Pharmacodynamics of 750 mg and 500 mg doses of levofloxacin against ciprofloxacin-resistant strains of Streptococcus pneumoniae

Pharmacodynamics of 750 mg and 500 mg doses of levofloxacin against ciprofloxacin-resistant strains of Streptococcus pneumoniae

Diagnostic Microbiology and Infectious Disease 44 (2002) 43– 49 www.elsevier.com/locate/diagmicrobio Pharmacodynamics of 750 mg and 500 mg doses of ...

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Diagnostic Microbiology and Infectious Disease 44 (2002) 43– 49

www.elsevier.com/locate/diagmicrobio

Pharmacodynamics of 750 mg and 500 mg doses of levofloxacin against ciprofloxacin-resistant strains of Streptococcus pneumoniae Philip D. Lister* Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, Nebraska 68178, USA Received 1 February 2002; accepted 29 May 2002 The data in this manuscript were presented at the 39th Annual Infectious Disease Society of America Conference, San Francisco, CA, October 2001. Abstract No. 570

Abstract An in vitro pharmacokinetic model (IVPM) was used to evaluate the pharmacodynamics of the 750 mg and 500 mg doses of levofloxacin against 4 ciprofloxacin-nonsusceptible Streptococcus pneumoniae. Levofloxacin MICs ranged from 1.4 to 3.2 ␮g/ml. Log-phase cultures (5 ⫻ 107 cfu/ml) were inoculated into the IVPM and exposed to the peak free-drug concentrations of levofloxacin achieved in human serum with each dose. Levofloxacin was dosed at 0 and 24 h, elimination pharmacokinetics were simulated, and viable counts were measured over 30 h. The 750 mg dose was rapidly bactericidal against all 4 strains, achieving eradication within 30 h. Against strains with levofloxacin MICs of 1.4 and 1.8 ␮g/ml, the 500 mg dose exhibited pharmacodynamics similar to the 750 mg dose. In contrast, against strains with levofloxacin MICs of 2.6 and 3.2 ␮g/ml, viable counts never fell below 104 cfu/ml. The rapid killing and eradication of these pneumococci by the 750 mg dose warrant the clinical evaluation of this new dose in the treatment of pneumococcal infections. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction Although fluoroquinolone resistance among S. pneumoniae remains relatively rare at this time, there are indications that resistance is increasing in some regions (Chen et al., 1999; Jones and Pfaller, 2000; Sahm et al., 2001). As resistance increases, the usefulness of the currently available fluoroquinolones will be threatened, with the 500 mg dose of levofloxacin being the most vulnerable based upon its potency compared to gatifloxacin and moxifloxacin (Bauernfeind, 1997). In addition to the 500 mg dose, levofloxacin is also available as an oral dose of 750 mg which provides increased peak levels of 8.6 ␮g/ml (Chien et al., 1998) in the serum of healthy volunteers compared to 5.7 ␮g/ml for the oral 500 mg dose (Physicians, 2001). Although the 750 mg dose is only approved for complicated skin and soft tissue infections at this time, the improved pharmacokinetics of this dose make it attractive for treating pneumococcal in* Corresponding author. Tel.: ⫹1-402-280-1224; fax: ⫹1-402-2801225. E-mail address: [email protected] (P.D. Lister).

fections as well. Of particular interest is the pharmacodynamic potential of the 750 mg dose against pneumococcal mutants that have lost their susceptibility to ciprofloxacin. In this study, a two-compartment in vitro pharmacokinetic model (IVPM) was used to simulate the pharmacokinetics of and compare the pharmacodynamics of the 500 mg and 750 mg oral doses of levofloxacin against 4 S. pneumoniae that had lost their susceptibility to ciprofloxacin.

2. Materials and methods 2.1. Bacterial strains and culture conditions Four strains of S. pneumoniae were selected for this study. S. pneumoniae 331 is a clinical isolate that was kindly provided by Dr. Alan Evangelista (Ortho-McNeil Pharmaceutical Company). The other 3 strains were mutants selected in our laboratory through in-agar exposure of susceptible clinical isolates S. pneumoniae 212, S. pneumoniae 256, and S. pneumoniae 257 to ciprofloxacin at concentrations fourfold above the MIC. The MICs of ciprofloxacin and levofloxacin against the panel used in these

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Table 1 MICs and pharmacodynamic parameters of levofloxacin against S. pneumoniae Strain Number

S. S. S. S.

pneumoniae pneumoniae pneumoniae pneumoniae a b

Ciprofloxacin Levofloxacin Peak/MIC Levofloxacin AUC/MIC Levofloxacin Peak/MIC Levofloxacin AUC/MIC Levofloxacin MIC MIC 500 mg dose 500 mg dose 750 mg dose 750 mg dose (␮g/ml)a (␮g/ml)b 212 4 331 4 256 16 257 16

1.4 1.8 3.2 2.6

3.1 2.4 1.3 1.7

33 29 14 18

4.6 3.6 2 2.5

49 43 22 27

Minimum inhibitory concentrations were measured by agar dilution. Minimum inhibitory concentrations were measured by macrobroth dilution in THB using 0.2 ␮g/ml increments.

studies are shown in Table 1. Logarithmic-phase cultures were prepared by suspending 10 colonies from a 14 h culture on Trypticase soy agar supplemented with 5% sheep blood (BBL Microbiology Systems, Cockeysville, MD) into 6 mL of Todd-Hewitt broth (Unipath/Oxoid, Ogdensburg, NY) (20). Viable bacterial counts after 10 h of incubation at 37°C in 5% CO2 ranged from 1 ⫻ 108 to 5 ⫻ 108 cfu/ml. 2.2. Antibiotic preparations Ciprofloxacin powder was supplied by Bayer Corporation, New Haven, CT. Levofloxacin powder was supplied by R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ. Prior to each experiment, ciprofloxacin was dissolved in sterile distilled water and levofloxacin was dissolved in 0.2 mL of 0.1 M NaOH. Each drug was then diluted to desired concentration with distilled water, and sterilized by passage through a 0.20-␮m-pore-size Acrodisc syringe filter membrane (Gelman Sciences, Ann Arbor, Mich.). 2.3. Antimicrobial susceptibility testing Susceptibility tests with ciprofloxacin were performed by agar dilution methodology (National Committee for Clinical Laboratory Standards, 1997). Levofloxacin MICs were evaluated by broth macrodilution methodology according to the procedure recommended by the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, 1997), with the following exceptions. THB was used as the test medium since it was the medium used for the pharmacodynamics studies. In addition, to more accurately define the AUC/MIC ratios, MICs were evaluated using 0.2 ␮g/ml increments, rather than the standard two-fold dilution methodology. 2.4. In vitro pharmacokinetic model The two-compartment IVPM used in these studies was a modification (Lister et al., 1998) of the original model described by Blaser and colleagues (Blaser et al., 1985). The hollow-fiber cartridges (Model# BR130; Unisyn Fibertech, San Diego, CA) used in these studies consisted of 2,250

cellulose acetate hollow-fibers contained within a polycarbonate housing, with each fiber having 30,000 MW pores within its wall. The surface area of exchange between the hollow fibers and the extracapillary space (peripheral compartment) was 1.5 square feet. Medium containing antibiotic was pumped through the lumen of the fibers at a flow rate of 20 mL/min using Masterflex computerized peristaltic pumps (Model 7550 –90; Cole-Parmer Instrument Company, Vernon Hills, IL) and Easy-Load pump heads (Model 7518 – 00; Cole-Parmer). In addition, the bacterial culture within the peripheral compartment was continuously circulated with similar peristaltic pumps at a rate of 20 mL/min through a loop of silicone tubing attached to two ports entering and exiting the peripheral compartment. The initial volume of culture circulated through the peripheral compartment and loop of silicone tubing was 35 to 40 mL. When samples were required from the peripheral compartment, 0.5 mL volumes were removed through a 4-way sterile stopcock (Medex, Hilliard, Ohio) positioned within the loop of silicone tubing. The volume of THB within the central reservoir was 500 mL. The rate of dilution and elimination was drug from the central reservoir was set to mimic an elimination half-life of 7 h. In drug-free control experiments, the volume of THB in the central reservoir was also 500 mL and the flow-rates for addition of fresh media and elimination from the central reservoir were 2 mL/min. 2.5. Pharmacodynamic studies in the IVPM Pharmacodynamic studies with S. pneumoniae were evaluated in THB at 37°C in ambient air. Logarithmicphase cultures (5 ⫻ 107 cfu/ml) were introduced into the peripheral compartment of the IVPM and were exposed to the free-drug peak concentrations of levofloxacin that are observed in human serum after doses of 500 mg and 750 mg. The total peak drug concentrations achieved in human serum with oral doses of levofloxacin are 5.7 ␮g/ml for the 500 mg dose (Physicians, 2001) and 8.6 ␮g/ml for the 750 mg dose (Chien et al., 1998). Protein binding of levofloxacin in human serum is approximately 25% (Physicians, 2001). Therefore, target peak concentrations accounting for protein binding were 4.2 ␮g/ml for the 500 mg dose and 6.5

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␮g/ml for the 750 mg dose, and an elimination half-life of 8 h was simulated for each dose. To establish the pharmacokinetics of the two doses in the model, samples were removed from the peripheral compartment at 0.5, 2, 4, 8, 12, and 24h, and concentrations of levofloxacin were measured by disk diffusion bioassay using a susceptible strain of Escherichia coli (Chapin-Robertson and Edberg, 1991). The linear range of the bioassay was 0.1 to 0.9 ␮g/ml, r2 ⫽ 0.98. The interday percent coefficient of variation was ⬍3%. Area under the concentration-time curve over 24 h (AUC0 –24) were calculated using the trapezoidal rule. The AUC/MIC ratios were calculated by dividing the AUC0 –24 by the MICs for specific strains of S. pneumoniae (Schentag et al., 1991). Peak concentrations of levofloxacin were dosed into the IVPM at 0 and 24 h. After each dose, elimination pharmacokinetics were simulated to mimic an elimination half-life of 8 h. To evaluate pharmacodynamic interactions, samples were removed from the peripheral compartment of the IVPM at 0, 2, 4, 8, 24, and 30 h, and viable bacterial counts were measured by plating serial 10-fold dilutions of each sample into Todd-Hewitt agar (BBL) and incubating plates overnight at 37°C in 5% CO2. The lowest dilution plated was 0.1 mL of undiluted sample from the peripheral compartment. Therefore, the lowest level of detection was 1 colony per plate or 10 cfu/ml. Antibiotic carryover was prevented in these experiments by first incubating samples taken from the IVPM with antibiotic-removal beads for 15 min (Zabinski et al., 1993). To evaluate for the selection of mutants exhibiting decreased susceptibility to fluoroquinolones, 30 h samples were also plated into Todd-Hewitt agar containing levofloxacin at a concentration fourfold above the MIC. Each experiment was performed in duplicate on separate days and data are presented as mean values from those duplicate experiments.

3. Results 3.1. Characterization of test strains and IVPM The MICs of ciprofloxacin and levofloxacin against the four S. pneumoniae isolates are shown in Table 1. MICs for ciprofloxacin ranged from 4 to 16 ␮g/ml. MICs for levofloxacin ranged from 1.4 to 3.8 ␮g/ml. Peak concentrations (mean ⫾ SD) of levofloxacin in the peripheral compartment at 0.5 h were 4.3 ⫾ 0.2 ␮g/ml for the 500 mg dose and 6.4 ⫾ 0.3 ␮g/ml for the 750 mg dose (Fig. 1). The AUC24 for the 500 mg dose was 46 ␮g.h/mL and for the 750 mg dose was 69 ␮g.h/mL. The AUC/MIC ratios for the 500 mg dose ranged from 14 to 33 for and ranged from 22 to 49 for the 750 mg dose (Table 1). Fig. 1 also graphically presents the relationship between the pharmacokinetics of the 500 mg and 750 mg doses of levofloxacin as measured in the IVPM and the MICs of levofloxacin against the 4 pneumococcal strains.

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Fig. 1. Pharmacokinetic profiles of simulated 750 mg and 500 mg doses of levofloxacin in the peripheral compartment of a two-compartment IVPM. Each datum point represents the mean concentration of levofloxacin for duplicate experiments. The horizontal dashed lines represent the levofloxacin MICs for the S. pneumoniae strains. The strain number is indicated to the right of each line.

3.2. Pharmacodynamic studies The pharmacodynamics of levofloxacin against the 4 S. pneumoniae strains are shown in Fig. 2. Drug-free control cultures of each strain grew to a maximum of approximately 5 ⫻ 108 cfu/ml by 12 to 24 h, and viable counts remained steady throughout the remainder of the 30 h experimental period. 3.2.1. S. pneumoniae 212 The 750 mg and 500 mg doses exhibited similar rates of killing of S. pneumoniae 212 over the initial 8 h, with both doses decreasing viable counts 4.5 logs (Fig. 2A). After 8h, the 750 mg dose (AUC/MIC ⫽ 49) continued to kill S. pneumoniae 212 and achieved eradication (viable counts ⬍10 cfu/ml) within the first 24 h dose interval. In contrast, viable counts changed very little between 8 and 24 h in cultures treated with the 500 mg dose (AUC/MIC ⫽ 33). Nevertheless, S. pneumoniae 212 was eradicated with the second 500 mg dose introduced into the IVPM at 24 h. 3.2.2. S. pneumoniae 331 In studies with S. pneumoniae 331, both the 750 mg and 500 mg doses of levofloxacin were rapidly bactericidal (Fig. 2B). By 8 h, the 750 mg (AUC/MIC ⫽ 43) dose had

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Fig. 2. Pharmacodynamics of levofloxacin against S. pneumoniae 212 (Panel A), S. pneumoniae 331 (Panel B), S. pneumoniae 256 (Panel C) and S. pneumoniae 257 (Panel D). Each datum point represents the mean number of viable bacteria (log10 cfu/ml) for duplicate experiments. Error bars represent standard deviations. Arrows on the X-axis represent the times of levofloxacin dosing into the model.

decreased viable counts 5 logs, compared to a 3.5-log decrease with the 500 mg dose (AUC/MIC ⫽ 29). Although the initial rate of killing with the 500 mg was slightly slower than the 750 mg dose, both of these doses achieved eradication of S. pneumoniae 313 within the first 24h dose interval.

3.2.3. S. pneumoniae 256 Over the initial 8 h, the rates of killing of S. pneumoniae 256 by the 750 mg and 500 mg doses of levofloxacin were similar (Fig. 2C). Both doses decreased viable counts approximately 3 to 4 logs. After 8h, the 750 mg dose (AUC/ MIC ⫽ 22) continued to decrease viable counts and

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achieved eradication of S. pneumoniae 256 within the first 6 h of the second dose interval. In contrast, viable counts of S. pneumoniae 256 increased 2 logs between 8 and 24 h when treated with the 500 mg dose (AUC/MIC ⫽ 14). This increase in viable counts was not due to the emergence of resistant subpopulations, as no growth was observed on drug-selection plates. Despite a second 500 mg dose at 24 h, viable counts remained above 105 cfu/ml at 30 h. 3.2.4. S. pneumoniae 257 The 750 mg dose of levofloxacin was rapidly bactericidal against S. pneumoniae 257, with viable counts decreasing over 5 logs by 8 h (Fig. 2D). Eradication of S. pneumoniae 257 was achieved within 8 to 24 h after the first 750 mg dose (AUC/MIC ⫽ 27). In contrast, viable counts of S. pneumoniae 257 only decreased 2.5 logs within the first 8 h after the 500 mg dose (AUC/MIC ⫽ 18), and viable counts changed very little between 8 and 30h, never falling below 104 cfu/ml despite a second 500 mg dose. No mutant subpopulations were detected on drug selection plates.

4. Discussion Fluoroquinolone resistance remains relatively rare among pneumococci at this time. From 1994 –1997, surveys of thousands of pneumococcal isolates from multiple medical centers yielded fluoroquinolone resistance rates of only 0.3 to 0.5% (Ballow et al., 1997; Brueggemann et al., 1997; Doern et al., 1998). In 1997, a study of 2,284 clinical pneumococcal isolates from the United States, Europe, Latin America, and Canada yielded only 13 (0.6%) fluoroquinolone-resistant strains (Odland et al., 1999). Analysis of pneumococcal isolates collected globally from two studies during 1997 to 2000 (n ⫽ 26,195 total isolates) yielded 116 (0.4%) fluoroquinolone resistant isolates (Hoban et al., 2001; Thornsberry et al., 2001). Breakdown of regional rates showed that rates were ⱕ0.5% in the United States, Canada, Latin America, and Europe and 0.7% in AsiaPacific. Although fluoroquinolone resistance is not a serious therapeutic threat at this time, recent data suggest we may just be seeing an increase in resistance in some regions. In a Canadian study of 7551 pneumococcal isolates recovered from 1988 to 1998, Chen and colleagues reported increasing levels of ciprofloxacin resistance among pneumococcal isolates starting in the mid 1990s and continuing through 1998 when the study was completed (Chen et al., 1999). Activity of the newer fluoroquinolones was also diminished, with only 60 –70% of ciprofloxacin-resistant isolates remaining susceptible to levofloxacin, moxifloxacin and gatifloxacin. Recently published data from the SENTRY and TRUST surveillance programs in the United States and Canada have shown that from 1997 to 1999, fluoroquinolone rates have increased significantly, even though they remain at or below 1% (Jones and Pfaller, 2000; Sahm et al., 2001).

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As resistance increases, the usefulness of all the currently available anti-pneumococcal fluoroquinolones will be threatened over time. Among the fluoroquinolones most commonly used to treat pneumococcal infections, the 500 mg dose of levofloxacin appears to be the most vulnerable based upon its potency compared to gatifloxacin and moxifloxacin (Bauernfeind, 1997). This study was designed to evaluate the pharmacodynamics of the larger 750 mg dose of levofloxacin against a panel of S. pneumoniae that had lost their susceptibility to ciprofloxacin and in some cases levofloxacin. Although the 750 mg dose is not approved for infections other than skin and soft tissue, the increased peak levels achieved with this dose (Chien et al., 1998) compared to the 500 mg dose (Physicians, 2001) make it attractive for treatment of S. pneumoniae infections in light of the increasing threat of resistance. In studies with the two strains that had lost their susceptibility to ciprofloxacin, but remained susceptible levofloxacin, the 500 mg and 750 mg simulated doses were similar in their pharmacodynamics and both doses achieved eradication of a 5 ⫻ 107 cfu/ml inoculum of these strains from the IVPM. The eradication of these two strains with both doses was not surprising since AUC/MIC ratios were approximately 30 to 50 in all experiments. Previous studies have demonstrated that levofloxacin and other fluoroquinolones can achieve eradication of S. pneumoniae from IVPMs when AUC/MIC ratios are approximately 30 to 50 (Lacy et al., 1999; Lister, 2002; Lister and Sanders, 1999a; Lister and Sanders, 1999b; Lister and Sanders, 2001). The 750 mg dose of levofloxacin also eradicated the two pneumococcal strains that had lost susceptibility to levofloxacin, despite an AUC/MIC ratio of only 22 for S. pneumoniae 256. Previous studies with gatifloxacin have suggested that once AUC/MIC ratios fall below 30 then the likelihood of eradication is diminished (Lister, 2002). However, in that study eradication was still observed with AUC/ MIC ratios as low as 27. In studies with S. pneumoniae 256, levofloxacin concentrations remained above the MIC for 8 h after the 750 mg dose (Fig. 1). The lack of inoculum regrowth during the first dose interval in these experiments, despite up to 16 h of subinhibitory concentrations of levofloxacin, suggests that a substantial post-antibiotic sub-MIC effect (PA-SME) was occurring. The PA-SME is an extension of the post-antibiotic effect period when sub-inhibitory concentrations of antibiotic remain in the environment (Cars and Odenholt-Tornqvist, 1993), and this pharmacodynamic phenomenon has been reported with levofloxacin against S. pneumoniae (Licata et al., 1997). In these studies with the 750 mg dose against S. pneumoniae 256 and in experiments with the 500 mg dose against the two susceptible strains where levofloxacin levels remained above the MIC for less than half of the 24 h dose interval, the PA-SME intervals appeared to be much longer than the 1.25 to 3.25 h reported by Licata and colleagues (Licata et al., 1997). However, this is not surprising since antibiotic was removed from the

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bacterial environment more gradually and by more natural processes in the IVPM, as opposed to centrifugation and washing techniques used by Licata and colleagues. In contrast to the 750 mg dose, the 500 mg dose was unable to decrease viable counts of the nonsusceptible strains below 104 cfu/ml. The decreased killing and lack of eradication observed with the 500 mg dose against these strains was not surprising since AUC/MIC ratios were only 14 and 18. Furthermore, levofloxacin levels from the 500 mg dose only remained above the MIC for 4 to 6 h. Although inoculum regrowth was observed in studies with S. pneumoniae 256, this was not due to the emergence of a more resistant subpopulation. The rapid regrowth observed after 8 h was most likely related to the fact that levofloxacin concentrations in the IVPM remained above the MIC for S. pneumoniae 256 for only about 4 h with the 500 mg dose. Therefore, the regrowth observed most likely reflected the natural regrowth of the culture after inhibitory concentrations were lost. Also of interest was the lack of any net change in viable bacterial counts between 24 and 30 h (first 6 h of second dose interval) in experiments with the 500 mg dose and S. pneumoniae 256 and 257. Previous studies with ␤-lactams and fluoroquinolones against S. pneumoniae and Pseudomonas aeruginosa have shown similar decreases in the antibacterial activity of second and subsequent doses in the IVPM, despite the absence of any stable resistant mutant subpopulations (Lister et al., 1998; Lister et al., 1999; Lister and Sanders, 1999a; Lister and Sanders, 1999b). In some of these studies, the second and subsequent doses of antibiotic were actually associated with gradual net increases in viable counts at the end of each dose interval, despite observed killing during the initial hours after each dose. Since no resistant mutants were detected on drug-selection plates in the current study or these previous studies, the decreased antibacterial activity observed may represent adaptive resistance, or the reversible decrease in susceptibility after first exposure to an antibiotic (Daikos et al., 1990; Daikos et al., 1991). It was of interest that no emergence of resistance occurred during any of these experiments, despite simulation of peak levels of levofloxacin that were often only 1- to threefold above the MIC (Table 1). In contrast to the staphylococci, strains of S. pneumoniae have been comparatively slow in their development of fluoroquinolone resistance, despite the wide-spread use of ciprofloxacin since the late 1980s. As already discussed, surveys of thousands of pneumococcal isolates from the United States between 1997 and 2000 have yielded fluoroquinolone resistance rates of only 0.5% (Hoban et al., 2001; Sahm et al., 2001; Thornsberry et al., 2001). One possible explanation for the relative lack of fluoroquinolone resistance among pneumococci may be the restricted use of fluoroquinolones in pediatric patients where long-term colonization by pneumococci presents an ideal environment for resistance selection. However, there are also some data that suggest S. pneumoniae lack the same propensity to mutate to resistance that staphylococci and

other species exhibit. In a recent study by Thomson and colleagues, cultures of 20 staphylococcal clinical isolates and 21 pneumococcal clinical isolates were exposed to ciprofloxacin and levofloxacin at concentrations 2- to eightfold above the MIC and selection of first-step mutants was evaluated (K. S. Thomson, E. S. Moland, and C. C. Sanders, unpublished data). In experiments with the staphylococci and ciprofloxacin, first-step mutants (⬎4-fold increase in MIC) were isolated from 19/20 (95%) of the clinical isolates. In contrast, first-step mutants were selected from only 5/21 (24%) pneumococcal isolates exposed to ciprofloxacin and 3/21 (14%) isolates exposed to levofloxacin, even when concentrations were only twofold above the MIC. These data in combination with the lack of resistance emergence in the current study suggest that pneumococci do not exhibit the same propensity to mutate to resistance as is seen with staphylococci and many Gram-negative pathogens. In conclusion, the pharmacodynamics exhibited by the 750 mg dose of levofloxacin against the fluoroquinoloneresistant S. pneumoniae in this study suggest that the 750 mg dose of levofloxacin may be effective in treating both wild-type and first-step fluoroquinolone resistant mutants. Since these studies focused mostly on mutants that were created within the laboratory environment, further studies with mutants that evolved in the clinical environment would be of interest. Furthermore, these data warrant the clinical evaluation of the 750 mg dose of levofloxacin for the treatment of pneumococcal infections. Acknowledgments This work was supported by a grant from Ortho-McNeil Pharmaceutical Company. I would like to thank Jennifer Black, M.T., for her excellent technical assistance on this project. References Ballow, C. H., Jones, R. N., Johnson, D. M., Deinhart, J. A., & Schentag, J. J. (1997). Comparative in vitro assessment of sparfloxacin activity and spectrum using results from over 14,000 pathogens isolated at 190 medial centers in the USA. Diagnostic Microbiology and Infectious Diseases, 29, 173–186. Bauernfeind, A. (1997). Comparison of the antibacterial activities of the quinolones BAY 12– 8039, gatifloxacin (AM 1155), trovafloxacin, clinafloxacin, levofloxacin and ciprofloxacin. Journal of Antimicrobial Chemotherapy, 40, 639 – 651. Blaser, J., Stone, B. B., & Zinner, S. H. (1985). Two compartment kinetic model with multiple artificial capillary units. Journal of Antimicrobial Chemotherapy, 15, 131–137. Brueggemann, A. B., Kugler, K. C., & Doern, G. V. (1997). In vitro activity of BAY 12– 8039, a novel 8-methoxyquinolone, compared to activities of six fluoroquinolones against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Antimicrobial Agents and Chemotherapy, 41, 1594 –1597. Cars, O., & Odenholt-Tornqvist, I. (1993). The post-antibiotic sub-MIC effect in vitro and. in vivo. Journal of Antimicrobial Chemotherapy, 31, 159 –166.

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