Pharmacodynamics of Doripenem Alone and in Combination with Relebactam in an In Vitro Hollow-Fiber Dynamic Model: Emergence of Resistance of Carbapenemase-Producing Klebsiella pneumoniae and the Inoculum Effect.

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.

Experimental Evaluation of the Hypersensitivity Reactions of a New Glycopeptide Antibiotic Flavancin in Animal Models.

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.

Testing the mutant selection window hypothesis with meropenem: In vitro model study with OXA-48-producing Klebsiella pneumoniae.

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.

A novel parameter to predict the effects of antibiotic combinations on the development of Staphylococcus aureus resistance: in vitro model studies at subtherapeutic daptomycin and rifampicin exposures.

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.

Resistance studies with Streptococcus pneumoniae using an in vitro dynamic model: amoxicillin versus azithromycin at clinical exposures.

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.

Concentration-dependent enrichment of resistant Enterococcus faecium exposed to linezolid in an in vitro dynamic model.

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.

Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to linezolid in an in vitro dynamic model.

OBJECTIVES: To test the mutant selection window (MSW) hypothesis applied to linezolid-exposed Staphylococcus aureus and to delineate the concentration-resistance relationship, a mixed inoculum of linezolid-susceptible S. aureus cells and linezolid-resistant mutants (RMs) was exposed to linezolid multiple dosing. METHODS: Three S. aureus strains (MIC of linezolid 2 mg/L), S. aureus 479, S. aureus 688 and S. aureus ATCC 700699, and their RMs (MIC 8 mg/L) selected by passaging on antibiotic-containing media were used in the study. RMs of S. aureus 479 and S. aureus ATCC 700699 contained a G2576T replacement (Escherichia coli numbering) in one of the copies of the 23S rRNA gene, which had been reported in clinically isolated mutants. Five-day treatments with twice-daily linezolid were simulated over a 32-fold range of the 24 h AUC (AUC24) to the MIC ratio. RESULTS: A pronounced enrichment of mutants resistant to 2×, 4× and 8× MIC was observed at AUC24/MIC ratios of 30 and 60 when linezolid concentrations were within the MSW for more than half of the dosing interval for each strain. The number of viable mutant cells decreased significantly at the simulated AUC24/MIC ratio of 120, while the AUC24/MIC ratio of 240 completely prevented mutant enrichment in vitro. Bell-shaped AUBCM-AUC24/MIC and AUBCM-AUC24/MPC relationships (r2 0.91 and 0.79, respectively) were strain independent. CONCLUSIONS: The bell-shaped pattern of AUC24/MIC and AUC24/MPC relationships with S. aureus resistance to linezolid is consistent with the MSW hypothesis. 'Antimutant' AUC24/MIC ratios were predicted based on the AUC24/MIC relationship with AUBCM.

Predicting effects of antibiotic combinations using MICs determined at pharmacokinetically derived concentration ratios: in vitro model studies with linezolid- and rifampicin-exposed Staphylococcus aureus.

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 (AUC24) 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 AUC24/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 AUC24/MIC on the same graph (r2 0.94). These findings suggest that the effects of linezolid-rifampicin combinations can be predicted by AUC24/MICs of linezolid using its MIC determined at pharmacokinetically derived linezolid-to-rifampicin concentration ratios.

Pharmacokinetically-based prediction of the effects of antibiotic combinations on resistant Staphylococcus aureus mutants: in vitro model studies with linezolid and rifampicin.

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.

Bacterial resistance studies using in vitro dynamic models: the predictive power of the mutant prevention and minimum inhibitory antibiotic concentrations.

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 (r(2) = 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.

The impact of duration of antibiotic exposure on bacterial resistance predictions using in vitro dynamic models.

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.