Exploration of the in-vitro pharmacodynamic activity of moxifloxacin for Staphylococcus aureus and streptococci of Lancefield Groups A and G

Alasdair P. MacGowan*, Karen E. Bowker, Mandy Wootton and H. Alan Holt

Bristol Centre for Antimicrobial Research and Evaluation, North Bristol NHS Trust and University of Bristol, Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The serum concentrations associated with the oral administration of 400 mg moxifloxacin every 24 h over 48 h in man were simulated in an in-vitro dilutional, continuous bacterial culture model of infection. The initial inoculum was 5 x 107–5 x 108 cfu/mL and all strains were tested on at least three occasions. Two strains of Staphylococcus aureus (one methicillin susceptible, the other resistant) with moxifloxacin MICs 0.14 mg/L and 0.06 mg/L and two strains of ß-haemolytic streptococci, Lancefield Group A, MIC 0.16 mg/L and Group G, MIC 0.4 mg/L were used. In addition, two laboratory-generated mutants with raised moxifloxacin MICs were also employed: methicillin-sensitive S. aureus (MSSA) MIC 1.0 mg/L and Group A streptococcus MIC 1.8 mg/L. The antibacterial effect of moxifloxacin was judged by changes in viable count over time, and the area under the bacterial-kill curve (AUBKC) after 24 and 48 h. For S. aureus MIC 0.14 mg/L the AUBKC24 (log cfu/mL.h) was 77.8 ± 4.6 and AUBKC48 92.0 ± 6.9. For its mutant, moxifloxacin MIC 1.0 mg/L, the AUBKC24 was 116.1 ± 15.6 and AUBKC48 211.9 ± 23.1, indicating decreased killing. AUBKC24 and AUBKC48 values of 110.7 ± 10.3 and 130.9 ± 21.3, respectively, were noted for the MRSA strain. The Group A streptococcus, MIC 0.16 mg/L, had an AUBKC24 of 91.4 ± 19.4 and AUBKC48 of 157.0 ± 70.9. The mutant, MIC 1.8 mg/L, had an AUBKC24 of 127.0 ± 1.9 and AUBKC48 of 205.1 ± 6.4. Despite a lower MIC (0.4 mg/L) the single strain of Group G streptococcus tested was killed poorly, AUBKC24 139.9 ± 3.6 and AUBKC48 252.3 ± 18.6. The pharmacodynamic parameters AUC/MIC, T > MIC, (AUC > MIC)/MIC (AUC = area under the curve, T = time) and WAUC ((AUC/MIC) (T > MIC/100)) (WAUC = weighted area under the curve) were related to AUBKC24 and AUBKC48 using an inhibitory sigmoid Emax model. T > MIC was poorly related to AUBKC (r = 0.36) while AUC/MIC, (AUC > MIC)/MIC and WAUC were strongly related to AUBKC24 (r = 0.75–0.79) and AUBKC48 (r = 0.78–0.84). The maximum antibacterial effect was achieved with an AUC/MIC ratio of 150–200. AUC-related pharmacodynamic parameters predicted antibacterial effect better than T > MIC.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Moxifloxacin (Bayer name: Bay 12-8039) is an 8-methoxyquinolone which, when compared with ciprofloxacin, has improved in-vitro activity against Gram-positive bacteria, atypical respiratory pathogens and anaerobes. The MIC90 values for methicillin- and ciprofloxacin-susceptible Staphylococcus aureus (MSSA) are 0.03 mg/L, but for methicillin- and ciprofloxacin-resistant S. aureus (MRSA) are 4 mg/L. MIC90s for Group A streptococci are 0.25 mg/L.1,2,3 Moxifloxacin is known to be rapidly bactericidal against S. aureus, but less so against Group A streptococci, when constant concentrations of 0.5x and 10 x MIC are used.4 In addition, at concentrations of 1x, 4x and 10 x MIC the post-antibiotic effect against S. aureus is 0.9–3.3 h and for Group A streptococci 0.3–3.3 h.4

Previous work using in-vitro models has concentrated on studying the antibacterial effect of moxifloxacin on respiratory tract pathogens such as Streptococcus pneumoniae5,6,7 or Haemophilus influenzae and Moraxella cattarhalis.8 This study focused on pathogens likely to be associated with skin and soft tissue infection, i.e. S. aureus or ß-haemolytic streptococci. The antibacterial effect of moxifloxacin was assessed by simulating changing concentrations of antibiotic in an in-vitro pharmacodynamic model of infection. As in previous studies the serum concentrations of moxifloxacin associated with doses of 400 mg administered once a day were modelled, as this is the dose of moxifloxacin used most commonly in human clinical trials.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In-vitro model

An in-vitro model (New Brunswick Bioflo 1000; Hatfield, UK) was used to simulate oral administration of moxifloxacin 400 mg every 24 h over 48 h; i.e. two simulated doses. The apparatus consists of a single central culture chamber connected via aluminium and silicone tubing first to a dosing chamber (which is in turn connected to a reservoir containing broth) and secondly to a vessel collecting outflow broth from the central chamber. The dosing chamber and central culture chamber were diluted with broth from the reservoir using a peristaltic pump (Ismatec Bennett & Co, Weston-super-Mare, UK) at a flow rate of 66 mL/h, to simulate the decline in antibiotic concentration. The temperature was maintained at 37°C and the broth in the dosing and central chambers was agitated by a magnetic stirrer.

Media

Brain Heart Infusion (BHI) broth (75%) (Unipath, Basingstoke, UK), supplemented with haemin 10 mg/L and ß-nicotinamide adenine dinucleotide 10 mg/L and L-histidine 10 mg/L, was used for the experiment with ß-haemolytic streptococci, and 6% IsoSensitest broth (ISB) (Unipath) was used for S. aureus. Preliminary investigations indicated that these broths supported a growth density of 5 x 107–5 x 108 cfu/mL 18 h after inoculation into the model. Magnesium chloride at 1% (BDH, Poole, UK) was incorporated into nutrient agar plates (Merck, Poole, UK) containing 5% whole horse blood (TCS Microbiology, Buckingham, UK) to neutralize moxifloxacin before viable counts were determined.

Strains

S. aureus SMH 13942, methicillin and ciprofloxacin sensitive; S. aureus SMH 251, methicillin resistant, ciprofloxacin sensitive; Group A streptococcus SMH 14200 and Group G streptococcus SMH 14230 were used. Strains 13942M and 14200M were laboratory-generated mutants of strains 13942 and 14200 that were produced by the method of Dalhoff et al.1 Briefly this method involves serial passage through antibiotic-containing broth, using bacterial isolates that grow at or around the MIC value.

Antibiotic

Moxifloxacin was obtained from Bayer AG, Wuppertal, Germany. Stock solutions were prepared according to The British Society of Antimicrobial Chemotherapy (BSAC) guidelines9 and stored at –70°C.

MICs

MICs were determined by the BSAC-defined standard broth dilution method9 modified to use moxifloxacin concentrations that decreased by 0.2 mg/L or 0.02 mg/L steps rather than doubling dilutions.

Pharmacokinetics and bacterial killing curves

The in-vitro activity of changing concentrations of moxifloxacin against the six strains was tested in the model described above. Concentrations were chosen to simulate serum concentrations in man after a 400 mg oral dose every 24 h (to give a target peak serum concentration of 2.5 mg/L at 2 h post dose and a 24 h post-dose concentration of 0.4 mg/L10). The area under the curve (AUC) was 24.4 mg/L.

For all the experiments, 100 µL of an overnight broth suspension of the test organism was inoculated into the central culture chamber via an entry port (initial inoculum of about 106 cfu/mL) and the model run for 18 h to allow equilibrium to be attained at a density of about 5 x 107–5 x 108 cfu/mL. Moxifloxacin (1332 µL of a 1 g/L solution) was then added to the dosing chamber at time 0, and at 24 h. Samples were taken from the central chamber via a port throughout the 48 h period (at times 0, 1, 2, 3, 4, 5, 6, 7, 10, 12, 22, 24, 25, 26, 27, 28, 29, 30, 31, 34, 36, 46 and 48 h) for assessment of viable bacterial count. The bacteria were counted without dilution and after 1/100 dilution using a spiral plater (Don Whitley Spiral Systems, Shipley, UK). The minimum detection level was 2 x 102 cfu/mL. In addition, aliquots were taken at the same time intervals and stored at –70°C for measurement of moxifloxacin concentration. Samples were assayed by bioassay using Escherichia coli NCTC 10418 as indicator organism.11 All standards and samples were prepared and diluted as necessary in the same concentration of BHI or ISB as that used in the model simulations. The limit of detection was 0.03 mg/L. All pharmacokinetic simulations and bacterial killing experiments were performed at least three times.

Pharmacokinetics, pharmacodynamic measurement of antibacterial effects and statistical analysis

Antibacterial activity was assessed by calculating the log change in viable count compared with time 0 at 12 h ({Delta}12), 24 h ({Delta}24), 36 h ({Delta}36) and 48 h ({Delta}48). In addition, the maximum reduction in viable count was recorded ({Delta}max). The area under the bacterial killing curve (AUBKC, log cfu/mL.h) was calculated, after the inoculum was standardized, by the log-linear trapezoidal rule for the time periods 0–24 h (AUBKC24) and 0–48 h (AUBKC48). For pharmacodynamic analysis the AUC/MIC ratio, the percentage of time the simulated concentration exceeded the MIC (T > MIC) (T = time), the AUC over MIC, MIC ratio ((AUC > MIC)/MIC) and weighted AUC: WAUC = (AUC/MIC x T > MIC/100) were also calculated.12

AUC/MIC, T > MIC, (AUC > MIC)/MIC and WAUC were related to AUBKC24 and AUBKC48 using an inhibitory sigmoid Emax model (WinNonlin, Microsoft Corp., USA). Goodness of fit was assessed by r and inspection of the plot of the residuals.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pharmacokinetic data

Moxifloxacin was detected at a concentration of 2.3 ± 0.5 mg/L at 2 h and 0.5 ± 0.1 mg/L at 24 h. These levels correlated well with those expected.

Bacterial killing curves

The killing curves for S. aureus after exposure to moxifloxacin are shown in Figures 1 and 2. For the two strains with moxifloxacin MIC <= 0.14 mg/L there was a marked reduction in viable count (6 log) at 36 h with no regrowth occurring up to 48 h. In contrast, killing of the laboratory-generated mutant (MIC 1.0 mg/L) was reduced (2 log reduction in count), with marked regrowth between 36 and 48 h. The AUBKC values were less for the more susceptible strains when compared with the mutant strains, especially AUBKC48. Results for the Group A streptococci tested showed that the more susceptible strain (MIC 0.16 mg/L) was killed more rapidly (3.5 log10) with no regrowth. The mutant strain (MIC 1.8 mg/L) showed marked regrowth (2 log) and was poorly killed between 0 and 24 h (Figure 3). For these simulations a significant between-experiment variability was exhibited (Figure 3 and Table). Killing of Group G streptococci (MIC 0.4 mg/L) was poor, with only a 2 log reduction in count over 48 h (Figure 4 and Table).



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Figure 1. Bactericidal effect of moxifloxacin at a simulated dose of 400 mg every 24 h on S. aureus strains 13942 parent ({blacksquare}) (MIC 0.14 mg/L) and 13942M mutant ({square}) (MIC 1.0 mg/L). Bacterial counts are means ± S.D.

 


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Figure 2. Bactericidal effect of moxifloxacin at a simulated dose of 400 mg every 24 h on methicillin-resistant S. aureus strain 251 (•) (MIC 0.06 mg/L). Bacterial counts are means ± S.D.

 


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Figure 3. Bactericidal effect of moxifloxacin at a simulated dose of 400 mg every 24 h on ß-haemolytic streptococcus (Group A) strains 14200 parent (MIC 0.16 mg/L) ({blacksquare}) and 14200M mutant (MIC 1.8 mg/L) ({square}). Bacterial counts are means ± S.D.

 

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Table. Antibacterial effect of moxifloxacin for S. aureus and ß-haemolytic streptococci
 


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Figure 4. Bactericidal effect of moxifloxacin at a simulated dose of 400 mg every 24 h on ß-haemolytic streptococcus (Group G) strain 14230 (MIC 0.4 mg/L). Bacterial counts are mean ± S.D.

 
Pharmacodynamics of the antibacterial effect

Curve fitting using an inhibitory Emax sigmoidal model indicated that T > MIC was poorly related to AUBKC24 and AUBKC48 (r <0.37). AUC/MIC, WAUC and (AUC > MIC)/MIC were strongly related to AUBKC24 (r = 0.75–0.79) and AUBKC48 (r = 0.78–0.84). The plot of AUC/MIC versus AUBKC24 is shown in Figure 5. Inspection of the residual plots was satisfactory. The maximum response, i.e. the lowest plateau AUBKC, occurs at an AUC/MIC ratio of >150.



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Figure 5. Moxifloxacin antibacterial activity against S. aureus and ß-haemolytic streptococci: relationship between AUBKC0–48h and AUC/MIC using a sigmoid inhibitory Emax model.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous data have indicated that moxifloxacin may be less bactericidal for Group A streptococci than for S. aureus even if allowance is made for differing MIC values.4 Our data support this view—killing of a Group A streptococcus (MIC 0.16 mg/L) was remarkably less than that of a strain of S. aureus with an MIC of 0.14 mg/L. At higher MICs, no differences were observed, probably because moxifloxacin was not especially bactericidal for either species. While other data from in-vitro models are mostly lacking for S. aureus or ß-haemolytic streptococci, it is clear that moxifloxacin at in-vitro doses of 400 mg/24 h is rapidly bactericidal for S. pneumoniae with MICs <= 0.5 mg/L.5,6 In these experiments ß-haemolytic streptococci, Lancefield Group A MIC 0.16 mg/L or Group G MIC 0.4 mg/L, were not eradicated after two exposures to moxifloxacin. In contrast, using the same model, we were able to demonstrate significant bactericidal action of moxifloxacin at up to 36 h, using S. pneumoniae strains with MICs <= 0.5 mg/L.5 Using simulations of 100 mg or 200 mg single doses, it has been shown previously that S. aureus can be eradicated from an in-vitro model within 12 h of dosing.13 Our data confirm that there is significant bactericidal activity at 36–48 h for S. aureus strains with MICs <= 0.14 mg/L.

Animal data, in which E. coli, Klebsiella pneumoniae, S. aureus and S. pneumoniae are used in neutropenic mouse models of lung or thigh infection, have indicated that the dose required to produce a net bacteriostatic effect over 24 h is unchanged, whether moxifloxacin is given every 3, 6 or 12 h. In mice, moxifloxacin has a serum half-life of <1 h compared with 12–15 h in man. This probably indicates that once-daily dosing will be as effective as twice daily or more frequent doses. Drug AUC correlated with antibacterial efficacy more strongly than did Cmax or T > MIC.14 Furthermore, in an experimental animal model of S. pneumoniae meningitis, the drug AUC in cerebrospinal fluid correlated with the change in viable count.15 Previous studies by this group, using the model system described here, have shown that the antibacterial effect of moxifloxacin correlated best with AUC/MIC for H. influenzae and M. catarrhalis.18 These data also show that AUC parameters correlate best with antimicrobial effect, whether this be AUC/MIC, (AUC > MIC)/MIC or WAUC. T > MIC is not a good predictor using an inhibitory sigmoidal Emax model, and WAUC here was not superior to the other parameters as has been postulated recently.12 This is perhaps not surprising, as T > MIC is an integral part of WAUC. As this study is not of a dose fractionation or escalation design, it was not possible to define the relative roles of Cmax/MIC ratio in comparison with AUC/MIC, despite recent interest in this area.16

In conclusion, moxifloxacin has marked bactericidal action against S. aureus with MICs <= 0.14 mg/L but may be less bactericidal against ß-haemolytic streptococci; AUC-based pharmacodynamic factors predict antibacterial effect better than does T > MIC.


    Acknowledgments
 
We wish to thank Professor A. Dalhoff and Bayer AG, Wuppertal, Germany for advice and financial support in performing this study. These data were partly published as an abstract (Wootton, M., Bowker, K. E., Holt H. A. & MacGowan, A. P. Bactericidal activity of moxifloxacin against Staphylococcus aureus and Group A streptococci explored using a pharmacodynamic model of infection. In Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, 1998.Abstract A-32, p. 10. American Society for Microbiology, Washington, DC).


    Notes
 
* Corresponding author. Tel: +44-117-959-5652; Fax: +44-117-959-3154; E-mail: macgowan_a{at}southmead.swest.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dalhoff, A., Petersen, U. & Endermann, R. (1996). In vitro activity of BAY 12-8039, a new 8-methoxyquinolone. Chemotherapy 42, 410–25.[ISI][Medline]

2 . Woodcock, J. M., Andrews, J. M., Boswell, F. J., Brenwald, N. P. & Wise, R. (1997). In vitro activity of BAY 12-8039, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 41, 101–6.[Abstract]

3 . 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, 639–51.[Abstract]

4 . Boswell, F. J., Andrews, J. M. & Wise, R. (1997). Pharmacodynamic properties of BAY 12-8039 on Gram-positive and Gram-negative organisms as demonstrated by studies of time–kill kinetics and post antibiotic effect. Antimicrobial Agents and Chemotherapy 41, 1377–9.[Abstract]

5 . Bowker, K. E., Wootton, M., Holt, H. A., Reeves, D. S. & MacGowan, A. P. (1997). Bactericidal activity of Bay 12-8039, against Streptococcus pneumoniae explored using an in-vitro continuous bacterial culture model. In Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract F-134, p. 169. American Society for Microbiology, Washington, DC.

6 . Lister, P. D. & Sanders, C. C. (1998). Pharmacodynamics of moxifloxacin against Streptococcus pneumoniae in an in-vitro pharmacokinetic model. In Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract A-21, p. 7. American Society for Microbiology, Washington, DC.

7 . Zinner, S., Gilbert, D., Simmons, K. & Sarlak, E. (1998). Moxifloxacin activity against S. pneumoniae in an in-vitro dynamic model. In Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract A-26, p. 8. American Society for Microbiology, Washington, DC.

8 . Bowker, K. E., Wootton, M., Holt, H. A. & MacGowan, A. P. (1998). In-vitro activity of moxifloxacin against Haemophilus influenzae and Moraxella catarrhalis investigated using a pharmacodynamic model of infection. In Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract E-207, p. 229. American Society for Microbiology, Washington, DC.

9 . British Society for Antimicrobial Chemotherapy. (1991). A Guide to Sensitivity Testing: Report of the Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. Academic Press, London.

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12 . Corvaisier, S., Maive, P. H., Bouvier d'Yvoire, M. Y., Barbaut, X., Bleyzac, N. & Jelliffe, R. W. (1998). Comparisons between antimicrobial pharmacodynamic indices and bacterial killing as described by using the Zhi model. Antimicrobial Agents and Chemotherapy 42,1731 –7.[Abstract/Free Full Text]

13 . Dalhoff, A. (1996). Antibacterial efficacy of fluctuating concentrations of Bay 12-8039 simulating human serum kinetics. In Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996. Abstract F-26, p. 104. American Society for Microbiology, Washington, DC.

14 . Vesga, O., Conklin, R., Stamstad, T. & Craig, W. A. (1996). Pharmacodynamic activity of Bay 12-8039 in animal infection model. In Abstracts of the Thirty-Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996. Abstract F-22, p. 103. American Society for Microbiology, Washington, DC.

15 . Ostergaard, C., Sorensen, T. K., Knudsen, J. D. & Frimodt-Moller, N. (1998). Evaluation of moxifloxacin, a new 8 methoxyquinolone, for treatment of meningitis caused by a penicillin-resistant pneumococcus in rabbits. Antimicrobial Agents and Chemotherapy 42,1706 –12.[Abstract/Free Full Text]

16 . Preston, S. L., Drusano, G. L., Berman, A. L., Fowler, C. L., Chow, A. T., Dornseif, B. et al. (1998). Pharmacodynamics of levofloxacin: a new paradigm for early clinical trials. Journal of the American Medical Association 279, 125–9.[Abstract/Free Full Text]

Received 5 February 1999; returned 5 May 1999; revised 25 May 1999; accepted 9 August 1999