Intracellular Activity of Poly (DL-Lactide-co-Glycolide) Nanoparticles Encapsulated with Prothionamide, Pyrazinamide, Levofloxacin, Linezolid, or Ethambutol on Multidrug-Resistant Mycobacterium tuberculosis.

BACKGROUND: Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is a major cause of death amongst tuberculosis patients. Nanomedicine avoids some limitations of conventional drug treatment and increases therapeutic efficacy against bacterial infections. However, the effect of anti-TB drug nanoparticle (NP) compounds in anti-TB regimens against MDR-TB remains unclear. OBJECTIVE: The objective of this article is to prepare levofloxacin, linezolid, ethambutol, prothionamide, and pyrazinamide encapsulated NPs and to evaluate their therapeutic efficacy against MDR-TB in macrophages. METHODS: Drug-loaded PLGA NPs were prepared by the multiple emulsion method. The colocalization, intracellular release, and anti-TB activity of these NPs were investigated on cultured macrophages. The immune phenotype of the macrophages, including their mitochondrial membrane potential, reactive oxygen species (ROS), and nitric oxide (NO) production, was evaluated following treatment with NPs or free drug compounds. RESULTS: All drug-loaded PLGA NPs were spherical in shape, 150 to 210 nm in size, and showed 14.22% to 43.51% encapsulation efficiencies and long-duration release. Drug-loaded PLGA NPs were mainly distributed in the cytoplasm of macrophages, showed high cellular compatibility, and maintained their concentration for at least 13 days. Compared with the free drug compounds, the number of colonies after exposure to PLGA NP compounds was significantly less. The enhanced antibacterial activity of the NP compounds may be due to the enhanced levels of ROS and NO and the increased early apoptosis stress within M. tuberculosis-infected macrophages additionally. CONCLUSION: The application of PLGA NP compounds not only enhances drug efficacy but also induces innate bactericidal events in macrophages, confirming this as a promising approach for MDR-TB therapy.

A recombinant selective drug-resistant M. bovis BCG enhances the bactericidal activity of a second-line anti-tuberculosis regimen.

Drug-resistant tuberculosis (DR-TB) poses a new threat to global health; to improve the treatment outcome, therapeutic vaccines are considered the best chemotherapy adjuvants. Unfortunately, there is no therapeutic vaccine approved against DR-TB. Our study assessed the therapeutic efficacy of a recombinant drug-resistant BCG (RdrBCG) vaccine in DR-TB. We constructed the RdrBCG overexpressing Ag85B and Rv2628 by selecting drug-resistant BCG strains and transformed them with plasmid pEBCG or pIBCG to create RdrBCG-E and RdrBCG-I respectively. Following successful stability testing, we tested the vaccine's safety in severe combined immune deficient (SCID) mice that lack both T and B lymphocytes plus immunoglobulins. Finally, we evaluated the RdrBCG's therapeutic efficacy in BALB/c mice infected with rifampin-resistant M. tuberculosis and treated with a second-line anti-TB regimen. We obtained M. bovis strains which were resistant to several second-line drugs and M. tuberculosis resistant to rifampin. Notably, the exogenously inserted genes were lost in RdrBCG-E but remained stable in the RdrBCG-I both in vitro and in vivo. When administered adjunct to a second-line anti-TB regimen in a murine model of DR-TB, the RdrBCG-I lowered lung M. tuberculosis burden by 1 log10. Furthermore, vaccination with RdrBCG-I adjunct to chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG-I showed almost the same virulence as its parent BCG Tice strain in SCID mice. Our findings suggested that the RdrBCG-I was stable, safe and effective as a therapeutic vaccine. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.

Bacterial Genome-Wide Association Identifies Novel Factors That Contribute to Ethionamide and Prothionamide Susceptibility in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, recent genome-wide association studies have identified a novel constellation of mutations that are correlated with high-level drug resistances. Interpreting the functional importance of the new resistance-associated mutations has been complicated, however, by a lack of experimental validation and a poor understanding of the epistatic factors influencing these correlations, including strain background and programmatic variation in treatment regimens. Here we perform a genome-wide association analysis in a panel of Mycobacterium tuberculosis strains from China to identify variants correlated with resistance to the second-line prodrug ethionamide (ETH). Mutations in a bacterial monooxygenase, Rv0565c, are significantly associated with ETH resistance. We demonstrate that Rv0565c is a novel activator of ETH, independent of the two known activators, EthA and MymA. Clinically prevalent mutations abrogate Rv0565c function, and deletion of Rv0565c confers a consistent fitness benefit on M. tuberculosis in the presence of partially inhibitory doses of ETH. Interestingly, Rv0565c activity affects susceptibility to prothionamide (PTH), the ETH analog used in China, to a greater degree. Further, clinical isolates vary in their susceptibility to both ETH and PTH, to an extent that correlates with the total expression of ETH/PTH activators (EthA, MymA, and Rv0565c). These results suggest that clinical strains considered susceptible to ETH/PTH are not equally fit during treatment due to both Rv0565c mutations and more global variation in the expression of the prodrug activators.IMPORTANCE Phenotypic antibiotic susceptibility testing in Mycobacterium tuberculosis is slow and cumbersome. Rapid molecular diagnostics promise to help guide therapy, but such assays rely on complete knowledge of the molecular determinants of altered antibiotic susceptibility. Recent genomic studies of antibiotic-resistant M. tuberculosis have identified several candidate loci beyond those already known to contribute to antibiotic resistance; however, efforts to provide experimental validation have lagged. Our study identifies a gene (Rv0565c) that is associated with resistance to the second-line antibiotic ethionamide at a population level. We then use bacterial genetics to show that the variants found in clinical strains of M. tuberculosis improve bacterial survival after ethionamide exposure.

Detection of novel mutations associated with independent resistance and cross-resistance to isoniazid and prothionamide in Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: Prothionamide, a structural analogue of isoniazid, is used mainly for treating multidrug-resistant tuberculosis (MDR-TB). Both drugs have a common target InhA, so prothionamide can be ineffective against isoniazid-resistant (INHR) Mycobacterium tuberculosis. We aimed to investigate the prevalence of mutations in katG, ethA, ndh, ethR, mshA, inhA and/or its promoter associated with independent resistance and cross-resistance to INHR and/or prothionamide-resistant (PTOR) M. tuberculosis isolates. METHODS: We sequenced the above genes in 206 M. tuberculosis isolates with susceptibility testing against ten drugs. RESULTS: Of the 173 INHR PTOR isolates, 170 (98.3%) harboured mutations in katG, 111 (64.2%) in ethA, 58 (33.5%) in inhA or its promoter, 5 (2.9%) in ndh, 3 (1.7 %) in ethR and 2 (1.2%) in mshA. Among the 18 INHR PTOS isolates, mutations in katG were found in all of them; one had a mutation in the inhA promoter and another in ndh. Of the five INHS PTOR isolates, four showed mutations in ethA and two in the inhA promoter. Notably, 55 novel non-synonymous mutations were found in them and 20.2% of the PTORM. tuberculosis isolates harboured no known mutations. CONCLUSIONS: This is the first report to investigate cross-resistance between INHR and/or PTOR isolates. Among INHR (94.4% MDR-TB) M. tuberculosis isolates, the high diversity of mutations for independent resistance and cross-resistance with prothionamide highlight the importance of both phenotypic susceptibility and genotypic diagnosis when using it to treat patients with INHR-TB. The high proportion (one-fifth) of PTORM. tuberculosis isolates showed no known mutation related to PTOR genes, so uncovered resistance mechanism(s) of prothionamide exist.

Predicting the Outcomes of New Short-Course Regimens for Multidrug-Resistant Tuberculosis Using Intrahost and Pharmacokinetic-Pharmacodynamic Modeling.

Short-course regimens for multidrug-resistant tuberculosis (MDR-TB) are urgently needed. Limited data suggest that the new drug bedaquiline (BDQ) may have the potential to shorten MDR-TB treatment to less than 6 months when used in conjunction with standard anti-TB drugs. However, the feasibility of BDQ in shortening MDR-TB treatment duration remains to be established. Mathematical modeling provides a platform to investigate different treatment regimens and predict their efficacy. We developed a mathematical model to capture the immune response to TB inside a human host environment. This model was then combined with a pharmacokinetic-pharmacodynamic model to simulate various short-course BDQ-containing regimens. Our modeling suggests that BDQ could reduce MDR-TB treatment duration to just 18 weeks (4 months) while still maintaining a very high treatment success rate (100% for daily BDQ for 2 weeks, or 95% for daily BDQ for 1 week during the intensive phase). The estimated time to bacterial clearance of these regimens ranges from 27 to 33 days. Our findings provide the justification for empirical evaluation of short-course BDQ-containing regimens. If short-course BDQ-containing regimens are found to improve outcomes, then we anticipate clear cost savings and a subsequent improvement in the efficiency of national TB programs.

Prothionamide susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTECMGIT 960 system.

Resazurin microtitre assay (RMA) has been successfully used to detect minimal inhibitory concentrations (MICs) of both first-line and several second-line drugs in drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). In this study, we firstly compared prothionamide (PTH) susceptibility testing of Mycobacterium tuberculosis (MTB) using resazurin microtitre assay (RMA) and MGIT. Overall, the sensitivity and specificity of RMA for detecting PTH susceptibility was 96.5% [95% confidence interval (CI): 91.7-100.0] and 93.2% (95% CI: 89.6-96.8) respectively. In addition, the median time to positivity was significantly shorter for RMA than for the automated MGIT 960 (RMA, 8 days [range: 8-8 days] vs MGIT, 10.1 days, [range: 5.0-13.0]; P < 0.01). Concordance rate for MICs between RMA and MGIT for PTH-resistant group was 64.3% (95% CI: 46.5-82.0), which was significantly lower than that of PTH-susceptible group (85.9%, 95% CI: 78.8-93.0; P= 0.01). In conclusion, our data demonstrated that RMA can be used as an acceptable alternative for determination of PTH susceptibility with shorter turn-around time. When compared with MGIT 960, RMA method was prone to produce higher MICs for PTH-resistant MTB strains.

Antibiotic Susceptibility Patterns of Mycobacterium tuberculosis Isolates from Guizhou Province of China Against 13 Antituberculosis Drugs.

A total of 92 Mycobacterium tuberculosis isolates were collected from patients with pulmonary tuberculosis (TB) in the Zunyi region between 2011 and 2012. Collected isolates were used to determine antibiotic susceptibility patterns against 13 anti-TB drugs: 4 first-line and 9 second-line (ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, para-aminosalicylic acid, amikacin, capreomycin, kanamycin, and prothionamide) drugs. Results showed that among 57 new cases of TB only 66.7% were susceptible to all four first-line anti-TB drugs and 64.9% were susceptible to fluoroquinolones and second-line injectables; 10.5% of new and 22.9% of previously treated cases were multidrug-resistant TB (MDR-TB); and 1.8% of new and 2.9% of previously treated cases were extensively drug-resistant TB (XDR-TB). In addition, 14.3% of MDR-TB cases (2 out of 14) were XDR-TB, which is higher than the average numbers in China (about 8%) and in the world (9.6%). This study confirms that primary transmission of drug-resistant TB, including MDR/XDR-TB, is a real threat to achieving effective control of drug-resistant TB in the Guizhou Province and indicates the necessity to determine antibiotic susceptibility patterns in patients with TB to improve treatment outcomes.

Polymorphisms in isoniazid and prothionamide resistance genes of the Mycobacterium tuberculosis complex.

Sequence analyses of 74 strains that encompassed major phylogenetic lineages of the Mycobacterium tuberculosis complex revealed 10 polymorphisms in mshA (Rv0486) and four polymorphisms in inhA (Rv1484) that were not responsible for isoniazid or prothionamide resistance. Instead, some of these mutations were phylogenetically informative. This genetic diversity must be taken into consideration for drug development and for the design of molecular tests for drug resistance.

Mechanism of thioamide drug action against tuberculosis and leprosy.

Thioamide drugs, ethionamide (ETH) and prothionamide (PTH), are clinically effective in the treatment of Mycobacterium tuberculosis, M. leprae, and M. avium complex infections. Although generally considered second-line drugs for tuberculosis, their use has increased considerably as the number of multidrug resistant and extensively drug resistant tuberculosis cases continues to rise. Despite the widespread use of thioamide drugs to treat tuberculosis and leprosy, their precise mechanisms of action remain unknown. Using a cell-based activation method, we now have definitive evidence that both thioamides form covalent adducts with nicotinamide adenine dinucleotide (NAD) and that these adducts are tight-binding inhibitors of M. tuberculosis and M. leprae InhA. The crystal structures of the inhibited M. leprae and M. tuberculosis InhA complexes provide the molecular details of target-drug interactions. The purified ETH-NAD and PTH-NAD adducts both showed nanomolar Kis against M. tuberculosis and M. leprae InhA. Knowledge of the precise structures and mechanisms of action of these drugs provides insights into designing new drugs that can overcome drug resistance.

A case of relapse with drug-susceptible M. leprae after multidrug therapy.

A male born in 1930 was diagnosed as smear-positive borderline leprosy in 1971, and was treated with dapsone and/or sulfamethoxypyridazine from 1972 to 1980 with clinical improvement. However, new skin lesions with smears strongly positive appeared in August 1980, and he was diagnosed as having downgraded to lepromatous (LL) leprosy, but the bacilli recovered from the skin biopsy were fully susceptible to both dapsone and rifampin by mouse foot pad technique. Between 1981 and 1983, the patient was treated with 24 months of rifampin 600 mg and dapsone 100 mg daily, supplemented with prothionamide 500 mg daily during the initial 3 months, and his skin lesions gradually improved during treatment with the combined regimen. Afterward, the patient was kept under surveillance without treatment. From 1984 to 1986, his skin smears were negative, and no bacilli could be found from a skin biopsy taken in 1985. Then in 1987, 52 months after stopping treatment, new skin lesions appeared with a high concentration of Mycobacterium leprae (2 x 10(6)/mg tissue). The drug-susceptibility test again demonstrated that the organisms were fully susceptible to both dapsone and rifampin. Apparently the relapse was due to remultiplication of drug-susceptible persisters.

Activity of ofloxacin against Mycobacterium leprae in the mouse.

Mice inoculated with 4800 Mycobacterium leprae in the left hind foot pad were treated from day 62 to day 150 after infection with 50 mg or 150 mg of ofloxacin per kg body weight, 150 mg pefloxacin per kg, or 50 mg prothionamide per kg. These drugs were administered by esophageal cannula 5 days weekly with dapsone (0.01 g per 100 g diet). Multiplication of M. leprae in the treated and in untreated control mice was assessed by monthly harvests. The treatment of mice with the smaller dosage ofloxacin, with pefloxacin, prothionamide, or dapsone uniformly resulted in a delay of multiplication of 4 months, compared to the multiplication of M. leprae in the untreated controls. The delay of multiplication (4 months) being 1 month longer than the duration of drug administration (3 months), all of the treatments may be considered as bacteriopausal or moderately bactericidal. In contast with these results, treatment of mice with 150 mg ofloxacin per kg resulted in no growth of the organisms whatever as late as 18 months after inoculation, strongly suggesting that, in that dosage, ofloxacin had killed all of the M. leprae. Such a profound killing activity has been observed only with rifampin. Although the pharmacokinetic characteristics of ofloxacin are different in man from those in the mouse, the daily dosage of 150 mg ofloxacin per kg body weight in the mouse is equivalent to 400 mg per day in man which is the usual therapeutic dosage; thus, the results obtained in the mouse may be extrapolated to man. Therefore, ofloxacin appears a very promising drug for the chemotherapy of leprosy.

Sensitivity to capreomycin and prothionamide in strains of Mycobacterium, Nocardia, Rhodococcus, and related taxa for taxonomical purposes.

Sensitivity to capreomycin and to prothionamide was analysed for 150 strains belonging to the genera Mycobacterium, Nocardia, Rhodococcus, and related taxa. The analyses showed, e.g., that strains of Mycobacterium chelonei and Nocardia brasiliensis were more resistant to capreomycin than the other strains tested and that M. farcinogenes differed from M. senegalense concerning sensitivity to this drug. The analysis showed, furthermore, that strains of Mycobacterium differ from those of Nocardia, Rhodococcus, "Mycobacterium album", and "Gordona aurantiaca" in being more sensitive to prothionamide than these organisms. Strains designated N. amarae were more sensitive to both drugs than were the other tested strains of Nocardia, which indicates that N. amarae diverges from the other species of this genus.

Antimycobacterial activity of isoniazid + prothionamide + dapsone against a number of randomly selected 'wild' strains of Mycobacterium tuberculosis.

46 strains of Mycobacterium tuberculosis were studied as to their susceptibility to isoniazid (INH) or to INH + prothionamide (PTH) in the presence or absence of dapsone (DDS) by microdilution transfer plate technique. Synergy with DDS as defined by Berenbaum in 1978 was seen in 78% with INH and in 91% with INH + PTH. It concerned the minimal inhibitory concentration, whereas the minimal bactericidal concentration (MBC) was unaffected in the case of INH and it was increased in only 52% with INH + PTH. The latter MBC proportion mentioned would be decreased to roughly 16% accepting an only twofold decrease in the MBC as possibly due to dilution errors. The possible effect of dimethyl sulphoxide on the reported results is discussed.

On the mechanism of the antimycobacterial activity of isoniazid + prothionamide + dapsone (Isoprodian).

Increased antimycobacterial activity of Isoprodian (isoniazid + prothionamide + dapsone) may be due to (i) decreased mutation rate for INH resistance provoked in mycobacteria by DDS (in 2 of 3 strains tested); (ii) leakage of K+, Na+ or Ca++ caused by INH and/or by PTH (in all 3 strains tested), and (iii) indicating damages which may increase the low level penetration of DDS into the cell in sub-MIC concentrations as shown by phage proliferation changes (tested in 1 strain with 1 phage).