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 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...

Does High-Dose Antimicrobial Chemotherapy Prevent the Evolution of Resistance?

“…If evolutionary theory does not support the use of MPC as a general approach then why does this nevertheless appear to work in some cases [ 34 , 53 ]? The theory presented here provides some possible explanations.…”

Impact on Bacterial Resistance of Therapeutically Nonequivalent Generics: The Case of Piperacillin-Tazobactam

“…A similar approach using mixed bacterial populations to study resistance selection has been employed with E . coli [ 49 ], Streptococcus pneumoniae [ 50 – 52 ], Staphylococcus aureus [ 53 ] and Mycobacterium tuberculosis [ 54 ].…”

Antibiotics can be used to contain drug-resistant bacteria by maintaining sufficiently large sensitive populations

“…In fact, recent work suggests that aggressive treatment strategies can hasten the emergence and spread of resistance [30][31][32][33][34][35][36][37][38][39][40][41]. In addition, numerous studies, both experimental and theoretical, provide evidence that less aggressive treatment strategies may be called for under some conditions [42][43][44][45][46][47][48][49][50], while several clinical trials have also demonstrated advantages of lower dose therapies [50][51][52][53][54][55][56][57][58].…”