Improvement in Health-Related Quality of Life Following Antibiotic Treatment in Nontuberculous Mycobacterial Pulmonary Disease: Initial Analysis of the NTM-KOREA Cohort.

BACKGROUND: Improving health-related quality of life (HRQOL) has emerged as a priority in the management of nontuberculous mycobacterial pulmonary disease (NTM-PD). We aimed to evaluate HRQOL and its changes after 6 months' treatment in patients with NTM-PD. METHODS: The NTM-KOREA is a nationwide prospective cohort enrolling patients initiating treatment for NTM-PD in 8 institutions across South Korea. We conducted the Quality of Life-Bronchiectasis (QOL-B) at 6-month intervals and evaluated baseline scores (higher scores indicate better quality of life) and changes after 6 months' treatment. Multivariate logistic regression was performed to identify factors associated with improvement in the QOL-B physical functioning and respiratory symptoms domains. RESULTS: Between February 2022 and August 2023, 411 patients were included in the analysis. Baseline scores (95% confidence interval [CI]) for physical functioning and respiratory symptoms were 66.7 (46.7-86.7) and 81.5 (70.4-92.6), respectively. Among 228 patients who completed the QOL-B after 6 months' treatment, improvements in physical functioning and respiratory symptoms were observed in 61 (26.8%) and 71 (31.1%) patients, respectively. A lower score (adjusted odds ratio; 95% CI) for physical functioning (0.93; 0.91-0.96) and respiratory symptoms (0.92; 0.89-0.95) at treatment initiation was associated with a greater likelihood of physical functioning and respiratory symptom improvement, respectively; achieving culture conversion was not associated with improvement in physical functioning (0.62; 0.28-1.39) or respiratory symptoms (1.30; 0.62-2.74). CONCLUSIONS: After 6 months of antibiotic treatment for NTM-PD, HRQOL improved in almost one-third, especially in patients with severe initial symptoms, regardless of culture conversion. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov identifier: NCT03934034.

Design, synthesis, biological evaluation study of spirocyclic POM analogues as novel MmpL3 anti-tubercular agent.

We present the development of a phenyl oxazole methyl (POM) core structure with spirocyclic derivatives as part of our efforts to discover innovative anti-tuberculosis agents. Derivatives of spirocyclic POM were synthesized and evaluated for their inhibitory effects on M.tuberculosis (M. tb) H37Rv. Notably, compound 5c displayed potent anti-tubercular activity with MIC value of 0.206 µM in culture broth medium. Furthermore MIC values of compound 5c against DS/MDR/pre-XDR clinical isolates ranged from 0.34 to 0.68 µg/mL, 0.17 to 0.68 µg/mL, and 0.17 to 0.34 µg/mL, respectively. Also, compound 5c with favorable ADME and PK properties was not cytotoxic to THP-1 human cells. Based on the spontaneous mutant generation, we have identified the target of compound 5c to be MmpL3. The computational docking study suggested its plausible binding mode against MmpL3. There is no approved drug targeting this target yet, and the outcomes of the presented research will contribute to the future discovery of novel anti-tuberculosis drugs.

Novel Antibacterial Activity of Febuxostat, an FDA-Approved Antigout Drug against Mycobacterium tuberculosis Infection.

Accumulating evidence suggests that drug repurposing has drawn attention as an anticipative strategy for controlling tuberculosis (TB), considering the dwindling drug discovery and development pipeline. In this study, we explored the antigout drug febuxostat and evaluated its antibacterial activity against Mycobacterium species. Based on MIC evaluation, we found that febuxostat treatment significantly inhibited mycobacterial growth, especially that of Mycobacterium tuberculosis (Mtb) and its phylogenetically close neighbors, M. bovis, M. kansasii, and M. shinjukuense, but these microorganisms were not affected by allopurinol and topiroxostat, which belong to a similar category of antigout drugs. Febuxostat concentration-dependently affected Mtb and durably mediated inhibitory functions (duration, 10 weeks maximum), as evidenced by resazurin microtiter assay, time-kill curve analysis, phenotypic susceptibility test, and the Bactec MGIT 960 system. Based on these results, we determined whether the drug shows antimycobacterial activity against Mtb inside murine bone marrow-derived macrophages (BMDMs). Notably, febuxostat markedly suppressed the intracellular growth of Mtb in a dose-dependent manner without affecting the viability of BMDMs. Moreover, orally administered febuxostat was efficacious in a murine model of TB with reduced bacterial loads in both the lung and spleen without the exacerbation of lung inflammation, which highlights the drug potency. Taken together, unexpectedly, our data demonstrated that febuxostat has the potential for treating TB.

Aconitate Decarboxylase 1 Deficiency Exacerbates Mouse Colitis Induced by Dextran Sodium Sulfate.

Ulcerative colitis is a complex inflammatory bowel disorder disease that can induce rectal and colonic dysfunction. Although the prevalence of IBD in Western countries is almost 0.5% of the general population, genetic causes are still not fully understood. In a recent discovery, itaconate was found to function as an immune-modulating metabolite in mammalian immune cells, wherein it is synthesized as an antimicrobial compound from the citric acid cycle intermediate cis-aconitic acid. However, the association between the Acod1 (Aconitate decarboxylase 1)-itaconate axis and ulcerative colitis has rarely been studied. To elucidate this, we established a DSS-induced colitis model with Acod1-deficient mice and then measured the mouse body weights, colon lengths, histological changes, and cytokines/chemokines in the colon. We first confirmed the upregulation of Acod1 RNA and protein expression levels in DSS-induced colitis. Then, we found that colitis symptoms, including weight loss, the disease activity index, and colon shortening, were worsened by the depletion of Acod1. In addition, the extent of intestinal epithelial barrier breakdown, the extent of immune cell infiltration, and the expression of proinflammatory cytokines and chemokines in Acod1-deficient mice were higher than those in wild-type mice. Finally, we confirmed that 4-octyl itaconate (4-OI) alleviated DSS-induced colitis in Acod1-deficient mice and decreased the expression of inflammatory cytokines and chemokines. To our knowledge, this study is the first to elucidate the role of the Acod1-itaconate axis in colitis. Our data clearly showed that Acod1 deletion resulted in severe DSS-induced colitis and substantial increases in inflammatory cytokine and chemokine levels. Our results suggest that Acod1 may normally play an important regulatory role in the pathogenesis of colitis, demonstrating the potential for novel therapies using 4-OI.

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.

Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

Pan-lineage Mycobacterium tuberculosis reference genome for enhanced molecular diagnosis.

In Mycobacterium tuberculosis (MTB) control, whole genome sequencing-based molecular drug susceptibility testing (molDST-WGS) has emerged as a pivotal tool. However, the current reliance on a single-strain reference limits molDST-WGS's true potential. To address this, we introduce a new pan-lineage reference genome, 'MtbRf'. We assembled 'unmapped' reads from 3,614 MTB genomes (751 L1; 881 L2; 1,700 L3; and 282 L4) into 35 shared, annotated contigs (54 coding sequences [CDSs]). We constructed MtbRf through: (1) searching for contig homologues among genome database that precipitate results uniquely within Mycobacteria genus; (2) comparing genomes with H37Rv ('lift-over') to define 18 insertions; and (3) filling gaps in H37Rv with insertions. MtbRf adds 1.18% sequences to H37rv, salvaging >60% of previously unmapped reads. Transcriptomics confirmed gene expression of new CDSs. The new variants provided a moderate DST predictive value (AUROC 0.60-0.75). MtbRf thus unveils previously hidden genomic information and lays the foundation for lineage-specific molDST-WGS.

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.

Isoegomaketone exhibits potential as a new Mycobacterium abscessus inhibitor.

Although the incidence of Mycobacterium abscessus infection has recently increased significantly, treatment is difficult because this bacterium is resistant to most anti-tuberculosis drugs. In particular, M. abscessus is often resistant to available macrolide antibiotics, so therapeutic options are extremely limited. Hence, there is a pressing demand to create effective drugs or therapeutic regimens for M. abscessus infections. The aim of the investigation was to assess the capability of isoegomaketone (iEMK) as a therapeutic option for treating M. abscessus infections. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of iEMK for both reference and clinically isolated M. abscessus strains. In addition to time-kill and biofilm formation assays, we evaluated iEMK's capability to inhibit M. abscessus growth in macrophages using an intracellular colony counting assay. iEMK inhibited the growth of reference and clinically isolated M. abscessus strains in macrophages and demonstrated effectiveness at lower concentrations against macrophage-infected M. abscessus than when used to treat the bacteria directly. Importantly, iEMK also exhibited anti-biofilm properties and the potential to mitigate macrolide-inducible resistance, underscoring its promise as a standalone or adjunctive therapeutic agent. Overall, our results suggest that further development of iEMK as a clinical drug candidate is promising for inhibiting M. abscessus growth, especially considering its dual action against both planktonic bacteria and biofilms.

Genetic stability of Mycobacterium abscessus during antibiotic treatment.

OBJECTIVES: Genetic changes in Mycobacterium abscessus during antibiotic treatment are not fully understood. This study aimed to investigate the genetic changes in M. abscessus in patients receiving antibiotic treatment, and their clinical implications. METHODS: Pretreatment and 12-month post-treatment M. abscessus isolates were obtained from patients with M. abscessus pulmonary disease. Isolates from each time point were separated into six groups based on their distinctive morphological characteristics. Twenty-four isolates, comprising 12 from patient A exhibiting progressive disease and 12 from patient B demonstrating stable disease, underwent sequencing. Subsequently, minimal inhibitory concentrations (MICs) for the administered antibiotics were measured. RESULTS: Persistent infection with a single strain was observed in patients A and B. During 12 months of treatment, MICs for administered drugs did not generally change over time in either patient and single nucleotide variations (SNV) associated with antimicrobial resistance (rrl, rrs, erm(41), gyrA, gyrB, whiB7 and hflX) were not mutated. Although not significant, 47 and 52 non-synonymous SNVs occurred in M. abscessus from patients A and B, respectively, and the accumulation of these SNVs differed in patients A and B, except for five SNVs. The most variable positions were within a probable NADH-dependent glutamate synthase gene and a putative YrbE family protein gene in patients A and B, respectively. CONCLUSIONS: Persistent infections by a single strain of M. abscessus were observed in two patients with different clinical courses. Genetic changes in M. abscessus during antibiotic treatment were relatively stable in these patients. CLINICAL TRIALS IDENTIFIER: NCT01616745 (ClinicalTrials.gov ID).

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.

The autophagy-targeting compound V46 enhances antimicrobial responses to Mycobacteroides abscessus by activating transcription factor EB.

Mycobacteroides abscessus (Mabc) is a rapidly growing nontuberculous mycobacterium that poses a considerable challenge as a multidrug-resistant pathogen causing chronic human infection. Effective therapeutics that enhance protective immune responses to Mabc are urgently needed. This study introduces trans-3,5,4'-trimethoxystilbene (V46), a novel resveratrol analogue with autophagy-activating properties and antimicrobial activity against Mabc infection, including multidrug-resistant strains. Among the resveratrol analogues tested, V46 significantly inhibited the growth of both rough and smooth Mabc strains, including multidrug-resistant strains, in macrophages and in the lungs of mice infected with Mabc. Additionally, V46 substantially reduced Mabc-induced levels of pro-inflammatory cytokines and chemokines in both macrophages and during in vivo infection. Mechanistic analysis showed that V46 suppressed the activation of the protein kinase B/Akt-mammalian target of rapamycin signaling pathway and enhanced adenosine monophosphate-activated protein kinase signaling in Mabc-infected cells. Notably, V46 activated autophagy and the nuclear translocation of transcription factor EB, which is crucial for antimicrobial host defenses against Mabc. Furthermore, V46 upregulated genes associated with autophagy and lysosomal biogenesis in Mabc-infected bone marrow-derived macrophages. The combination of V46 and rifabutin exerted a synergistic antimicrobial effect. These findings identify V46 as a candidate host-directed therapeutic for Mabc infection that activates autophagy and lysosomal function via transcription factor EB.

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.

Differential Immune Responses and Underlying Mechanisms of Metabolic Reprogramming in Smooth and Rough Variants of Mycobacterium peregrinum Infections.

Mycobacterium peregrinum (Mpgm) is a rapidly growing mycobacteria that is classified as a nontuberculous mycobacterium (NTM) and is commonly found in environmental sources such as soil, water, and animals. Mpgm is considered an opportunistic pathogen that causes infection in immunocompromised individuals or those with underlying medical conditions. Although there have been clinical reports on Mpgm, reports of the immune response and metabolic reprogramming have not been published. Thus, we studied standard Mpgm-ATCC and two clinical strains (Mpgm-S and Mpgm-R) using macrophages and mouse bone marrow-derived cells. Mpgm has two types of colony morphologies: smooth and rough. We grew all strains on the 7H10 agar medium to visually validate the morphology. Cytokine levels were measured via ELISA and real-time PCR. The changes in mitochondrial function and glycolysis in Mpgm-infected macrophages were measured using an extracellular flux analyzer. Mpgm-S-infected macrophages showed elevated levels of inflammatory cytokines, including interleukin (IL)-6, IL-12p40, and tumor necrosis factor (TNF)-α, compared to Mpgm-ATCC- and Mpgm-R-infected macrophages. Additionally, our findings revealed metabolic changes in Mpgm-ATCC and two clinical strains (Mpgm-S and Mpgm-R) during infection; significant changes were observed in the mitochondrial respiration, extracellular acidification, and the oxygen consumption of BMDMs upon Mpgm-S infection. In summary, within the strains examined, Mpgm-S displayed greater virulence, triggered a heightened immune response, and induced more profound shifts in bioenergetic metabolism than Mpgm-ATCC and Mpgm-R. This study is the first to document distinct immune responses and metabolic reorganization following Mpgm infection. These findings lay a crucial foundation for further investigations into the pathogenesis of Mpgm.

Chemical modulation of SQSTM1/p62-mediated xenophagy that targets a broad range of pathogenic bacteria.

The N-degron pathway is a proteolytic system in which the N-terminal degrons (N-degrons) of proteins, such as arginine (Nt-Arg), induce the degradation of proteins and subcellular organelles via the ubiquitin-proteasome system (UPS) or macroautophagy/autophagy-lysosome system (hereafter autophagy). Here, we developed the chemical mimics of the N-degron Nt-Arg as a pharmaceutical means to induce targeted degradation of intracellular bacteria via autophagy, such as Salmonella enterica serovar Typhimurium (S. Typhimurium), Escherichia coli, and Streptococcus pyogenes as well as Mycobacterium tuberculosis (Mtb). Upon binding the ZZ domain of the autophagic cargo receptor SQSTM1/p62 (sequestosome 1), these chemicals induced the biogenesis and recruitment of autophagic membranes to intracellular bacteria via SQSTM1, leading to lysosomal degradation. The antimicrobial efficacy was independent of rapamycin-modulated core autophagic pathways and synergistic with the reduced production of inflammatory cytokines. In mice, these drugs exhibited antimicrobial efficacy for S. Typhimurium, Bacillus Calmette-Guérin (BCG), and Mtb as well as multidrug-resistant Mtb and inhibited the production of inflammatory cytokines. This dual mode of action in xenophagy and inflammation significantly protected mice from inflammatory lesions in the lungs and other tissues caused by all the tested bacterial strains. Our results suggest that the N-degron pathway provides a therapeutic target in host-directed therapeutics for a broad range of drug-resistant intracellular pathogens.Abbreviations: ATG: autophagy-related gene; BCG: Bacillus Calmette-Guérin; BMDMs: bone marrow-derived macrophages; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CFUs: colony-forming units; CXCL: C-X-C motif chemokine ligand; EGFP: enhanced green fluorescent protein; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; LIR: MAP1LC3/LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PB1: Phox and Bem1; SQSTM1/p62: sequestosome 1; S. Typhimurium: Salmonella enterica serovar Typhimurium; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; UBA: ubiquitin-associated.

Arginine-mediated gut microbiome remodeling promotes host pulmonary immune defense against nontuberculous mycobacterial infection.

Nontuberculous mycobacterial pulmonary diseases (NTM-PDs) are emerging as global health threats with issues of antibiotic resistance. Accumulating evidence suggests that the gut-lung axis may provide novel candidates for host-directed therapeutics against various infectious diseases. However, little is known about the gut-lung axis in the context of host protective immunity to identify new therapeutics for NTM-PDs. This study was performed to identify gut microbes and metabolites capable of conferring pulmonary immunity to NTM-PDs. Using metabolomics analysis of sera from NTM-PD patients and mouse models, we showed that the levels of l-arginine were decreased in sera from NTM-PD patients and NTM-infected mice. Oral administration of l-arginine significantly enhanced pulmonary antimicrobial activities with the expansion of IFN-γ-producing effector T cells and a shift to microbicidal (M1) macrophages in the lungs of NTM-PD model mice. Mice that received fecal microbiota transplants from l-arginine-treated mice showed increased protective host defense in the lungs against NTM-PD, whereas l-arginine-induced pulmonary host defense was attenuated in mice treated with antibiotics. Using 16S rRNA sequencing, we further showed that l-arginine administration resulted in enrichment of the gut microbiota composition with Bifidobacterium species. Notably, oral treatment with either Bifidobacterium pseudolongum or inosine enhanced antimicrobial pulmonary immune defense against NTM infection, even with multidrug-resistant clinical NTM strains. Our findings indicate that l-arginine-induced gut microbiota remodeling with enrichment of B. pseudolongum boosts pulmonary immune defense against NTM infection by driving the protective gut-lung axis in vivo.

Efficient differentiation of Mycobacterium abscessus complex isolates to the species level by a novel PCR-based variable-number tandem-repeat assay.

A novel duplex PCR method based on variable-number tandem-repeat targets to discriminate among Mycobacterium abscessus complex isolates was developed and evaluated in 85 clinical isolates. The assay accuracy was confirmed by a multiple-target sequence analysis. The duplex PCR assay is a one-step, reliable, and accurate assay for discriminating M. abscessus species.

Conversion of Mycobacterium smegmatis to a pathogenic phenotype via passage of epithelial cells during macrophage infection.

Mycobacteria encounter many different cells during infection within their hosts. Although alveolar epithelial cells play an essential role in host defense as the first cells to be challenged upon contact with mycobacteria, they may contribute to the acquisition of mycobacterial virulence by increasing the expression of virulence or adaptation factors prior to being ingested by macrophages on the side of pathogens. From this aspect, the enhanced virulence of nonpathogenic Mycobacterium smegmatis (MSM) passed through human alveolar A549 epithelial cells (A-MSM) was compared to the direct infection of MSM (D-MSM) in THP-1 macrophages and mouse models. The intracellular growth rate and cytotoxicity of A-MSM were significantly increased in THP-1 macrophages. In addition, compared to D-MSM, A-MSM induced relatively greater interleukin (IL)-1ß, IL-6, IL-8, IL-12, TNF-α, MIP-1α, and MCP-1 in THP-1 macrophages. As a next step, a more persistent A-MSM infection was observed in a murine infection model with the development of granulomatous inflammation. Finally, 58 genes induced specifically in A-MSM were partially identified by differential expression using a customized amplification library. These gene expressions were simultaneously maintained in THP-1 infection but no changes were observed in D-MSM. Bioinformatic analysis revealed that these genes are involved mainly in bacterial metabolism including energy production and conversion, carbohydrate, amino acid, and lipid transport, and metabolisms. Conclusively, alveolar epithelial cells promoted the conversion of MSM to the virulent phenotype prior to encountering macrophages by activating the genes required for intracellular survival and presenting its pathogenicity.

Disseminated infection due to Mycobacterium avium subsp. avium in an Asian elephant (Elephas maximus).

A disseminated infection caused by Mycobacterium avium subspecies avium (MAA) was diagnosed in a 57-yr-old male Asian elephant (Elephas maximus) housed at the Seoul Zoo, Gyeonggi, Republic of Korea. An apparent granulomatous inflammation with central caseous necrosis was evident in the lung sections. To confirm mycobacterial infection, polymerase chain reaction-restriction enzyme polymorphism analysis (PCR-RFLP) of the rpoB and hsp65 genes was performed from multiple organs and cultured bacteria. The PCR-RFLP revealed a M. avium subspecies. MAA was identified by multiplex PCR for detection of IS901 and IS1311. Thus, it is believed that MAA caused the disseminated infection in this case. Although the source of infection was not determined, the elephant may have become infected through contamination of soil and feed by free-living birds infected with MAA. This is the first reported case of disseminated infection due to MAA in a captive elephant in the Republic of Korea.

Tuberculosis: A persistent unpleasant neighbour of humans.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, has long been an unpleasant neighbour of humans. Following transmission of the bacterium from patients with active infection, new hosts do not immediately develop symptoms, as M. tuberculosis initially remains quiescent. However, it is eventually triggered, leading to the infection of other individuals. Humans are the exclusive host, and the rapid proliferation of the human population worldwide along with increasing globalisation have contributed to the pathogen's persistence, as have the survival strategies employed by M. tuberculosis, especially its resistance to several antimicrobials. Defeating this enemy will require novel approaches.