1 Department of Medicine, Mount Auburn Hospital, Harvard Medical School, Cambridge, MA; 3 Roger Williams Medical Center, Providence, RI; 4 Public Health Research Institute, Newark, NJ, USA; 2 Department of Pharmacokinetics & Pharmacodynamics, Gause Institute of New Antibiotics, 11 Bolshaya Pirogovskaya Street, Moscow, 119021 Russia
Received 13 March 2003; returned 12 May 2003; revised 1 July 2003; accepted 6 July 2003
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Abstract |
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Methods: Daily administration of moxifloxacin for 3 consecutive days was mimicked using a two-compartment dynamic model with peripheral units containing a starting inoculum of 108 cfu/mL S. pneumoniae. Changes in susceptibility were examined by repeated MIC determinations and by plating the specimens on agar containing zero, 2 x MIC, 4 x MIC and 8 x MIC of moxifloxacin.
Results: Both in terms of the MIC and resistance frequency, S. pneumoniae resistance developed at concentrations that fell inside the MSW [ratios of 24 h area under the curve (AUC24) to MIC between 24 and 47 h]. A Gaussian-like function fitted the AUC24/MIC-dependent increases in MIC and resistance frequency with central points at AUC24/MICs of 38 and 42 h, respectively, where resistant mutants are enriched selectively. Selective enrichment of resistant mutants was not seen at AUC24/MICs <10 h or >100 h.
Conclusions: These data suggest that AUC24/MICs >100 h may protect against the selection of resistant S. pneumoniae mutants. Since the usual 400 mg dose of moxifloxacin provides much higher AUC24/MIC (270 h), it is expected to prevent mutant selection at clinically achievable concentrations. Also, these data provide further support for the MSW hypothesis.
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Introduction |
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The present study is aimed at further examination of the MSW hypothesis with moxifloxacin-exposed Streptococcus pneumoniae. To express S. pneumoniae resistance better, a population analysis of resistance frequency is provided, along with timekill dynamics and MIC time courses.
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Materials and methods |
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Moxifloxacin powder was provided by Bayer Corporation (West Haven, CT, USA). S. pneumoniae ATCC 49619 (MIC 0.1 mg/L; MPC 0.5 mg/L)3 was selected for the study.
Susceptibility testing was performed in duplicate on bacteria obtained before and 24 h after each moxifloxacin dose (0, 24 and 48 h). The MICs were determined using broth microdilution techniques with S. pneumoniae grown in MuellerHinton broth (MHB) supplemented with lysed horse blood (2% v/v). The inoculum size was 106 cfu/mL.
MPC was determined as described elsewhere.1 Briefly, the tested microorganisms were cultured in MHB and incubated for 24 h. The suspension was centrifuged (4000g for 10 min) and re-suspended in MHB to yield a concentration of 1010 cfu/mL. A series of agar plates containing known moxifloxacin concentrations was then inoculated with 1010 cfu of S. pneumoniae. The inoculated plates were incubated for 48 h at 37°C and screened visually for growth. To estimate MPC, logarithms of bacterial numbers were plotted against moxifloxacin concentrations. MPC was taken as the point where the plot intersected the x-axis, i.e. the lowest fluoroquinolone concentration that inhibited growth completely.
In vitro dynamic model and simulated pharmacokinetic profiles
The in vitro dynamic model used in this study has been described elsewhere.4 For all experiments, the bacterial inoculum was prepared from previously frozen inocula by thawing, diluting with an equal part of fresh MHB supplemented with lysed horse blood (LHB; 2% v/v) and incubating for 90 min at 37°C to bring the organisms into growth phase. This mixture was then inoculated into each peripheral compartment, which also contained MHB/LHB 2%, via an entry port, and incubated to a density of 108 cfu/mL, at which time the antibiotic was introduced into the central compartment (time zero). Given a 20 mL volume of the peripheral compartment, the total number of organisms in the starting inoculum reached
2 x 109 cfu. Antibiotic-free, sterile MHB (no horse serum was added to the MHB) was infused and eliminated at flow rates selected to mimic the half-life of moxifloxacin (12 h) that corresponds to values reported in humans: 1114 h.5 All dynamic model experiments were performed in triplicate.
A series of monoexponential profiles that mimicked daily administration of moxifloxacin for 3 consecutive days was simulated over a 32-fold range of the AUC24/MIC ratio, from 8256 h. At the end of a 60 min infusion, the drug concentration reached a maximum, analogous to peak concentrations that are reached after oral administration of the quinolone. As the antimicrobial effect depends on quinolone concentration in peripheral compartments (where the organisms contact antibiotic), peripheral compartments were sampled to determine moxifloxacin concentrations by bioassay using well plates seeded with BBL Bacillus subtilis spore suspension, origin ATCC 6633.
Quantification of the timekill curves and antimicrobial effect
In each experiment, the peripheral compartments were sampled to determine bacterial concentrations. To determine the number of surviving organisms, a sample was serially diluted in cold sterile saline and 20 µL was inoculated in triplicate onto MuellerHinton agar (MHA) supplemented with 5% sheep blood (SB). A small number of bacteria were counted by placing 100 µL of sample into 10 mL of cold sterile saline. This mixture was then passed through a 0.45 µm filter and then placed on MHASB. After overnight incubation at 37°C, the resulting bacterial colonies were counted, and the numbers of cfu/mL calculated. The detection limit was 10 cfu/mL. The time taken by antibiotic-exposed bacteria (after the last dose) to reach the same maximum numbers as observed in the absence of antibiotic (109 cfu/mL) defined the duration of the experiments, but experiments were continued for at least 192 h if re-growth did not occur.
Based on the timekill data, the area between the control curve and the timekill curve (ABBC)6 was calculated within the first, second and third 24 h interval: ABBC1, ABBC2 and ABBC3, respectively. The upper limit of bacterial numbers, i.e. the cut-off level on the re-growth and control growth curves used to determine ABBC, was 109 cfu/mL. The computation of ABBC1, ABBC2 and ABBC3 is depicted graphically in Figure 1.
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To reveal changes in susceptibility, moxifloxacin MICs for bacterial cultures sampled from the model were determined 24, 48 and 72 h after beginning treatment and at the end of the observation period if it was longer than 72 h. The final MIC (MICfinal) was then related to the initial value (MICinitial). The stability of resistance in each of these specimens was determined by consecutive passaging of S. pneumoniae onto antibiotic-free agar plates for 310 consecutive days. MICs were determined frequently during this time as described above.
To determine resistance frequency (f) in experiments where bacterial regrowth occurred, each sample was plated onto agar plates containing 2 x MIC, 4 x MIC and 8 x MIC of moxifloxacin (detection limit 2 x 102 cfu/mL). At a given time, f was expressed by the ratio of bacterial number observed in the presence of antibiotic to that in the absence of antibiotic (f2 x MIC, f4 x MIC and f8 x MIC, respectively). Then, the respective ratios of the final f (ffinal) to the initial value (finitial) were calculated.
To relate the increase in the MIC and f to the simulated AUC24/MICs, a Gaussian type function was used:
Y = Y0 + a exp [ (x xc)2/b] (Equation 1)
where Y is the MICfinal/MICinitial or ffinal/finitial ratio, Y0 is the minimal value of Y, x is log10 AUC24/MIC, xc is log10 AUC24/MIC that corresponds to the maximal value of MICfinal/MICinitial or ffinal/finitial, and a and b are parameters.
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Results |
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To relate increases in MIC to AUC24/MIC for the entire data set (18 AUC24/MIC values), the MICs observed at the end of each treatment were normalized to their respective initial MIC values. As seen in Figure 3, a Gaussian-like function [Equation (1)] fitted the MICfinal/MICinitial versus log AUC24/MIC relationship (r2 0.90). The central point was at an AUC24/MIC of 38 h, where the loss in pneumococcal susceptibility was maximal. No resistance was observed at AUC24/MICs < 10 h or AUC24/MICs > 100 h.
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Discussion |
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The present study demonstrates good concordance between S. pneumoniae resistance expressed by susceptibility testing and by population analysis. The selective enrichment of resistant S. pneumoniae exposed to moxifloxacin occurred at similar AUC24/MIC ratios, both in terms of loss in the susceptibility and increases in resistance frequency. Moreover, both methods showed similar AUC24/MIC relationships of resistance that were reflected by bell-shaped curves having a maximum at similar AUC24/MICs (38 h with the MIC data and 42 h with the resistance frequency data). A similar relationship between AUC24/MIC and MICfinal/MICinitial was delineated in our recent study with quinolone-exposed S. aureus.2 In that case a maximum was also seen at an AUC24/MIC of 43 h. Further studies are required to determine whether this pattern of AUC24/MIC resistance relationship occurs with other antibioticpathogen pairs.
Like S. aureus exposed to four quinolones including moxifloxacin,2 selection of resistant S. pneumoniae occurred when moxifloxacin concentrations were inside the MSW (TMSW) for >20% of the dosing interval (AUC24/MICs 6100 h). No selection was seen with shorter times inside the MSW (TMSW < 20% of the dosing interval), i.e. at AUC24/MICs < 10 h and > 100 h.
Changes in resistance frequency and susceptibility of moxifloxacin-exposed S. pneumoniae were accompanied by respective changes in the anti-pneumococcal effect. Its loss during treatment (ABBC3 < ABBC2) was observed at moxifloxacin concentrations that fall inside the MSW (AUC24/MIC 2447 h), where selection of resistant mutants occurred. No erosion in effectiveness was seen at concentrations that were outside the MSW. Similar correlations were reported in our study with moxifloxacin- and levofloxacin-exposed S. aureus.17 There the loss of the antimicrobial effect was observed at AUC24/MICs of 28 h (moxifloxacin) and 3161 h (levofloxacin).
As with S. aureus, the most pronounced losses in S. pneumoniae susceptibility and the highest resistance frequencies were observed at moxifloxacin concentrations that fell inside the MSW (AUC24/MIC from 24 to 47 h). At concentrations <MIC or >MPC (AUC24/MIC < 10 h or > 100 h), no resistance was observed. Based on these data, an AUC24/MIC ratio of 100 h might protect against pneumococcal resistance. This value is readily achievable in patients treated with moxifloxacin: much higher ratios of AUC (33 mg x h/L)5 to MIC50 (0.125 mg/L)18, i.e. 33/0.125 = 270 h, are provided by its usual 400 mg clinical dose.
Overall, these findings suggest that selection of resistant mutants can be observed using in vitro pharmacokinetic simulations in which resistance may be monitored by either the frequency of mutations or increases in MICs. Also, these data support the MSW hypothesis1 that predicts selection of resistant mutants at antibiotic concentrations >MIC and <MPC.
By supporting the MSW hypothesis, the data presented above challenge a central assumption of antimicrobial therapy, that resistant mutants are enriched selectively when antimicrobial concentrations are <MIC. This assumption may lead to dosing recommendations, such as achieving an AUC/MIC of 306016,19,20 that place antimicrobial concentrations inside the MSW. According to the MSW hypothesis, many traditional dosing regimens may constitute misuse of antimicrobial agents.
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Acknowledgements |
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Footnotes |
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References |
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