Common Variants in the Glycerol Kinase Gene Reduce Tuberculosis Drug Efficacy

Despite the administration of multiple drugs that are highly effective in vitro, tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify Mycobacterium tuberculosis genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the glpK gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of M. tuberculosis to antibiotics in vitro, and glpK-deficient bacteria persisted during antibiotic treatment in vivo, particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the glpK gene were found to be a specific marker of multidrug resistance in clinical M. tuberculosis isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the glpK coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance. IMPORTANCE: TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for M. tuberculosis mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the glpK gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance

Tuberculosis drugs' distribution and emergence of resistance in patient's lung lesions: A mechanistic model and tool for regimen and dose optimization.

BackgroundThe sites of mycobacterial infection in the lungs of tuberculosis (TB) patients have complex structures and poor vascularization, which obstructs drug distribution to these hard-to-reach and hard-to-treat disease sites, further leading to suboptimal drug concentrations, resulting in compromised TB treatment response and resistance development. Quantifying lesion-specific drug uptake and pharmacokinetics (PKs) in TB patients is necessary to optimize treatment regimens at all infection sites, to identify patients at risk, to improve existing regimens, and to advance development of novel regimens. Using drug-level data in plasma and from 9 distinct pulmonary lesion types (vascular, avascular, and mixed) obtained from 15 hard-to-treat TB patients who failed TB treatments and therefore underwent lung resection surgery, we quantified the distribution and the penetration of 7 major TB drugs at these sites, and we provide novel tools for treatment optimization.Methods and findingsA total of 329 plasma- and 1,362 tissue-specific drug concentrations from 9 distinct lung lesion types were obtained according to optimal PK sampling schema from 15 patients (10 men, 5 women, aged 23 to 58) undergoing lung resection surgery (clinical study NCT00816426 performed in South Korea between 9 June 2010 and 24 June 2014). Seven major TB drugs (rifampin [RIF], isoniazid [INH], linezolid [LZD], moxifloxacin [MFX], clofazimine [CFZ], pyrazinamide [PZA], and kanamycin [KAN]) were quantified. We developed and evaluated a site-of-action mechanistic PK model using nonlinear mixed effects methodology. We quantified population- and patient-specific lesion/plasma ratios (RPLs), dynamics, and variability of drug uptake into each lesion for each drug. CFZ and MFX had higher drug exposures in lesions compared to plasma (median RPL 2.37, range across lesions 1.26-22.03); RIF, PZA, and LZD showed moderate yet suboptimal lesion penetration (median RPL 0.61, range 0.21-2.4), while INH and KAN showed poor tissue penetration (median RPL 0.4, range 0.03-0.73). Stochastic PK/pharmacodynamic (PD) simulations were carried out to evaluate current regimen combinations and dosing guidelines in distinct patient strata. Patients receiving standard doses of RIF and INH, who are of the lower range of exposure distribution, spent substantial periods (>12 h/d) below effective concentrations in hard-to-treat lesions, such as caseous lesions and cavities. Standard doses of INH (300 mg) and KAN (1,000 mg) did not reach therapeutic thresholds in most lesions for a majority of the population. Drugs and doses that did reach target exposure in most subjects include 400 mg MFX and 100 mg CFZ. Patients with cavitary lesions, irrespective of drug choice, have an increased likelihood of subtherapeutic concentrations, leading to a higher risk of resistance acquisition while on treatment. A limitation of this study was the small sample size of 15 patients, performed in a unique study population of TB patients who failed treatment and underwent lung resection surgery. These results still need further exploration and validation in larger and more diverse cohorts.ConclusionsOur results suggest that the ability to reach and maintain therapeutic concentrations is both lesion and drug specific, indicating that stratifying patients based on disease extent, lesion types, and individual drug-susceptibility profiles may eventually be useful for guiding the selection of patient-tailored drug regimens and may lead to improved TB treatment outcomes. We provide a web-based tool to further explore this model and results at http://saviclab.org/tb-lesion/

Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis

Background. Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. Methods. In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. Results. Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. Conclusions. The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea

Polymorphisms Associated with Resistance and Cross-Resistance to Aminoglycosides and Capreomycin in Mycobacterium tuberculosis Isolates from South Korean Patients with Drug-Resistant Tuberculosis ▿

The aminoglycosides streptomycin, amikacin, and kanamycin and the cyclic polypeptide capreomycin are all widely used in second-line therapy for patients who develop multidrug-resistant tuberculosis. We have characterized a set of 106 clinical isolates of Mycobacterium tuberculosis using phenotypic drug susceptibility testing (DST) to determine the extent of resistance to each agent and cross-resistance between agents. These results were compared with polymorphisms in the DNA sequences of ribosome-associated genes previously implicated in resistance and with the clinical outcomes of subjects from whom these isolates were obtained. Thirty-six (34%) of these isolates displayed resistance to one or more of these agents, and the majority of these (20 of 36) showed cross-resistance to one or more agents. Most (33 of 36) of the resistant isolates showed polymorphisms in the 16S ribosome components RpsL and rrs. Three resistant strains (3 of 36) were identified that had no known polymorphisms in ribosomal constituents. For kanamycin and streptomycin, molecular DST significantly outperformed phenotypic DST using the absolute concentration method for predicting 4-month sputum conversion (likelihood ratios of 4.0 and 2.0, respectively) and was equivalent to phenotypic DST using the National Committee for Clinical Laboratory Standards (NCCLS)-approved agar proportion method for estimating MIC (likelihood ratio, 4.0). These results offer insight into mechanisms of resistance and cross-resistance among these agents and suggest that the development of rapid molecular tests to distinguish polymorphisms would significantly enhance clinical utility of this important class of second-line antituberculosis drugs

Host-Mediated Bioactivation of Pyrazinamide: Implications for Efficacy, Resistance, and Therapeutic Alternatives

Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resistant, active, and latent tuberculosis (TB). Despite widespread recognition of its therapeutic importance, the sterilizing properties of this 60-year-old drug remain an enigma given its rather poor activity in vitro. Here we revisit longstanding paradigms and offer pharmacokinetic explanations for the apparent disconnect between in vitro activity and clinical impact. We show substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA), in TB patients and in animal models. We demonstrate favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. In standardized growth inhibition experiments, we show that POA exhibits superior in vitro potency compared to PZA, indicating that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing the apparent discrepancy between in vitro and in vivo activity. However, the results also raise the possibility that subinhibitory concentrations of POA generated by the host could fuel the emergence of resistance to both PZA and POA. In contrast to widespread expectations, we demonstrate good oral bioavailability and exposure in preclinical species in pharmacokinetic studies of oral POA. Baseline exposure of oral POA can be further increased by the xanthine oxidase inhibitor and approved gout drug allopurinol. These promising results pave the way for clinical investigations of oral POA as a therapeutic alternative or an add-on to overcome PZA resistance and salvage this essential TB drug. [Image: see text