[PK/PD Modeling as a Tool for Predicting Bacterial Resistance to Antibiotics: Alternative Analyses of Experimental Data].

Postexposure number of mutants (NM) is a conventional endpoint in bacterial resistance studies using in vitro dynamic models that simulate antibiotic pharmacokinetics. To compare NM with a recently introduced integral parameter AUBC(M), the area under the time course of resistance mutants, the enrichment of resistant Staphylococcus aureus was studied in vitro by simulation of mono(daptomycin, doxycycline) and combined treatments (daptomycin + rifampicin, rifampicin + linezolid). Differences in the time courses of resistant S. aureus could be reflected by AUBC(M) but not N(M). Moreover, unlike AUBC(M), N(M) did not reflect the pronounced differences in the time courses of S. aureus mutants resistant to 2x, 4x, 8x and 16xMIC of doxycycline and rifampicin. The findings suggested that AUBC(M) was a more appropriate endpoint of the amplification of resistant mutants than N(M).

The antistaphylococcal pharmacodynamics of linezolid alone and in combination with doxycycline in an in vitro dynamic model.

To delineate the possible advantages of linezolid/doxycycline combinations over either drug alone, the in vitro pharmacodynamics of linezolid, doxycycline and linezolid plus doxycycline were studied with Staphylococcus aureus.S. aureus ATCC 43300 and a clinical isolate S. aureus 479 were exposed to twice-daily linezolid and once-daily doxycycline, alone and in combination, for five consecutive days. Three dosing regimens were simulated with each drug alone: linezolid (AUC(24)/MIC 30, 60 and 200 h-L30, L60 and L200, respectively) and doxycycline (AUC(24)/MIC 90, 180 and 520 h - D90, D180 and D520, respectively) and in combination: linezolid plus doxycycline (L30+D90; L60+D180 and L200+D520).With both S. aureus ATCC 43300 and S. aureus 479 exposed to linezolid or doxycycline, the area between the line crossing each time-kill curve at the level of 10(8) CFU/mL and the respective time-kill curve (I(E)) increased with increasing simulated AUC(24)/MIC ratios. Each of the combined treatments produced greater I(E)s than the sum of linezolid and doxycycline I(E)s observed in the respective single drug treatments.This study suggests that linezolid combinations with doxycycline may be synergistic in treating staphylococcal infections.

Simulated in vitro quinolone pharmacodynamics at clinically achievable AUC/MIC ratios: advantage of I E over other integral parameters.

To compare the antimicrobial effects of clinically achievable ratios of area under the curve (AUC) to MIC, a clinical isolate of Moraxella catarrhalis was selected with MICs corresponding to the MIC(50)s of four quinolones. Monoexponentially declining concentrations observed in human plasma after oral administration of 1,000 mg of ciprofloxacin (as two 500-mg doses at a 12-hour interval), 320 mg gemifloxacin, 500 mg levofloxacin or 400 mg moxifloxacin (each as a single dose) and were simulated in an in vitro dynamic model. The respective half-lives were 4, 7.4, 6.8 and 12.1 h, and the AUC/MICs were 730, 1,130, 920 and 690 h. The time-kill/regrowth curves yielded similar patterns with the four quinolones: a rapid reduction in bacterial numbers followed by bacterial regrowth that occurred later with moxifloxacin than with ciprofloxacin, gemifloxacin, and levofloxacin. The total antimicrobial effect of moxifloxacin as expressed by the I(E) parameter (area between the control growth and time- kill curves from time zero to the time when bacterial counts on the regrowth curve achieve the same maximal numbers as in the absence of antimicrobial) was 30, 55, and 120% greater than gemifloxacin, levofloxacin and ciprofloxacin, respectively. Unlike I(E), the other integral indices determined over a fixed time (24 h) - the area between the control growth and time-kill curves, area above the time-kill curve and area under the time-kill curve were similar for the four fluoroquinolones, thus precluding their differentiation.

Comparative anti-staphylococcal effects of gemifloxacin and trovafloxacin in an in vitro dynamic model in terms of AUC/MIC and dose relationships.

To compare the antimicrobial effects of gemifloxacin and trovafloxacin on Staphylococcus aureus, their pharmacodynamics were studied in an in vitro dynamic model. A series of pharmacokinetic profiles of gemifloxacin and trovafloxacin with half-lives of 7.4 and 9.2 h, respectively, were simulated in vitro over an eightfold range of area under the curve (AUC)-to-MIC ratio, from 58 to 466 h. The relationships observed between the intensity of antimicrobial effect (I(E)) and log AUC/MIC were linear, species- and strain-independent and were distinct (not superimposed) for both gemifloxacin and trovafloxacin (r(2) = 0.99 in both cases). At AUC/MICs > 100 h, trovafloxacin had greater effects than gemifloxacin. For example, at an AUC/MIC of 250 h, the antimicrobial effect of trovafloxacin was 17% higher than gemifloxacin. However, due to its higher intrinsic activity, gemifloxacin may be as efficient as trovafloxacin at their clinical doses (320 and 200 mg, respectively): the I(E)s on a hypothetical strain of S. aureus with gemifloxacin's and trovafloxacin's MICs corresponding to the MIC(50)s were similar-290 and 310 (log CFU/mL)x h, respectively. This analysis suggests that both AUC/MIC and dose relationships of the antimicrobial effect are needed for comprehensive comparisons of fluoroquinolone pharmacodynamics.

Relationships of the area under the curve/MIC ratio to different integral endpoints of the antimicrobial effect: gemifloxacin pharmacodynamics in an in vitro dynamic model.

Most integral endpoints of the antimicrobial effect are determined over an arbitrarily chosen time period, such as the dosing interval (tau), regardless of the actual effect duration. Unlike the tau-related endpoints, the intensity of the antimicrobial effect (I(E)) does consider its duration-from time zero to the time when bacterial counts on the regrowth curve achieve the same maximal numbers as in the absence of the antimicrobial. To examine the possible impact of this fundamental difference on the relationships of the antimicrobial effect to the ratio of the area under the concentration-time curve (AUC) to the MIC, a clinical isolate of Staphylococcus aureus was exposed to simulated gemifloxacin pharmacokinetics over a 40-fold range of AUC/MIC ratios, from 11 to 466 h. In each run, I(E) and four tau-related endpoints, including the area under the time-kill curve (AUBC), the area above the curve (AAC), the area between the control growth and time-kill curves (ABBC), and the ABBC related to the area under the control growth curve (AUGC), were calculated for tau = 24 h. Unlike the I(E), which displayed pseudolinear relationships with the AUC/MIC ratio; each tau-related endpoint showed a distinct saturation at potentially therapeutic AUC/MIC ratios (116 to 466 h) when the antimicrobial effect persisted longer than tau. This saturation results from the underestimation of the true effect and may be eliminated if ABBC, AAC, and AUBC (but not AUGC) are modified and determined in the same manner as the I(E) to consider the actual effect duration. These data suggest a marginal value of the tau-related endpoints as indices of the total antimicrobial effect. Since all of them respond to AUC/MIC ratio changes less than the I(E), the latter is preferable in comparative pharmacodynamic studies.

Comparative pharmacodynamics of moxifloxacin and levofloxacin in an in vitro dynamic model: prediction of the equivalent AUC/MIC breakpoints and equiefficient doses.

To demonstrate the impact of the different pharmacokinetics of moxifloxacin and levofloxacin on their antimicrobial effects (AMEs), killing and regrowth kinetics of two clinical isolates of Staphylococcus aureus and one each of Escherichia coli and Klebsiella pneumoniae were studied. With each organism, a series of monoexponential pharmacokinetic profiles of single doses of moxifloxacin (T:1/2 = 12.1 h) and levofloxacin (T:(1/2) = 6.8 h) were simulated. The respective eight-fold ranges of the ratios of area under the concentration-time curve (AUC) to the MIC were 58-475 and 114-934. Species- and strain-independent linear relationships observed between the intensity of AME (I:(E)) and log AUC/MIC were not superimposed for moxifloxacin and levofloxacin (r(2) = 0.99 in both cases). The predicted AUC/MIC ratios for moxifloxacin and levofloxacin that might be equivalent to Schentag's AUC/MIC breakpoint for ciprofloxacin (125) were estimated at 80 and 130, respectively. The respective equivalent MIC breakpoints were 0.41 mg/L (for a 400 mg dose of moxifloxacin) and 0.35 mg/L (for a 500 mg dose of levofloxacin). Based on the I:(E)-log AUC/MIC relationships, equiefficient 24 h doses (D:(24)s) of moxifloxacin and levofloxacin were calculated for hypothetical strains of S. aureus, E. coli and K. pneumoniae with MICs equal to the respective MIC50s (weighted geometric means of reported values). To provide an 'acceptable' I:(E) = 200 (log cfu/mL)*h, the D:(24)s of moxifloxacin for all three organisms were much lower (150, 30 and 60 mg, respectively) than the clinically proposed 400 mg dose. Although the usual dose of levofloxacin (500 mg) would be in excess for E. coli and K. pneumoniae (D:(24) = 36 and 220 mg, respectively), it might be insufficient for S. aureus (the estimated D:(24) = 850 mg). Moreover, to provide the same effect as a 400 mg D:(24) of moxifloxacin against staphylococci, levofloxacin would have to be given in a 5000 mg D:(24), which is 10-fold higher than its clinically accepted dose. The described method of generalization of data obtained with specific organisms to other representatives of the same species might be useful to predict the AMEs of new quinolones.

Prediction of the effects of inoculum size on the antimicrobial action of trovafloxacin and ciprofloxacin against Staphylococcus aureus and Escherichia coli in an in vitro dynamic model.

The effect of inoculum size (N0) on antimicrobial action has not been extensively studied in in vitro dynamic models. To investigate this effect and its predictability, killing and regrowth kinetics of Staphylococcus aureus and Escherichia coli exposed to monoexponentially decreasing concentrations of trovafloxacin (as a single dose) and ciprofloxacin (two doses at a 12-h interval) were compared at N0 = 10(6) and 10(9) CFU/ml (S. aureus) and at N0 = 10(6), 10(7), and 10(9) CFU/ml (E. coli). A series of pharmacokinetic profiles of trovafloxacin and ciprofloxacin with respective half-lives of 9.2 and 4 h were simulated at different ratios of area under the concentration-time curve (AUC) to MIC (in [micrograms x hours/milliliter]/[micrograms/milliliter]): 58 to 466 with trovafloxacin and 116 to 932 with ciprofloxacin for S. aureus and 58 to 233 and 116 to 466 for E. coli, respectively. Although the effect of N0 was more pronounced for E. coli than for S. aureus, only a minor increase in minimum numbers of surviving bacteria and an almost negligible delay in their regrowth were associated with an increase of the N0 for both organisms. The N0-induced reductions of the intensity of the antimicrobial effect (IE, area between control growth and the killing-regrowth curves) were also relatively small. However, the N0 effect could not be eliminated either by simple shifting of the time-kill curves obtained at higher N0s by the difference between the higher and lowest N0 or by operating with IEs determined within the N0-adopted upper limits of bacterial numbers (IE's). By using multivariate correlation and regression analyses, linear relationships between IE and log AUC/MIC and log N0 related to the respective mean values [(log AUC/MIC)average and (log N0)average] were established for both trovafloxacin and ciprofloxacin against each of the strains (r2 = 0.97 to 0.99). The antimicrobial effect may be accurately predicted at a given AUC/MIC of trovafloxacin or ciprofloxacin and at a given N0 based on the relationship IE = a + b [(log AUC/MIC)/(log AUC/MIC)average] - c [(log N0)/(log N0)average]. Moreover, the relative impacts of AUC/MIC and N0 on IE may be evaluated. Since the c/b ratios for trovafloxacin and ciprofloxacin against E. coli were much lower (0.3 to 0.4) than that for ampicillin-sulbactam as examined previously (1.9), the inoculum effect with the quinolones may be much less pronounced than with the beta-lactams. The described approach to the analysis of the inoculum effect in in vitro dynamic models might be useful in studies with other antibiotic classes.

MIC-based interspecies prediction of the antimicrobial effects of ciprofloxacin on bacteria of different susceptibilities in an in vitro dynamic model.

Multiple predictors of fluoroquinolone antimicrobial effects (AMEs) are not usually examined simultaneously in most studies. To compare the predictive potentials of the area under the concentration-time curve (AUC)-to-MIC ratio (AUC/MIC), the AUC above MIC (AUCeff), and the time above MIC (Teff), the kinetics of killing and regrowth of four bacterial strains exposed to monoexponentially decreasing concentrations of ciprofloxacin were studied in an in vitro dynamic model. The MICs of ciprofloxacin for clinical isolates of Staphylococcus aureus, Escherichia coli 11775 (I) and 204 (II), and Pseudomonas aeruginosa were 0.6, 0.013, 0.08, and 0.15 microg/ml, respectively. The simulated values of AUC were designed to provide similar 1,000-fold (S. aureus, E. coli I, and P. aeruginosa) or 2, 000-fold (E. coli II) ranges of the AUC/MIC. In each case except for the highest AUC/MIC ratio, the observation periods included complete regrowth in the time-kill curve studies. The AME was expressed by its intensity, IE (the area between the control growth and time-kill and regrowth curves up to the point where the viable counts of regrowing bacteria are close to the maximum values observed without drug). For most AUC ranges the IE-AUC curves were fitted by an Emax (maximal effect) model, whereas the effects observed at very high AUCs were greater than those predicted by the model. The AUCs that produced 50% of maximal AME were proportional to the MICs for the strains studied, but maximal AMEs (IEmax) and the extent of sigmoidicity (s) were not related to the MIC. Both Teff and log AUC/MIC correlated well with IE (r2 = 0.98 in both cases) in a species-independent fashion. Unlike Teff or log AUC/MIC, a specific relationship between IE and log AUCeff was inherent in each strain. Although each IE and log AUCeff plot was fitted by linear regression (r2 = 0.97 to 0.99), these plots were not superimposed and therefore are bacterial species dependent. Thus, AUC/MIC and Teff were better predictors of ciprofloxacin's AME than AUCeff. This study suggests that optimal predictors of the AME produced by a given quinolone (intraquinolone predictors) may be established by examining its AMEs against bacteria of different susceptibilities. Teff was shown previously also to be the best interquinolone predictor, but unlike AUC/MIC, it cannot be used to compare different quinolones. AUC/MIC might be the best predictor of the AME in comparisons of different quinolones.

A new approach to in vitro comparisons of antibiotics in dynamic models: equivalent area under the curve/MIC breakpoints and equiefficient doses of trovafloxacin and ciprofloxacin against bacteria of similar susceptibilities.

Time-kill studies, even those performed with in vitro dynamic models, often do not provide definitive comparisons of different antimicrobial agents. Also, they do not allow determinations of equiefficient doses or predictions of area under the concentration-time curve (AUC)/MIC breakpoints that might be related to antimicrobial effects (AMEs). In the present study, a wide range of single doses of trovafloxacin (TR) and twice-daily doses of ciprofloxacin (CI) were mimicked in an in vitro dynamic model. The AMEs of TR and CI against gram-negative bacteria with similar susceptibilities to both drugs were related to AUC/MICs that varied over similar eight-fold ranges [from 54 to 432 and from 59 to 473 (microg . h/ml)/(microg/ml), respectively]. The observation periods were designed to include complete bacterial regrowth, and the AME was expressed by its intensity (the area between the control growth in the absence of antibiotics and the antibiotic-induced time-kill and regrowth curves up to the point where viable counts of regrowing bacteria equal those achieved in the absence of drug [IE]). In each experiment monoexponential pharmacokinetic profiles of TR and CI were simulated with half-lives of 9.2 and 4.0 h, respectively. Linear relationships between IE and log AUC/MIC were established for TR and CI against three bacteria: Escherichia coli (MIC of TR [MICTR] = 0.25 microg/ml; MIC of CI [MICCI] = 0.12 microg/ml), Pseudomonas aeruginosa (MICTR = 0.3 microg/ml; MICCI = 0.15 microg/ml), and Klebsiella pneumoniae (MICTR = 0.25 microg/ml; MICCI = 0.12 microg/ml). The slopes and intercepts of these relationships differed for TR and CI, and the IE-log AUC/MIC plots were not superimposed, although they were similar for all bacteria with a given antibiotic. By using the relationships between IE and log AUC/MIC, TR was more efficient than CI. The predicted value of the AUC/MIC breakpoint for TR [mean for all three bacteria, 63 (microg . h/ml)/(microg/ml)] was approximately twofold lower than that for CI. Based on the IE-log AUC/MIC relationships, the respective dose (D)-response relationships were reconstructed. Like the IE-log AUC/MIC relationships, the IE-log D plots showed TR to be more efficient than CI. Single doses of TR that are as efficient as two 500-mg doses of CI (500 mg given every 12 h) were similar for the three strains (199, 226, and 203 mg). This study suggests that in vitro evaluation of the relationships between IE and AUC/MIC or D might be a reliable basis for comparing different fluoroquinolones and that the results of such comparative studies may be highly dependent on their experimental design and datum quantitation.