Alexander A. Firsov

In vitro pharmacodynamic evaluation of the mutant selection window hypothesis using four fluoroquinolones against Staphylococcus aureus.

ABSTRACT To study the hypothesis of the mutant selection window (MSW) in a pharmacodynamic context, the susceptibility of a clinical isolate of methicillin-resistant Staphylococcus aureus exposed to moxifloxacin (MOX), gatifloxacin (GAT), levofloxacin (LEV), and ciprofloxacin (CIP) was tested daily by using an in vitro dynamic model that simulates human pharmacokinetics. A series of monoexponential pharmacokinetic profiles that mimic once-daily administration of MOX (half-life, 12 h), GAT (half-life, 7 h), and LEV (half-life, 6.8 h) and twice-daily administration of CIP (half-life, 4 h) provided peak concentrations (Cmax) that either equaled the MIC, fell between the MIC and the mutant prevention concentration (MPC) (i.e., within or “inside” the MSW), or exceeded the MPC. The respective ratios of the area under the curve (AUC) over a 24-h dosing interval (AUC24) to the MIC varied from 13 to 244 h, and the starting inoculum was 108 CFU/ml (6 × 109 CFU per 60-ml central compartment). With all four quinolones, the greatest increases in MIC were observed at those AUC24/MIC values (from 24 to 62 h) that corresponded to quinolone concentrations within the MSW over most of the dosing interval (>20%). Less-pronounced increases in MIC were associated with the smallest simulated AUC24/MIC values (15 to 16 h) of GAT and CIP, whose Cmax exceeded the MICs. No such increases were observed with the smallest AUC24/MIC values (13 to 17 h) of MOX and LEV, whose Cmax were close to the MICs. Also, less pronounced but significant increases in MIC occurred at AUC24/MIC values (107 to 123 h) that correspond to quinolone concentrations partly overlapping the MIC-to-MPC range. With all four drugs, no change in MIC was seen at the highest AUC24/MIC values (201 to 244 h), where quinolone concentrations exceeded the MPC over most of the dosing interval. These “protective” AUC24/MIC ratios correspond to 66% of the usual clinical dose of MOX (400 mg), 190% of a 400-mg dose of GAT, 220% of a 500-mg dose of LEV, and 420% of two 500-mg doses of CIP. Thus, MOX may protect against resistance development at subtherapeutic doses, whereas GAT, LEV, and CIP provide similar effects only at doses that exceed their usual clinical doses. These data support the concept that resistant mutants are selectively enriched when antibiotic concentrations fall inside the MSW and suggest that in vitro dynamic models can be used to predict the relative abilities of quinolones to prevent mutant selection.

Predictors of effect of ampicillin-sulbactam against TEM-1 beta-lactamase-producing Escherichia coli in an in vitro dynamic model: enzyme activity versus MIC.

The clinical outcome in patients treated with ampicillin-sulbactam may not always be predictable by disc susceptibility testing or with the MIC as determined with a constant level (4 micrograms/ml) of the beta-lactamase inhibitor (MIC1). The enzyme activities (EA) and the MICs estimated at a constant ratio of ampicillin to sulbactam of 2:1 (MIC2) for 15 TEM-1 beta-lactamase-producing strains of Escherichia coli were examined as alternatives to MIC1 as predictors of the antibacterial effects of this combined drug as studied in an in vitro model which simulates ampicillin-sulbactam pharmacokinetic profiles observed in human peripheral tissues. Integral parameters describing the area under the bacterial count-time curve (AUBC), the area between the normal growth curve, and the killing curve of bacteria exposed to antibiotic (ABBC), and the second parameter expressed as a percentage of its maximal hypothetical value (ABBC/ABBCmax) were calculated. All three parameters correlated well with EA (AUBC, r = 0.93; ABBC, r = -0.88; ABBC/ABBCmax, r = -0.91) and with MIC2 (r = 0.94, -0.94, and -0.95, respectively) but not with MIC1. Both EA and MIC2 can be considered reliable predictors of the antibacterial effect of ampicillin-sulbactam in an in vitro model. These correlations suggest that in vitro kinetic-dynamic models might be useful to reexamine established susceptibility breakpoints obtained with data based on the MIC1 (MICs obtained with constant levels of beta-lactamase inhibitors). These data also suggest that quantitative determinations of bacterial beta-lactamase production and MICs based on the component concentration ratio observed in vivo might be useful predictors of the effect of ampicillin-sulbactam and other beta-lactam-inhibitor combinations.

Enrichment of resistant Staphylococcus aureus at ciprofloxacin concentrations simulated within the mutant selection window: bolus versus continuous infusion.

Enrichment of resistant mutants at antibiotic concentrations above the minimum inhibitory concentration (MIC) but below the mutant prevention concentration (MPC), i.e. within the mutant selection window (MSW), might be dependent on the shape of the pharmacokinetic profile. To address this issue, two strains of Staphylococcus aureus were exposed to fluctuating (bolus administration) and constant (continuous infusion) concentrations of ciprofloxacin. Staphylococcus aureus ATCC 43300 and ATCC 6538 exhibiting different MPC/MIC ratios (4 and 16, respectively) were exposed to ciprofloxacin twice daily by bolus administration and continuous infusion for 3 days. With each organism and mode of administration, a series of pharmacokinetic profiles was simulated to have the same 24-h area under the concentration-time curve (AUC24) to MIC ratio. The simulated AUC24/MIC ratios were designed to provide ciprofloxacin concentrations within the MSW over most of the dosing interval (bolus administration) or over the entire dosing interval (continuous infusion). In all simulations, ciprofloxacin-resistant staphylococci were enriched in a concentration-dependent manner, i.e. the higher the AUC24/MIC, the later the onset of mutant selection and the smaller the area under the bacterial mutant curve (AUBC M). The relationships between AUC24/MIC and AUBC M were independent of the shape of the simulated pharmacokinetic profiles that corresponded to the different modes of ciprofloxacin administration. For mutants resistant to > or = 4 x MIC of ciprofloxacin, the AUC24/MIC was less predictive of the AUBC M than the AUC24/MPC ratio. This study indicates the mode of ciprofloxacin administration does not influence selection of resistant staphylococci, which is better predicted by AUC24/MPC than by AUC24/MIC.

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

ABSTRACT Most integral endpoints of the antimicrobial effect are determined over an arbitrarily chosen time period, such as the dosing interval (τ), regardless of the actual effect duration. Unlike the τ-related endpoints, the intensity of the antimicrobial effect (IE) 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 ofStaphylococcus aureus was exposed to simulated gemifloxacin pharmacokinetics over a 40-fold range of AUC/MIC ratios, from 11 to 466 h. In each run, IE and four τ-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 τ = 24 h. Unlike the IE, which displayed pseudolinear relationships with the AUC/MIC ratio; each τ-related endpoint showed a distinct saturation at potentially therapeutic AUC/MIC ratios (116 to 466 h) when the antimicrobial effect persisted longer than τ. 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 IE to consider the actual effect duration. These data suggest a marginal value of the τ-related endpoints as indices of the total antimicrobial effect. Since all of them respond to AUC/MIC ratio changes less than theIE, the latter is preferable in comparative pharmacodynamic studies.

Comparative pharmacodynamics of gatifloxacin and ciprofloxacin in an in vitro dynamic model: prediction of equiefficient doses and the breakpoints of the area under the curve/MIC ratio.

ABSTRACT To demonstrate the impact of the pharmacokinetics of gatifloxacin (GA) relative to those of ciprofloxacin (CI) on the antimicrobial effect (AME), the killing and regrowth kinetics of two differentially susceptible clinical isolates each of Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae were studied. With each organism, a series of monoexponential pharmacokinetic profiles of GA (half-life [t1/2], 7 h) and CI (t1/2 = 4 h) were simulated to mimic different single doses of GA and two 12-h doses of CI. The respective eightfold ranges of the ratios of the area under the concentration-time curve (AUC) to the MIC were 58 to 466 and 116 to 932 (μg · h/ml)/(μg/ml). The species- and strain-independent linear relationships observed between the intensity of AME (IE) and log AUC/MIC were not superimposed for GA and CI (r2 = 0.99 in both cases). The predicted AUC/MIC ratio for GA that might be equivalent to a clinically relevant AUC/MIC breakpoint for CI was estimated to be 102 rather than 125 (μg · h/ml)/(μg/ml). The respective MIC breakpoints were 0.32 μg/ml (for a 400-mg dose of GA) and 0.18 μg/ml (for two 500-mg doses of CI). On the basis of the IE-log AUC/MIC relationships, equiefficient 24-h doses (D24hs) of GA and CI were calculated for hypothetical strains of S. aureus, E. coli, andK. pneumoniae for which the MICs were equal to the MICs at which 50% of isolates are inhibited. To provide an “acceptable”IE equal to 200 (log CFU/ml) · h, i.e., the IE provided by AUC/MIC of 125 (μg · h/ml)/(μg/ml) for ciprofloxacin, theD24hs of GA for all three organisms were much lower (115, 30, and 60 mg) than the clinically proposed 400-mg dose. Although the usual dose of CI (two doses of 500 mg) would be in excess for E. coli and K. pneumoniae(D24h = two doses of 40 mg and two doses of 115 mg, respectively), even the highest clinical dose of CI (two doses of 750 mg) might be insufficient for S. aureus(D24h, > two doses of 1,000 mg). The method of generalization of data obtained with specific organisms to other representatives of the same species described in the present report might be useful for prediction of the AMEs of new quinolones.

Selection of linezolid-resistant Enterococcus faecium in an in vitro dynamic model: protective effect of doxycycline

OBJECTIVES To relate the enrichment of linezolid-resistant Enterococcus faecium with linezolid pharmacokinetics, the pharmacodynamics of linezolid and its ability to prevent the selection of resistant mutants were studied in an in vitro model that simulates antibiotic concentrations in and out of the mutant selection window (MSW), i.e. the concentration range from the MIC to the mutant prevention concentration (MPC). METHODS A clinical isolate of E. faecium (MIC 1.8 mg/L and MPC 7 mg/L) at a starting inoculum of 8 log cfu/mL was exposed to twice-daily linezolid, alone and in combination with once-daily doxycycline (MIC 0.2 mg/L and MPC 3.4 mg/L), for 3 consecutive days in a hollow-fibre two-compartment model. RESULTS The ratios of 24 h area under the curve (AUC24) to MIC of linezolid were estimated at 70, 100 and 230 h and those of doxycycline were estimated at 230 and 720 h. At the two lower AUC24/MIC ratios of linezolid given alone, E. faecium resistant to 2 x MIC-16 x MIC and 2 x MIC-8 x MIC of linezolid, respectively, were selectively enriched with a concomitant slight loss in susceptibility. Neither growth on linezolid-containing media nor changes in susceptibility occurred at the high AUC24/MIC ratio. A similar protective effect was observed with the minimal AUC24/MIC ratio of linezolid (70 h) combined with doxycycline at an AUC24/MIC of 230 h. CONCLUSIONS This study suggests that selection of linezolid-resistant enterococci can be predicted from the MSW concept and can be prevented by linezolid given in combination with doxycycline, each at suboptimal AUC24/MIC ratios.

Bacterial Resistance Studies Using In Vitro Dynamic Models: the Predictive Power of the Mutant Prevention and Minimum Inhibitory Antibiotic Concentrations

ABSTRACT In light of the concept of the mutant selection window, i.e., the range between the MIC and the mutant prevention concentration (MPC), MPC-related pharmacokinetic indices should be more predictive of bacterial resistance than the respective MIC-related indices. However, experimental evidence of this hypothesis remains limited and contradictory. To examine the predictive power of the ratios of the area under the curve (AUC24) to the MPC and the MIC, the selection of ciprofloxacin-resistant mutants of four Escherichia coli strains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. Resistant E. coli was intensively enriched at AUC24/MPCs from 1 to 10 h (AUC24/MIC from 60 to 360 h) but not at the lower or higher AUC24/MPC and AUC24/MIC ratios. AUC24/MPC and AUC24/MIC relationships of the areas under the time courses of ciprofloxacin-resistant E. coli (AUBCM) were bell-shaped. A Gaussian-like function fits the AUBCM-AUC24/MPC and AUBCM-AUC24/MIC data combined for all organisms (r2 = 0.69 and 0.86, respectively). The predicted anti-mutant AUC24/MPC ratio was 58 ± 35 h, and the respective AUC24/MIC ratio was 1,080 ± 416 h. Although AUC24/MPC was less predictive of strain-independent E. coli resistance than AUC24/MIC, the established anti-mutant AUC24/MPC ratio was closer to values reported for Staphylococcus aureus (60 to 69 h) than the respective AUC24/MIC ratio (1,080 versus 200 to 240 h). This implies that AUC24/MPC might be a better interspecies predictor of bacterial resistance than AUC24/MIC.

Enrichment of Fluoroquinolone-Resistant Staphylococcus aureus: Oscillating Ciprofloxacin Concentrations Simulated at the Upper and Lower Portions of the Mutant Selection Window

ABSTRACT The time inside the mutant selection window (MSW), TMSW, appears to be less predictive of the selection of fluoroquinolone-resistant Staphylococcus aureus than is the ratio of the area under the concentration-time curve (AUC) to the MIC. This observation might be attributed to the fact that TMSW does not consider the actual position of simulated antibiotic concentrations inside the MSW, which also might influence the amplification of resistant mutants. To test this hypothesis, the enrichment of ciprofloxacin-resistant S. aureus was studied at ciprofloxacin (CIP) concentrations that oscillate near the mutant prevention concentration (MPC), i.e., closer to the top of the MSW (“upper case”), and closer to the MIC, i.e., at the lower limit of the MSW (“lower case”) at the same TMSW. Two methicillin-resistant strains of S. aureus, ATCC 6538 and ATCC 43300 (MICs of 0.25 and 0.5 mg/liter, respectively, and MPCs of 4 and 2 mg/liter, respectively), were exposed to twice-daily CIP treatments for three consecutive days. With S. aureus ATCC 6538, the simulated ratios of the AUC at 24 h (AUC24) to the MIC were 50 and 260 h (TMSW 75% of the dosing interval). With S. aureus ATCC 43300, the simulated AUC24/MICs were 30 and 100 h (TMSW 56%). With each organism, mutants resistant to CIP were enriched in an AUC24/MIC-dependent manner: the higher the AUC24/MIC ratio, the lower the growth on CIP-containing plates. For example, the area under the time-kill curve of mutants resistant to 4× MIC of CIP in the upper case was three times smaller than that in the lower case for both S. aureus strains. Similar differences were seen at the higher (8× MIC) and lower (2× MIC) CIP concentrations. These data highlight differences in the selection of resistant S. aureus, depending on the position of simulated concentrations inside the MSW at a given TMSW. This explains why TMSW-based predictions of resistance are less accurate than those based on AUC/MIC and AUC/MPC.

Antistaphylococcal Effect Related to the Area under the Curve/MIC Ratio in an In Vitro Dynamic Model: Predicted Breakpoints versus Clinically Achievable Values for Seven Fluoroquinolones

ABSTRACT Prediction of the relative efficacies of different fluoroquinolones is often based on the ratios of the clinically achievable area under the concentration-time curve (AUC) to the MIC, usually with incorporation of the MIC50 or the MIC90 and with the assumption of antibiotic-independent patterns of the AUC/MIC-response relationships. To ascertain whether this assumption is correct, the pharmacodynamics of seven pharmacokinetically different quinolones against two clinical isolates of Staphylococcus aureus were studied by using an in vitro model. Two differentially susceptible clinical isolates of S. aureus were exposed to two 12-h doses of ciprofloxacin (CIP) and one dose of gatifloxacin (GAT), gemifloxacin (GEM), grepafloxacin (GRX), levofloxacin (LVX), moxifloxacin (MXF), and trovafloxacin (TVA) over similar AUC/MIC ranges from 58 to 932 h. A specific bacterial strain-independent AUC/MIC relationship with the antimicrobial effect (IE) was associated with each quinolone. Based on the IE-log AUC/MIC relationships, breakpoints (BPs) that are equivalent to a CIP AUC/MIC ratio of 125 h were predicted for GRX, MXF, and TVA (75 to 78 h), GAT and GEM (95 to 103 h) and LVX (115 h). With GRX and LVX, the predicted BPs were close to those established in clinical settings (no clinical data on other quinolones are available in the literature). To determine if the predicted AUC/MIC BPs are achievable at clinical doses, i.e., at the therapeutic AUCs (AUCthers), the AUCther/MIC50 ratios were studied. These ratios exceeded the BPs for GAT, GEM, GRX, MXF, TVA, and LVX (750 mg) but not for CIP and LVX (500 mg). AUC/MIC ratios above the BPs can be considered of therapeutic potential for the quinolones. The highest ratios of AUCther/MIC50 to BP were achieved with TVA, MXF, and GEM (2.5 to 3.0); intermediate ratios (1.5 to 1.6) were achieved with GAT and GRX; and minimal ratios (0.3 to 1.2) were achieved with CIP and LVX.

In Vitro Resistance Studies with Bacteria That Exhibit Low Mutation Frequencies: Prediction of “Antimutant” Linezolid Concentrations Using a Mixed Inoculum Containing both Susceptible and Resistant Staphylococcus aureus

ABSTRACT Bacterial resistance studies using in vitro dynamic models are highly dependent on the starting inoculum that might or might not contain spontaneously resistant mutants (RMs). To delineate concentration-resistance relationships with linezolid-exposed Staphylococcus aureus, a mixed inoculum containing both susceptible cells and RMs was used. An RM selected after the 9th passage of the parent strain (MIC, 2 μg/ml) on antibiotic-containing media (RM9; MIC, 8 μg/ml) was chosen for the pharmacodynamic studies, because the mutant prevention concentration (MPC) of linezolid against the parent strain in the presence of RM9 at 102 (but not at 104) CFU/ml did not differ from the MPC value determined in the absence of the RMs. Five-day treatments with twice-daily linezolid doses were simulated at concentrations either between the MIC and MPC or above the MPC. S. aureus RMs (resistant to 2× and 4× MIC but not 8× and 16× MIC) were enriched at ratios of the 24-h area under the concentration-time curve (AUC24) to the MIC that provide linezolid concentrations between the MIC and MPC for 100% (AUC24/MIC, 60 h) and 86% (AUC24/MIC, 120 h) of the dosing interval. No such enrichment occurred when linezolid concentrations were above the MIC and below the MPC for a shorter time (37% of the dosing interval; AUC24/MIC, 240 h) or when concentrations were consistently above the MPC (AUC24/MIC, 480 h). These findings obtained using linezolid-susceptible staphylococci supplemented with RMs support the mutant selection window hypothesis. This method provides an option to delineate antibiotic concentration-resistance relationships with bacteria that exhibit low mutation frequencies.