Service des Maladies Infectieuses, Microbiologie Médicale et Moléculaire, Hôpital du Bocage, 21000 Dijon Cedex, France
Received 14 April 2004; returned 3 June 2004; revised 28 June 2004; accepted 2 July 2004
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Abstract |
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Materials and methods: Five pneumococcal strains were tested and were defined as follows [MIC of ciprofloxacin (mg/L)/MIC of gatifloxacin (mg/L)/MPC of gatifloxacin (mg/L)/involved quinolone resistance mechanisms]: strain 16089=0.5/0.25/0.25/wild-type; strain MS1A=2/0.5/1/efflux; strain MS2A=8/1/8/parC S79F; strain MR3B4=10/1/8/parC S79T; strain Gyr-1207=6/4/4/gyrA S81F.
Results: A 48 h human-like treatment with gatifloxacin was significantly bactericidal on pneumonia induced by strain 16089 ( > 6 log10 killing) as well as the efflux derivative strain MS1A ( > 5 log10 killing). However, a small number of parCgyrA mutants were recovered in 26% of the animals infected with this efflux strain. As expected, no decrease in viable bacteria counts was observed when pneumonia was induced by the gyrA resistant strain. In contrast, because of the enrichment of highly resistant mutants in 100% of the animals, no significant bacterial reduction was observed after treatment of pneumonia induced by the two susceptible parC mutated strains. A classification and regression tree (CART) analysis identified TMSW (percentage of the time during which gatifloxacin serum concentrations are inside the MSW) and AUCMSW (area under curve between MIC and MPC values) as the best parameters associated with the enrichment of resistant pneumococci.
Conclusions: This study shows that the acquisition of a low level of fluoroquinolone resistance (especially a parC mutation and to a lesser extent an efflux mechanism) is associated with a clearly lower potential for preventing resistance development. These data support the concept that resistant mutants are selectively enriched when antibiotic concentrations fall inside the mutant selection window and suggest that in vivo dynamic models have to be used to predict the relative abilities of quinolones to prevent mutant selection.
Keywords: parC , pneumococcal pneumonia , mutant selection window , mutant prevention concentration
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Introduction |
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Materials and methods |
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Five penicillin-resistant pneumococcal strains18 were used in this study (Table 1). They included one parent ciprofloxacin-susceptible strain (strain 16089) and four strains with different ciprofloxacin resistance phenotypes (efflux, parC and gyrA). Bacteria were grown either in brain heart infusion (BHI) broth (bioMérieux, Marcy l'Étoile, France) or on sheep blood agar plates (bioMérieux) in 5% CO2. Bacterial stocks were kept frozen at 70°C in a 15% (v/v) glycerol-supplemented BHI. Ciprofloxacin was provided by SigmaAldrich (Steinheim, Switzerland) and gatifloxacin was kindly supplied by Grünenthal (Levallois Perret, France). The drugs were reconstituted according to the manufacturers' instructions. Stock solutions were used as fresh preparations.
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All determinations were done in triplicate and the results were identical.
Determination of the MICs
MICs were determined by the standard serial two-fold dilutions method in agar, with an inoculum of 5 x 105 cfu/mL.19 Results were recorded after 18 h of incubation in 5% CO2 at 37°C.
Determination of the MPCs
MPCs were determined by plating the bacterial suspension (1010 cfu/mL) on tryptic soy agar plates containing 5% of defibrinated sheep blood and various concentrations of gatifloxacin. MPCs were recorded as the lowest concentration of quinolone that allowed no growth of mutant strains after 72 h of incubation in 5% CO2 at 37°C.13
Timekill curves
The in vitro bactericidal activity of gatifloxacin on the different strains was evaluated as described previously.20 The bacterial growth in test and control tubes was counted at 0, 3, 6, 12 and 24 h after incubation at 37°C. The initial inoculum size was 5 x 105 cfu/mL. The concentrations of gatifloxacin corresponded approximately to the maximal blood concentration observed in humans.
Determination of quinolone resistance mechanisms
PCR amplification of quinolone resistance-determining regions (QRDRs) and DNA sequencing
PCR was used to amplify parC, parE, gyrA and gyrB genes21 with primers that were those previously reported.22 After amplification, PCR products were sequenced (Génôme Express S.A., Grenoble, France) with an automated sequencer (ABI Prism 377 DNA sequencer).
Presence of quinolone efflux mechanism
MICs were determined in the presence and absence of 10 µg/mL of reserpine (Sigma Chemicals, St Louis, MO, USA) as described previously.2325 By definition, an efflux mechanism existed when there was at least a four-fold reduction in MIC in the presence of reserpine.
Production and human-like treatment of experimental pneumococcal pneumonia in rabbits
Animals
Male immunocompetent New Zealand rabbits (weight, 2.73.0 kg; Elevage Scientifique des Dombes, Romans, France) were used for all studies. Animals were kept in accordance with current recommendations.
Lung infection model
The production of pneumonia in immunocompetent rabbits and the installation of the central venous catheters were carried out as previously described.18,26 Briefly, 24 h after jugular catheterization, bacterial pneumonia was induced by endobronchial challenge of the animals with 0.5 mL of saline containing 1010 cfu/mL of either tested strain. Human-like treatment with gatifloxacin was started 5 h after bacterial challenge and lasted for 2 days. Gatifloxacin was delivered through the first central venous catheter at changing infusion rates obtained by a computer-controlled electric pump and at doses that simulated antibiotic kinetics observed in human serum after a standard dose of 400 mg once a day.
Pharmacokinetic (PK) analysis
For each animal, the concentrations of gatifloxacin in the serum were determined on iterative blood samples, obtained through the second central catheter by a standard microbiological assay method with antibiotic diffusion medium II (Difco Laboratories) and Escherichia coli NIJJHC2 as the indicator organism.27 The level of protein binding in the serum of infected rabbits was determined ex vivo by a membrane ultrafiltration method.28 The degree of binding was measured by using gatifloxacin concentrations of 0.56 mg/L. Pharmacokinetics data were analysed using Kinetica software (Innaphase, Philadelphia, PA, USA).
Evaluation of infection
The rabbits were anaesthetized and killed 2 h after the end of the antibiotic infusion. Untreated control rabbits were killed just before treatment or after 48 h. The assessment of primary efficacy was based on the analysis of the spleen and each pulmonary lobe, which were removed and processed to determine the numbers of cfu/g. Then residual bacteria were examined for fluoroquinolone susceptibility on agar plates containing gatifloxacin at 2x and 4x MIC. Resistant mutants were examined for their drug susceptibility and their gene mutations in the QRDR.
Pharmacokineticpharmacodynamic (PKPD) analysis
By using the individual pharmacokinetics data obtained for each animal, the relationship between drug exposure and bacterial response was expressed according to the following parameters (expressed as the unbound fraction): maximal concentration over MIC (Cmax/MIC) or MPC (Cmax/MPC), area under the curve of serum concentration versus time divided by MIC (AUC024/MIC) or MPC (AUC024/MPC), time of serum concentration above MIC (T > MIC) or MPC (T > MPC), time of serum concentration within MIC and MPC (TMSW) and area of serum concentration within MIC and MPC versus time (AUCMSW). Emax, sigmoid and peak formulas were obtained from Sigma Plot Software (Version 7.101).
Statistical analysis
The results were expressed as the mean±S.D. Quantitative variables were compared with MannWhitney or analysis of variance (ANOVA), eventually completed by a post-hoc analysis using Bonferroni's test. Percentages were compared using the 2 test with Yates correction or with the Fisher exact test. The correlation between antimicrobial efficacy and each of the PKPD parameters (Cmax/MIC, Cmax/MPC, AUC024/MIC, AUC024/MPC, T > MIC, T > MPC, TMSW, AUCMSW) was determined by non-linear least-squares regression (Sigma Plot Software, Version 7.101). To define the PKPD zones associated with the occurrence of mutants, a qualitative analysis was carried out using the classification and regression tree (CART) methodology.29,30 For all the tests, a P value <0.05 was considered indicative of statistical significance.
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Results |
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The MICs of ciprofloxacin and gatifloxacin, and MPCs of gatifloxacin for the five strains studied are shown in Table 1. All these strains, except for strain 16089, were resistant to ciprofloxacin. As expected, gatifloxacin retained a better in vitro activity than ciprofloxacin against all strains tested. All strains except strain Gyr-1207 (MIC=4 mg/L) were susceptible to gatifloxacin. The presence of a gyrA mutation (strain Gyr-1207) was associated with a 16-fold increase in the MIC of gatifloxacin. However, the presence of only one ciprofloxacin efflux mechanism (strain MS1A) or of a parC mutation (strains MS2A and MR3B4) slightly increased the MICs of gatifloxacin. In contrast, the presence of a parC mutation (strains MS2A and MR3B4), and to a lesser extent an efflux mechanism (strain MS1A), was clearly associated with an increase in MPC values, thus these strains exhibited an in vitro mutant selection window since their MPCs were higher than their MICs.
In vitro bactericidal activity of gatifloxacin
Timekill curves for the five pneumococcal strains tested for gatifloxacin at the simulated serum concentration after oral dosing of 400 mg are shown in Figure 1. Gatifloxacin rapidly reduced bacteria below the limit of detection within 6 h after exposure for strains 16089 (wild-type) and MS1A (efflux) (P0.01 compared to the other strains). In contrast, for strains MS2A (parC), MR3B4 (parC) and Gyr-1207 (gyrA), decreases in viable bacterial counts slowed (18 to 24 h) (P<0.01 compared to strains 16089 and MS1A) and gatifloxacin showed a minimum bactericidal activity at 24 h for the resistant strain Gyr-1207 (gyrA).
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Figure 2 shows that total serum concentrations observed in rabbits (n=51) mirrored the human serum concentration curve observed after the administration of 400 mg of gatifloxacin. The level of gatifloxacin protein binding in infected rabbits was 25% at drug concentrations from 0.5 to 6 mg/L. This is similar to the low degree of binding reported for gatifloxacin in other animal species and in human serum.28,31,32 Free drug levels were considered in pharmacokinetic calculations throughout the study. The pharmacokinetic parameters obtained in rabbits were as follows (expressed as the free fraction): AUC024, 29.01±8.5 mg·h/L; Cmax, 2.66±0.5 mg/L; Cmin24, 0.46±0.28mg/L; Cmin48, 0.56±0.35 mg/L. The exposure to gatifloxacin (AUC024) was similar for the groups as defined by strains (P > 0.5).
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Bacterial reduction after treatment
At the start of therapy, the mean pulmonary bacterial concentration was 8.5±0.54 log10 cfu/g with no difference between groups of animals. As shown in Figure 3, gatifloxacin, at a dose equivalent to the standard regimen of 400 mg once a day in humans, was associated with a sharp decrease in viable bacterial counts in lungs when pneumonia was induced by strain 16089 (1.00±0.00 log10 cfu/g, P<0.0001) and its derivative efflux strain MS1A (2.46±1.50 log10 cfu/g, P<0.0001) after 48 h of drug exposure, compared to untreated controls. A significant difference in efficacy was found between these two groups (P=0.01, MannWhitney test).
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Emergence in vivo of resistant mutants
No gatifloxacin-resistant mutants were recovered after 48 h of antibiotic exposure when the experimental pneumonia was induced by either the ciprofloxacin-susceptible strain 16089 or the gatifloxacin-resistant gyrA mutated strain Gyr-1207. In contrast, resistant mutants (MIC for gatifloxacin = 4 mg/L), which had acquired an additional gyrA mutation (gyrA Ser-81Phe), were recovered in 100% and 80% of the animals infected by the two parC mutated strains MS2A and MR3B4, respectively. The mean pulmonary concentrations of resistant mutants for these two strains were 8.31±0.43 log10 cfu/g and 7.42±1.11 log10 cfu/g, respectively. Gatifloxacin standard regimen was also associated with the emergence of double mutated gyrAparC resistant mutants when pneumonia was induced by the efflux strain (MS1A), but in only four of 15 treated rabbits (26%) and at a very low concentration (1.1±0.8 log10 cfu/g).
Pharmacodynamic analysis
Table 2 lists all mean MIC- and MPC-related parameters (unbound fraction) obtained for each group of animals as defined by strains. All these values decreased as the MIC of the infecting strain increased; for example, AUC024/MIC ratios ranged from 7.8±1.05 to 93.2±16. The relationships between the microbiological effect in animals and each of the MIC- and MPC-related parameters were studied. Thus, the residual bacterial population in the lungs after 48 h of antibiotic exposure was split into the population with and the population without an increase in MIC to gatifloxacin (Figure 4ag).
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Thresholds of the mutant selection window (MSW) (Table 3)
In order to approach the limits within which mutants appear in vivo, a classification and regression tree (CART) analysis was tested for each PKPD parameter. This analysis identified TMSW (percentage of the time during which gatifloxacin serum concentrations are inside the MSW) and AUCMSW (area under curve between MIC and MPC values) as the best parameters statistically associated with the emergence of resistant mutants; however, only AUCMSW was significant in the multivariate analysis. When this analysis was done only for rabbits infected with parC strains, the selection window was narrow and again only AUCMSW was found significantly associated with the in vivo emergence of mutants (not shown).
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Discussion |
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In this human-like animal model, a standard gatifloxacin treatment is associated with a sharp bacterial reduction when the animals were infected by the wild-type pneumococcal strain (strain 16089) and its efflux derivative (strain MS1A). This efficacy was associated with a large drug exposure as the AUC/MIC ratio was on average 90 and 58, respectively (Table 2). However, a few resistant mutants were detected (26% of the treated animals) when the pneumonia was induced with the efflux derivative strain. This result was not anticipated by the in vitro killing curve or the in vitro model39 and also gatifloxacin is considered insensitive to efflux by clinical strains.40 However, for this specific strain, a mutant selection window did exist since its MPC was twice the MIC. In vivo, gatifloxacin concentration was within this window for approximately 30% of the time compared to zero for the wild-type strain infection (Table 2). Although this finding could confirm the MSW concept in vivo, only one efflux strain was tested in this study; so we are unable to anticipate the real role of efflux on the eventual emergence of fluoroquinolone resistance. Of note, the residual concentration of these mutants after treatment was very low.
As expected, no decrease in viable bacterial counts was observed when pneumonia was induced by the gyrA resistant strain which thus validates our humanized animal model. Furthermore, no mutants were detected in any animals; of note this strain was not only resistant to gatifloxacin but also did not exhibit any mutation selection window since its MPC and MIC were equal.
Concerning parC induced pneumonia, the occurrence of resistant mutants was very high since 80100% of the treated animals harboured such double parCgyrA mutants in their lungs. Furthermore, the proportion of the mutated bacterial population was also high (Figure 4); these in vivo findings are concordant with the increased numbers of a second mutation found in vitro in parC-harbouring pneumococci.41,42
This finding also confirms the MSW concept; indeed the MPC of the two parC strains were eight times the MIC and consequently, in animals, the percentage of time gatifloxacin concentration was between these two concentrations (TMSW) and AUCMSW was high. The CART analysis revealed the boundaries of this in vivo mutant selection window (Table 3); clearly, when TMSW is above 45%, the risk of mutation is 100%. Of note, no mutant appeared when the Cmax/MIC ratio was above 6. This result is close to other experimental findings;28 however, the corresponding threshold for the AUC/MIC ratio was 90; this value is higher than both the usually considered range of 3050 for the efficacy of fluoroquinolones on pneumococcal infections28 and 33 for clinical pneumococcal eradication in humans.31 To explain this result, it is important to point out that our study focused on mutant occurrence using several strains with various fluoroquinolone susceptibility profiles and not only on the global concentrationeffect relationship. The other explanation could be that the PKPD values could be somewhat specific to a bugdrug couple in a particular model.43 However, these results are of the same order of magnitude as those obtained in previous studies.14,16,17
Even considering these limitations, our study showed that an AUC/MIC ratio of 50 is associated with a 100% bacterial reduction in lungs (in fact, a reduction of more than 6 log10 cfu), which is probably equivalent to clinical cure with eradication, if the initial causative pneumococcus was fully susceptible to fluoroquinolones. This is concordant with pharmacodynamic estimations for gatifloxacin.44 On the other hand, if the initial causative pneumococcus harboured a low level of resistance to gatifloxacin (parC mutation and to a lesser extent efflux), the risk of both emerging resistance and failure to decrease the infecting burden in the lung with a standard regimen of gatifloxacin 400 mg per day is high. Since the current frequency of infection with parC pneumococci remains low,45 this latter risk is also low. However, from a clinical point of view, this risk is much higher when patients have previously received a second generation fluoroquinolone treatment.46
In conclusion, our findings have shown the excellent in vivo efficacy of a human-like regimen of 400 mg gatifloxacin on experimental pneumococcal pneumonia due to a ciprofloxacin-susceptible strain. However, mutants emerged when the pneumonia was induced by pneumococci with a low level of resistance to fluoroquinolones, especially for parC strains. These findings could be anticipated using the in vitro mutation selection window concept that our results translate in vivo.
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Acknowledgements |
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Footnotes |
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References |
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2 . Watanakunakorn, C. & Bailey, T. (1997). Adult bacteremic pneumococcal pneumonia in a community teaching hospital, 19921996. A detailed analysis of 108 cases. Archives of Internal Medicine 157, 196571.[Abstract]
3 . Mufson, M. & Stanek, R. (1999). Bacteremic pneumococcal pneumonia in one American city: a 20-year longitudinal study, 19781997. American Journal of Medicine 107, 3443S.
4 . Musher, D. (1992). Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clinical Infectious Diseases 14, 8019.[ISI][Medline]
5 . Kalin, M., Ortqvist, A., Almela, M. et al. (2000). Prospective study of prognostic factors in community-acquired bacteremic pneumococcal disease in 5 countries. Journal of Infectious Diseases 182, 8407.[CrossRef][ISI][Medline]
6
.
Ho, P., Yung, R., Tsang, D. et al. (2001). Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong multicentre study in 2000. Journal of Antimicrobial Chemotherapy 48, 65965.
7
.
Chen, D., McGeer, A., de Azavedo, J. et al. (1999). Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada. New England Journal of Medicine 341, 2339.
8
.
Linares, J., de la Campa, A. & Pallares, R. (1999). Fluoroquinolone resistance in Streptococcus pneumoniae. New England Journal of Medicine 341, 15467.
9
.
Perez-Trallero, E., Garcia-Rey, C., Martin-Sanchez, A. et al. (2002). Activities of six different quinolones against clinical respiratory isolates of Streptococcus pneumoniae with reduced susceptibility to ciprofloxacin in Spain. Antimicrobial Agents and Chemotherapy 46, 26657.
10 . Baquero, F. & Negri, M. (1997). Strategies to minimize the development of antibiotic resistance. Journal of Chemotherapy 9, 2937.[ISI][Medline]
11 . Zhao, X. & Drlica, K. (2002). Restricting the selection of antibiotic-resistant mutant bacteria: measurement and potential use of the mutant selection window. Journal of Infectious Diseases 185, 5615.[CrossRef][ISI][Medline]
12 . Zhao, X. & Drlica, K. (2001). Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. Clinical Infectious Diseases 33, Suppl. 3, S14756.[CrossRef][ISI][Medline]
13
.
Blondeau, J., Zhao, X., Hansen, G. et al. (2001). Mutant prevention concentrations of fluoroquinolones for clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 45, 4338.
14
.
Zinner, S. H., Lubenko, I. Y., Gilbert, D. et al. (2003). Emergence of resistant Streptococcus pneumoniae in an in vitro dynamic model that simulates moxifloxacin concentrations inside and outside the mutant selection window: related changes in susceptibility, resistance frequency and bacterial killing. Journal of Antimicrobial Chemotherapy 52, 61622.
15
.
Allen, G., Kaatz, G. & Rybak, M. (2003). Activities of mutant prevention concentration-targeted moxifloxacin and levofloxacin against Streptococcus pneumoniae in an in vitro pharmacodynamic model. Antimicrobial Agents and Chemotherapy 47, 260614.
16
.
Firsov, A. A., Vostrov, S. N., Lubenko, I. Y. et al. (2003). In vitro pharmacodynamic evaluation of the mutant selection window hypothesis using four fluoroquinolones against Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 47, 160413.
17
.
Croisier, D., Etienne, M., Bergoin, E. et al. (2004). Mutant selection window in levofloxacin and moxifloxacin treatments of experimental pneumococcal pneumonia in a rabbit model of human therapy. Antimicrobial Agents and Chemotherapy 48, 1699707.
18
.
Croisier, D., Chavanet, P., Lequeu, C. et al. (2002). Efficacy and pharmacodynamics of simulated human-like treatment with levofloxacin on experimental pneumonia induced with penicillin-resistant pneumococci with varying susceptibilities to fluoroquinolones. Journal of Antimicrobial Chemotherapy 50, 34960.
19 . Comité de l'Antibiogramme de la Société Française de Microbiologie. (1996). 1996 report of the Comité de l'Antibiogramme de la Société Française de Microbiologie. Technical recommendations for in vitro susceptibility testing. Clinical Microbiology and Infection 2S1, 1125.
20 . National Committee for Clinical Laboratory Standards. (1992). Methods for Determining Bactericidal Activity of Antimicrobial Agents Tentative Guideline 771 E. NCCLS, Villanova, PA, USA.
21 . Pan, X., Ambler, J., Mehtar, S. et al. (1996). Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 23216.[Abstract]
22
.
Morrissey, I. & George, J. (1999). Activities of fluoroquinolones against Streptococcus pneumoniae type II topoisomerase purified as recombinant proteins. Antimicrobial Agents and Chemotherapy 43, 257985.
23
.
Brenwald, N., Gill, M. & Wise, R. (1998). Prevalence of a putative efflux mechanism among fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 20325.
24
.
Gill, M., Brenwald, N. & Wise, R. (1999). Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 1879.
25
.
Markham, P. (1999). Inhibition of the emergence of ciprofloxacin resistance in Streptococcus pneumoniae by the multidrug efflux inhibitor reserpine. Antimicrobial Agents and Chemotherapy 43, 9889.
26
.
Piroth, L., Martin, L., Coulon, A. et al. (1999). Development of a new experimental model of penicillin-resistant Streptococcus pneumoniae pneumonia and amoxicillin treatment by reproducing human pharmacokinetics. Antimicrobial Agents and Chemotherapy 43, 248492.
27 . Chapin-Robertson, K. & Edberg, S. (1991). Measurement of antibiotics in human body fluids: techniques and significance. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 295366. Williams and Wilkins, Baltimore, MD, USA.
28
.
Andes, D. & Craig, W. (2002). Pharmacodynamics of the new fluoroquinolone gatifloxacin in murine thigh and lung infection models. Antimicrobial Agents and Chemotherapy 46, 166570.
29 . Breiman, L., Friedman, J., Olshen, R., et al. (1984). Classification and Regression Trees. Wadsworth, Belmont, CA, USA.
30 . Loh, W. & Shih, Y. (1997). Split selection methods for classification trees. Statistica Sinica 7, 81540.[ISI]
31
.
Ambrose, P., Grasela, D., Grasela, T. et al. (2001). Pharmacodynamics of fluoroquinolones against Streptococcus pneumoniae in patients with community-acquired respiratory tract infections. Antimicrobial Agents and Chemotherapy 45, 27937.
32 . Sullivan, J., McElroy, A. & Honsinger, R. (1999). Treating community-acquired pneumonia with once-daily gatifloxacin vs once-daily levofloxacin. Journal of Respiratory Diseases 47, 2926.
33 . Arguedas, A., Sher, L., Lopez, E. et al. (2003). Open label, multicenter study of gatifloxacin treatment of recurrent otitis media and acute otitis media treatment failure. Clinical Infectious Diseases 22, 94956.
34 . Gotfried, M., DeAbate, C., Fogarty, C. et al. (2001). Comparison of 5-day, short-course gatifloxacin therapy with 7-day gatifloxacin therapy and 10-day clarithromycin therapy for acute exacerbation of chronic bronchitis. Clinical Therapeutics 23, 97107.[CrossRef][ISI][Medline]
35 . Nicodemo, A. (2003). An open label, multicenter, non-comparative study of the efficacy and safety of oral gatifloxacin in the treatment of community-acquired pneumonia: a Brazilian study in five centers. Brazilian Journal of Infectious Diseases 7, 628.[Medline]
36 . Sher, L., McAdoo, M., Bettis, R. et al. (2002). A multicenter, randomized, investigator-blinded study of 5- and 10-day gatifloxacin versus 10-day amoxicillin-clavulanate in patients with acute bacterial sinusitis. Clinical Therapeutics 24, 26981.[CrossRef][ISI][Medline]
37 . Solèr, M., Lode, H., Baldwin, R. et al. (2003). Randomised double-blind comparison of oral gatifloxacin and co-amoxiclav for acute exacerbation of chronic bronchitis. European Journal of Clinical Microbiology and Infectious Diseases 22, 14450.[ISI][Medline]
38
.
Drlica, K. (2003). The mutant selection window and antimicrobial resistance. Journal of Antimicrobial Chemotherapy 52, 1117.
39
.
Zhanel, G., Roberts, D., Waltky, A. et al. (2002). Pharmacodynamic activity of fluoroquinolones against ciprofloxacin-resistant Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 49, 80712.
40 . Zhanel, G., Walkty, A., Nichol, K. et al. (2003). Molecular characterization of fluoroquinolone resistant Streptococcus pneumoniae clinical isolates obtained from across Canada. Diagnostic Microbiology and Infectious Disease 45, 637.[CrossRef][ISI][Medline]
41
.
Fukuda, H., Kishii, R., Takei, M. et al. (2001). Contributions of the 8-methoxy group of gatifloxacin to resistance selectivity, target preference, and antibacterial activity against Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 45, 164953.
42
.
Li, X., Zhao, X. & Drlica, K. (2002). Selection of Streptococcus pneumoniae mutants having reduced susceptibility to moxifloxacin and levofloxacin. Antimicrobial Agents and Chemotherapy 46, 5224.
43
.
MacGowan, A. P., Rogers, C., Holt, H. A. et al. (2003). Activities of moxifloxacin against, and emergence of resistance in, Streptococcus pneumoniae and Pseudomonas aeruginosa in an in vitro pharmacokinetic model. Antimicrobial Agents and Chemotherapy 47, 108895.
44 . Nicolau, D. & Ambrose, P. (2001). Pharmacodynamic profiling of levofloxacin and gatifloxacin using Monte Carlo simulation for community-acquired isolates of Streptococcus pneumoniae. American Journal of Medicine 111, Suppl. 9A, 13S18S.
45
.
Jones, R., Rubino, C., Bhavnani, S. et al. (2003). Worldwide antimicrobial susceptibility patterns and pharmacodynamic comparisons of gatifloxacin and levofloxacin against Streptococcus pneumoniae: report from the Antimicrobial Resistance Rate Epidemiology Study Team. Antimicrobial Agents and Chemotherapy 47, 2926.
46 . Scheld, W. (2003). Maintaining fluoroquinolone class efficacy: review of influencing factors. Emerging Infectious Diseases 9, 19.[ISI][Medline]