Comparison of Macrophage Immune Responses and Metabolic Reprogramming in Smooth and Rough Variant Infections of Mycobacterium mucogenicum.

Mycobacterium mucogenicum (Mmuc), a rapidly growing nontuberculous mycobacterium (NTM), can infect humans (posttraumatic wound infections and catheter-related sepsis). Similar to other NTM species, Mmuc exhibits colony morphologies of rough (Mmuc-R) and smooth (Mmuc-S) types. Although there are several case reports on Mmuc infection, no experimental evidence supports that the R-type is more virulent. In addition, the immune response and metabolic reprogramming of Mmuc have not been studied on the basis of morphological characteristics. Thus, a standard ATCC Mmuc strain and two clinical strains were analyzed, and macrophages were generated from mouse bone marrow. Cytokines and cell death were measured by ELISA and FACS, respectively. Mitochondrial respiration and glycolytic changes were measured by XF seahorse. Higher numbers of intracellular bacteria were found in Mmuc-R-infected macrophages than in Mmuc-S-infected macrophages. Additionally, Mmuc-R induced higher levels of the cytokines TNF-α, IL-6, IL-12p40, and IL-10 and induced more BMDM necrotic death. Furthermore, our metabolic data showed marked glycolytic and respiratory differences between the control and each type of Mmuc infection, and changes in these parameters significantly promoted glucose metabolism, extracellular acidification, and oxygen consumption in BMDMs. In conclusion, at least in the strains we tested, Mmuc-R is more virulent, induces a stronger immune response, and shifts bioenergetic metabolism more extensively than the S-type. This study is the first to report differential immune responses and metabolic reprogramming after Mmuc infection and might provide a fundamental basis for additional studies on Mmuc pathogenesis.

The Impact of Trehalose Dimycolate on the Clinical Course of Mycobacterium avium Complex Pulmonary Disease.

Rationale: The clinical implications of trehalose 6,6'-dimycolate (TDM) in nontuberculous mycobacterial pulmonary disease have not been studied. Objectives: To examine the presence of TDM in clinical isolates obtained from patients with Mycobacterium avium complex (MAC) pulmonary disease (PD) and its impact on disease severity and treatment outcomes. Methods: We analyzed clinical isolates from patients with diagnoses of MAC PD at Seoul National University Hospital between January 1, 2019, and December 31, 2021. The lipids were extracted from clinical isolates obtained at the time of diagnosis using mass spectrometry. Mass peaks between 300 and 3,500 m/z were obtained, and the peak patterns of the total lipids were analyzed. Results: TDM was identified in clinical isolates from 176 of 343 patients. Cavities were more prevalent in patients with TDM-negative isolates (19.8%) than in those with TDM-positive isolates (10.2%) (P = 0.015). The time to antibiotic treatment was shorter in patients with TDM-negative isolates (4 mo [interquartile range, 2-10 mo]) than in those with TDM-positive isolates (7 mo [interquartile range, 3-16 mo]) (P = 0.032). Patients with TDM-negative isolates had a significantly lower proportion of culture conversions (P = 0.012). TDM was associated with higher likelihood of culture conversion (adjusted hazard ratio, 2.29; P = 0.035). Conclusions: TDM-negative isolates were linked to a higher occurrence of cavities, earlier initiation of treatment, and worse treatment outcome in patients with MAC PD.

High-dose rifampicin for 3 months after culture conversion for drug-susceptible pulmonary tuberculosis.

BACKGROUND: This study aimed to determine whether a shorter high-dose rifampicin regimen is non-inferior to the standard 6-month tuberculosis regimen. METHOD: This multicenter, randomized, open-label, non-inferiority trial enrolled participants with respiratory specimen positivity by Xpert MTB/RIF assay or Mycobacterium tuberculosis culture without rifampicin-resistance. Participants were randomized at 1:1 to the investigational or control group. The investigational group received high-dose rifampicin (30 mg/kg/day), isoniazid, and pyrazinamide until culture conversion, followed by high-dose rifampicin and isoniazid for 12 weeks. The control group received the standard 6-month regimen. The primary outcome was the rate of unfavorable outcomes at 18 months post-randomization. The non-inferiority margin was set at <6% difference in unfavorable outcomes rates. RESULTS: Between 4 November 2020 and 3 January 2022, 76 participants were enrolled. Of these, 58 were included in the modified intention-to-treat analysis. Unfavorable outcomes occurred in 10 (31.3%) of 32 in the control group and 10 (38.5%) of 26 in the investigational group. The difference was 7.2% (95% confidence interval, ∞ to 31.9%), failing to prove non-inferiority. Serious adverse events and grade 3 or higher adverse events did not differ between the groups. CONCLUSIONS: The shorter high-dose rifampicin regimen failed to demonstrate non-inferiority but had an acceptable safety profile. Trial registration ClinicalTrials.gov NCT04485156.

Dimethyl itaconate is effective in host-directed antimicrobial responses against mycobacterial infections through multifaceted innate immune pathways.

BACKGROUND: Itaconate, a crucial immunometabolite, plays a critical role in linking immune and metabolic functions to influence host defense and inflammation. Due to its polar structure, the esterified cell-permeable derivatives of itaconate are being developed to provide therapeutic opportunities in infectious and inflammatory diseases. Yet, it remains largely uncharacterized whether itaconate derivatives have potentials in promoting host-directed therapeutics (HDT) against mycobacterial infections. Here, we report dimethyl itaconate (DMI) as the promising candidate for HDT against both Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria by orchestrating multiple innate immune programs. RESULTS: DMI per se has low bactericidal activity against Mtb, M. bovis Bacillus Calmette-Guérin (BCG), and M. avium (Mav). However, DMI robustly activated intracellular elimination of multiple mycobacterial strains (Mtb, BCG, Mav, and even to multidrug-resistant Mtb) in macrophages and in vivo. DMI significantly suppressed the production of interleukin-6 and -10, whereas it enhanced autophagy and phagosomal maturation, during Mtb infection. DMI-mediated autophagy partly contributed to antimicrobial host defenses in macrophages. Moreover, DMI significantly downregulated the activation of signal transducer and activator of transcription 3 signaling during infection with Mtb, BCG, and Mav. CONCLUSION: Together, DMI has potent anti-mycobacterial activities in macrophages and in vivo through promoting multifaceted ways for innate host defenses. DMI may bring light to new candidate for HDT against Mtb and nontuberculous mycobacteria, both of which infections are often intractable with antibiotic resistance.

Minimal Inhibitory Concentration of Clofazimine Among Clinical Isolates of Nontuberculous Mycobacteria and Its Impact on Treatment Outcome.

BACKGROUND: Clofazimine has been regarded as a promising agent for the treatment of nontuberculous mycobacteria pulmonary disease (NTM-PD). However, its overall effectiveness in vitro and in the clinic remains unknown. RESEARCH QUESTION: What is the minimal inhibitory concentration (MIC) of clofazimine in clinical isolates and the association between MICs and treatment outcome? STUDY DESIGN AND METHODS: MICs for clofazimine were measured in clinical isolates from NTM-PD patients who participated in a prospective study at Seoul National University Hospital. The MIC was determined by using the broth microdilution concentration method. Correlation between MIC and conversion to negative of sputum culture with clofazimine was determined. RESULTS: Of a total 189 isolates, 133 strains were Mycobacterium avium complex (MAC) and 40 strains were M abscessus. Although the clofazimine MICs for MAC ranged from 0.031 mg/L to 8 mg/L, the values obtained for M abscessus ranged from 0.031 mg/L to 16 mg/L. Of 20 patients who were treated with a regimen including clofazimine, eight achieved negative conversion of sputum culture. All patients with isolates exhibiting clofazimine MIC values ≤ 0.25 mg/L achieved culture conversion. The likelihood of culture conversion in patients with MIC value ≤ 0.25 mg/L was much higher than that of patients with MIC value > 0.5 mg/L (OR, 39.3; P = .021). INTERPRETATION: The MICs of clofazimine varied widely in clinical isolates from patients with NTM-PD. Negative conversion of sputum culture with clofazimine use was associated with a lower MIC value. Clofazimine use could be considered in patients with NTM-PD when the MIC value is ≤ 0.25 mg/L. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01616745; URL: www.clinicaltrials.gov.

Detection of resistance to fluoroquinolones and injectable drugs among antituberculosis drugs by allele-specific primer extension on a microsphere-based platform.

Molecular drug susceptibility testing (DST) for antituberculosis drugs is important for improving the efficacy of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) treatment. In this study, we developed a molecular high-throughput assay system based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres, referred to here as TAG-ASPE, which can detect mutations related to resistance to injectable second-line drugs and fluoroquinolones. Target genes were amplified by multiplex PCR using DNA from H37Rv and 190 clinical Mycobacterium tuberculosis strains and extended by ASPE using 22 ASPE primers. ASPE products were then sorted on the TAG-ASPE array and detected using a Luminex 200 system. The performance of the TAG-ASPE method was compared with that of sequencing and phenotypic DST. Comparison of the TAG-ASPE method with sequencing showed that the sensitivity and specificity of the TAG-ASPE method were 100% [95% confidence interval (CI), 96.38-100%] and 100% (95% CI, 95.70-100%) for the rrs gene and 100% (95% CI, 96.90-100%) and 100% (95% CI, 95.07-100%) for the gyrA gene, respectively. Compared with phenotypic DST, the sensitivity and specificity of the TAG-ASPE method for detecting drug-resistance mutations against injectable second-line drugs were 92.52% (95% CI, 85.8-96.72%) and 98.7% (95% CI, 92.98-99.97%), respectively. Additionally, the sensitivity and specificity for fluoroquinolone-resistance detection were 85.4% (95% CI, 78.36-90.85%) and 100% (95% CI, 92.38-100%), respectively. The results of this study demonstrate that the TAG-ASPE method can effectively detect mutations conferring resistance to second-line antituberculosis drugs in numerous clinical specimens.

Delamanid, Bedaquiline, and Linezolid Minimum Inhibitory Concentration Distributions and Resistance-related Gene Mutations in Multidrug-resistant and Extensively Drug-resistant Tuberculosis in Korea.

BACKGROUND: Delamanid, bedaquiline, and linezolid have recently been approved for the treatment of multidrug- and extensively drug-resistant (MDR and XDR, respectively) tuberculosis (TB). To use these drugs effectively, drug susceptibility tests, including rapid molecular techniques, are required for accurate diagnosis and treatment. Furthermore, mutation analyses are needed to assess the potential for resistance. We evaluated the minimum inhibitory concentrations (MICs) of these three anti-TB drugs for Korean MDR and XDR clinical strains and mutations in genes related to resistance to these drugs. METHODS: MICs were determined for delamanid, bedaquiline, and linezolid using a microdilution method. The PCR products of drug resistance-related genes from 420 clinical Mycobacterium tuberculosis strains were sequenced and aligned to those of M. tuberculosis H37Rv. RESULTS: The overall MICs for delamanid, bedaquiline, and linezolid ranged from ≤0.025 to >1.6 mg/L, ≤0.0312 to >4 mg/L, and ≤0.125 to 1 mg/L, respectively. Numerous mutations were found in drug-susceptible and -resistant strains. We did not detect specific mutations associated with resistance to bedaquiline and linezolid. However, the Gly81Ser and Gly81Asp mutations were associated with resistance to delamanid. CONCLUSIONS: We determined the MICs of three anti-TB drugs for Korean MDR and XDR strains and identified various mutations in resistance-related genes. Further studies are needed to determine the genetic mechanisms underlying resistance to these drugs.

Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform: Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit.

Tuberculosis (TB), caused by infections of the Mycobacterium tuberculosis (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by N-acetyl-l-cysteine-2% NaOH and then extracted using three methods: "in-house" 5% Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing.

A molecular epidemiological analysis of tuberculosis trends in South Korea.

Molecular epidemiological data are needed to assess tuberculosis (TB)-management policy outcomes in South Korea. IS6110 restriction fragment-length polymorphism (IS6110-RFLP) and mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) analyses are major molecular epidemiological tools for investigating the transmission or reactivation of active TB. Here, we determined trends in the clustering rate (i.e., the prevalence of Mycobacterium tuberculosis isolates with identical genotype patterns) of active TB and related differences between the 1990s and 2000s in Korea. M. tuberculosis isolates (1,007) of nationwide origins were analyzed by IS6110-RFLP and 24-locus standardized MIRU-VNTR genotyping. The clustering rate was measured by IS6110-RFLP, 24-locus MIRU-VNTR, and both analytical methods in combination. IS6110-RFLP, 24-locus MIRU-VNTR typing, and the combined method revealed 882, 754, and 983 distinct profiles; 809, 651, and 961 unique isolates; and 198, 356, and 46 clustered isolates grouped into 73, 103, and 22 clusters, respectively. In addition, we confirmed that the clustering rates in the 2000s decreased by 11.2%, 2.1%, and 3.1% relative to that in the 1990s using the three methods, respectively. Furthermore, in multivariate analysis, the younger-age group (<30) clustered more frequently than the older-age group (>50), based on all the three methods. Our study is the first report to provide nationwide molecular epidemiological information on TB in Korea.