Pharmacodynamic studies of trovafloxacin and grepafloxacin in vitro against Gram-positive and Gram-negative bacteria

Inga Odenholt*, Thomas Cars and Elisabeth Löwdin

Antibiotic Research Unit, Department of Infectious Diseases and Clinical Microbiology, University Hospital, S-751 85 Uppsala, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Grepafloxacin and trovafloxacin are two novel fluoroquinolones with extended Gram-positive bacterial spectra compared with older quinolones. The aim of the present study was to investigate the different pharmacodynamic parameters of grepafloxacin in comparison with those of trovafloxacin. The following studies were performed against various Gram-positive and Gram-negative bacteria: (i) determination of the rate and extent of killing at a concentration corresponding to the 1 h non-protein-bound human serum level following an oral dose of 800 mg grepafloxacin and 300 mg trovafloxacin; (ii) determination of the rate and extent of killing of the two quinolones at different concentrations; (iii) determination of the post-antibiotic effects (PAEs); (iv) determination of the post-antibiotic sub-MIC effects (PA SMEs); (iv) determination of the rate and extent of killing in an in vitro kinetic model. It was shown that both grepafloxacin and trovafloxacin exhibited concentration-dependent killing against both Gram-positive and Gram-negative bacteria. Grepafloxacin exhibited a slower bactericidal effect against all the Gram-positive strains investigated in comparison with trovafloxacin in spite of a similar Cmax/MIC in the static experiments and a similar AUC/MIC ratio in the kinetic experiments. No major differences in the extent and rate of killing were noted against the Gram-negative strains, which were killed almost completely after 3 h except for Pseudomonas aeruginosa. A PAE of both quinolones was noted for all strains investigated. Trovafloxacin induced longer PAEs against the Gram-positive strains but shorter PAEs in comparison with those of grepafloxacin against the Gram-negative strains. A prolonging of the PAEs was noted for all bacteria when exposed to sub-MICs in the post-antibiotic phase. With a similar AUC/MIC of 310 for the penicillin-sensitive strain of Streptococcus pneumoniae and 143 for the penicillin-resistant strain, the time for 99.9% eradication for both strains was 2 h for trovafloxacin and 6 h for grepafloxacin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The fluoroquinolone antimicrobial agents were introduced to the market at the end of the 1980s and have thereafter been used extensively, owing to a broad Gram-negative spectrum including Pseudomonas aeruginosa. However, despite widespread use, there have been concerns about the activity of these agents against Gram-positive bacteria.1 During the past years several agents with enhanced activity against Gram-positive bacteria have been developed. Grepafloxacin and trovafloxacin are two novel quinolones that have a bacterial spectrum that includes Gram-positive bacteria such as Streptococcus pneumoniae, both penicillin-sensitive and penicillin-resistant, and Streptococcus pyogenes.2,3

The pharmacodynamics of antibiotics have become increasingly important for determining optimal dosing schedules. It has been shown that the relationship between pharmacokinetic and pharmacodynamic parameters is different for different classes of antibiotics.4 For the quinolones, the 24 h area under the serum concentration curve (AUC)/MIC has been suggested to correlate with efficacy both in animal studies and in humans.5,6 Also the peak concentration/MIC has been correlated with higher survival rates in some animal studies, especially when the peak concentration/MIC is >=10/1.7 Several investigators have also studied the pharmacodynamics of quinolones in in vitro kinetic models and have shown that some quinolones have pharmacodynamic advantages over others.810 The purpose of this study was to investigate basic pharmacodynamic parameters such as time–kill curves at a fixed concentration, correlating with the Cmax in humans, killing at different concentrations, the post-antibiotic effect (PAE) and post-antibiotic sub-MIC effect (PA SME) of trovafloxacin and grepafloxacin, and also to determine the rate and extent of killing by the two quinolones in an in vitro kinetic model. The strains studied were S. pneumoniae (penicillin-sensitive, penicillin-intermediate and penicillin-resistant), S. pyogenes, Escherichia coli, Klebsiella pneumoniae and P. aeruginosa.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics

Grepafloxacin was provided by Glaxo Wellcome, Gothenburg, Sweden and trovafloxacin by Pfizer AB, Täby, Sweden. The antibiotics were obtained as reference powders with known potency. All substances were dissolved in 0.1 M NaOH and thereafter diluted in the same broth as was used to grow the organisms. The solutions were made on the same day as the experiments were performed.

Bacterial strains and media

The strains used in the study included S. pneumoniae ATCC 6306 (penicillin sensitive), clinical isolates of S. pneumoniae 1020 (penicillin intermediate), S. pneumoniae R-2151 (penicillin resistant) and S. pyogenes group A, NCTC P1800. The Gram-negative strains studied were E. coli ATCC 25922, K. pneumoniae ATCC 29655 and P. aeruginosa ATCC 27853. The clinical strains were obtained from the Clinical Microbiological Laboratory, Uppsala, Sweden. The Gram-negative strains were grown in Mueller–Hinton (M-H) broth (Difco Laboratories, Detroit, MI, USA), supplemented with 50 mg/L Ca2+ and 25 mg/L Mg2+, for 6 h at 37°C, yielding an initial inoculum of approximately 5 x 108 cfu/mL. S. pyogenes and S. pneumoniae were grown in Todd-Hewitt broth (T-H; Difco), for 6 h at 37°C, resulting in approximately 109 cfu/mL.

Determination of MICs

MICs were determined in liquid media by a macro-dilution technique in triplicate on different occasions according to NCCLS guidelines.11 Two-fold serial dilutions of the antibiotics were added to broth which was inoculated with a final inoculum of the test strain of approximately 105 cfu/mL and incubated at 37°C for 24 h. The MIC was defined as the lowest concentration of the antibiotic allowing no visible growth.

Determination of the rate and extent of killing of grepafloxacin and trovafloxacin at a fixed concentration

A concentration of 1.5 mg/L was used, corresponding to the 1 h non-protein-bound human serum level following an oral dose of 800 mg grepafloxacin and 300 mg trovafloxacin.12,13 Tubes containing medium with antibiotic were inoculated with a suspension of the test strain, giving a final bacterial count of approximately 5 x 105 cfu/mL, and incubated at 37°C. Samples were withdrawn at 0, 3, 6, 9, 12 and 24 h and, if necessary, diluted in phosphate-buffered saline (PBS). Additional samplings were also performed after 1 and 2 h in the experiments, where a rapid initial killing was noted. Three dilutions of each sample (1/10, 1/100 and 1/1000) were spread on blood agar plates (Colombia agar base with 5% horse blood; Acumedia Manufacturers, Inc., Baltimore, MD, USA) incubated at 37°C and colonies were counted after 24 h. The sensitivity of the viable count is estimated at 101–5 x 101 cfu/mL. The antibiotics were tested against all the strains used in this study. Two experiments were performed for each antibiotic/bacterium combination.

Determination of the rate and extent of killing by grepafloxacin and trovafloxacin at different concentrations

Tubes containing 4 mL of the broth with the addition of the antibiotic at 2, 4, 8, 16 and 32 x MIC, respectively, were inoculated with a suspension of the test strain, giving a final bacterial count of approximately 5 x 105 cfu/mL. The tubes were incubated at 37°C and samples were withdrawn and seeded as described above. Both antibiotics were tested against S. pneumoniae ATCC 6306, S. pneumoniae 2151, E. coli ATCC 25922 and P. aeruginosa ATCC 27853. Two experiments were performed for each strain.

Determination of the PAE

The PAEs of trovafloxacin and grepafloxacin were investigated against S. pneumoniae ATCC 6306, S. pneumoniae R-2151, E. coli ATCC 25922 and P. aeruginosa ATCC 27853. After incubation for 6 h, the test strains in exponential growth phase were diluted 10–1 to obtain a starting inoculum of 5 x 107–108 cfu/mL. All strains except P. aeruginosa were then exposed to 10 x MIC of the antibiotic for 2 h at 37°C. Owing to the higher MIC for P. aeruginosa, and to have any clinical relevance, the PAE was induced with 2 x MIC of the antibiotics. To eliminate the antibiotics, the cultures were washed three times by centrifuging for 5 min at 1400g and diluting 10–1 in fresh medium. Depending on the killing, some of the cultures were thereafter further diluted to obtain an inoculum of approximately 105 cfu/mL. The unexposed control strains were washed similarly and diluted 10–3 in order to obtain an inoculum as close to that of the exposed strains as possible. The cultures with bacteria in the post-antibiotic phase and the controls were thereafter divided into four different tubes. In order to determine the PAE, one tube of each culture was reincubated at 37°C for another 22 h. Samples were withdrawn at 0 and 2 h (before and after dilution), at 3, 4, 5, 6, 8, 11 and at 24 h and if necessary diluted in PBS. In some experiments, samples were also taken at 14 h. Three dilutions of each sample were seeded on blood agar plates and colonies counted for determination of the number of cfu. The PAE was defined according to the following formula:

where T is the time required for the viable counts of the antibiotic-exposed cultures to increase by one log10 above the counts observed immediately after washing and C is the corresponding time for the controls.14 All antibiotic/ bacterium combinations were investigated in duplicate.

Determination of the PA SMEs

After washing and dilution, the remaining three tubes of the control cultures and the cultures in the post-antibiotic phase were exposed to 0.1, 0.2 and 0.3 x MIC, respectively, of the same antibiotic as used for the induction of the PAE and reincubated at 37°C for another 22 h. Samples were withdrawn and viable bacteria were determined as described above.

The PA SME was defined according to the following formula:

where TPA is the time required for the previously antibiotic-exposed cultures, which thereafter had been exposed to different sub-MICs, to increase by one log10 above the counts observed immediately after washing and C is the corresponding time for the unexposed control.15 The bacteria investigated were the same as those used in the determination of PAEs. All antibiotic/bacterium combinations were investigated in duplicate.

Determination of the rate and extent of killing in an in vitro kinetic model

A recently described model was used in these experiments.16 It consists of a spinner flask with a 0.45 mm filter membrane and a prefilter fitted in between the upper and the lower parts. A magnetic stirrer ensures homogeneous mixing of the culture and prevents membrane pore blockage. In one of the sidearms of the culture vessel, a silicon membrane is inserted to enable repeated sampling. The other arm is connected by thin plastic tubing to a vessel containing fresh medium. The medium is removed from the culture flask, through the filter, at a constant rate with a pump. Fresh sterile medium is sucked into the flask at the same rate by the negative pressure built up inside the culture vessel. The antibiotic was added to the vessel and eliminated at a constant rate according to the first order kinetics C = C0 x ekt where C0 is the initial antibiotic level, C the antibiotic level at the time t, k the rate of elimination and t the time elapsed since the addition of antibiotic. The apparatus was placed in a thermostatic room at 37°C during the experiments. The culture vessel was sterilized by autoclaving between experiments. An initial concentration of 1.5 mg/L was used for both grepafloxacin and trovafloxacin. The simulated half-life was 11 h for both trovafloxacin and grepafloxacin. The bacteria investigated were S. pneumoniae ATCC 6306 and S. pneumoniae 2151. Two experiments were performed for each strain.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs

The MICs and Cmax/MIC for the various strains are shown in Table IGo. Low MICs were noted for all strains except for P. aeruginosa. The differences in MICs between the two quinolones were no more than one dilution-step. The MICs found were similar to those previously published.2,3


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Table I. The Cmax/MIC and MIC (mg/L) of grepafloxacin and trovafloxacin for the strains investigated
 
Rate and extent of killing by grepafloxacin and trovafloxacin at a concentration of 1.5 mg/L

At similar Cmax/MIC ratios, trovafloxacin exhibited more favourable bactericidal activity than grepafloxacin against all Gram-positive strains investigated (Figure 1Go). Against the penicillin-sensitive and intermediate strains of S. pneumoniae, there was a >3 log10 difference in cfu after 3 h and 2.2 and 1.5 log10 difference, respectively, after 6 h. At 9 h, there was still a >1 log10 cfu difference in favour of trovafloxacin. Against the penicillin-resistant strain, there was a difference of 1.5, 1.0 and 0.7 log10 cfu after 3, 6 and 9 h, respectively. Against S. pyogenes the corresponding figures were 1.4, 1.4 and 1.1 log10 cfu. No major differences between quinolones in the extent and rate of killing were noted against K. pneumoniae, which was killed almost completely after 3 h. The killing of E. coli by trovafloxacin was similar. Although there was a marked killing of E. coli by grepafloxacin within 3 h, complete killing was not observed until 24 h. The slowest killing was noted for P. aeruginosa by both trovafloxacin and grepafloxacin, with a decrease of 3 log10 cfu after 9 h, but neither drug was able to eradicate this strain at achievable serum concentrations after 24 h (Figure 2Go).



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Figure 1. Killing curves of trovafloxacin (filled symbols) and grepafloxacin (open symbols) at a constant concentration of 1.5 mg/L against Streptococcus pneumoniae ATCC 6306 ({blacksquare}, {square}), S. pneumoniae I-1020 ({blacktriangleup}, {triangleup}), S. pneumoniae R-2151 (•, {circ}), Streptococcus pyogenes ({diamondsuit}, {diamond}). Controls without antibiotic: S. pneumoniae ATCC 6306 ({blacksquare}), S. pneumoniae I-1020 ({oplus}), S. pneumoniae R-2151 (•) and S. pyogenes (*).

 


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Figure 2. Killing curves of trovafloxacin (filled symbols) and grepafloxacin (open symbols) at a constant concentration of 1.5 mg/L against Escherichia coli ATCC 25922 (squares), Klebsiella pneumoniae ATCC 29655 (circles) and Pseudomonas aeruginosa ATCC 27853 (triangles). Controls without antibiotic: E. coli ATCC 25922 ({blacksquare}), K. pneumoniae ATCC 29655 ({oplus}), P. aeruginosa ATCC 27853 (*).

 
Rate and extent of killing of grepafloxacin and trovafloxacin at different concentrations

A concentration-dependent killing was seen in all experiments with the highest killing reached at approximately 16 x MIC for both grepafloxacin and trovafloxacin against the Gram-positive and Gram-negative strains (Figures 3 and 4GoGo, respectively). It was noted here that grepafloxacin had a slower bactericidal effect against the Gram-positive bacteria than trovafloxacin.



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Figure 3. Killing curves of (a) grepafloxacin against Streptococcus pneumoniae ATCC 6306; (b) trovafloxacin against S. pneumoniae ATCC 6306; (c) grepafloxacin against S. pneumoniae R-2151; (d) trovafloxacin against S. pneumoniae R-2151. {square} = 2 x MIC, {blacksquare} = 4 x MIC, {circ} = 8 x MIC, • = 16 x MIC, * = 32 x MIC, + = control without antibiotic.

 


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Figure 4. Killing curves of (a) grepafloxacin against Escherichia coli ATCC 25922; (b) trovafloxacin against E. coli ATCC 25922; (c) grepafloxacin against Pseudomonas aeruginosa ATCC 278532; (d) trovafloxacin against P. aeruginosa ATCC 278532. Drug concentrations were {square} = 2 x MIC, {blacksquare} = 4 x MIC, {circ} = 8 x MIC, • = 16 x MIC, * = 32 x MIC, + = control without antibiotic.

 
PAEs and PA SMEs

Trovafloxacin exhibited significantly longer PAEs in comparison with grepafloxacin against both the penicillin-sensitive and penicillin-resistant strains of S. pneumoniae (Table IIGo). The PAE of trovafloxacin for the sensitive strain was 7.3 h compared with 1.8 h for grepafloxacin, and for the resistant strain 3.0 h and 0.4 h, respectively. However, grepafloxacin exhibited longer PAEs against both E. coli (3.3 h versus 1.0 h for trovafloxacin) and P. aeruginosa (0.9 h versus 3.3 h). Very long PA SMEs were noted for trovafloxacin at 0.3 x MIC except for P. aeruginosa. This was in contrast to grepafloxacin, which induced long PA SMEs against P. aeruginosa but shorter values were found for the other strains investigated (Table IIGo).


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Table II. The post-antibiotic effects (PAEs) and post-antibiotic sub-MIC effects (PA SMEs) of trovafloxacin and grepafloxacin
 
Killing in the in vitro kinetic model

With a similar AUC of 18.6 mg.h/L for both quinolones and an AUC/MIC of 310 for the penicillin-sensitive strain of S. pneumoniae and 143 for the penicillin-resistant strain, the time for 99.9% eradication of both strains was 2 h for trovafloxacin and 6 h for grepafloxacin. All bacteria were eradicated after 4 h exposure to trovafloxacin and after 24 h exposure to grepafloxacin (Figure 5Go).



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Figure 5. Killing curves of trovafloxacin (filled symbols) and grepafloxacin (open symbols) against Streptococcus pneumoniae ATCC 6306 ({blacksquare}, {square}) and S. pneumoniae R-2151 (•, {circ}) in the in vitro kinetic model. Controls without antibiotic: S. pneumoniae ATCC 6306 ({blacktriangleup}); S. pneumoniae R-2151 ({triangleup}).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been shown both in vitro and in animal studies that fluoroquinolones display a concentration-dependent bactericidal effect.17,18 In the present study, this was confirmed for grepafloxacin and trovafloxacin, which exerted an increased bactericidal effect towards both Gram-positive and Gram-negative strains with increased concentrations, maximal killing being reached at 16 x MIC. Grepafloxacin exhibited a lower reduction of the initial inoculum of the Gram-positive strains investigated at 3, 6 and 9 h than trovafloxacin in spite of similar Cmax/MIC ratios. These differences in killing rates for the two quinolones were also confirmed in the kinetic model. In earlier studies of the killing activity of different quinolones we demonstrated that levofloxacin exhibited a significantly greater bactericidal effect on Gram-positive bacteria than sparfloxacin and ciprofloxacin. As seen in the present study, no differences were noted against Gram-negative bacteria.18

One pharmacodynamic factor that is most often investigated for new antibiotics is the PAE, i.e. the inhibition of bacterial growth after short exposure to antibiotics.14 In general, quinolones induce a PAE in both Gram-positive and Gram-negative bacteria.14,19,20 In the present study, trovafloxacin exhibited longer PAEs than grepafloxacin against both strains of S. pneumoniae. In contrast, grepafloxacin showed a longer PAE against E. coli and P. aeruginosa. Pankuch et al.19 recently reported a PAE of trovafloxacin against different Gram-positive and Gram-negative strains of approximately 1–4 h. They reported shorter PAEs against S. pneumoniae than in our study. This can be explained partly by their use of bacteria in stationary phase in contrast to our experiments, where bacteria in logarithmic phase were exposed to the antibiotic. Boswell et al.20 investigated the PAE of trovafloxacin against different strains of P. aeruginosa and found a PAE ranging from 0.6 to 1.4 h, which is similar to our results.

We have investigated previously the influence of sub-MICs on bacteria in the post-antibiotic phase and have found a very long delay in bacterial regrowth for many antibiotic classes and different bacterial species.15,21,22 For example, in a study of sparfloxacin, a PA SME at 0.3 x MIC of 5–6 h was found for the Gram-positive strains and between 1.7 and 13 h for the Gram-negative strains.22 Licata et al.23 investigated the PAEs and PA SMEs of levofloxacin and ciprofloxacin against several strains of S. pneumoniae and S. aureus and found a PAE of 0.5–2 h. They also found, as seen in our earlier studies, a pronounced prolonging of the PAE when bacteria in the post-antibiotic phase were re-exposed to sub-MICs. In the present study, the PA SMEs for trovafloxacin were very long at 0.3 x MIC, except against P. aeruginosa, which had a PA SME at 0.3 x MIC of 4.3 h. In contrast, grepafloxacin had an overall shorter PA SME against the strains investigated but a long PA SME at 0.3 x MIC against P. aeruginosa.

Several authors have tried to correlate different pharmacodynamic parameters of the quinolones such as AUC/ MIC, T > MIC and peak/MIC with clinical and bacteriological outcome.24 In an in vitro pharmacodynamic model, Madaras-Kelly et al.25 suggested that AUC/MIC24 was the most descriptive measurement of antibacterial effect of ciprofloxacin and ofloxacin against P. aeruginosa. They also suggested a value of 100 SIT–1.h as a breakpoint to prevent selection of resistant mutants. Drusano et al.7 showed in an animal model with lomefloxacin that peak values of >=10:1 were associated with better outcome but when a peak value of 10:1 was not reached, the AUC/MIC ratio was a better predictor of outcome. Craig found in animal infection models that the 24 h AUC/MIC ratio is the parameter that correlates best with the efficacy of quinolones.4 To produce a bacteriostatic effect they found that this ratio should be >35. However, an AUC/MIC ratio of >100 was associated with almost zero mortality. Forrest and coworkers6 found a similar ratio to correlate with clinical and microbiological cure in critically ill patients with nosocomial pneumonia. In our study, high AUC/ MIC (>100) ratios were obtained for both the penicillinsensitive and penicillin-resistant strains of S. pneumoniae for both grepafloxacin and trovafloxacin when human kinetics were simulated in the in vitro kinetic model, and no regrowth was noted in any of the experiments. However, a 99.9% reduction in bacterial count was obtained earlier with trovafloxacin than with grepafloxacin (2 h versus 6 h). All bacteria were eradicated after 4 h exposure to trovafloxacin and after 24 h exposure to grepafloxacin. Lister and Sanders10 described similar results in an in vitro kinetic model examining trovafloxacin, ofloxacin and ciprofloxacin. They noted a 99.9% reduction in bacterial count with trovafloxacin against S. pneumoniae after 1–3 h, which was faster than with the other two agents. The AUC/MIC value for trovafloxacin in their experiments was 300–600.

In conclusion, we showed that both grepafloxacin and trovafloxacin exhibited a concentration-dependent killing against both Gram-positive and Gram-negative bacteria. Grepafloxacin exhibited a slower bactericidal effect against all the Gram-positive strains investigated than trovafloxacin, in spite of a similar Cmax/MIC in the static experiments and similar AUC/MIC ratios in the kinetic experiments. A PAE of both quinolones was noted against all strains investigated. Trovafloxacin induced longer PAEs than grepafloxacin against the Gram-positive strains but shorter PAEs than grepafloxacin against the Gram-negative strains. A prolonging of the PAEs was noted for all bacteria exposed to sub-MICs in the post-antibiotic phase. With a similar AUC/MIC of 310 for the penicillin-sensitive strain of S. pneumoniae and 143 for the penicillin-resistant strain, the time for 99.9% eradication for both strains was 2 h for trovafloxacin and 6 h for grepafloxacin.


    Acknowledgments
 
This material was presented in part at the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, 24–27 September 1998, and was supported by grants from Glaxo Wellcome, Göteborg, Sweden and Pfizer AB, Täby Sweden.


    Notes
 
* Corresponding author. Tel: +46-40-922855; Fax: +46-40-331000; E-mail: inga.odenholt{at}inf.mas.lu.se Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 4 June 1999; returned 27 October 1999; revised 20 December 1999; accepted 11 February 2000