Background In the treatment of tuberculosis, ethambutol is used in case there is isoniazid resistance. We examined for the emergence of drug resistance to ethambutol monotherapy in pharmacokinetic-pharmacodynamic studies in the hollow fiber system. Methods Dose–effect studies and dose-scheduling studies were carried out with ethambutol and log-phase growth Mycobacterium tuberculosis to identify exposures and schedules linked to optimal kill and resistance suppression. In one study, after 7 days of ethambutol 300 mg/day isoniazid was administered to each system to determine its early bactericidal activity (EBA). Results Efflux-pump blockage reduced the mutation frequency to ethambutol 64-fold. In dose-effect studies, ethambutol had a maximal EBA of 0.22 log10 CFU/ml/day, as encountered in patients. By day 7, resistance had arisen to both ethambutol and isoniazid. Prior exposure to ethambutol abolished isoniazid EBA. Daily therapy, as opposed to more intermittent therapy, was associated with the least proportion of efflux-pump driven resistance, consistent with time driven effect. Microbial kill was best explained by the ratio of area under the concentration-time curve to minimal inhibitory effect (r2=0.90). Conclusion Induction of an efflux-pump that reduces effect to multiple drugs provides an alternative pathway to sequential acquisition of mutations in the development of multiple drug resistance.

Linezolid has an excellent sterilizing effect in tuberculosis patients but high adverse event rates. The dose that would maximize efficacy and minimize toxicity is unknown. We performed linezolid dose-effect and dose-scheduling studies in the hollow fiber system model of tuberculosis (HFS-TB) for sterilizing effect. HFS-TB units were treated with several doses to mimic human-like linezolid intrapulmonary pharmacokinetics and repetitively sampled for drug concentration, total bacterial burden, linezolid-resistant subpopulations, and RNA sequencing over 2 months. Linezolid-resistant isolates underwent whole-genome sequencing. The expression of genes encoding efflux pumps in the first 1 to 2 weeks revealed the same exposure-response patterns as the linezolid-resistant subpopulation. Linezolid-resistant isolates from the 2nd month of therapy revealed mutations in several efflux pump/transporter genes and a LuxR-family transcriptional regulator. Linezolid sterilizing effect was linked to the ratio of unbound 0- to 24-h area under the concentration-time curve (AUC0–24) to MIC. Optimal microbial kill was achieved at an AUC0–24/MIC ratio of 119. The optimal sterilizing effect dose for clinical use was identified using Monte Carlo simulations. Clinical doses of 300 and 600 mg/day (or double the dose every other day) achieved this target in 87% and >99% of 10,000 patients, respectively. The susceptibility breakpoint identified was 2 mg/liter. The simulations identified that a 300-mg/day dose did not achieve AUC0–24s associated with linezolid toxicity, while 600 mg/day achieved those AUC0–24s in <20% of subjects. The linezolid dose of 300 mg/day performed well and should be compared to 600 mg/day or 1,200 mg every other day in clinical trials.

Treatment of disseminated tuberculosis in children ≤ 6 years has not been optimized. The pyrazinamide-containing combination regimen used to treat disseminated tuberculosis in babies and toddlers was extrapolated from adult pulmonary tuberculosis. Due to hepatotoxicity worries, there are no dose–response studies in children. We designed a hollow fiber system model of disseminated intracellular tuberculosis with co-perfused three-dimensional organotypic liver modules to simultaneously test for efficacy and toxicity. We utilized pediatric pharmacokinetics of pyrazinamide and acetaminophen to determine dose-dependent pyrazinamide efficacy and hepatotoxicity. Acetaminophen concentrations that cause hepatotoxicity in children led to elevated liver function tests, while 100 mg/kg pyrazinamide did not. Surprisingly, pyrazinamide did not kill intracellular Mycobacterium tuberculosis up to fourfold the standard dose as monotherapy or as combination therapy, despite achieving high intracellular concentrations. Host-pathogen RNA-sequencing revealed lack of a pyrazinamide exposure transcript signature in intracellular bacteria or of phagolysosome acidification on pH imaging. Artificial intelligence algorithms confirmed that pyrazinamide was not predictive of good clinical outcomes in children ≤ 6 years who had extrapulmonary tuberculosis. Thus, adding a drug that works inside macrophages could benefit children with disseminated tuberculosis. Our in vitro model can be used to identify such new regimens that could accelerate cure while minimizing toxicity.

fThe treatment of pulmonary Mycobacterium abscessus disease is associated with very high failure rates and easily acquired drug resistance. Amikacin is the key drug in treatment regimens, but the optimal doses are unknown. No good preclinical model exists to perform formal pharmacokinetics/pharmacodynamics experiments to determine these optimal doses. We developed a hollow-fiber system model of M. abscessus disease and studied amikacin exposure effects and dose scheduling. We mimicked amikacin human pulmonary pharmacokinetics. Both amikacin microbial kill and acquired drug resistance were linked to the peak concentration-to-MIC ratios; the peak/MIC ratio associated with 80% of maximal kill (EC 80 ) was 3.20. However, on the day of the most extensive microbial kill, the bacillary burden did not fall below the starting inoculum. We performed Monte Carlo simulations of 10,000 patients with pulmonary M. abscessus infection and examined the probability that patients treated with one of 6 doses from 750 mg to 4,000 mg would achieve or exceed the EC 80 . We also examined these doses for the ability to achieve a cumulative area under the concentration-time curve of 82,232 mg · h/liter ؋ days, which is associated with ototoxicity. The standard amikacin doses of 750 to 1,500 mg a day achieved the EC 80 in <21% of the patients, while a dose of 4 g/day achieved this in 70% of the patients but at the cost of high rates of ototoxicity within a month or two. The susceptibility breakpoint was an MIC of 8 to 16 mg/liter. Thus, amikacin, as currently dosed, has limited efficacy against M. abscessus. It is urgent that different antibiotics be tested using our preclinical model and new regimens developed. Mycobacterium abscessus is a rapidly growing mycobacterium (RGM) responsible for about 80% of all pulmonary infections caused by RGM (1). It is one of the most drug-resistant microorganisms encountered in the clinic, far worse than extensively and totally drug-resistant Mycobacterium tuberculosis; this is a reason why it is considered the "new antibiotic nightmare" (2). The current treatment for M. abscessus diseases varies according to the infecting subspecies (3); in general, it involves a backbone of amikacin in combination with clarithromycin and either cefoxitin or imipenem early during therapy, followed by subsequent use of oral antibiotics, which is analogous to the initial and continuation phases of tuberculosis treatment (1). Unfortunately, at least half of the patients either fail this therapy, relapse, or die; there is no reliable antibiotic regimen that cures M. abscessus lung disease (1, 4). Several other regimens have been tried and found wanting. In such regimens and the standard regimen, the doses were chosen based on what has worked well in mundane Gramnegative bacilli. No formal antimicrobial pharmacokinetic/pharmacodynamic (PK/PD) analyses have been performed with M. abscessus. Our time-kill assays in the past demonstrated that amikacin was the most active antibiotic compared to cefoxitin and clarithromycin (5)....

Multidrug resistant-tuberculosis is a pressing problem. One of the major mechanisms proposed to lead to the emergence of drug resistance is pharmacokinetic mismatch. Stated as a falsifiable hypothesis, the greater the pharmacokinetic mismatch between rifampin and isoniazid, the higher the isoniazid-and rifampinresistant subpopulation sizes become with time. To test this, we performed hollow-fiber-system studies for both bactericidal and sterilizing effects in experiments of up to 42 days. We mimicked pharmacokinetics of 600-mg/day rifampin and 300-mg/day isoniazid administered to patients. Rifampin was administered first, followed by isoniazid 0, 6, 12, and 24 h later. The treatment was for drug-susceptible Mycobacterium tuberculosis in some experiments and hollow fiber systems with inoculum preseeded with isoniazid-and rifampin-resistant isogenic Mycobacterium tuberculosis strains in others. Analysis of variance revealed that the 12-h and 24-h-mismatched regimens always killed better than the matched regimens during both bactericidal and sterilizing effects (P < 0.05). This means that either the order of scheduling or the sequential administration of drugs in combination therapy may lead to significant improvement in microbial killing. Rifampin-resistant and isoniazid-resistant subpopulations were not significantly higher with increased mismatching in numerous analysis-of-variance comparisons. Thus, the pharmacokinetic mismatch hypothesis was rejected. Instead, sequential administration of anti-tuberculosis (TB) drugs (i.e., deliberate mismatch) following particular schedules suggests a new paradigm for accelerating M. tuberculosis killing. We conclude that current efforts aimed at better pharmacokinetic matching to decrease M. tuberculosis resistance emergence are likely futile and counterproductive.In the early days of chemotherapy, monotherapy was administered for the treatment of tuberculosis (TB). This practice led to the rapid emergence of drug resistance (2, 24, 30). Combination therapy regimens that could suppress drug resistance were therefore developed and are now the standard of care. Short-course treatment regimens of isoniazid, rifampin, pyrazinamide, and ethambutol are highly effective for the treatment of drug-susceptible TB (6,23,27). Despite these advances, however, a significant upsurge in drug resistance in Mycobacterium tuberculosis has been reported (28), with the most consequential being resistance to isoniazid and rifampin. Simultaneous resistance to both isoniazid and rifampin defines multidrug-resistant TB. Risk factors for emergence of drugresistant M. tuberculosis include high bacillary burden, cavitation, and immunodeficiency (5, 9, 18, 25, 26). Several mechanisms for the emergence of drug resistance have been proposed, with one of the most important being pharmacokinetic mismatching (18,23).Pharmacokinetic mismatching is encountered in combination therapy including drugs with a long half-life and postantibiotic effect as well as some with a short half-life (18, 23). The drugs with a...