Linezolid

Bacterial strains, plasmids, and primers are listed in Supplementary Tables S1–S3. For cloning and genetic manipulation, E. coli was cultivated in LB medium. S. aureus strains with the pRB473-XylR-Brx-roGFP2 plasmids were cultivated in LB medium with 1% xylose to ensure constitutive expression of the biosensor. For stress experiments, S. aureus cells were grown in LB until an optical density at 540 nm (OD₅₄₀) of 1.0 and were transferred to Belitsky minimal medium (BMM) with 1% xylose. The fully reduced control cells were treated with 10 mM DTT and the fully oxidized control was treated with 5 mM diamide for 20 min each, harvested with 10 mM N-ethylmaleimide (NEM) to block the biosensor redox state, and transferred to the microplate wells. The samples for stress exposure were transferred to the microplates, and different oxidants were injected into the wells of microplates. The Brx-roGFP2 biosensor fluorescence emission was measured at 510 nm after excitation at 405 and 488 nm using the CLARIOstar microplate reader (BMG Labtech) as described next for the in vitro measurements. Three biological tests were performed for each stress experiment.
For the survival assays, S. aureus COL wild type and the bshA mutant were treated with NaOCl and H₂O₂ at an OD₅₀₀ of 1.0 in BMM and serial dilutions were plated on LB agar plates and counted for colony-forming units. The survival assays were performed in three biological replicates for each strain.
For the determination of the growth-inhibitory and sub-lethal antibiotics concentrations, S. aureus was cultivated in RPMI medium and the antibiotics erythromycin, rifampicin, vancomycin, ciprofloxacin, gentamicin, ampicillin, fosfomycin, lincomycin, linezolid, or oxacillin were added at an OD₅₀₀ of 0.5 to monitor the reduction in growth as previously described (8 (link)). The measurements of the biosensor responses after antibiotics treatment were performed in S. aureus Brx-roGFP2 cells that were grown in RPMI medium and treated with sub-lethal antibiotics doses that reduced the growth rate. Cells were harvested after different times of antibiotics treatment, washed with phosphate-buffered saline (PBS), and blocked with NEM before the microplate reader measurements. Four biological replicates were performed for each antibiotics stress experiment. Sodium hypochlorite, diamide, DTT, H₂O₂ (35% w/v), and antibiotics (erythromycin, rifampicin, vancomycin, ciprofloxacin, gentamicin, ampicillin, fosfomycin, lincomycin, linezolid, and oxacillin) were purchased from Sigma-Aldrich.

The basic construction of the HFS was similar to the adult HFS model for extracellular tuberculosis, which was recently qualified by the European Medicines Agency and endorsed by the US Food and Drug Administration given its high forecasting accuracy [16 (link)–18 (link)]. We have adapted this to a HFS model of intracellular tuberculosis, by changing the media to RPMI/FBS and inoculating HFS with infected THP-1 cells, as recently described in detail [9 (link)]. We inoculated 20 mL of Mtb-infected THP-1 cells into the peripheral compartment of each of 16 HFSs that had been preconditioned with RPMI/FBS and maintained in incubators at 37°C for at least 72 hours. Linezolid was administered by computer-programmed syringe pumps via an infusion port into the central compartment. A combined exposure-effect and dose-scheduling study was performed wherein 9 of the 16 HFSs were dosed with linezolid to achieve AUC_0–24 exposure of 0, 3, 6,12, 24, 31, 48, 77.5, and 118 mg × hour/L, and the remaining 7 systems were dosed with linezolid twice daily to achieve AUC _0–24 exposures that are twice the once-daily schedule, but at the same peak concentrations. This allows the breaking of the co-linearity of pharmacokinetic/pharmacodynamic (PK/PD) indices that would otherwise occur with dose changes. PK/PD indices under study included the AUC_0–24/MIC ratio, peak concentration to MIC ratio (peak/MIC), and percentage of time concentration persists above MIC (%T_MIC). The duration of therapy was 28 days. Fresh media were pumped into and out of the HFSs at predefined rates to mimic the linezolid half-life of 3 hours encountered in infants [3 (link)]. Concentration-time profiles of linezolid achieved in the HFS were substantiated by sampling the central compartment of each HFS at 7 time points over a 24-hour period post–drug infusion based on optimal sampling theory [19 (link)]. The peripheral compartments were sampled on days 0, 7, 14, 21, and 28 for quantification of the Mtb population and THP-1 cells. Samples were also cultured on agar supplemented with 3 times the linezolid MIC to capture the linezolid-resistant subpopulation at each time point.