Background/Objectives: Assessing antibiotic MICs at high bacterial counts is likely to disclose hidden bacterial resistance and the inoculum effect if present and therefore also reveal potential decreased antibiotic effectiveness. In the current study, we evaluated the predictive potential of MICs determined at high bacterial inocula to evaluate meropenem effectiveness and emergence of resistance in Klebsiella pneumoniae. Methods: Nine carbapenemase-free or carbapenemase-producing K. pneumoniae strains were exposed to meropenem in an in vitro hollow-fiber infection model (HFIM). The treatment effects were correlated with simulated antibiotic ratios of the area under the concentration–time curve (AUC) to the MIC (AUC/MIC) and to MICs determined at high inocula (AUC/MICHI). Results: Based on MICs determined at standard inocula, meropenem effects at different AUC/MIC ratios for both carbapenemase-free and carbapenemase-producing K. pneumoniae strains were stratified and could not be described by a single relationship. In contrast, when AUC/MICHI ratios were used, a single relationship with the antibiotic effect was obtained for all tested strains. Similarly, the emergence of meropenem resistance in HFIM was concordant with AUC/MICHI, but not with AUC/MIC ratios. Conclusions: MICs determined at high bacterial inocula enable the prediction of meropenem effects both for carbapenemase-free and for carbapenemase-producing K. pneumoniae strains. Also, MICs at standard and high inocula can identify carbapenemase-producing strains by revealing the inoculum effect.

Glycopeptide antibiotics are still in demand in clinical practice for treating infections caused by resistant gram-positive pathogens; however, their use is limited due to severe adverse reactions. Their predominant types of side effects are immunoglobulin E-mediated or nonmediated hypersensitivity reactions. Therefore, the development of new glycopeptide antibiotics with improved toxicity profiles remains an important objective in advancing modern antimicrobial agents. We investigated a new eremomycin aminoalkylamide flavancin, its anaphylactogenic properties, influence on histamine levels in blood plasma, pseudoallergic inflammatory reaction on concanavalin A and the change in the amount of flavancin in the blood plasma after administration. It has been shown that flavancin does not demonstrate anaphylactogenic properties. The injection of flavancin resulted in a level of histamine in the blood three times lower than that caused by vancomycin. The therapeutic dose of vancomycin led to a statistically significant increase in the concanavalin A response index compared to flavancin (54% versus 3.7%). Thus, flavancin does not cause a pseudo-allergic reaction. The rapid decrease in flavancin concentration in the blood and the low levels of histamine in the plasma lead us to assume that any pseudoallergic reactions resulting from flavancin application, if they do occur in clinical practice, will be significantly less compared to the use of vancomycin.

OXA-48 carbapenemases are frequently expressed by Klebsiella pneumoniae clinical isolates; they decrease the effectiveness of carbapenem therapy, particularly with meropenem. Among these isolates, meropenem-susceptible carbapenemase-producers may show decreased meropenem effectiveness. However, the probability of the emergence of resistance in susceptible carbapenemase-producing isolates and its dependence on specific K . pneumoniae meropenem MICs is not completely known. It is also not completely clear what resistance patterns will be exhibited by these bacteria exposed to meropenem, if they would follow the patterns of non-beta-lactamase-producing bacteria and other than beta-lactams antibiotics. These issues might be clarified if patterns of meropenem resistance related to the mutant selection window (MSW) hypothesis. To test the applicability of the MSW hypothesis to meropenem, OXA-48-carbapenemase-producing K . pneumoniae clinical isolates with MICs in a 64-fold range (from susceptible to resistant) were exposed to meropenem in a hollow-fiber infection model; epithelial lining fluid meropenem pharmacokinetics were simulated following administration of 2 grams every 8 hours in a 3-hour infusion. Strong bell-shaped relationships between the meropenem daily dose infused to the model as related to the specific isolate MIC and both the antimicrobial effect and the emergence of resistance were observed. The applicability of the MSW hypothesis to meropenem and carbapenemase producing K . pneumoniae was confirmed. Low meropenem efficacy indicates very careful prescribing of meropenem to treat K . pneumoniae infections when the causative isolate is confirmed as an OXA-48-carbapenemase producer.

The emergence of bacteria resistant to beta-lactam/beta-lactamase inhibitor combinations is insufficiently studied, wherein the role of the inoculum effect (IE) in decreased efficacy is unclear. To address these issues, 5-day treatments with doripenem and doripenem/relebactam combination at different ratios of the agents were simulated in a hollow-fiber dynamic model against carbapenemase-producing K. pneumoniae at standard and high inocula. Minimal inhibitory concentrations (MICs) of doripenem alone and in the presence of relebactam at two inocula were determined. Combination MICs were tested using traditional (fixed relebactam concentration) and pharmacokinetic-based approach (fixed doripenem-to-relebactam concentration ratio equal to the therapeutic 24-h area under the concentration-time curve (AUC) ratio). In all experiments, resistant subpopulations were noted, but combined simulations reduced their numbers. With doripenem, the IE was apparent for both K. pneumoniae isolates in combined treatments for one strain. The pharmacokinetic-based approach to combination MIC estimation compared to traditional showed stronger correlation between DOSE/MIC and emergence of resistance. These results support (1) the constraint of relebactam combined with doripenem against the emergence of resistance and IE; (2) the applicability of a pharmacokinetic-based approach to estimate carbapenem MICs in the presence of an inhibitor to predict the IE and to describe the patterns of resistance occurrence.

The pharmacokinetics of amphamide, a new semisynthetic antifungal antibiotic, are studied after intravenous injection to rats and rabbits. The amphamide concentration in biological samples was determined by HPLC. Amphamide pharmacokinetics in rabbits were nonlinear. Amphamide levels in animal blood plasma decreased in a triphasic manner. The total clearance and steady-state distribution volume were greater in rats than in rabbits (0.85 vs. 0.3 – 0.5 mL ∙ min –1 ∙ kg–1 and 1.5 vs. 0.6 – 0.9 L/kg, respectively). The mean residence times of amphamide in rats and rabbits were similar (30 vs. 27 – 43 h, respectively). The elimination half-lives calculated using a three-compartment model were 24 h for rats and 31 h for rabbits. The availability of rat tissue for amphamide ranged from 100% (brain) to 1700% (kidneys).

The pharmacokinetics and acute toxicity of the new anthra[2, 3- b ]furan-3-carboxamide derivative anthrafuran (AF) were studied after intravenous (i.v.), intraperitoneal (i.p.), and peroral administrations (p.o.) to rats. AF blood-plasma concentrations in the animals were determined by HPLC. The total clearance, steady-state distribution volume, and mean residence time of AF were 17.4 mL·min –1 ·kg –1 , 6.6 L/kg, and 6.3 h, respectively. The absolute AF bioavailabilities after i.p. and p.o. administration were 96 and 31%, respectively. The elimination half-life of AF was 5 – 6 h. The LD 50 values in the rats after i.v., i.p., and p.o. administration were 17.1, 64.6, and 353.6 mg/kg, respectively. Correlations were found between LD 50 or the maximum tolerated dose (MTD) for the studied AF administration pathways and the logarithms of the areas under the curve (AF-concentration— time) or maximum AF concentration and natural times to reach it.

The pharmacokinetics of olivamide, a semisynthetic antitumor derivate of olivomycin A, were studied in rats and rabbits after bolus administration. Plasma concentrations of olivamide were determined by an HPLC assay. The decrease in olivamide levels in blood plasma was biphasic in both rats and rabbits. Olivamide levels declined slowly for up to 24 hours after a sharp drop in drug concentration during the first 15 minutes due to the rapid redistribution of olivamide between blood and peripheral tissues. The tissue availability of olivamide in rats ranged from 5700% (spleen) to 450% (liver). Eight-hour cumulative renal excretion of unchanged olivamide in rats was 18% of the dose. A three-exponential equation was used to describe pharmacokinetic profiles of olivamide in rat and rabbit plasma. The total clearance and steady-state volume of distribution in rats were higher than in rabbits (17.9-22.0 vs. 16.2 ml/(minXkg) and 7.8-8.7 vs. 5.1 l/kg, respectively). The mean retention time of olivamide in rabbits was less than in rats (5.3 vs 6.3-7.3 h). Olivamide pharmacokinetics in rats were linear.

Current knowledge of the emergence of Streptococcus pneumoniae resistance during treatment with aminopenicillins and macrolides is limited. In particular, clinical reports on isolation of azithromycin-resistant mutants do not relate their enrichment to the actual antibiotic concentrations in blood. In the present work, the selection of amoxicillin- and azithromycin-resistant S. pneumoniae mutants at therapeutic and subtherapeutic antibiotic exposures was studied in an in vitro dynamic model. There was no enrichment of S. pneumoniae mutants resistant to amoxicillin, while azithromycin-resistant mutants were enriched in all simulations. This difference was related to the different times above the mutant prevention concentration: 60-100% of the dosing interval for amoxicillin versus zero percentage for azithromycin. These findings are in concordance with the mutant selection window hypothesis.

The search for optimal predictors of anti-mutant effects remains a pressing problem in studies of antibiotic-associated bacterial resistance. To relate the emergence of bacterial resistance with the antibiotic mutant prevention concentration (MPC), a novel integral parameter - the area around the resistance threshold, i.e. MPC level (AAMPC) is proposed. The AAMPC is the algebraic sum of the area under the antibiotic concentration-time curve that is above the MPC (positive area) and the area above the concentration-time curve that is under the MPC (negative area). To assess the predictive performance of AAMPC, the enrichment of resistant Staphylococcus aureus was studied by simulating treatment with daptomycin and rifampicin alone and in combination in an in vitro dynamic model. The enhanced anti-mutant effects of the antibiotic combinations were due to lowering the negative 24-h AAMPCs. These findings suggest that a novel MPC-related parameter is a reliable predictor of mutant enrichment.

To explore the relationship between pharmacokinetic variables and enterococcal resistance to linezolid, a vancomycin-resistant strain whose mutant prevention concentration (MPC) exceeded the MIC by two fold was selected among six clinical isolates of Enterococcus faecium. The selected strain was exposed to simulated pharmacokinetics of twice-daily linezolid for five days. Mutants resistant to 2 × MIC of the antibiotic were enriched at ratios of the 24-h area under the concentration-time curve (AUC24) to the MIC of 15 and 30 h but not at 60 and 120 h. These observations could be explained by the different times when antibiotic concentrations exceed the MPC (T>MPC): 0 to 14, 63 and 100% of the dosing interval. Using the area under the bacterial mutant concentration-time curve (AUBCM) determined in this study and in previous work with other E. faecium strains (MPC/MIC 4), a strain-independent T>MPC relationship with mutant enrichment was established.

To predict the effects of combined use of antibiotics on their pharmacodynamics, the susceptibility of Staphylococcus aureus to linezolid-rifampicin combinations was tested at concentration ratios equal to the ratios of 24-area under the concentration-time curve (AUC (24)) simulated in an in vitro dynamic model. The linezolid MICs in combination with rifampicin decreased 8-to 67-fold. The rifampicin MICs were similar with or without linezolid. The enhanced activity of linezolid combined with rifampicin increased the AUC (24)/MIC ratios and provided more pronounced antibacterial effects compared with single treatments. The areas between the control growth and time-kill curves (ABBCs) determined in combined and single treatments with linezolid were plotted against AUC (24)/MIC on the same graph (r(2) 0.94). These findings suggest that the effects of linezolid-rifampicin combinations can be predicted by AUC 24 / MICs of linezolid using its MIC determined at pharmacokinetically derived linezolidto-rifampicin concentration ratios.

To explore if combinations of linezolid (L) with rifampicin (R) are able to restrict Staphylococcus aureus resistance, the enrichment of L- and R-resistant mutants was studied in an in vitro dynamic model. L- and R-resistant mutants were enriched in all single drug treatments. In contrast, L-resistant mutants were not enriched and R-resistant mutants were similar to baseline amounts with only minimal regrowth at the end of the combination treatments. These effects appear to be explained by lowering the mutant prevention concentration (MPC) for L+R combinations (MPCL+R) compared to the MPCs of L and R alone (MPCL and MPCR) and thereby the longer times above MPCL+R (73-100% of the dosing interval for L and 42-58% for R) compared to the times above MPCL (0-44%) and MPCR (0%). These findings provide an opportunity to predict the selection of S. aureus resistance in L+R treatments using MPCL+Rs.

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 (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 /MPCs from 1 to 10 h (AUC 24 /MIC from 60 to 360 h) but not at the lower or higher AUC 24 /MPC and AUC 24 /MIC ratios. AUC 24 /MPC and AUC 24 /MIC relationships of the areas under the time courses of ciprofloxacin-resistant E. coli (AUBC M ) were bell-shaped. A Gaussian-like function fits the AUBC M -AUC 24 /MPC and AUBC M -AUC 24 /MIC data combined for all organisms ( r 2 = 0.69 and 0.86, respectively). The predicted anti-mutant AUC 24 /MPC ratio was 58 ± 35 h, and the respective AUC 24 /MIC ratio was 1,080 ± 416 h. Although AUC 24 /MPC was less predictive of strain-independent E. coli resistance than AUC 24 /MIC, the established anti-mutant AUC 24 /MPC ratio was closer to values reported for Staphylococcus aureus (60 to 69 h) than the respective AUC 24 /MIC ratio (1,080 versus 200 to 240 h). This implies that AUC 24 /MPC might be a better interspecies predictor of bacterial resistance than AUC 24 /MIC.

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) over tilde (E)) increased with increasing simulated AUC24/MIC ratios. Each of the combined treatments produced greater (I) over tilde (E)s than the sum of linezolid and doxycycline (I) over tilde (E)s observed in the respective single drug treatments.This study suggests that linezolid combinations with doxycycline may be synergistic in treating staphylococcal infections.

The pharmacokinetics of N-(5-oxynicotinoyl)-L-glutamate (ONG) was studied in rats (doses, 20, 100 and 500 mg/kg) and rabbits (50 mg/kg) after bolus administration of calcium salt of N-(5-oxynicotinoyl)-L-glutamic acid (Ampasse preparation). The ONG concentration in the blood serum was determined by HPLC assay with fluorimetric detection. The lower limit of accurate detection for ONG was 100 ng/ml. The ONG pharmacokinetics in rats was linear at relatively small doses (20 - 100 mg/kg) but nonlinear at a large dose (500 mg/kg). The ONG concentration decay had a two-phase character in both rats and rabbits, so that the pharmacokinetic profiles were fitted to a biexponential equation of the two-compartment model. Systemic pharmacokinetic parameters determined in rats and rabbits, respectively, were as follows: total clearance, 18 and 15 ml/(min kg); steady state distribution volume, 330 and 880 ml/kg; mean retention time, 0.3 and 1.0 h; half-life, 0.73 and 2.3 h. Using the allometric approach to the interspecies extrapolation of the pharmacokinetic data, the half-life of ONG in humans is predicted to be 4 h.

The pharmacokinetics of N-(5-oxynicotinoyl)-L-glutamate (ONG) was studied in rats (doses, 20, 100 and 500 mg/kg) and rabbits (50 mg/kg) after bolus administration of calcium salt of N-(5-oxynicotinoyl)-L-glutamic acid (Ampasse preparation). The ONG concentration in the blood serum was determined by HPLC assay with fluorimetric detection. The lower limit of accurate detection for ONG was 100 ng/ml. The ONG pharmacokinetics in rats was linear at relatively small doses (20-100 mg/kg) but nonlinear at a large dose (500 mg/kg). The ONG concentration decay had a two-phase character in both rats and rabbits, so that the pharmacokinetic profiles were fitted to a biexponential equation of the two-compartment model. Systemic pharmacokinetic parameters determined in rats and rabbits, respectively, were as follows: total clearance, 18 and 15 ml/(min kg); steady state distribution volume, 330 and 880 ml/kg; mean retention time, 0.3 and 1.0 h; half-life, 0.73 and 2.3 h. Using the allometric approach to the interspecies extrapolation of the pharmacokinetic data, the half-life of ONG in humans is predicted to be 4 h.

OBJECTIVES To explore whether the duration of in vitro simulated antibiotic exposure influences bacterial resistance, time-dependent amplification of resistant subpopulations of Staphylococcus aureus was studied in 10 day simulations in a dynamic model with daptomycin as a prototypic agent. METHODS S. aureus ATCC 43300 was exposed to once-daily dosing of daptomycin at subtherapeutic ratios of 24 h area under the curve (AUC(24)) to the MIC (32 and 64 h). To provide an integral presentation of the time course of mutants grown on agar plates containing 2x and 4x the MIC of daptomycin, areas under the bacterial mutant kinetic curves (AUBC(M)s) were calculated. RESULTS Daptomycin-resistant S. aureus mutants were enriched gradually over the entire treatment duration, with systematic increases in AUBC(M) and concomitant decreases in susceptibility. AUBC(M) analyses were also applied to resistance data reported from other studies with S. aureus exposed to daptomycin and garenoxacin over a wide range of AUC(24)/MIC ratios. Although the maximal AUBC(M)s were greater with longer than with shorter exposures, the treatment or observation durations did not influence the predicted anti-mutant AUC(24)/MIC ratios. CONCLUSIONS These findings suggest that the duration of in vitro simulated antibiotic exposure is important for estimates of the maximal enrichment of resistant mutants but not for the prediction of the anti-mutant AUC(24)/MIC ratio.

OBJECTIVESTo extend the mutant selection window (MSW) hypothesis to include antibiotics in addition to fluoroquinolones, the pharmacodynamics of daptomycin (DAP) and vancomycin (VAN) and their ability to prevent the selection of resistant Staphylococcus aureus were studied in an in vitro model that simulates antibiotic concentrations below the MIC, between the MIC and the mutant prevention concentration (MPC), and above the MPC.METHODSTwo clinical isolates of S. aureus, S. aureus 866 (MIC(DAP) 0.35, MIC(VAN) 0.7, MPC(DAP) 1.1, MPC(VAN) 2.4 mg/L) and S. aureus 10 (MIC(DAP) 1.1, MIC(VAN) 1.3, MPC(DAP) 5.5, MPC(VAN) 11 mg/L), were exposed for five consecutive days to once-daily daptomycin (half-life 9 h) and twice-daily vancomycin (half-life 6 h) at the ratio of 24 h area under the concentration-time curve (AUC24) to MIC that varied over a 16- to 30-fold range. The cumulative antimicrobial effect was expressed by its intensity (I(E)). Changes in susceptibility and numbers of surviving organisms on agar plates containing 2x and 4x MIC of daptomycin or vancomycin were monitored daily.RESULTSThe I(E)-log AUC24/MIC plots were bacterial strain- and antibiotic-independent. This allowed combination of data obtained with both antibiotics and both organisms. Based on the sigmoid relationship between I(E) and the AUC24/MIC (r2 = 0.9), the antistaphylococcal effect of the therapeutic doses of daptomycin (4 and 6 mg/kg) against a hypothetical S. aureus with MIC equal to the MIC90 (AUC24/MIC90 380 and 570 h, respectively) was predicted to be similar to the effect of two 1 g doses of vancomycin given at a 12 h interval (AUC24/MIC90 200 h). AUC24/MIC relationships of the final-to-initial MIC ratio and logarithm of the ratio of maximal-to-initial numbers of organisms resistant to 2x and 4x MIC of daptomycin or vancomycin were bell-shaped and bacterial strain- and antibiotic-independent. Based on these relationships, an AUC24/MIC ratio that protects against the selection of resistant mutants was predicted at > or = 200 h. This protective value is less than the AUC24/MIC90s provided by the 4 mg/kg dose and considerably less than the 6 mg/kg dose of daptomycin, but it is close to the AUC24/MIC90 provided by two 1 g doses of vancomycin.CONCLUSIONSThese findings support the MSW hypothesis and suggest comparable antistaphylococcal effects of clinically achievable AUC24/MIC90s of daptomycin and vancomycin but slightly better prevention against the selection of resistant S. aureus by daptomycin.

A complete constructive description is given for the groups whose all nonnormal cyclic subgroups generate a nontrivial proper subgroup.

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