Experimental Therapeutics

Linezolid Kills Acid-Phase and Nonreplicative-Persister-Phase Mycobacterium tuberculosis in a Hollow-Fiber Infection Model

G. L. Drusano, Jenny Myrick, Michael Maynard, Jocelyn Nole, Brandon Duncanson, David Brown, Stephan Schmidt, Michael Neely, C. A. Scanga, Charles Peloquin, Arnold Louie
DOI: 10.1128/AAC.00221-18

ABSTRACT

The therapy for treatment of Mycobacterium tuberculosis infections is long and arduous. It has been hypothesized that the therapy duration is driven primarily by populations of organisms in different metabolic states that replicate slowly or not at all (acid-phase and nonreplicative-persister [NRP]-phase organisms). Linezolid is an oxazolidinone antimicrobial with substantial activity against Log-phase M. tuberculosis. Here, we examined organisms in acid-phase growth and nonreplicative-persister-phenotype growth and determined the effect of differing clinically relevant exposures to linezolid in a hollow-fiber infection model (HFIM). The endpoints measured were bacterial kill over 29 days and whether organisms that were less susceptible to linezolid could be recovered during that period. In addition, we evaluated the effect of administration schedule on linezolid activity, contrasting daily administration with administration of twice the daily dose every other day. Linezolid demonstrated robust activity when administered daily against both acid-phase and NRP-phase organisms. We demonstrated a clear dose response, with 900 mg of linezolid daily generating ≥3 Log(CFU/ml) killing of acid-phase and NRP-phase M. tuberculosis over 29 days. Amplification of a population less susceptible to linezolid was not seen. Activity was reduced with every 48-h dosing, indicating that the minimum concentration (Cmin)/MIC ratio drove the microbiological effect. We conclude that once-daily linezolid dosing has substantial activity against M. tuberculosis in acid-phase and NRP-phase metabolic states. Other studies have shown activity against Log-phase M. tuberculosis. Linezolid is a valuable addition to the therapeutic armamentarium for M. tuberculosis and has the potential for substantially shortening therapy duration.

INTRODUCTION

The treatment of Mycobacterium tuberculosis infections is a long process, taking at least 6 months to complete. It has been hypothesized that a part of the explanation for this long duration may be the existence of M. tuberculosis in multiple metabolic states (1). Three of the most commonly recognized metabolic states are the Log-growth phase, the acid-growth phase, and the nonreplicative-persister phenotype. Log-phase M. tuberculosis has a doubling time of 20 to 24 h, while acid-phase organisms have a prolonged doubling time. Nonreplicative-persister (NRP)-phenotype M. tuberculosis turns over very slowly or not at all (2–4). From this point forward, when referring to NRP organisms, we employ the phrase “streptomycin-starved strain 18b” (SS18b). For most anti-infective agents, the rapidity of bacterial cell kill is related to the rate at which the organism replicates (5). Cells replicating more slowly and exposed to most antimicrobials are killed at a lower rate than a more rapidly growing population (6). The prolonged duration of M. tuberculosis therapy required for cure may be driven by the slower killing of these acid-phase and SS18b organisms.

Linezolid (LZD) is an oxazolidinone antimicrobial with activity against M. tuberculosis, especially Log-growth-phase organisms. The outcomes for patients with extensively drug-resistant (XDR) tuberculosis (TB) (7, 8) were promising in a trial where patients on a failing regimen had linezolid added as a single agent. This supports the hypothesis that linezolid may have activity against slower-replicating M. tuberculosis, such as acid-phase and SS18b organisms.

To test this hypothesis, we performed 29-day experiments in our hollow-fiber infection model (HFIM) against strain H37Rv for acid-phase organisms and M. tuberculosis strain 18b (streptomycin auxotroph) for SS18b.

RESULTS

MIC and mutational frequency to resistance.For H37Rv ATCC 27294, the linezolid MIC was 1 mg/liter in both the Log phase and the acid phase. The mutational frequency to resistance was −6.309 Log(CFU). M. tuberculosis strain 18b is a streptomycin-resistant, streptomycin auxotroph that exists in Log-phase growth when incubated with streptomycin but exists in an NRP state when streptomycin starved. When the organism is grown in the presence of streptomycin (Log-phase growth), the linezolid MIC was 1 mg/liter. When it is starved for streptomycin, the MIC value cannot be determined.

Linezolid activity against acid-phase M. tuberculosis H37Rv.The activity of 300, 600, and 900 mg of linezolid administered once daily and of 600, 1,200, and 1,800 mg of linezolid administered every 48 h is shown in Fig. 1. The 900-mg daily dose mediated a 3.93 Log10(CFU/ml) bacterial reduction over the 29-day experiment relative to the no-treatment control. The every-48-h dosing regimen (at twice the daily dose) underperformed the daily dosing regimen. This demonstrates that the minimum concentration (Cmin)/MIC ratio drives acid-phase M. tuberculosis killing for linezolid, as it does for Log-phase organisms (9).

FIG 1
FIG 1

Activity of 3 daily (q24h) doses of linezolid (LIN) and 3 doses of linezolid administered every 48 h (q48h) plus a no-treatment control against acid-phase Mycobacterium tuberculosis (MTB) H37Rv examined in a HFIM over 29 days.

The effect of each LZD regimen on the M. tuberculosis subpopulation with reduced susceptibilities to LZD was assessed by quantitatively culturing bacterial suspensions collected from the HFIM onto agar supplemented with 2.5× MIC of LZD. We isolated 0.29 Log10(CFU)/ml at baseline (time zero). No other, less-susceptible isolates were identified on any other sampling occasion except day 1, where the 300-mg daily regimen had 0.7 Log10(CFU)/ml recovered. The MIC value for the organisms recovered from drug-containing plates was 32 mg/liter in all instances (baseline value, 1 mg/liter).

Mathematical modeling of acid-phase study data.We modeled the system outputs (drug concentration-time profile and total bacterial burden) simultaneously for all regimens using a variant of the model that we had previously described (9). In this variant, we also included a natural-death-rate term (KNat_Death [h−1]) because of the lack of bacterial burden increase over time in the untreated control.

The fit of the model to the data was acceptable when the predicted-observed plots and measures of bias and precision were examined. The pre-Bayesian (population) and Bayesian (individual) regressions are shown for both outputs in Fig. 2. In the pre-Bayesian regression, the mean or median parameter vector is employed to generate the predicted values in the predicted-observed plot, while in the Bayesian analysis, the Bayesian parameter values for each hollow-fiber tube are employed to generate the predicted values. It is expected that the Bayesian estimates would perform better in terms of bias and imprecision than the pre-Bayesian estimates.

FIG 2
FIG 2

Predicted-observed plots for drug concentration and total bacterial burden for Mycobacterium tuberculosis H37Rv. (A and B) Predicted linezolid concentrations (A) and predicted M. tuberculosis colony counts (B), pre-Bayesian regression. (C and D) Predicted linezolid concentrations (C) and predicted M. tuberculosis colony counts (D), Bayesian regression. PK, pharmacokinetic.

The mean and median parameter vectors and their standard deviations are displayed in Table 1. When the Bayesian model parameters were employed for simulation, the bacterial kill attributable to 900 mg of linezolid daily was 3.50 Log10(CFU/ml) relative to the no-treatment control [note that the observed data indicated 3.9 Log10(CFU/ml) bacterial cell kill].

View this table:
TABLE 1

Mean, median, and standard deviation estimates for the parameter values for the effect of linezolid on acid-phase H37Rv

Activity against SS18b M. tuberculosis.The activity of 300, 600, and 900 mg of linezolid administered once daily and 600, 1,200, and 1,800 mg of linezolid administered every 48 h is shown in Fig. 3. The 900-mg daily dose mediated a 3.37 Log10(CFU/ml) kill at day 21, when eradication was achieved. The decline in the no-treatment control was due to the substantial natural death rate for an organism in this metabolic state. It should be noted that the substantial decline in the no-treatment control was greater than had been seen previously in static in vitro experiments (10) but was consistent with previous experiments in the HFIM where the restricted replication rate was induced by Wayne-Hayes level II anaerobiosis (11). The every-48-h dosing regimen (at twice the daily dose) substantially underperformed the daily dosing regimen. This again demonstrates that the Cmin/MIC ratio drives the NRP cell kill for linezolid, as it does for Log-phase M. tuberculosis organisms (9) and for acid-phase organisms (see above).

FIG 3
FIG 3

Activity of 3 daily doses of linezolid and 3 doses of linezolid administered every 48 h against NRP-phenotype Mycobacterium tuberculosis strain 18b (streptomycin starved) plus a no-treatment control examined in a HFIM over 29 days.

With each determination of M. tuberculosis burden, we looked for a less-susceptible subpopulation by plating on linezolid-containing plates at 2.5× MIC. None of these evaluations identified any less-susceptible isolates. This is not surprising, as these organisms were in the NRP state due to streptomycin starvation. Even if there had been resistant isolates, it would have been difficult to find them, given the sampling volume relative to the volume of the total HFIM.

Mathematical modeling of NRP study data.As described above, we modeled the two system outputs simultaneously for all regimens using a variant of the model we had previously described (9) but where we also included a natural-death-rate term (KNat_Death [h−1]) because of the lack of bacterial burden increase over time in the untreated controls.

The fit of the model to the data was acceptable with respect to the predicted-observed plots and measures of bias and precision. The pre-Bayesian (population) and Bayesian (individual) regressions are shown for all outputs in Fig. 4.

FIG 4
FIG 4

Predicted-observed plots for drug concentration and total bacterial burden for Mycobacterium tuberculosis 18b. (A and B) Predicted linezolid concentrations (A) and predicted M. tuberculosis colony counts (B), pre-Bayesian regression. (C and D) Predicted linezolid concentrations (C) and predicted M. tuberculosis colony counts (D), Bayesian regression.

The mean and median parameter vectors and the standard deviations are listed in Table 2. When the Bayesian model parameters were employed for simulation, the bacterial kill attributable to 900 mg of linezolid daily was 3.00 Log10(CFU/ml) relative to the no-treatment control [note that the observed data indicated a 3.37 Log10(CFU/ml) bacterial cell kill].

View this table:
TABLE 2

Mean, median, and standard deviation estimates for the parameter values for the effect of linezolid on M. tuberculosis 18b

DISCUSSION

The duration of therapy for pulmonary M. tuberculosis is 6 months in patients with a fully susceptible pathogen, with the current standard regimen consisting of 2 months of isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) followed by 4 months of isoniazid and rifampin. The duration of the regimen is likely dictated by the burden of a portion of the population, whose members are in different metabolic states, particularly the portion represented by those with slow or virtually no growth. These organisms are more difficult to kill because of their low rate of turnover and minimal metabolic activity. It is of interest that in the standard regimen (HRZE), only rifampin is active against SS18b organisms during the last 4 months of therapy and at a dose which is unlikely to generate maximal kill rates for these organisms (12–13).

Shortening the duration of therapy has been a long-standing goal of many organizations such as NIH, WHO, and the Bill and Melinda Gates Foundation, among others. We hypothesize that finding more anti-M. tuberculosis agents that are active against the slower-growing or nongrowing organisms is of paramount importance in order to achieve this goal.

In previous HFIM studies, we demonstrated that linezolid has good activity against Log-growth-phase M. tuberculosis H37Rv, providing 3 Log CFU/ml of killing at 21 days of treatment relative to time zero (9). However, linezolid monotherapy amplified the less-susceptible M. tuberculosis subpopulation (9).

In this set of experiments, we demonstrated that linezolid has robust activity against both acid-phase organisms and SS18b M. tuberculosis. The acid-phase experiment demonstrated a clear-cut exposure-response relationship when the agent was administered either daily or twice the dosage every 48 h. The latter schedule of administration was employed to determine if intermittent dosing could maintain antimicrobial activity. We had previously demonstrated in Log-phase M. tuberculosis that the platelet toxicity of linezolid as measured in vitro by inhibition of cytochrome c oxidase complex 4 was driven by trough linezolid concentrations (9). Consequently, if microbiological activity could be maintained with intermittent linezolid administration against bacteria in a different metabolic state, the platelet toxicity of linezolid could be ameliorated by the lower trough concentrations associated with alternate-day dosing. Unfortunately, administration on this schedule resulted in a substantial loss of microbiological effect compared to daily dosing, regardless of the total 48-h dose. This is displayed in Table 3 for both acid-phase and SS18b organisms.

View this table:
TABLE 3

Loss of microbiological activity by administration of a linezolid dose daily versus twice the dose every 48 h

The activity seen with administration on a daily basis was promising. Figure 1 demonstrates that the 900-mg daily dose drove a kill of M. tuberculosis of 3.9 Log10(CFU/ml) over the 29 days of the experiment. The 600-mg daily dose drove a 3.3 Log10(CFU/ml) kill. In contrast, Gumbo et al. looked at pyrazinamide in the HFIM over 28 days (14). By observation, using larger doses of pyrazinamide (2-fold to 4-fold the current recommended maximal daily dose), they were able to attain about 1.7 Log10(CFU/ml) bacterial kill beyond stasis. These data were somewhat unusual in that there was substantial no-treatment control growth even at pH of 5.8. Compared to the no-treatment M. tuberculosis burden on day 28, the net kill (no-treatment control burden minus pyrazinamide-exposed burden) was about 3.6 Logs. In either event, linezolid demonstrated acid-phase bacterial killing activity at least as great as that seen with pyrazinamide. As pyrazinamide is credited with playing a major role in the shortening of M. tuberculosis therapy to its current 6-month standard (15–17), the finding of substantial killing activity for linezolid against acid-phase M. tuberculosis is important, particularly so as the prevalence of pyrazinamide resistance has increased over time (18).

Examining the acid-phase M. tuberculosis parameter values (Table 1), it should be noted that the Kgrowth rate constant is almost exactly balanced by the Knat rate constant (the rate of natural death attributed to residence in the acid-phase population). The bacterial kill attributable to linezolid is then proportional to the drug concentration and the Kkill rate constant. This makes plain the reason for our observed exposure response, as this rate constant drove a larger bacterial kill over time as the dose escalated from 300 mg to 600 mg to 900 mg daily, with corresponding increases in concentrations.

It is likely that the next description of a regimen that substantially shortens M. tuberculosis therapy will involve the introduction of a therapeutic agent with additional activity against acid-phase and SS18b M. tuberculosis. Our data indicate that linezolid should be added to the list of agents with substantial activity against NRP M. tuberculosis. Figure 3 demonstrates that the concentration-time profiles of linezolid employing clinically relevant dosages were associated with substantial bacterial kill of SS18b organisms. For 600 mg daily, the observed kill levels relative to the no-treatment control were 1.67 Log10(CFU/ml) on day 21 and 2.29 Log10(CFU/ml) on day 29. When the dose was escalated to 900 mg daily, the observed M. tuberculosis burden was eradicated on day 21. The observed no-treatment control had 3.37 Log10(CFU/ml) at that time point. Calculating the burdens from the Bayesian parameter estimates for the no-treatment control and the 600-mg and 900-mg daily doses, the bacterial kill levels for 600 mg daily were 1.74 Log10(CFU/ml) on day 21 and 2.23 Log10(CFU/ml) on day 29. With dose escalation to 900 mg daily, the modeled bacterial kill levels were 2.79 Log10(CFU/ml) on day 21 and 3.00 Log10(CFU/ml) on day 24.

At both 600 mg and 900 mg given daily, there was substantial bacterial cell kill attributable to linezolid. The levels ranged from 1.7 to 2.25 Log10(CFU/ml) for 600 mg daily (observed versus modeled values). For 900 mg daily, there was 2.79 to 3.37 Log10(CFU/ml) bacterial kill (modeled versus observed values). These values are promising in that as a single agent, linezolid will likely produce multi-Log bacterial kill over a 3-to-4-week period for M. tuberculosis in both the acid phase and the NRP phase. The choice of 600 mg versus 900 mg daily is driven by the patient's condition and the duration of therapy that is anticipated. Patients with lower bone marrow reserves (who are perhaps more prone to thrombocytopenia) may find the 600-mg dose more tolerable. If there is to be an effort at shortening therapy, the 900-mg dose may be preferred, particularly in patients with better overall health.

Linezolid represents an agent with substantial activity against Log-phase organisms (9), acid-phase organisms, and SS18b M. tuberculosis. The immediate issue is how to employ this agent in a combination regimen to suppress resistance emergence while obtaining the maximal bacterial kill to (hopefully) achieve the goal of shortening therapy for M. tuberculosis.

The Log-phase M. tuberculosis population is by far the largest at therapy initiation. These rapidly dividing organisms are the most susceptible to kill by antimicrobials, but they also represent the population with the highest likelihood of generating resistant mutants. Therefore, since the drugs with activity against slowly dividing or nondividing bacteria are fewer than the agents with good Log-phase activity, it may be prudent to employ these agents later in the therapeutic course, where they can be administered in higher doses but for shorter periods of time.

If we choose to employ linezolid at a later time, what would be the optimal drug to pair it with? The standard regimen (HRZE2/HR4) has only rifampin with substantial activity against the slowly growing/nongrowing organisms for the last 4 months of therapy. The hypothesis to be tested would be that the use of a second agent (such as linezolid) with activity against these organisms might increase the rate of death and shorten therapy, as long as the agents are not mutually antagonistic against organisms in these phases. We have shown previously that rifampin and moxifloxacin are mutually antagonistic for SS18b M. tuberculosis (11). This combination was evaluated in the REMOX trial (19). It failed to meet its endpoint of 4 months of therapy. It should be noted that the moxifloxacin-containing regimen was significantly superior to the standard over the first 8 weeks of therapy, suggesting that the failure to meet the endpoint might have been the result of the antagonism for SS18b organisms. The immediate choices that come to mind for a partner to linezolid for the consolidation phase are high-dose rifampin and bedaquiline. We will evaluate these combinations in future work. It should be noted that the NIX-TB trial, which has demonstrated promising results, includes pretomanid, linezolid, and bedaquiline in the regimen (ClinicalTrials.gov registration no. NCT02333799). It should be noted that we had previously evaluated the combination of linezolid plus rifampin in a HFIM (20) and found that the interaction was additive but with a slight tendency to antagonism for Log-phase M. tuberculosis.

Evaluation of linezolid plus rifampin against Log-phase M. tuberculosis also demonstrated that 2-drug therapy is not a guarantee of resistance suppression (20). We had examined a range of linezolid and rifampin exposures (although not in the truly high-dose rifampin range), and a substantial number of the combination arms allowed the emergence of resistance to one or the other agent. This also provides the lesson that if linezolid is to be employed later in therapy, any organisms that have had emergence of resistance to earlier drug combinations in the regimen should be fully susceptible to linezolid and its partner agent (i.e., orthogonal resistance mechanisms).

In conclusion, linezolid's activity against three metabolic states of M. tuberculosis may prove valuable for identifying a regimen to significantly shorten M. tuberculosis therapy duration.

MATERIALS AND METHODS

Bacteria.M. tuberculosis strains H37Rv (ATCC 27294) and 18b (21) (kindly provided by Stewart Cole) were used. Stocks of the bacteria were stored at −80°C. For each experiment, an aliquot of the bacterial stock was inoculated into filter-capped T-flasks containing 7H9 Middlebrook broth that was supplemented with 0.05% Tween 80 and 10% albumin, dextrose, and catalase (ADC). The culture was incubated at 37°C and 5% CO2 on a rocker platform for 4 to 5 days to achieve Log-phase growth. For strain 18b (a streptomycin auxotroph), 50 mg/liter of streptomycin was added to the medium to allow growth. To generate acidic-growth-phase M. tuberculosis, the following procedures were used.

Acidic-growth-phase H37Rv M. tuberculosis.M. tuberculosis in acidic-phase growth was generated by transferring Log-growth-phase M. tuberculosis (propagated in 7H9 broth with 0.05% Tween and 10% ADC) into T-flasks containing 7H9 broth acidified to pH 6.0 with citric acid. After approximately 7 days of incubation at 37°C and 5% CO2 on a rocking platform, the replicating bacteria were transferred to hollow-fiber systems that were preconditioned with prewarmed ADC broth that had been adjusted to pH 6.0 with citric acid. The media in the hollow-fiber systems were continuously replaced with fresh acidified media over the course of the 29-day experiments. The bacteria inoculated into the hollow-fiber systems were incubated at 37°C and 5% CO2, and serial optical density measurements were conducted over time to confirm that the bacteria were replicating in the acidified medium prior to the start of an experiment. The pH of the medium in which the bacteria were incubating and the pH of the fresh acidified medium that was infused into the hollow-fiber systems were measured throughout the experiments.

SS18b strain 18b M. tuberculosis growth.M. tuberculosis 18b in SS18b growth was generated by washing 18b in Log-growth phase (propagated in 7H9 broth with 50 mg/liter of streptomycin, 0.05% Tween, and 10% ADC) 3 times in phosphate-buffered saline (PBS)–0.05% Tween to remove the streptomycin. The washed 18b was resuspended in 7H9 broth without streptomycin and then transferred into T-flasks. After approximately 7 days of incubation at 37°C and 5% CO2 on a rocking platform, the SS18b bacteria were transferred to hollow-fiber systems that had been preconditioned with prewarmed ADC broth.

Drugs.Pharmaceutical-grade linezolid was purchased from CuraScript (Orlando, FL) as a solution for injection and stored at room temperature in the dark. Prior to experimental use, linezolid was diluted to the desired concentrations in sterile deionized water.

Susceptibility testing and mutation frequency determination.Susceptibility studies for linezolid were conducted with Log-growth-phase H37v and 18b M. tuberculosis using the agar proportional method described by the CLSI (21) and the absolute serial dilution method on 7H10 agar–10% oleic acid, albumin, dextrose, and catalase (OADC) (with 50 mg/liter of streptomycin for M. tuberculosis 18b). Briefly, a 104-CFU volume of H37Rv in Log-phase growth was plated on Middlebrook 7H10 agar (Becton Dickinson Microbiology Systems, Sparks, MD) supplemented with 10% OADC (Becton Dickinson Microbiology Systems) containing 2-fold dilutions of linezolid. The cultures were incubated at 37°C and 5% CO2. After 4 weeks of incubation, the MICs were determined by identifying the lowest drug concentration at which there was no bacterial growth on the agar plate. For the agar proportional method, the lowest concentration of a drug that provided a 99% reduction in the bacterial density relative to the no-drug control was read as the MIC. For the absolute serial dilution method, the MIC was read as the lowest concentration of drug for which there was no growth on the agar plate. For susceptibility studies using acidic-phase M. tuberculosis, determinations of MICs for linezolid were conducted on 7H10 agar (without OADC) that had been adjusted to a pH of 6.0 with citric acid.

The mutation frequencies of the H37Rv strain and the 18b strain were evaluated using methods that are described elsewhere (9). Briefly, H37Rv and 18b (streptomycin-supplemented) cultures in Log-phase growth were inoculated onto plates containing Middlebrook 7H10 agar plus 10% Middlebrook OADC with linezolid at a concentration equivalent to 2.5× the MIC. The mutation frequency was identified after 4 weeks of incubation at 37°C and 5% CO2.

HFIM 29-day studies using clinically relevant drug exposures for acidic-growth-phase M. tuberculosis.The goals of the continuous infusion studies were to delineate the time course of killing of M. tuberculosis in acidic-phase growth by linezolid using clinically relevant exposures of this compound and to determine if resistance to linezolid could develop and whether resistance amplification could be suppressed with large exposures. Further, we wished to determine if twice the daily dose administered every other day would generate microbiological activity similar to that seen with daily administration.

The experimental arms for the studies using acid-growth-phase M. tuberculosis H37Rv were as follows: arm A, no-treatment growth control; arms B to D, linezolid administered once daily, with the area under the concentration-time curve (AUC) at the steady state for free drug matching doses of 300 mg, 600 mg, and 900 mg; arms E to G, linezolid administered every 48 h, with the AUC at the steady state for free drug matching doses of 600 mg, 1,200 mg, and 1,800 mg.

The experiment with the seven arms was performed once.

HFIM 29-day studies using clinically relevant drug exposures for nonreplicative-persister-growth-phase M. tuberculosis.As described above, the goals of this experiment were to look at bacterial cell kill, resistance suppression, and the influence of the schedule of administration on linezolid's microbiological activity. The experimental arms were identical to those used in the acid-growth-phase study but employed the 18b strain in SS18b.

The experiment with seven arms was performed once.

Pharmacokinetic and protein binding data.Human pharmacokinetic parameters and protein binding values for linezolid were obtained from the Zyvox package insert (22).

Detection of resistance amplification.Serial bacterial specimens were collected from the hollow-fiber system arms over the course of the 29-day experiments. The samples were washed and then quantitatively plated onto antibiotic-free agar and antibiotic-supplemented agar to characterize the effect of each treatment regimen on the total bacterial and less-susceptible bacterial populations. A volume of 200 μl was removed from the peripheral compartment and was streaked onto the zero-dilution plate. Another 100-μl volume was used to perform serial 10-fold dilutions to obtain accurate countable numbers. This was done both for antibiotic-free plates (total bacterial burden) and antibiotic-containing plates (linezolid-resistant organisms).

For detection of mutants resistant to linezolid, the agar (pH 7.0 [for SS18b experiments]) was supplemented with 10% OADC. For the acid-phase experiments, the pH of the agar was adjusted (pH 6.0). The agar plates were read after 6 weeks of incubation at 37°C in a 5% CO2 atmosphere. Linezolid concentrations incorporated into the agar were 2.5× baseline MIC value.

Achievement of target exposure profiles.Serial samples of media were collected from the hollow-fiber treatment arms for assay of drug content by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by a previously published technique (9) to confirm that the targeted concentration-time profiles were achieved.

Population pharmacokinetic/pharmacodynamic mathematical model.We simultaneously modeled 3 system outputs for the analysis of the acid-phase and SS18b M. tuberculosis data. These were performed as separate analyses. The system outputs were as follows: concentration of linezolid, total M. tuberculosis burden, and burden of M. tuberculosis that was less susceptible to linezolid. Population modeling was performed employing the Non-Parametric Adaptive Grid (NPAG) program of Leary et al. (23). Modeling choices (weighting, etc.) and goodness-of-fit evaluations were performed as previously published (9). Simulation was performed using the ADAPT V Program of D'Argenio et al. (24) using Bayesian posterior parameter estimates.

ACKNOWLEDGMENTS

This work was supported by P01AI123036 from NIAID.

The content is solely our responsibility and does not necessarily represent the official views of the National Institutes of Health.

FOOTNOTES

    • Received 2 February 2018.
    • Returned for modification 6 March 2018.
    • Accepted 29 May 2018.
    • Accepted manuscript posted online 4 June 2018.

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KEYWORDS

acid phase
hollow-fiber infection model
linezolid
nonreplicative-persister phase

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