / > / / / / / /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

4 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 Levofloxacin 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, 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, 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, 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, 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 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, 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,

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