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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 (AUC 24 ) 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 AUC 24 /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 AUC 24 T he concept of the mutant selection window (MSW), i.e., the range between the MIC and the mutant prevention concentration (MPC) (1), has contributed to an understanding of why antibiotic-resistant mutants are enriched in some but not all treatments. In light of this hypothesis, MPC-related pharmacokinetic indices should be more predictive of bacterial resistance than the respective MIC-related indices. However, the advantages of the former over the latter indices have been reported in only a few in vitro studies with ciprofloxacin-exposed (2) and levofloxacin-exposed(3) Staphylococcus aureus and with ciprofloxacin-exposed Escherichia coli (4). On the other hand, the predictive potentials of the MPC-and MIC-related indices were shown to be similar in an in vivo study of Streptococcus pneumoniae-infected rabbits treated with gatifloxacin using a human-like dosing regimen (5) and in an in vitro study with marbofloxacin-and enrofloxacin-exposed E. coli (6). In our in vitro study with S. aureus exposed to daptomycin and vancomycin, the ratio of the 24-h area under the concentration-time curve (AUC 24 ) to the MPC was less predictive of bacterial resistance than the AUC 24 /MIC ratio (7).To further examine the predictive power of AUC 24 /MPC and AUC 24 /MIC, the selection of ciprofloxacin-resistant mutants of four E. coli strains at various MPC/MIC ratios was studied over a wide range of simulated AUC 24 /MICs. MATERIALS AND METHODSAntimicrobial agents, bacterial strains, and susceptibility testing. Ciprofloxacin powder was purchased from AppliChem Biochemica Chemical Synthesis Services, Darmstadt, Germany.E. coli ATCC 25922, E. coli clinical isolates 4300 and 4454, and E. coli laboratory strain mutator GM2995 (ES1578) (8) were selected for the study. The MICs were determined prior to and after 3-day simulated treatments with ciprofloxacin. Susceptibility testing was performed at least in duplicate by broth microdilution techniques at 24 h postexposure with organisms grown in Ca 2ϩ -and Mg 2ϩ -supplemented Mueller-Hinton broth (MHB) at an inoculum size of 10 6 CFU/ml. The MICs of ciprofloxacin were 0.008 g/ml with E. coli strains ATCC 25922, 4300, and 4454 and 0.016 g/ml with E. coli strain GM2995.Th...

The time inside the mutant selection window (MSW), T MSW , 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 T MSW 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 T MSW . 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 (AUC 24 ) to the MIC were 50 and 260 h (T MSW 75% of the dosing interval). With S. aureus ATCC 43300, the simulated AUC 24 /MICs were 30 and 100 h (T MSW 56%). With each organism, mutants resistant to CIP were enriched in an AUC 24 /MIC-dependent manner: the higher the AUC 24 /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 T MSW . This explains why T MSW -based predictions of resistance are less accurate than those based on AUC/MIC and AUC/MPC.

A new family of protic ammonium ionic liquids (ILs) with various inorganic anions was synthesized from bioderived 5-HMF. Starting with cellulose biomass, a complete preservation of the C 6 unit was achieved throughout the synthetic sequence (no carbon loss). Evaluation of green metrics showed a significant advantage of the developed bio-derived pathway to access ILs from a natural renewable source, depending on feasible routes to 5-HMF manufacturing. The reduced number of synthetic steps and availability of the starting materials were the key advantages. Experimental physicochemical and biological studies, as well as computational modeling revealed a unique multifunctional intrinsic organization of these bio-derived ILs. The nature of interactions between the cations and anions of the novel ILs was mapped at the molecular level. The substituents in the cationic core and the nature of the original building blocks had a prominent impact on cytotoxicity of the novel ILs. The obtained results suggest possible sustainable applications of the least toxic ILs, while the regulation of biological activity of the ILs via the corresponding structural adjustments can find biological and medicinal applications. The 5-HMF-derived IL with a sulfate anion demonstrated potentially useful properties in dissolution of microcrystalline cellulose.

To explore if the time inside the mutant selection window (T) is a reliable predictor of emergence of bacterial resistance to linezolid, mixed inocula of each of three methicillin-resistant Staphylococcus aureus strains (MIC of linezolid 2 μg ml) and their previously selected resistant mutants (MIC 8 μg ml) were exposed to linezolid pharmacokinetics using an in vitro dynamic model. In five-day treatments simulated over a wide range of the 24-h area under the concentration-time curve (AUC) to the MIC ratio, mutants resistant to 4 × MIC of antibiotic were enriched in a T-dependent manner. With each strain, T relationships with the area under the bacterial mutant concentration-time curve (AUBC) exhibited a hysteresis loop, with the upper portion corresponding to the time above the mutant prevention concentration (MPC; T) of 0 and the lower portion-to the T > 0. Using AUBC related to the maximal value observed with a given strain (normalized AUBC) at T > 0, a strain-independent sigmoid relationship was established between AUBC and T, as well as T (r 0.99 for both). AUC/MIC and AUC/MPC relationships with normalized AUBC for combined data on the three studied S. aureus strains were bell-shaped (r 0.85 and 0.80, respectively). These findings suggest that T at T > 0, T, AUC/MIC and AUC/MPC are useful bacterial strain-independent predictors of the emergence of staphylococcal resistance to linezolid.

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 g/ml) on antibiotic-containing media (RM9; MIC, 8 g/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 10 2 (but not at 10 4 ) 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. A specific mechanism of oxazolidinone action blocks functional initiation complexes in bacterial translation systems. Given the lack of analogues among available antimicrobials, a low probability of preexisting, naturally occurring resistance mechanisms has been hypothesized (1, 2). For example, resistance of Staphylococcus aureus rarely occurred through spontaneous mutations at frequencies of only 10 Ϫ9 to 10 Ϫ11 (3-5). However, the first report on the detection of a clinical isolate of S. aureus with a linezolid MIC of Ͼ32 g/ml was published as early as 2001 (6). Analysis of 23S rRNA encoding DNA sequences showed that linezolid-resistant S. aureus had a G-to-T mutation at position 2576 (Escherichia coli numbering). The same point mutations also were observed in S. aureus isolated in other clinical studies (7-14). Later, amino acid substitutions in ribosomal protein L3 (50S large-subunit ribosomal protein) were associated with oxazolidinone resistance (linezolid MIC, 8 g/ml) in a clinical S. aureus strain (15). Thus, clinical data indicate that resistance to linezolid mediated by mutations in ribosomal genes may emerge more readily than was initially predicted by routine in vitro studies performed early in the development of oxazolidinones, because they were not specifically designed to simulate antibiotic exposures that would allow the enrichment of resistant mutants.The resistance studies with linezolid cited above were not supported by concomitant pharmacokinetic studies to relate antibiotic concentrations to the selection of resistant mutants. To be sure, some in vitro model studies of the enrichment of resistant S. aureus simulated linezolid pharmacokinetics (16, 17).However, these attempts were unsuccessful because resistant mutants were not enriched, at least in simulations of oscillating antibiotic concentrations that mimic the usual linezolid dosing in humans. Linezolid-resistant staphylococci also were not enriched in other in vitro studies that were not designed to establish concentration-resistance relationships (18)(19)(20), probably because of the lack of spontaneous mutants in the starting inocula.To more clearly delineate concentration-resistance rel...