Intracellular iron accumulation facilitates mycobacterial infection in old mouse macrophages.

Aging has a significant impact on the immune system, leading to a gradual decline in immune function and changes in the body's ability to respond to bacterial infections. Non-tuberculous mycobacteria (NTM), also known as atypical mycobacteria or environmental mycobacteria, are commonly found in soil, water, and various environmental sources. While many NTM species are considered opportunistic pathogens, some can cause significant infections, particularly in individuals with compromised immune systems, such as older individuals. When mycobacteria enter the body, macrophages are among the first immune cells to encounter them and attempt to engulf mycobacteria through a process called phagocytosis. Some NTM species, including Mycobacterium avium (M. avium) can survive and replicate within macrophages. However, little is known about the interaction between NTM and macrophages in older individuals. In this study, we investigated the response of bone marrow-derived macrophage (BMMs) isolated from young (5 months) and old (25 months) mice to M. avium serotype 4, one of the main NTM species in patients with pulmonary NTM diseases. Our results demonstrated that BMMs from old mice have an increased level of intracellular iron and are more susceptible to M. avium serotype 4 infection compared to BMMs from young mice. The whole-cell proteomic analysis indicated a dysregulated expression of iron homeostasis-associated proteins in old BMMs regardless of mycobacterial infection. Deferoxamine, an iron chelator, significantly rescued mycobacterial killing and phagolysosome maturation in BMMs from old mice. Therefore, our data for the first time indicate that an intracellular iron accumulation improves NTM survival within macrophages from old mice and suggest a potential application of iron-chelating drugs as a host-directed therapy for pulmonary NTM infection in older individuals.

Treatment of the Less Common Nontuberculous Mycobacterial Pulmonary Disease.

Nontuberculous mycobacterial pulmonary disease caused by the less common nontuberculous mycobacteria have distinct features depending on the species. Diagnostic evaluation follows the established criteria for all nontuberculous mycobacteria, but with certain qualifications given species-specific and regional differences in pathogenicity. Clinicians should first institute nonpharmacologic management and evaluate clinical, radiologic, and microbiologic factors in the decision regarding antimycobacterial therapy. Treatment is challenging, and evidence-based recommendations are limited for most species. Drug susceptibility testing is used to help with regimen selection; however, this approach is imperfect given the uncertain correlation between in vitro activity and clinical response for most drugs.

Spatial Epidemiologic Analysis and Risk Factors for Nontuberculous Mycobacteria Infections, Missouri, USA, 2008-2019.

Nontuberculous mycobacteria (NTM) infections are caused by environmental exposure. We describe spatial distribution of NTM infections and associations with sociodemographic factors and flooding in Missouri, USA. Our retrospective analysis of mycobacterial cultures reported to the Missouri Department of Health and Social Services surveillance system during January 1, 2008-December 31, 2019, detected geographic clusters of infection. Multilevel Poisson regression quantified small-area geographic variations and identified characteristics associated with risk for infection. Median county-level NTM infection rate was 66.33 (interquartile range 51-91)/100,000 persons. Risk of clustering was significantly higher in rural areas (rate ratio 2.82, 95% CI 1.90-4.19) and in counties with >5 floodings per year versus no flooding (rate ratio 1.38, 95% CI 1.26-1.52). Higher risk for NTM infection was associated with older age, rurality, and more flooding. Clinicians and public health professionals should be aware of increased risk for NTM infections, especially in similar environments.

The roles of neutrophils in non-tuberculous mycobacterial pulmonary disease.

Non-tuberculous Mycobacterial Pulmonary Disease (NTM-PD) is an increasingly recognised global health issue. Studies have suggested that neutrophils may play an important role in controlling NTM infection and contribute to protective immune responses within the early phase of infection. However, these cells are also adversely associated with disease progression and exacerbation and can contribute to pathology, for example in the development of bronchiectasis. In this review, we discuss the key findings and latest evidence regarding the diverse functions of neutrophils in NTM infection. First, we focus on studies that implicate neutrophils in the early response to NTM infection and the evidence reporting neutrophils' capability to kill NTM. Next, we present an overview of the positive and negative effects that characterise the bidirectional relationship between neutrophils and adaptive immunity. We consider the pathological role of neutrophils in driving the clinical phenotype of NTM-PD including bronchiectasis. Finally, we highlight the current promising treatments in development targeting neutrophils in airways diseases. Clearly, more insights on the roles of neutrophils in NTM-PD are needed in order to inform both preventative strategies and host-directed therapy for these important infections.

Colloidal silver against macrophage infections and biofilms of atypical mycobacteria.

Skin and soft tissue infection (SSTI) caused by atypical mycobacteria such as Mycobacterium abscessus and Mycobacterium avium intracellulare complex (MAIC) have increased in recent years. Current therapeutic options are limited, and hence new and better therapies are urgently required. Colloidal Silver (CS) has been identified for its widespread antibacterial properties and silver-impregnated dressings have been used for SSTIs caused by various pathogens. The efficacy of Green Synthesized Colloidal Silver (GSCS) was investigated for bacterial growth inhibition (BGI) using a microdilution method and minimum biofilm eradication concentration (MBEC) using resazurin assay and confocal scanning laser microscopy (CSLM) of M. abscessus (n = 5) and MAIC (n = 5). The antibacterial effect of GSCS against M. abscessus infected macrophages was also evaluated. The in vitro cytotoxicity of GSCS on a human keratinocyte cell line (HaCaT) and neonatal foreskin fibroblasts was analyzed by the crystal violet proliferation assay. Average BGI and MBEC of GSCS varied between 0.7 and 22 ppm for M. abscessus and MAIC. The concentration of 3 ppm reduced M. abscessus-infection in macrophages significantly. GSCS was not cytotoxic to HaCaT and neonatal foreskin fibroblast cells at concentrations < 3 ppm up to 2 h exposure time. GSCS therefore, has the potential for topical application against atypical mycobacterial SSTI.

Outcomes of HIV-positive patients with non-tuberculous mycobacteria positive culture who received anti-tuberculous treatment in Botswana: Implications of using diagnostic algorithms without non-tuberculous mycobacteria.

BACKGROUND: Patients with non-tuberculous mycobacteria (NTM) or Mycobacterium tuberculosis (MTB) pulmonary disease may have similar clinical presentation. The potential for misdiagnosis and inappropriate treatment exists in settings with limited testing capacity for Xpert® MTB/RIF (Xpert), phenotypic culture and NTM speciation. We describe treatment outcomes among people living with HIV (PLHIV) who received anti-tuberculosis treatment and were found to have NTM or MTB positive sputum cultures. METHODS: PLHIV attending one of the 22 participating HIV clinics, who screened positive for ≥1 tuberculosis (TB) symptoms (cough, fever, night sweats, or weight loss) were asked to submit sputa for culture and speciation from August 2012 to November 2014. The national intensified TB case finding algorithms were followed: initially symptomatic patients were evaluated by testing sputum samples using a smear (smear-based TB diagnostic algorithm) and, after GeneXpert instruments were installed, by testing with Xpert (Xpert-based TB diagnostic algorithm). Within the study period, TB diagnostic algorithms used for MTB did not include screening, diagnosis, and management of NTM. Despite MTB negative culture, some symptomatic patients, including those with NTM positive culture, received empirical anti-TB treatment at the discretion of treating clinicians. Per the World Health Organization treatment outcomes classification: died, treatment failure or loss-to-follow-up were classified as unfavorable (unsuccessful) outcome; cured and treatment completed were classified as favorable (successful) outcome. Empiric treatment was defined as initiating treatment without or before receiving a test result indicating MTB. We compare treatment outcomes and characteristics among patients with NTM or MTB positive culture who received anti-TB treatment. RESULTS: Among 314 PLHIV, who were found co-infected with TB, 146 cases had microbiological evidence; and for 131/146 MTB positive cultures were reported. One-hundred fifty-two of the 314 were clinically diagnosed with TB and treated empirically. Among those empirically treated for TB, 36/152 had culture results positive for NTM, and another 43/152 had culture results positive for MTB, reported after patients received empirical anti-TB treatment. Overall, MTB positive culture results were reported for 174 (131 plus 43) patients. Treatment outcomes were available for 32/36 NTM and 139/174 MTB; unfavorable outcomes were 12.5% and 8.7% for NTM and MTB, respectively, p = 0.514, respectively. For 34/36 tested NTM patients, all Xpert results indicated 'no MTB'. Among patients who initially received empiric anti-TB treatment and ultimately were found to have MTB positive culture, the unfavorable outcome was 11.8% (4/34), compared to 12.5% (4/32) of patients with NTM positive culture, Fisher's exact test p = 1.00. CONCLUSIONS: While the higher unfavorable outcome was non statistically significant, the impact of inappropriate treatment among NTM patients should not be overlooked. Our findings suggest that Xpert has the potential to rapidly rule-out NTM and avoid sub-optimal treatment; further research is needed to evaluate such potential.

Of tuberculosis and non-tuberculous mycobacterial infections - a comparative analysis of epidemiology, diagnosis and treatment.

Pulmonary diseases due to mycobacteria cause significant morbidity and mortality to human health. In addition to tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), recent epidemiological studies have shown the emergence of non-tuberculous mycobacteria (NTM) species in causing lung diseases in humans. Although more than 170 NTM species are present in various environmental niches, only a handful, primarily Mycobacterium avium complex and M. abscessus, have been implicated in pulmonary disease. While TB is transmitted through inhalation of aerosol droplets containing Mtb, generated by patients with symptomatic disease, NTM disease is mostly disseminated through aerosols originated from the environment. However, following inhalation, both Mtb and NTM are phagocytosed by alveolar macrophages in the lungs. Subsequently, various immune cells are recruited from the circulation to the site of infection, which leads to granuloma formation. Although the pathophysiology of TB and NTM diseases share several fundamental cellular and molecular events, the host-susceptibility to Mtb and NTM infections are different. Striking differences also exist in the disease presentation between TB and NTM cases. While NTM disease is primarily associated with bronchiectasis, this condition is rarely a predisposing factor for TB. Similarly, in Human Immunodeficiency Virus (HIV)-infected individuals, NTM disease presents as disseminated, extrapulmonary form rather than as a miliary, pulmonary disease, which is seen in Mtb infection. The diagnostic modalities for TB, including molecular diagnosis and drug-susceptibility testing (DST), are more advanced and possess a higher rate of sensitivity and specificity, compared to the tools available for NTM infections. In general, drug-sensitive TB is effectively treated with a standard multi-drug regimen containing well-defined first- and second-line antibiotics. However, the treatment of drug-resistant TB requires the additional, newer class of antibiotics in combination with or without the first and second-line drugs. In contrast, the NTM species display significant heterogeneity in their susceptibility to standard anti-TB drugs. Thus, the treatment for NTM diseases usually involves the use of macrolides and injectable aminoglycosides. Although well-established international guidelines are available, treatment of NTM disease is mostly empirical and not entirely successful. In general, the treatment duration is much longer for NTM diseases, compared to TB, and resection surgery of affected organ(s) is part of treatment for patients with NTM diseases that do not respond to the antibiotics treatment. Here, we discuss the epidemiology, diagnosis, and treatment modalities available for TB and NTM diseases of humans.

Whole genome sequencing of Nontuberculous Mycobacterium (NTM) isolates from sputum specimens of co-habiting patients with NTM pulmonary disease and NTM isolates from their environment.

BACKGROUND: Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is thought to be caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis. RESULTS: We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting patients with NTM-PD who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium-PD, a couple with M. intracellulare-PD, and a second couple, one of whom was infected with M. intracellulare and the other of whom was infected with M. abscessus. Whole genome sequencing was performed using patients' NTM isolates as well as environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs). By comparison with the genetic distances among 78 publicly available NTM genomes, NTM isolates derived from the two pairs of patients infected with the same NTM species were not closely related to each other. In phylogenetic analysis, the NTM isolates from patients with M. avium-PD clustered with isolates from different environmental sources. CONCLUSIONS: In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

Exploring how structural changes to new Licarin A derivatives effects their bioactive properties against rapid growing mycobacteria and biofilm formation.

Several species of rapidly growing mycobacteria (RGM) have been associated with biofilms in areas such as biomedical devices, water distribution systems, cosmetic surgery, and catheter-related blood infections. Biofilms which exhibit antimicrobial resistance such as those formed by the genus Mycobacterium pose a significant risk to health and are of particular interest to researchers. Licarin A (a neolignan found in numerous plant species e.g. nutmeg) has been reported to show a wide range of biological actions including anti-inflammatory, antioxidant, and antibacterial properties. The aim of this study was to prepare a set of Licarin A derivatives and investigate the impact of specific structural changes on its antimycobacterial ability, and its effect on the biofilm formation of RGM species. Initially, the phenolic sub-unit and alkenyl side chain of Licarin A were modified to create derivatives with a higher partition coefficient; as the activity of a compound against mycobacteria seems to be strongly influenced by its hydrophobicity. Further, polar groups were inserted into the side chain to change the hydrophilic-lipophilic profile of the molecules. Results showed variability in the susceptibility profile of mycobacteria against the Licarin A derivatives under analysis. A number of the derivatives showed significant inhibitory activity of planktonic growth of the three strains of mycobacteria used, with even lower MIC values than those observed with reference drugs and Licarin A itself. Cytotoxicity assays showed they also have low toxicity, confirming that structural modifications to the Licarin A have made improvements to its antimycobacterial properties.

Serum Anti-interferon-γ Autoantibody Titer as a Potential Biomarker of Disseminated Non-tuberculous Mycobacterial Infection.

PURPOSE: In the past decade, the relationship between naturally occurring interferon-γ-neutralizing autoantibodies (IFNγ-Ab) and disseminated non-tuberculous mycobacteria (NTM) infection has been established. Furthermore, immune suppressive therapy aimed at the suppression of antibody production has shown efficacy as a supportive treatment. However, the nature of antibody behavior and antibody titer during the course of this disease, as well as the pathophysiological significance of IFNγ-Ab, has not yet been fully elucidated. METHODS: Thirteen Japanese subjects suffering from disseminated NTM (dNTM) infection with IFNγ-Ab were evaluated. The fluctuation of IFNγ-Ab titer and the neutralizing capacity against IFN-γ during the course of the disease were retrospectively analyzed. IFNγ-Ab titers in the sera were quantified using an enzyme-linked immunosorbent assay; neutralizing capacity was evaluated via flow cytometry. RESULTS: Serum antibody titers were not constant during the treatment period and varied over the course of the disease. The antibody titer decreased when the disease was improved by anti-mycobacterial treatment (p < 0.01) and increased as the disease progressed (p < 0.05). Even after the antibody titer decreased, the neutralizing capacity against IFN-γ was maintained by individual sera. CONCLUSIONS: Despite the improvement in the pathological condition via treatment, the patients' sera maintained neutralizing capacity against IFN-γ. Antibody titer fluctuated over the course of the disease and exhibited potential as a biomarker for judgment of the disease state.

Retrospective analysis of patients with non-tuberculous mycobacteria from a primary hospital in Southeast China.

To achieve a comprehensive understanding of the characteristics of patients with non-tuberculous mycobacteria (NTM), patients with NTM between January 2016 and June 2019 were recruited from a primary hospital. NTM were identified based on the MBP64 protein assay. The clinical records and laboratory assay results were retrospectively reviewed. A total of 204 patients with NTM were included in the final analysis. The patients with multiple isolations were more likely accompanied with chronic obstructive pulmonary disease (COPD) (p = 0.029) and arthritis (p = 0.049), but showed a lower percentage of positive T-spot results (p = 0.022). In addition, patients with multiple isolations showed a higher rate of positive acid-fast staining results and their symptom duration was more likely longer than 30 days (p = 0.019). Patients with a positive response in T-spot assay showed a higher proportion of nodular manifestation on computed tomography (CT) than those with a negative response. Compared with male patients with NTM, female patients showed lower rates of positive acid-fast staining results (p = 0.03), but were more likely accompanied with COPD (p < 0.0001). The positive acid-fast staining results were closely associated with pulmonary cavities and tuberculosis antibody. Patients with different NTM isolation frequencies were closely associated with coexisting diseases and examination results.

Nontuberculous Mycobacteria Show Differential Infectivity and Use Phospholipids to Antagonize LL-37.

Comparisons of infectivity among the clinically important nontuberculous mycobacteria (NTM) species have not been explored in great depth. Rapid-growing mycobacteria, including Mycobacterium abscessus and M. porcinum, can cause indolent but progressive lung disease. Slow-growing members of the M. avium complex are the most common group of NTM to cause lung disease, and molecular approaches can now distinguish between several distinct species of M. avium complex including M. intracellulare, M. avium, M. marseillense, and M. chimaera. Differential infectivity among these NTM species may, in part, account for differences in clinical outcomes and response to treatment; thus, knowing the relative infectivity of particular isolates could increase prognostication accuracy and enhance personalized treatment. Using human macrophages, we investigated the infectivity and virulence of nine NTM species, as well as multiple isolates of the same species. We also assessed their capacity to evade killing by the antibacterial peptide cathelicidin (LL-37). We discovered that the ability of different NTM species to infect macrophages varied among the species and among isolates of the same species. Our biochemical assays implicate modified phospholipids, which may include a phosphatidylinositol or cardiolipin backbone, as candidate antagonists of LL-37 antibacterial activity. The high variation in infectivity and virulence of NTM strains suggests that more detailed microbiological and biochemical characterizations are necessary to increase our knowledge of NTM pathogenesis.

Nontuberculous mycobacterial and Nocardia infections mimicking pulmonary tuberculosis: a retrospective study of a general hospital patient population in China.

Purpose. In this study, we differentiated between tuberculosis (TB) and infections caused by nontuberculous mycobacteria (NTM) or Nocardia in a tertiary general hospital in China. Differences in clinical manifestations and factors between respiratory infections associated with these organisms were also investigated.Methodology. A retrospective analysis was conducted for suspected pulmonary TB patients with positive bacterial culture results under treatment at Wangjing Hospital, a tertiary general hospital, between January 2014 and June 2017. Sputum samples were submitted for liquid culture and species identification by mass spectrometry.Results. Between January 2014 and June 2017, a total of 3981 suspected TB cases were analysed, of which 151 (3.8 %) exhibited positive mycobacterial culture results. Using mass spectrometry, the 151 isolates were classified into three groups: Mycobacterium tuberculosis (MTB) (n=112; 74.2 %), NTM (n=21 13.9 %) and Nocardia (n=18; 11.9 %). The NTM and Nocardia prevalence rates were significantly higher amongst elderly patients [aged ≥65 years; odds ratio (95 % confidence interval): 3.89 (1.05-14.38) for NTM; odds ratio (95 % confidence interval): 5.10 (1.09-23.91) for Nocardia]. In addition, treatment with immunosuppressive therapy [odds ratio (95 % confidence interval): 3.92 (1.16-13.27)] was identified as a risk factor for Nocardia infection in these patients.Conclusion. Our results demonstrated that a quarter of culture-positive 'suspected TB patients' harboured NTM or Nocardia infections. Notably, nearly all patients with non-TB infections presented with clinical syndromes mimicking pulmonary TB. Individuals receiving immunosuppressive therapy were at greater risk of acquiring Nocardia infections.

The influence of non-tuberculous mycobacteria on the interaction of opportunistic microorganisms with erythrocytes.

Getting into a weakened organism, non-tuberculosis mycobacteria (NTMB) contact not only with the cells of the microorganism but also with the microflora of the human body; however, these interactions are poorly understood. The purpose of this work was to study the effect of NTMB supernatants on the properties of conditionally pathogenic microorganisms in their interaction with red blood cells. We used strains of non-tuberculous mycobacteria Mycobacterium iranicum and M. rutilum, as well as strains of Staphylococcus epidermidis and Escherichia coli. Using the fluorescence in situ hybridisation (FISH) method, the processes of adhesion to the surface of erythrocytes and the intra-erythrocyte penetration of cells of S. epidermidis and E. coli under the influence of NTMB supernatants were studied. To study changes in the haemoglobin molecule under the action of the supernatants of NTMB, spectral analysis was performed. Statistical processing was performed using STATISTIKA 6.0. The results showed that the supernatants of M. iranicum and M. rutilum increased the adhesion of conditionally pathogenic bacteria with a low level of AntiHbA to the surface of erythrocytes by 3-4 times. It also increased the intra-erythrocyte penetration of cells of S. epidermidis and E. coli relative to the control values. As a result of studying the haemoglobin spectrum of erythrocytes under the influence of M. iranicum, a decrease in the optical density values of oxyhaemoglobin by a factor of 2 relative to the values in the control sample was noted. Thus, the supernatants of NTMB have a multidirectional effect on the interaction of opportunistic microorganisms with erythrocytes, increasing the adhesive activity and the penetration of cells into the erythrocytes, as well as reducing the optical density of oxyhaemoglobin.

An improved simple method for the identification of Mycobacteria by MALDI-TOF MS (Matrix-Assisted Laser Desorption- Ionization mass spectrometry).

The aim of this study was to establish a simple method for the rapid identification of Mycobacteria species by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass spectrometry) using the Bruker MALDI-TOF Biotyper system (Bruker Daltonik, Bremen, Germany). A multicentre, prospective, and single blind study was performed in three European Hospitals, two Spanish and one UK hospital from May to August 2018. The BD BACTEC MGIT (Becton Dickinson, Berks, UK) liquid culture system was used in all three centres for the growth of Mycobacteria. When signal positive, tubes were removed from the analyser and in addition to standard laboratory procedures were subcultured on blood agar plates for MALDI-TOF analysis. Plates were incubated aerobically for 1 to 7 days at 37 °C and inspected every day. Once any growth was visible, it was transferred to the steel target plate, overlaid with 1 µl of neat formic acid and 1 µl HCCA matrix (alpha hydroxyl 4 cinnamic acid), and analysed in a Bruker Biotyper MALDI-TOF. Results given by MALDI-TOF were compared with the reference methods used for identification in the different centres. At two Spanish hospitals, identification by MALDI-TOF was only attempted on presumptive non-tuberculosis mycobacteria (NTM) and the results were initially compared with the results obtained by a commercial reverse hybridisation assay, GenoType CM/AS (Hain Lifescience, Tübingen, Germany). At the UK Hospital, identification of any presumptive mycobacteria was attempted and compared with the results obtained by whole genome sequencing (WGS). Overall in 142/167 (85%) of cases the identifications obtained were concordant; all Mycobacterium tuberculosis (MTB) isolates 43/43 (100%), 57/76 (75%) of the rapid growing nontuberculous mycobacteria (NTM), and 42/48 (85%) slow growing NTM tested were identified correctly. We report a new, easy, cheap and quick method for isolation and identification of Mycobacterium spp. without the need for additional steps or equipment and this method is in routine used in all three centres.

Assessment of Interferon-Gamma Release Assay in Patients with Non-Tuberculous Mycobacteria Pulmonary Disease.

BACKGROUND: Interferon-gamma release assay (T-SPOT.TB) has the theoretical possibility of discriminating TB from most non-tuberculous mycobacteria (NTM) infections, but there are limited reports on the use of T-SPOT.TB for diseases due to NTM in high TB burden country. The aim of the present study was to assess the utility of T-SPOT.TB in patients with NTM pulmonary disease. METHODS: Clinical parameters and laboratory characteristics of patients with NTM pulmonary disease between July 2011 and Jan 2017 were investigated retrospectively and comprehensively reviewed. RESULTS: A total of 127 patients with NTM pulmonary disease were retrospectively reviewed. Seven NTM species were isolated from 115 patients, and the most common species were M. intracellulare (48.7%, 56/115) and M. abscessus (34.8%, 40/115). NTM isolates were mainly prevalent in people aged 50 years or older (73.0%). The overall positive rate of T-SPOT.TB test was 29.6% (24/81). In patients infected with NTM sharing the RD1 region of Mycobacterium tuberculosis (M. TB), 50% (3/6) were positive in the T-SPOT.TB test, whereas 28.0% (21/75) was positive in the group with NTM not sharing the RD1 region of M. TB. No significant difference was detected in the positive rate of T-SPOT.TB between definite (28.3%, 15/53) and probable disease (32.1%, 9/28). CONCLUSIONS: Our data indicated a relatively high positive rate of T-SPOT.TB test in patients infected with NTM not sharing the RD1 region of M. TB. Thus, T-SPOT.TB test displays a limited ability in differentiating TB infection from NTM disease in a high TB burden country.

Mycobacterium indicus pranii therapy induces tumor regression in MyD88- and TLR2-dependent manner.

OBJECTIVES: Mycobacterium indicus pranii (MIP) is an atypical mycobacterium species with potent antitumor efficacy. Macrophages and dendritic cells (DCs) are antigen-presenting cells, playing key roles in the activation of antitumor immunity. We have previously shown the potent activation of macrophages and DCs by MIP, which is mediated by MyD88-TLR2 signaling axis. In the present study, we further examined the role of MyD88 and TLR2 in MIP-mediated tumor regression. RESULTS: Wild-type and MyD88-/- mice were implanted with B16F10 tumor cells, treated with MIP or phosphate-buffered saline (PBS) and monitored for tumor growth. As expected, MIP therapy led to significant tumor regression in wild-type mice. However, antitumor efficacy of MIP was lost in MyD88-/- animals. Both PBS-treated (control) and MIP-treated MyD88-/- mice developed tumors with comparable volume. Since MyD88 relays TLR engagement signals, we analyzed the antitumor efficacy of MIP in TLR2-/- and TLR4-/- mice. It was observed that MIP therapy reduced tumor burden in wild-type and TLR4-/- mice but not in TLR2-/- mice. Tumor volume in MIP-treated TLR2-/- mice were comparable with those in PBS-treated wild-type animals. These results implicated the MyD88-TLR2 signaling axis in the antitumor efficacy of MIP.

Aggregation of Nontuberculous Mycobacteria Is Regulated by Carbon-Nitrogen Balance.

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens that colonize household water systems and cause chronic lung infections in susceptible patients. The ability of NTM to form surface-attached biofilms in the nonhost environment and corded aggregates in vivo is important to their ability to persist in both contexts. Underlying the development of these multicellular structures is the capacity of mycobacterial cells to adhere to one another. Unlike most other bacteria, NTM spontaneously and constitutively aggregate in vitro, hindering our ability to understand the transition between planktonic and aggregated cells. While culturing a model NTM, Mycobacterium smegmatis, in rich medium, we fortuitously discovered that planktonic cells accumulate after ∼3 days of growth. By providing selective pressure for bacteria that disperse earlier, we isolated a strain with two mutations in the oligopeptide permease operon (opp). A mutant lacking the opp operon (Δopp) disperses earlier than wild type (WT) due to a defect in nutrient uptake. Experiments with WT M. smegmatis revealed that growth as aggregates is favored when carbon is replete, but under conditions of low available carbon relative to available nitrogen, M. smegmatis grows as planktonic cells. By adjusting carbon and nitrogen sources in defined medium, we tuned the cellular C/N ratio such that M. smegmatis grows either as aggregates or as planktonic cells. C/N-mediated aggregation regulation is widespread among NTM with the possible exception of rough-colony Mycobacterium abscessus isolates. Altogether, we show that NTM aggregation is a controlled process that is governed by the relative availability of carbon and nitrogen for metabolism.IMPORTANCE Free-living bacteria can assemble into multicellular structures called biofilms. Biofilms help bacteria tolerate multiple stresses, including antibiotics and the host immune system. Nontuberculous mycobacteria are a group of emerging opportunistic pathogens that utilize biofilms to adhere to household plumbing and showerheads and to avoid phagocytosis by host immune cells. Typically, bacteria regulate biofilm formation by controlling expression of adhesive structures to attach to surfaces and other bacterial cells. Mycobacteria harbor a unique cell wall built chiefly of long-chain mycolic acids that confers hydrophobicity and has been thought to cause constitutive aggregation in liquid media. Here we show that aggregation is instead a regulated process dictated by the balance of available carbon and nitrogen. Understanding that mycobacteria utilize metabolic cues to regulate the transition between planktonic and aggregated cells reveals an inroad to controlling biofilm formation through targeted therapeutics.