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.

Comparative Meropenem Pharmacodynamics and Emergence of Resistance against Carbapenem-Susceptible Non-Carbapenemase-Producing and Carbapenemase-Producing Enterobacterales: A Pharmacodynamic Study in a Hollow-Fiber Infection Model.

Resistance to carbapenems has become a problem due to Klebsiella pneumoniae (K. pneumoniae), harboring carbapenemases. Among them, there are isolates that are recognized as carbapenem-susceptible; however, these carbapenemase-producing strains with low meropenem minimal inhibitory concentrations (MICs) may pose a threat to public health. We aimed to investigate the impact of the ability to produce carbapenemases by a bacterial isolate on the effectiveness of meropenem in the hollow-fiber infection model. K. pneumoniae and Escherichia coli (E. coli) strains with equal meropenem MICs but differing in their ability to produce carbapenemases were used in pharmacodynamic simulations with meropenem. In addition to standard MIC determination, we assessed the MICs against tested strains at high inoculum density to test if the inoculum effect occurs. According to pharmacodynamic data, the carbapenemase-producing strains were characterized with a relatively decreased meropenem effectiveness compared to non-producers. Meanwhile, the effect of meropenem perfectly correlated with the meropenem exposure expressed as the DOSE/MIC ratio when high-inoculum (HI) MICs but not standard-inoculum (SI) MICs were used for regression analysis. It could be concluded that meropenem-susceptible carbapenemase-producing strains may not respond to meropenem therapy; the antibiotic inoculum effect (IE) may have a prognostic value to reveal the meropenem-susceptible Enterobacterales that harbor carbapenemase genes.

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.

Fluorescence Microscopy: Determination of Meropenem Activity against Klebsiella pneumoniae.

The development and implementation of diagnostic methods that allow rapid assessment of antibiotic activity against pathogenic microorganisms is an important step towards antibiotic therapy optimization and increase in the likelihood of successful treatment outcome. To determine whether fluorescence microscopy with acridine orange can be used for rapid assessment (≤8 h) of the meropenem activity against Klebsiella pneumoniae, six isolates including three OXA-48-carbapenemase-producers were exposed to meropenem at different levels of its concentration (0.5 × MIC, 1 × MIC, 8 or 16 µg/mL) and the changes in the viable counts within 24 h were evaluated using fluorescence microscopy and a control culture method. The approach was to capture the regrowth of bacteria as early as possible. Within the first 8 h fluorescence microscopy allowed to categorize 5 out of 6 K. pneumoniae strains by their meropenem susceptibility (based on the MIC breakpoint of 8 mg/L), but meropenem activity against three isolates, two of which were OXA-48-producers, could not be accurately determined at 8 h. The method proposed in our study requires improvement in terms of accelerating the bacterial growth and regrowth for early meropenem MIC determination. Volume-dependent elevation in meropenem MICs against OXA-48-producers was found and this phenomenon should be studied further.

Meropenem MICs at Standard and High Inocula and Mutant Prevention Concentration Inter-Relations: Comparative Study with Non-Carbapenemase-Producing and OXA-48-, KPC- and NDM-Producing Klebsiella pneumoniae.

The minimal inhibitory concentration (MIC) is conventionally used to define in vitro levels of susceptibility or resistance of a specific bacterial strain to an antibiotic and to predict its clinical efficacy. Along with MIC, other measures of bacteria resistance exist: the MIC determined at high bacterial inocula (MICHI) that allow the estimation of the occurrence of inoculum effect (IE) and the mutant prevention concentration, MPC. Together, MIC, MICHI and MPC represent the bacterial "resistance profile". In this paper, we provide a comprehensive analysis of such profiles of K. pneumoniae strains that differ by meropenem susceptibility, ability to produce carbapenemases and specific carbapenemase types. In addition, we have analyzed inter-relations between the MIC, MICHI and MPC for each tested K. pneumoniae strain. Low IE probability was detected with carbapenemase-non-producing K. pneumoniae, and high IE probability was detected with those that were carbapenemase-producing. MICs did not correlate with the MPCs; significant correlation was observed between the MICHIs and the MPCs, indicating that these bacteria/antibiotic characteristics display similar resistance properties of a given bacterial strain. To determine the possible resistance-related risk due to a given K. pneumoniae strain, we propose determining the MICHI. This can more or less predict the MPC value of the particular strain.

Klebsiella pneumoniae Susceptibility to Carbapenem/Relebactam Combinations: Influence of Inoculum Density and Carbapenem-to-Inhibitor Concentration Ratio.

The inoculum effect (IE) is a well-known phenomenon with beta-lactams. At the same time, the IE has not been extensively studied with carbapenem/carbapenemase inhibitor combinations. The antibiotic-to-inhibitor concentration ratio used in susceptibility testing can influence the in vitro activity of the combination. To explore the role of these factors, imipenem/relebactam and doripenem/relebactam MICs were estimated against six Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae strains at standard inocula (SI) and high inocula (HI) by two methods: with a fixed relebactam concentration and with a fixed, pharmacokinetic-based carbapenem-to-relebactam concentration ratio. The combination MICs at HI, compared to SI, increased with most of the tested strains. However, the IE occurred with only two K. pneumoniae strains regardless of the MIC testing method. The relationship between the MICs at SI and the respective inoculum-induced MIC changes was observed when the MICs were estimated at pharmacokinetic-based carbapenem-to-relebactam concentration ratios. Thus, (1) IE was observed with both carbapenem/relebactam combinations regardless of the MIC testing method; however, IE was not observed frequently among tested K. pneumoniae strains. (2) At HI, carbapenem/relebactam combination MICs increased to levels associated with carbapenem resistance. (3) Combination MICs determined at pharmacokinetic-based carbapenem-to-inhibitor concentration ratios predict susceptibility elevations at HI in KPC-producing K. pneumoniae.

Predicting the Effects of Carbapenem/Carbapenemase Inhibitor Combinations against KPC-Producing Klebsiella pneumoniae in Time-Kill Experiments: Alternative versus Traditional Approaches to MIC Determination.

Traditionally, the antibacterial activity of ß-lactam antibiotics in the presence of ß-lactamase inhibitors is determined at the fixed inhibitor concentration. This traditional approach does not consider the ratio of antibiotic-to-inhibitor concentrations achieved in humans. To explore whether an alternative pharmacokinetically based approach to estimate MICs in combinations is predictive of antimicrobial efficacy, the effects of imipenem and doripenem alone and in combination with relebactam were studied in time-kill experiments against carbapenemase-producing Klebsiella pneumoniae. The carbapenem-to-relebactam concentration ratios in time-kill assays were equal to the therapeutic 24-h area under the concentration-time curve (AUC) ratios of the drugs (1.5/1). The simulated levels of carbapenem and relebactam were equal to their concentrations achieved in humans. When effects of combined regimens were plotted against respective C/MICs, a sigmoid relationship was obtained only with MICs determined by pharmacokinetically based method. The effectiveness of both carbapenems in the presence of relebactam was comparable by the results of time-kill experiments. These findings suggest that (1) antibiotic/inhibitor MICs determined at a pharmacokinetically based concentration ratio allow an adequate assessment of carbapenem susceptibility in carbapenemase-producing K. pneumoniae strains and can be used to predict antibacterial effects; (2) in time-kill experiments, the effects of imipenem and doripenem in the presence of relebactam are comparable.

MPC-Based Prediction of Anti-Mutant Effectiveness of Antibiotic Combinations: In Vitro Model Study with Daptomycin and Gentamicin against Staphylococcus aureus.

To explore whether combined treatments with daptomycin and gentamicin can prevent the development of Staphylococcus aureus resistance, and whether the possible restriction is associated with changes in antibiotic mutant prevention concentrations (MPCs), the enrichment of daptomycin- and gentamicin-resistant mutants was studied by simulating 5-day single and combined treatments in an in vitro dynamic model. The MPCs of the antibiotics in the combination were determined at concentration ratios equal to the ratios of 24 h areas, under the concentration-time curve (AUCs) of the antibiotics, as simulated in pharmacodynamic experiments. The MPCs of both daptomycin and gentamicin decreased in the presence of each other; this led to an increase in the time when antibiotic concentrations were above the MPC (T>MPC). The increases in T>MPCs were concurrent with increases of the anti-mutant effects of the combined antibiotics. When anti-mutant effects of the antibiotics in single and combined treatments were plotted against the T>MPCs, significant sigmoid relationships were obtained. These findings suggest that (1) daptomycin-gentamicin combinations prevent the development of S. aureus resistance to each antibiotic; (2) the anti-mutant effects of antibiotic combinations can be predicted using MPCs determined at pharmacokinetic-based antibiotic concentration ratios; (3) T>MPC is a reliable predictor of the anti-mutant efficacy of antibiotic combinations.

Anti-mutant efficacy of antibiotic combinations: in vitro model studies with linezolid and daptomycin.

OBJECTIVES: To explore whether linezolid/daptomycin combinations can restrict Staphylococcus aureus resistance and if this restriction is associated with changes in the mutant prevention concentrations (MPCs) of the antibiotics in combination, the enrichment of resistant mutants was studied in an in vitro dynamic model. METHODS: Two MRSA strains, vancomycin-intermediate resistant ATCC 700699 and vancomycin-susceptible 2061 (both susceptible to linezolid and daptomycin), and their linezolid-resistant mutants selected by passaging on antibiotic-containing medium were used in the study. MPCs of antibiotics in combination were determined at a linezolid-to-daptomycin concentration ratio (1:2) that corresponds to the ratio of 24 h AUCs (AUC24s) actually used in the pharmacokinetic simulations. Each S. aureus strain was supplemented with respective linezolid-resistant mutants (mutation frequency 10-8) and treated with twice-daily linezolid and once-daily daptomycin, alone and in combination, simulated at therapeutic and sub-therapeutic AUC24s. RESULTS: Numbers of linezolid-resistant mutants increased at therapeutic and sub-therapeutic AUC24s, whereas daptomycin-resistant mutants were enriched only at sub-therapeutic AUC24 in single drug treatments. Linezolid/daptomycin combinations prevented the enrichment of linezolid-resistant S. aureus and restricted the enrichment of daptomycin-resistant mutants. The pronounced anti-mutant effects of the combinations were attributed to lengthening the time above MPC of both linezolid and daptomycin as their MPCs were lowered. CONCLUSIONS: The present study suggests that (i) the inhibition of S. aureus resistant mutants using linezolid/daptomycin combinations can be predicted by MPCs determined at pharmacokinetically derived antibiotic concentration ratios and (ii) T>MPC is a reliable predictor of the anti-mutant efficacy of antibiotic combinations as studied using in vitro dynamic models.

Verification of a Novel Approach to Predicting Effects of Antibiotic Combinations: In Vitro Dynamic Model Study with Daptomycin and Gentamicin against Staphylococcus aureus.

To explore whether susceptibility testing with antibiotic combinations at pharmacokinetically derived concentration ratios is predictive of the antimicrobial effect, a Staphylococcus aureus strain was exposed to daptomycin and gentamicin alone or in combination in multiple dosing experiments. The susceptibility of the S. aureus strain to daptomycin and gentamicin in combination was tested at concentration ratios equal to the ratios of 24 h areas under the concentration-time curve (AUC24s) of antibiotics simulated in an in vitro dynamic model in five-day treatments. The MICs of daptomycin and gentamicin decreased in the presence of each other; this led to an increase in the antibiotic AUC24/MIC ratios and the antibacterial effects. Effects of single and combined treatments were plotted against the AUC24/MIC ratios of daptomycin or gentamicin, and a significant sigmoid relationship was obtained. Similarly, when the effects of single and combined treatments were related to the total exposure of both drugs (the sum of AUC24/MIC ratios (∑AUC24/MIC)), a significant sigmoid relationship was obtained. These findings suggest that (1) the effects of antibiotic combinations can be predicted by AUC24/MICs using MICs of each antibacterial determined at pharmacokinetically derived concentration ratios; (2) ∑AUC24/MIC is a reliable predictor of the antibacterial effects of antibiotic combinations.

Predicting the antistaphylococcal effects of daptomycin-rifampicin combinations in an in vitro dynamic model.

To predict the effects of combined use of antibiotics on their pharmacodynamics, the susceptibility of Staphylococcus aureus to daptomycin-rifampicin combinations was tested at concentration ratios equal to the ratios of daptomycin and rifampicin 24-h areas under the concentration-time curve (AUC24s) simulated in an in vitro dynamic model. In combination with rifampicin, daptomycin MICs decreased 2- to 31-fold, whereas rifampicin MICs were similar with or without daptomycin. The enhanced susceptibility of S. aureus to daptomycin combined with rifampicin resulted in both an increase of the actual AUC24/MIC ratios and also more pronounced antibacterial effects compared with single treatments. The areas between the control growth and time-kill curves (ABBCs) determined in combined and single daptomycin treatments were plotted against AUC24/MIC on the same graph (r2 0.90). These findings suggest that the effects of daptomycin-rifampicin combinations can be predicted by AUC24/MICs of daptomycin using its MIC determined at pharmacokinetically derived daptomycin-to-rifampicin concentration ratios.

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.

Predicting antibiotic combination effects on the selection of resistant Staphylococcus aureus: In vitro model studies with linezolid and gentamicin.

To explore whether combinations of linezolid with gentamicin restrict Staphylococcus aureus resistance, the enrichment of resistant mutants was studied in an in vitro dynamic model. A clinical isolate S. aureus 10 and its linezolid-resistant mutant selected by passaging on antibiotic-containing media were used in the study. The minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC) of antibiotics in combination were determined at a linezolid-to-gentamicin concentration ratio corresponding to the ratio of 24-h areas under the concentration-time curve (AUC24s) used in the pharmacokinetic simulations. Five-day treatments of S. aureus 10 supplemented with linezolid-resistant mutants (mutation frequency 10-8) with twice-daily linezolid and once-daily gentamicin, alone and in combination, were simulated at therapeutic and subtherapeutic AUC24s. Numbers of linezolid-resistant mutants increased at both therapeutic and subtherapeutic AUC24s, whereas gentamicin-resistant mutants were enriched only at the subtherapeutic AUC24 in single drug treatments. Linezolid-gentamicin combinations prevented the enrichment of linezolid-resistant S. aureus and restricted the enrichment of gentamicin-resistant mutants. The pronounced anti-mutant effect of the combinations was attributed to lengthening the time above MPC of both linezolid and gentamicin as their MPCs were lowered. Unlike resistant S. aureus, killing of the total bacterial burden exposed to linezolid-gentamicin combinations was less than in treatments with gentamicin alone, but greater than with linezolid alone. These findings indicate that (1) the anti-mutant effects of antibiotic combinations can be predicted by MPC determinations at pharmacokinetically-derived concentration ratios, and (2) a given antibiotic combination may be optimal against resistant but not susceptible subpopulations.

Time inside the mutant selection window as a predictor of staphylococcal resistance to linezolid.

To explore if the time inside the mutant selection window (TMSW) 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-1) and their previously selected resistant mutants (MIC 8 µg ml-1) 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 (AUC24) to the MIC ratio, mutants resistant to 4 × MIC of antibiotic were enriched in a TMSW-dependent manner. With each strain, TMSW relationships with the area under the bacterial mutant concentration-time curve (AUBCM) exhibited a hysteresis loop, with the upper portion corresponding to the time above the mutant prevention concentration (MPC; T>MPC) of 0 and the lower portion-to the T>MPC > 0. Using AUBCM related to the maximal value observed with a given strain (normalized AUBCM) at T>MPC > 0, a strain-independent sigmoid relationship was established between AUBCM and TMSW, as well as T>MPC (r2 0.99 for both). AUC24/MIC and AUC24/MPC relationships with normalized AUBCM for combined data on the three studied S. aureus strains were bell-shaped (r2 0.85 and 0.80, respectively). These findings suggest that TMSW at T>MPC > 0, T>MPC, AUC24/MIC and AUC24/MPC are useful bacterial strain-independent predictors of the emergence of staphylococcal resistance to linezolid.

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.

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.

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 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. 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.