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Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pidot et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rationale: Nontuberculous mycobacteria (NTM) are a diverse group of environmental organisms that infrequently cause human disease. Understanding of the epidemiologic and clinical characteristics associated with NTM disease is needed to refine diagnostic and treatment strategies, particularly among the less commonly isolated species.Objectives: To improve knowledge of geographic variance of NTM species, to correlate detailed clinical information with isolation of specific NTM, and to examine the decision to treat and outcomes for specific NTM.Methods: Mycobacterial cultures submitted to the University of Washington mycobacterial laboratory from 1998 to 2011 were examined. We report isolation frequency and demographic information from all samples with clinical variables. We also examined treatment decisions and outcomes in a subset of patients with Mycobacterium abscessus complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium porcinum, and Mycobacterium xenopi.Results: Cultures of NTM were available from 3,470 patients, 937 of whom had clinical data available. When we compared patients born within or outside Washington State, we found that the mycobacterial species frequency varied. Among 168 patients with one of the studied environmental mycobacteria, 72% had major comorbid conditions. Bronchiectasis was common among patients with pulmonary isolation of any NTM, including those with nonpathogenic M. gordonae. Although mortality was high (37%), few deaths were directly attributable to mycobacterial infection. Among 56 patients who met American Thoracic Society criteria for NTM lung disease, 22 were treated, and 19 of those had M. abscessus complex and M. kansasii. The treatment regimens used tended to follow published guidelines.Conclusions: Isolation of NTM varied by geographic region of origin and location within Washington State. Several clinical risk factors were specific to individual species. Comorbid conditions were common in patients with and without mycobacterial disease. Among patients with one of the studied organisms, there was a high mortality rate more frequently related to comorbid conditions than to mycobacterial disease. [ABSTRACT FROM AUTHOR]

Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology-including the tendency to clump, and "differential" culturability-and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique., (© 2021. The Author(s).)

Abstract: Cured leprosy patients have special physical conditions, which could pose challenges for safety and immunogenicity after immunization. We performed an observational clinical study aimed to identify the safety and immunogenicity of influenza vaccine in cured leprosy patients. A total of 65 participants from a leprosarium were recruited into leprosy cured group or control group, and received a 0.5 ml dose of the inactivated split-virion trivalent influenza vaccine and a follow-up 28 days proactive observation of any adverse events. Hemagglutination and hemagglutination inhibition test was performed to evaluate serum antibody titer, flow cytometry was conducted to screen of cytokines level. The total rate of reactogenicity was 0.0% [0/41] in leprosy cured group and 37.5% [9/24] in control group. The seroconversion rate for H1N1 was difference between leprosy cured group and control group (41.83% vs 79.17%, P = .0082), but not for H3N2 (34.25% vs 50.00%, P = .4468). At day 0, leprosy cured group have relatively high concentration of interleukin-6, interleukin-10, tumor necrosis factor, interferon-γ, and interleukin-17 compared to control group. The interleukin-2 concentration increased 2 weeks after vaccination compared to pre-vaccination in leprosy cured group, but declined in control group (0.92 pg/ml vs -0.02 pg/ml, P = .0147). Leprosy cured group showed a more rapid down-regulation of interleukin-6 when influenza virus was challenged compared to control group (-144.38 pg/ml vs -11.52 pg/ml, P < .0001). Subgroup analysis revealed that the immunization administration declined interleukin-17 concentration in Tuberculoid type subgroup, but not in Lepromatous type subgroup or control group. Clinically cured leprosy patients are relatively safe for influenza vaccine. Leprosy cured patient have immune deficit in producing antibody. Interleukin-6 and interleukin-17 were 2 sensitive indicators in immune response for leprosy affected patients. The identification of indicators might be help management of leprosy and used as predictive markers in leprosy early symptom monitoring., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Abstract: The experience and causes of pain in patients with nontuberculous mycobacterial pulmonary disease (NTM-PD) have not been clarified.This study aimed to determine the prevalence and severity of bodily pain (BP) in patients with NTM-PD. We also investigated the clinical indicators that contribute to pain.We used a retrospective cross-sectional study design. The participants were 114 NTM-PD patients (109 women) with a mean age of 65 years. The prevalence and severity of pain were measured using 2 items from the 36-Item Short Form Survey version 2 (SF-36), and the BP score was calculated. Functional limitation due to dyspnea was quantified using the Modified Medical Research Council Dyspnea Scale (mMRC), depression was assessed using the Center for Epidemiological Studies Depression Scale (CES-D), sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI); health-related quality of life was assessed using the Leicester Cough Questionnaire (LCQ), and exercise tolerance was measured using the Incremental Shuttle Walk Test (ISWT).Pain was reported by 70.2% of the patients (n = 80), and of these, 35.7% (n = 25) reported moderate to very severe pain. NTM-PD patients with high levels of pain had significantly higher scores on the mMRC, CES-D, and PSQI scores, and significantly lower performance on the ISWT and LCQ. Multiple regression analysis identified ISWT, CES-D, and PSQI as independent factors that affected BP scores.Our findings suggest that pain significantly impacts daily life associated with reduced exercise tolerance, the presence of depressive symptoms, and poor sleep quality in patients with NTM-PD., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background/purpose: The World Health Organization has recommended commercial urine-sourced lipoarabinomannan (LAM) detection as a tool for screening HIV patients with suspected TB, but more sensitive immunodetection assays would help to identify HIV-negative TB patients. Here, we aimed to develop novel rabbit monoclonal antibodies (mAbs) against LAM for immunodetection purposes., Methods: Rabbits were immunized with cell-wall components from the Mycobacterium tuberculosis (Mtb) H37Rv strain. An immune single-chain fragment variable (scFv) phage display library was generated. The scFv mAbs to LAM were identified through ELISA screening. The light and heavy chain variable region genes from the selected clones were sequenced. Vectors containing the full-length light and heavy chains were constructed and co-expressed in 293 T cells to generate whole IgG antibodies. The performances and binding characteristics of the mAbs against purified LAM from M.tb H37Rv, multiple mycobacteria species (M.tb H37Rv, M. bovis and non-tuberculous mycobacteria (NTM) strains), and mycobacteria clinical isolates (Mtb and NTM isolates) were determined using various immunoassay methods., Results: We obtained five rabbit mAbs against LAM, four of which had high sensitivities (100 pg/ml) and affinities (1.16-1.73 × 10 -9  M) toward LAM. They reacted with M.tb H37Rv, M. bovis, and slow-growing NTM, but not with rapid-growing NTM. Similar results were obtained with mycobacterium isolates, where 96% of the Mtb isolates and 90% of the M. avium-intracellulare isolates were successfully identified., Conclusion: The novel rabbit LAM-specific mAbs performed well at recognizing LAM from slow-growing pathogenic mycobacteria, which support their future clinical application., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro Here, we show that in vivo , mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis , M. marinum , or M. leprae In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo , translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals. IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis ., (Copyright © 2021 van der Niet et al.)

The genus Mycobacterium includes a wide range of species of both slow and rapid growth under major pathogens, opportunists, and saprophytes. The number and combination of sigma factors are extremely diversified among various species of Mycobacterium. The comparative genome analysis illustrates that SigC, SigD, SigG, SigH, SigK and SigI are dominant among the pathogens. Evolutionary analysis using Bayesian inference on 16S rRNA and MLST-based phylogeny using 14 housekeeping genes distinctly differentiate the slow-growing Mycobacterium from fast growers and segregate pathogens from opportunists and saprophytes. Based on the similarity coefficient upon the allotment of sigma factors in mycobacterial species through UPGMA dendrogram analysis, it is apparent that the pathogens are grouped separately following the similar trend observed from the evolutionary approach. Predominance of a set of sigma factors particularly the pathogenic Mycobacterium co-exists with the distribution of six well-known virulence factors of Mycobacterium (PhoP, PcaA, FbpA, Mce1B, KatG and PE&#95;PGRS30). The pathogenicity responsible sigma factors elicit close resemblance with few notable characters of the known virulence factors. Thus the analysis renders that the distribution of sigma factors of different species of Mycobacterium can be a potential tool to predict their pathogenicity index.

The mycobacterial cell wall glycolipid trehalose-6,6-dimycolate (TDM) activates macrophages through the C-type lectin receptor MINCLE. Regulation of innate immune cells relies on miRNAs, which may be exploited by mycobacteria to survive and replicate in macrophages. Here, we have used macrophages deficient in the microprocessor component DGCR8 to investigate the impact of miRNA on the response to TDM. Deletion of DGCR8 in bone marrow progenitors reduced macrophage yield, but did not block macrophage differentiation. DGCR8-deficient macrophages showed reduced constitutive and TDM-inducible miRNA expression. RNAseq analysis revealed that they accumulated primary miRNA transcripts and displayed a modest type I IFN signature at baseline. Stimulation with TDM in the absence of DGCR8 induced overshooting expression of IFNβ and IFN-induced genes, which was blocked by antibodies to type I IFN. In contrast, signaling and transcriptional responses to recombinant IFNβ were unaltered. Infection with live Mycobacterium bovis Bacille Calmette-Guerin replicated the enhanced IFN response. Together, our results reveal an essential role for DGCR8 in curbing IFNβ expression macrophage reprogramming by mycobacteria., (© 2021 Killy et al.)

Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis , in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus , M. avium , M. kansasii , M. leprae , M. marinum , M. tuberculosis , and M. ulcerans IMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community., (Copyright © 2021 Judd et al.)

Mycobacterial pathogens pose a sustained threat to human health. There is a critical need for new diagnostics, therapeutics, and vaccines targeting both tuberculous and nontuberculous mycobacterial species. Understanding the basic mechanisms used by diverse mycobacterial species to cause disease will facilitate efforts to design new approaches toward detection, treatment, and prevention of mycobacterial disease. Molecular, genetic, and biochemical approaches have been widely employed to define fundamental aspects of mycobacterial physiology and virulence. The recent expansion of genetic tools in mycobacteria has further increased the accessibility of forward genetic approaches. Proteomics has also emerged as a powerful approach to further our understanding of diverse mycobacterial species. Detection of large numbers of proteins and their modifications from complex mixtures of mycobacterial proteins is now routine, with efforts of quantification of these datasets becoming more robust. In this review, we discuss the "genetic proteome," how the power of genetics, molecular biology, and biochemistry informs and amplifies the quality of subsequent analytical approaches and maximizes the potential of hypothesis-driven mycobacterial research. Published proteomics datasets can be used for hypothesis generation and effective post hoc supplementation to experimental data. Overall, we highlight how the integration of proteomics, genetic, molecular, and biochemical approaches can be employed successfully to define fundamental aspects of mycobacterial pathobiology., Competing Interests: The authors have declared that no competing interests exist.

Vaccination of goats against tuberculosis (TB) has been promoted as an ancillary tool for controlling the disease in infected livestock herds. A three-year trial to assess the efficacy of BCG vaccine was carried out in five goat herds. At the beginning of the trial (month 0), all animals were tested for TB using thee different diagnostic tests. Animals negative to all tests were vaccinated with BCG and all replacement goat kids were also systematically vaccinated throughout the trial. All animals were tested by Interferon-gamma release assay (IGRA) using vaccine compatible reagents at months 6, 12, 24, and 36. The risk factors for TB infection were also evaluated. At the end of the study, four out of five farms showed variable reductions of the initial prevalence (93.5%, 28.5%, 23.2%, and 14.3% respectively), and an overall incidence reduction of 50% was observed in BCG vaccinated goats, although adult vaccinated goats showed higher incidences than vaccinated goat kids. The unvaccinated positive animals remaining in herds and adult BCG vaccinated goats significantly enhanced the risk of infection in vaccinated animals. A systematic vaccination of goats with BCG, together with the removal of positive unvaccinated animals, may contribute to reducing the TB prevalence in goat herds.

Calcium plays an important role in regulating cell physiology and immune responses to various pathogens. Our recent work has highlighted the crucial role for calcium homeostasis in dendritic cells and macrophages during various infections. Here we investigated the effect of calcium homeostasis in regulating T cell activation and function during mycobacterial infection. Results show that calcium homeostasis had varied effects in regulating T cell activation and function during mycobacterial infection. This included regulation of the expression of co-stimulatory molecules, cytokine profiles and effector function. A net negative role for Voltage Gated Calcium Channel (VGCC) was observed. Inhibiting VGCC in mycobacteria primed T cells induced increased production of pro-inflammatory cytokines and an increased effector phenotype. Infected macrophages when incubated with VGCC inhibited T cells, induced increased expression of co-stimulatory molecule expression on macrophages, increased the production of pro-inflammatory cytokines and increased autophagy and apoptosis. This collectively led to reduced survival of mycobacteria inside macrophages. The data point towards a fine regulation of protective responses by routes of calcium influx and release that mediate pathogen survival or clearance., (Copyright © 2020 Elsevier Inc. All rights reserved.)

One hundred and twenty-two Mycobacterium chimaera strains isolated in Italy from cardiac surgery-related patients, cardiac surgery-unrelated patients and from heater-cooler units, were submitted to whole-genome sequencing and to subsequent SNP analysis. All but one strains isolated from cardiac surgery-related patients belonged to Subgroup 1.1 (19/23) or Subgroup 1.8 (3/23). Only 28 out of 79 strains isolated from heater-cooler units belonged to groupings other than 1.1 and 1.8. The strains isolated from cardiac surgery-unrelated patients were instead distributed across the phylogenetic tree. Our data, the first on isolates from Italy, are in agreement with a recent large genomic study suggesting a common source, represented by strains belonging to Subgroups 1.1 and 1.8, of cardiac surgery-related Mycobacterium chimaera infections. The strains belonging to groupings other than 1.1 and 1.8 isolated from heather-cooler units evidently resulted from contaminations at hospital level and had no share in the Mycobacterium chimaera outbreak. One Mycobacterium chimaera strain investigated in this study proved distant from every previously known Mycobacterium chimaera Groups (1, 2, 3 and 4) and we propose to assign to a novel group, named "Group 5"., Competing Interests: The authors have declared that no competing interests exist.

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. Affected patients are highly and selectively susceptible to weakly virulent mycobacteria, such as environmental mycobacteria and Bacillus Calmette-Guérin vaccines. Since 1996, disease-causing mutations have been reported in 15 genes, with allelic heterogeneity leading to 30 genetic disorders. Here, we briefly review the progress made in molecular, cellular, immunological, and clinical studies of MSMD since the last review published in 2018. Highlights include the discoveries of new genetic etiologies of MSMD: autosomal recessive (AR) complete deficiencies of (1) SPPL2a, (2) IL-12Rβ2, and (3) IL-23R, and (4) homozygosity for TYK2 P1104A, resulting in selective impairment of responses to IL-23. The penetrance of SPPL2a deficiency for MSMD is high, probably complete, whereas that of IL-12Rβ2 and IL-23R deficiencies, and TYK2 P1104A homozygosity, is incomplete, and probably low. SPPL2a deficiency has added weight to the notion that human cDC2 and Th1* cells are important for antimycobacterial immunity. Studies of IL-12Rβ2 and IL-23R deficiencies, and of homozygosity for P1104A TYK2, have shown that both IL-12 and IL-23 are required for optimal levels of IFN-γ. These recent findings illustrate how forward genetic studies of MSMD are continuing to shed light on the mechanisms of protective immunity to mycobacteria in humans.

In reverse vaccinology approaches, complete proteomes of bacteria are submitted to multiple computational prediction steps in order to filter proteins that are possible vaccine candidates. Most available tools perform such analysis only in a single strain, or a very limited number of strains. But the vast amount of genomic data had shown that most bacteria contain pangenomes, i.e., their genomic information contains core, conserved genes, and random accessory genes specific to each strain. Therefore, in reverse vaccinology methods it is of the utmost importance to define core proteins and core epitopes. EpitoCore is a decision-tree pipeline developed to fulfill that need. It provides surfaceome prediction of proteins from related strains, defines core proteins within those, calculate their immunogenicity, predicts epitopes for a given set of MHC alleles defined by the user, and then reports if epitopes are located extracellularly and if they are conserved among the core homologs. Pipeline performance is illustrated by mining peptide vaccine candidates in Mycobacterium avium hominissuis strains. From a total proteome of ~4,800 proteins per strain, EpitoCore predicted 103 highly immunogenic core homologs located at cell surface, many of those related to virulence and drug resistance. Conserved epitopes identified among these homologs allows the users to define sets of peptides with potential to immunize the largest coverage of tested HLA alleles using peptide-based vaccines. Therefore, EpitoCore is able to provide automated identification of conserved epitopes in bacterial pangenomic datasets., (Copyright © 2020 Fiuza, Lima and de Souza.)

Bovine tuberculosis (bTB) is a chronic illness in animals, especially in cattle, leading to loss in the productivity and signifies a crucial public health risk. Regardless of the zoonotic threat and significant economic costs associated with the disease, precise estimates of bTB prevalence are deficient in many countries, including India, where national control programs are yet to be instigated. The true burden of the disease remains unknown due to lack of routine surveillance data from most of the developing countries. India is progressing well towards attaining the End TB goal, yet bTB continues to remain largely hidden. Moreover, the paucity of literature on bTB in India might lead to undue complacency and hence has to be scrupulously guarded and prevented from gaining any misconceptions in the minds of the common people. Preventing and controlling bTB at the animal interface is pivotal to evade transmission to human, increase food safety and guard the livelihood of the people. To attain this goal, implementation of strategies based on international norms and a multi-sectoral approach will empower enhanced surveillance and diagnosis of disease in animals and subsequently reduce the risk for humans. As an initiative, we step forward to address this review which briefly summarizes the available data in the literature from early 20th century to date to assess the status of bTB in India. We have discussed in detail, the epidemiology, transmission and diagnosis pertaining to bTB. The review also focuses on the interconnection between the health of people and animal, discuss the preventions and control strategies and recommend the use of vaccination in cattle to reduce the spread of infection among other animals and humans. Implementing One Health approach in India, which recognizes the interdependence of the health of people and animals will help the nation in the fight against TB., Competing Interests: Declaration of competing interest There is no conflict of interest associated with this publication., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

Background: Mycobacterial species other than Mycobacterium tuberculosis and Mycobacterium leprae are generally free-living organisms and Mycobacterium simiae is one of the slowest growing Non-tuberculous mycobacteria. This is the first case report of Mycobacterium simiae infection in Sri Lanka and only very few cases with extrapulmonary manifestation reported in the literature., Case Presentation: A 24-year-old, previously healthy Sri Lankan male presented with generalized lymphadenopathy with discharging sinuses, evening pyrexia, weight loss, poor appetite and splenomegaly. Lymph node biopsies showed sheets of macrophages packed with organisms in the absence of granulomata. Ziehl Neelsen, Wade Fite and Giemsa stains revealed numerous red coloured acid-fast bacilli within foamy histiocytes. Slit skin smear for leprosy was negative and tuberculosis, fungal and bacterial cultures of the lymph node and bone marrow did not reveal any growth. Later he developed watery diarrhea and colonoscopy revealed multiple small polyps and ulcers throughout the colon extending up to the ileum, Which was confirmed to be due to cytomegalovirus confirmed by PCR and successfully treated with ganciclovir. Positron emission tomography scan guided biopsies of the gut and lymph nodes confirmed presence of mycobacterial spindle cell pseudo-tumours and PCR assays revealed positive HSP65. The culture grew Mycobacterium Simiae. Flow cytometry analysis on patient's blood showed extremely low T and B cell counts and immunofixation revealed low immunoglobulin levels. His condition was later diagnosed as adult onset immunodeficiency due to anti- interferon - gamma autoantibodies. He was initially commenced on empirical anti-TB treatment with atypical mycobacterial coverage. He is currently on a combination of daily clarithromycin, ciprofloxacin, linezolid with monthly 2 g/kg/intravenous immunoglobulin to which, he had a remarkable clinical response with complete resolution of lymphadenopathy and healing of sinuses., Conclusions: This infection is considered to be restricted to certain geographic areas such as mainly Iran, Cuba, Israel and Arizona and this is the first case report from Sri lanka. Even though the infection is mostly seen in the elderly patients, our patient was only 24 years old. In the literature pulmonary involvement was common presentation, but in this case the patient had generalized lymphadenopathy and colonic involvement without pulmonary involvement.

Since 2011, invasive infections due to Mycobacterium chimaera have been diagnosed in patients with previous cardiac surgery. The use of heating/cooling devices (heater-cooler units), necessary to regulate the blood temperature in extra-corporeal circulation, has been identified as a source of contaminated aerosol. Adhering to a strict maintenance program according to the manufacturer's instructions for use including sanitizing is essential to reduce the risk. Current manufacturer guidelines and national recommendations for detection and decontamination of Mycobacterium chimaera and other non-tuberculosis mycobacteria should organically update units' policies and protocols as they are published.

Adhesion to extracellular matrix proteins is an important first step in host invasion, employed by many bacterial pathogens. In mycobacteria, the secreted Ag85 complex proteins, involved in the synthesis of the cell envelope, are known to bind to fibronectin (Fn) through molecular forces that are currently unknown. In this study, single-molecule force spectroscopy is used to study the strength, kinetics and thermodynamics of the Ag85-Fn interaction, focusing on the multidrug-resistant Mycobacterium abscessus species. Single Ag85 proteins bind Fn with a strength of ~75 pN under moderate tensile loading, which compares well with the forces reported for other Fn-binding proteins. The binding specificity is demonstrated by using free Ag85 and Fn peptides with active binding sequences. The Ag85-Fn rupture force increases with mechanical stress (i.e., loading rate) according to the Friddle-Noy-de Yoreo theory. From this model, we extract thermodynamic parameters that are in good agreement with previous affinity determinations by surface plasmon resonance. Strong bonds (up to ~500 pN) are observed under high tensile loading, which may favor strong mycobacterial attachment in the lung where cells are exposed to high shear stress or during hematogenous spread which leads to a disseminated infection. Our results provide new insight into the pleiotropic functions of an important mycobacterial virulence factor that acts as a stress-sensitive adhesin.

Mycobacteria are important causes of disease in human and animal hosts. Diseases caused by mycobacteria include leprosy, tuberculosis (TB), nontuberculous mycobacteria (NTM) infections and Buruli Ulcer. To better understand and treat mycobacterial disease, clinicians, veterinarians and scientists use a range of discipline-specific approaches to conduct basic and applied research, including conducting epidemiological surveys, patient studies, wildlife sampling, animal models, genetic studies and computational simulations. To foster the exchange of knowledge and collaboration across disciplines, the Many Hosts of Mycobacteria (MHM) conference series brings together clinical, veterinary and basic scientists who are dedicated to advancing mycobacterial disease research. Started in 2007, the MHM series recently held its 8th conference at the Albert Einstein College of Medicine (Bronx, NY). Here, we review the diseases discussed at MHM8 and summarize the presentations on research advances in leprosy, NTM and Buruli Ulcer, human and animal TB, mycobacterial disease comorbidities, mycobacterial genetics and 'omics, and animal models. A mouse models workshop, which was held immediately after MHM8, is also summarized. In addition to being a resource for those who were unable to attend MHM8, we anticipate this review will provide a benchmark to gauge the progress of future research concerning mycobacteria and their many hosts., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The emergence of antibiotic-resistant bacteria in the last century is alarming and calls for alternative, nonchemical treatment strategies. Thermal medicine uses heat for the treatment of infectious diseases but its use in facultative and obligate intracellular bacteria remains poorly studied. In this review, we summarize previous research on reducing the infectious burden of Mycobacterium ulcerans and Chlamydia trachomatis by using water-filtered infrared A-radiation (wIRA), a special form of heat radiation with high tissue penetration and low thermal load on the skin surface. Mycobacterium ulcerans is a thermosensitive bacterium causing chronic necrotizing skin disease. Therefore, previous data on wIRA-induced improvement of wound healing and reduction of wound infections is summarized first. Then, pathogenesis and treatment of infections with M. ulcerans causing Buruli ulcer and of those with C. trachomatis infecting the ocular conjunctiva and resulting in blinding trachoma are discussed. Both bacteria cause neglected tropical diseases and have similar geographical distributions. Results of previous in vitro and in vivo studies using wIRA on M. ulcerans and C. trachomatis infections are presented. Finally, technical aspects of using wIRA in patients are critically reviewed and open questions driving future research are highlighted. In conclusion, wIRA is a promising tool for reducing infectious burden due to intracellular bacteria such as M. ulcerans and C. trachomatis .

Mycobacterium tuberculosis (Mtb) is a major causal pathogen of human tuberculosis (TB), which is a serious health burden worldwide. The demand for the development of an innovative therapeutic strategy to treat TB is high due to drug-resistant forms of TB. Autophagy is a cell-autonomous host defense mechanism by which intracytoplasmic cargos can be delivered and then destroyed in lysosomes. Previous studies have reported that autophagy-activating agents and small molecules may be beneficial in restricting intracellular Mtb infection, even with multidrug-resistant Mtb strains. Recent studies have revealed the essential roles of host nuclear receptors (NRs) in the activation of the host defense through antibacterial autophagy against Mtb infection. In particular, we discuss the function of estrogen-related receptor (ERR) α and peroxisome proliferator-activated receptor (PPAR) α in autophagy regulation to improve host defenses against Mtb infection. Despite promising findings relating to the antitubercular effects of various agents, our understanding of the molecular mechanism by which autophagy-activating agents suppress intracellular Mtb in vitro and in vivo is lacking. An improved understanding of the antibacterial autophagic mechanisms in the innate host defense will eventually lead to the development of new therapeutic strategies for human TB.

Background: Nontuberculous mycobacteria belonging to the Mycobacterium avium complex are recognized as opportunistic pathogens to humans. Mycobacterium arosiense is one of the novel members of the Mycobacterium avium complex. The organism has only rarely been reported in human clinical cases and may be routinely misidentified., Case Presentation: An adult male with a history of a discus prolapse and sarcoidosis presented with high fever and a strong back pain with projection to the extremities. A Magnetic Resonance Imaging scan of columna revealed a tumor suspect process at thoracic vertebrae 11/12 with changes at the second lumbar vertebra, which was partly removed by laminectomy. Biopsy smears revealed acid-fast bacilli and turned out to be Mycobacterium tuberculosis complex PCR negative. The routine line probe assay INNO-LiPa v2 (INNOGENETICS NV, Gent), which differentiates 16 mycobacterial species indicated the presence of a not readily identifiable NTM species. Whereas, the GenoType Mycobacterium CM v2.0 (HAIN Lifescience GmbH) that routinely differentiates 14 clinically relevant mycobacteria revealed a Mycobacterium intracellulare species. However, additional diagnostic sequencing of the 16S rRNA gene confirmed the presence of a Mycobacterium arosiense species., Conclusions: This is the second unusual case of osteomyelitis with clinical significance ever to be reported, caused by Mycobacterium arosiense and complicated by an underlying sarcoidosis. Mycobacterium arosiense has rarely been reported clinically and the first description of the species was identified as the cause of osteomyelitis in a child with a hereditary partial interferon gamma deficiency. Symptoms attributed to sarcoidosis waned on Mycobacterium arosiense treatment and it is inconclusive whether the patient ever suffered from sarcoidosis. Mycobacterium arosiense was misidentified by the GenoType as Mycobacterium intracellulare and implicates that the diagnosis requires supplemental sequencing of the 16S rRNA gene.

Background: Mycobacterium canettii forms part of the Mycobacterium tuberculosis complex. Mycobacterium canettii infections are mainly described in the Horn of Africa. The permanent presence of French soldiers in Djibouti raises the question of the risk of being infected with M. canettii. Here, we describe M. canettii infections among French military and their families between 1998 and 2015., Methods: This retrospective study relied on 3 sources of data: the reference center for mycobacteria in the Biology Department at Percy Military Hospital in Paris, the French Military Center for Epidemiology and Public Health, and the scientific literature. After an exhaustive census of the strains, we studied the epidemiological data on 20 cases among French soldiers and their families., Results: Twenty cases of M. canettii infections are reported, including 5 unpublished cases. Adenitis predominates (n = 15), especially in the cervico facial area and among children; 1 case was observed 1 month after dental care in Djibouti. The pulmonary forms were less frequent (n = 6), and 3 atypical forms are described. All patients had stayed in Djibouti., Conclusions: Cases of M. canettii infection among the French military consisted mainly of adenitis; disseminated forms were possible with immunodeficiency. Their evolution under specific treatments was comparable to that of tuberculosis. The presumed origin of the infection seemed to be environmental, possibly a water reservoir, and not due to human-to-human contagion., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Precise knowledge of the health status of experimental fish is crucial to obtain high scientific and ethical standards in biomedical research. In addition to the use of sentinel fish, the examination of diseased fish is a fundamental part of all health monitoring concepts. PCR assays offer excellent sensitivity and the ability to test a broad variety of pathogenic agents in different sample types. Recently, it was shown that analysis of environmental samples such as water, sludge or detritus from static tanks can complement PCR analysis of fish and is actually more reliable for certain pathogens. In our study, we investigated whether the analysis of filtered water mixed with detritus of tanks including fish showing clinical signs of illness is suitable to complement health monitoring programs in recirculating systems. The obtained data indicate that pathogens such as Pseudoloma neurophilia or Myxidium streisingeri were exclusively or mainly found in fish, while mycobacteria were predominantly present in environmental samples. A combination of both sample types seems to be required for the detection of a broad range of infectious agents in zebrafish colonies using real-time PCR technology., Competing Interests: The authors have declared that no competing interests exist.

Incidentally discovered solitary pulmonary nodules (SPN) caused by nontuberculous mycobacteria (NTM) is uncommon, and its optimal treatment strategy remains uncertain. This cohort study determined the clinical characteristics and outcome of asymptomatic patients with NTM-SPN after surgical resection. Resected SPNs with culture-positive for NTM in six hospitals in Taiwan during January, 2010 to January, 2017 were identified. Asymptomatic patients without a history of NTM-pulmonary disease (PD) or same NTM species isolated from the respiratory samples were selected. All were followed until May 1, 2019. A total of 43 patients with NTM-SPN were enrolled. Mycobacterium avium complex (60%) and M. kansasii (19%) were the most common species. The mean age was 61.7 ± 13.4. Of them, 60% were female and 4% had history of pulmonary tuberculosis. The NTM-SPN was removed by wedge resection in 38 (88%), lobectomy in 3 (7%) and segmentectomy in 2 (5%). Caseating granuloma was the most common histologic feature (58%), while chronic inflammation accounts for 23%. Mean duration of the follow-up was 5.2 ± 2.8 years (median: 4.2 years [2.5-7.0]), there were no mycobacteriology recurrence or NTM-PD development. In conclusion, surgical resection is likely to curative for incidentally discovered NTM-SPN in asymptomatic patients without culture evidence of the same NTM species from respiratory specimens, and routine mycobacterium culture for resected SPN might be necessary for differentiating pulmonary tuberculosis and NTM because further treatment differs., Competing Interests: The authors have declared that no competing interests exist.

Over the long course of evolution from a probable environmental reservoir, the pathogen that we know today as Mycobacterium tuberculosis has become fully capable of adapting to the life inside host cells by evading and modifying their responses to infection. Factors contributing to the success of this pathogen are numerous and thanks to a large body of work accumulated over the past decades, we are closer to understanding the remarkable complexity of tuberculosis pathogenesis. The unique type VII secretion systems and various complex lipids of the cell envelope have emerged as some of the most important and most studied factors in this regard. This review attempts to summarize recent findings on these and other virulence factors, while discussing their evolution in different closely related tuberculosis-causing bacteria as well, with the aim of exploring the processes which led M. tuberculosis to becoming one of the deadliest infections agents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Models on how bacterial lineages differentiate increase our understanding of early bacterial speciation events and the genetic loci involved. Here, we analyze the population genomics events leading to the emergence of the tuberculosis pathogen. The emergence is characterized by a combination of recombination events involving core pathogenesis functions and purifying selection on early diverging loci. We identify the phoR gene, the sensor kinase of a two-component system involved in virulence, as a key functional player subject to pervasive positive selection after the divergence of the Mycobacterium tuberculosis complex from its ancestor. Previous evidence showed that phoR mutations played a central role in the adaptation of the pathogen to different host species. Now, we show that phoR mutations have been under selection during the early spread of human tuberculosis, during later expansions, and in ongoing transmission events. Our results show that linking pathogen evolution across evolutionary and epidemiological time scales points to past and present virulence determinants.

Mycobacterium cosmeticum is a nontuberculous Mycobacterium recovered from different water sources including household potable water and water collected at nail salon. Individual cases of this bacterium have been reported to be associated with gastrointestinal tract infections. Here we present the first whole-genome study and comparative analysis of two new clinically-derived Mycobacterium sp. UM&#95;RHS (referred as UM&#95;RHS after this) and Mycobacterium sp. UM&#95;NYF (referred as UM&#95;NYF after this) isolated from patients in Indonesia and Malaysia respectively to have a better understanding of the biological characteristic of these isolates. Both strains are likely Mycobacterium cosmeticum as supported by the evidence from molecular phylogenetic, comparative genomic and Average Nucleotide Identity (ANI) analyses. We found the presence of a considerably large number of putative virulence genes in the genomes of UM&#95;RHS and UM&#95;NYF. Interestingly, we also found a horizontally transferred genomic island carrying a putative dsz operon proposing that they may have potential to perform biodesulfization of dibenzothiophene (DBT) that may be effective in cost reduction and air pollution during fuel combustion. This comparative study may provide new insights into M. cosmeticum and serve as an important reference for future functional studies of this bacterial species., Competing Interests: The authors have declared that no competing interests exist.

Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.

A collection of over 1600 sequenced bacteriophages isolated on a single host strain, Mycobacterium smegmatis mc 2 155, can be grouped into over two dozen types that have little or no nucleotide sequence similarity to each other. One group, Cluster K, can be divided into several subclusters, and the well-characterized and much exploited phage TM4 lies in Subcluster K2. Many of the Cluster K phages have broad host ranges and infect both fast- and slow-growing mycobacterial strains. Here we describe phage ZoeJ, a new Subcluster K2 member, which infects a broad spectrum of mycobacterial hosts including M. smegmatis, Mycobacterium tuberculosis, and Mycobacterium avium. ZoeJ has extensive sequence similarity to TM4, and comparative analysis reveals the precise deletion conferring the lytic phenotype of TM4. The ZoeJ immunity repressor was identified as gene 45, which is prophage-expressed, is required for lysogeny, and is sufficient to confer superinfection immunity to ZoeJ. ZoeJ gp45 also confers immunity to Subcluster K2 phage Milly, and Subcluster K1 phages Adephagia and CrimD, but surprisingly not to TM4. RNAseq analysis reveals the temporal pattern of early and late gene expressions in ZoeJ lytic growth and suggests a role for the ESAS motifs for gene regulation., (Copyright © 2019. Published by Elsevier Ltd.)

Context: Correctional facilities provide unique opportunities to diagnose and treat persons with latent tuberculosis infection (LTBI). Studies have shown that 12 weekly doses of isoniazid and rifapentine (INH-RPT) to treat LTBI resulted in high completion rates with good tolerability., Objective: To evaluate completion rates and clinical signs or reported symptoms associated with discontinuation of 12 weekly doses of INH-RPT for LTBI treatment., Setting/participants: During July 2012 to February 2015, 7 Federal Bureau of Prisons facilities participated in an assessment of 12 weekly doses of INH-RPT for LTBI treatment among 463 inmates., Main Outcome Measures: Fisher exact test was used to assess the associations between patient sociodemographic characteristics and clinical signs or symptoms with discontinuation of treatment., Results: Of 463 inmates treated with INH-RPT, 424 (92%) completed treatment. Reasons for discontinuation of treatment for 39 (8%) inmates included the following: 17 (44%) signs/symptoms, 9 (23%) transfer or release, 8 (21%) treatment refusal, and 5 (13%) provider error. A total of 229 (49.5%) inmates reported experiencing at least 1 sign or symptom during treatment; most frequently reported were fatigue (16%), nausea (13%), and abdominal pain (7%). Among these 229 inmates, signs/symptoms significantly associated with discontinuation of treatment included abdominal pain (P < .001), appetite loss (P = .02), fever/chills (P = .01), nausea (P = .03), sore muscles (P = .002), and elevation of liver transaminases 5× upper limits of normal or greater (P = .03)., Conclusions: The LTBI completion rates were high for the INH-RPT regimen, with few inmates discontinuing because of signs or symptoms related to treatment. This regimen also has practical advantages to aid in treatment completion in the correctional setting and can be considered a viable alternative to standard LTBI regimens.

Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The association of Mycobacterium chimaera infection in patients undergoing cardiopulmonary bypass (CPB) with the use of heater-cooler units (HCU) has been reported in various literature. We described microbiological monitoring and the extent of microbiological contamination of HCUs utilized in our centre and strategies employed to reduce the high microbial load., Methods: Since August 2016, we have been following the new Instructions for Use from the manufacturer for the cleaning and disinfection of three units of Stöckert 3T and four units of Stöckert 1T HCU at the National Heart Centre Singapore. Microbiological monitoring began in January 2017 and included acid-fast bacilli (AFB) culture, Pseudomonas aeruginosa, total colony and total coliform count. Methods, such as increasing disinfection frequency and making the HCU inactive by keeping it empty in storage, were used to reduce the high colony count., Results: All three units of Stöckert 3T and two units of Stöckert 1T were contaminated with Mycobacterium chimaera. Pseudomonas aeruginosa and total coliform count were consistently <1 colony-forming unit (CFU)/100 mL in every water sample of each HCU. High colony counts were encountered initially in all units. Step-up frequency of disinfection was found to be not as effective as keeping the HCU inactive in bringing the total colony count to an acceptable level., Conclusions: All monitoring and maintenance measures of HCUs need to be established and maintained to mitigate potential infection risks to patients. Strict adherence to all cleaning and disinfection processes and keeping the HCU inactive maintained the water quality of the HCU at acceptable levels.

Yifei Tongluo (YFTL) is a traditional Chinese medicine (TCM) formulation which has been shown clinical efficacy in treatment of patients with multidrug-resistant tuberculosis in China. However, the underlying mechanisms of the effects of YFTL are lacking. This study investigated the effects of YFTL on immune regulation with a mouse lung infection model with Bacille Calmette-Guérin (BCG). We found that compared with untreated mice, the lung mycobacterial load in YFTL-treated mice was significantly reduced, accompanied by alleviated pulmonary inflammation with reduction of pro-inflammatory cytokines and increase of prostaglandin E2 (PGE2). Flow cytometry analyses showed that Th1 cells were significantly higher in the lungs of YFTL-treated mice at early infection time. The results suggest that YFTL-treatment down-regulates pulmonary inflammation, which facilitates a rapid infiltration of Th1 cells into the lungs. Moreover, the Th1 cells in the lungs were resolved faster at later time concomitant with increased the regulatory T cells (Tregs). The reduction of mycobacterial burden associated with improved tissue pathology, faster Th1 cell trafficking, and accelerated resolution of Th1 cells in the lungs of YFTL-treated mice indicates that YFTL improves mycobacterial clearance by maintaining lung homeostasis and dynamically regulating T cells in the lung parenchyma, and suggests that YFTL can be used as host-directed therapies that target immune responses to mycobacterial infection., Competing Interests: The authors have declared that no competing interests exist.

Background: The rapid diagnosis of extrapulmonary tuberculosis in children remains challenging. The presence of enlarged lymph nodes provides an opportunity to obtain diagnostic material through fine needle aspiration biopsy (FNAB). Mycobacterial culture, traditionally the reference standard, has a slow turnaround time and PCR-based methods are not widely available in developing countries. Direct visualization of mycobacteria on microscopy can be a rapid method to confirm the diagnosis. This study compared three staining methods to visualize mycobacteria., Methods: Hundred FNAB specimens from persistently enlarged lymph nodes in children, clinically suspicious for tuberculosis, were evaluated for the presence of mycobacteria by three staining methods: Papanicolaou induced fluorescence (PIF) and Auramine O staining using fluorescence microscopy and Ziehl-Neelsen (ZN) staining using conventional light microscopy. These methods were evaluated against mycobacterial culture., Results: PIF positivity was 30%, with 38% and 48% for Auramine O and ZN respectively. The combined ZN/PIF positivity was 56%. The highest diagnostic accuracy (73%) was demonstrated by ZN alone and in combination with PIF, with PIF alone showing the lowest (49%) accuracy. Although the combined test showed the highest sensitivity, it had the lowest specificity, while ZN was significantly more sensitive than both other staining modalities. No statistical difference in specificity was seen among the tests., Conclusion: This study suggests that Auramine O staining on previously ZN stained slides does not significantly improve diagnostic accuracy. While currently widely available methods of direct visualization of mycobacteria suffer from low sensitivity, the ZN stain remains a useful diagnostic test, particularly in resource-constrained countries., (© 2018 Wiley Periodicals, Inc.)

A systems approach was used to explore the genome and transcriptome of Mycobacterium shigaense, a new opportunistic pathogen isolated from a patient with a skin infection, and the host response transcriptome was assessed using a macrophage infection model. The M. shigaense genome comprises 5,207,883 bp, with 67.2% G+C content and 5098 predicted coding genes. Evolutionarily, the bacterium belongs to a cluster in the phylogenetic tree along with three target opportunistic pathogenic strains, namely, M. avium, M. triplex and M. simiae. Potential virulence genes are indeed expressed by M. shigaense under culture conditions. Phenotypically, M. shigaense had similar infection and replication capacities in a macrophage model as the opportunistic species compared to M. tuberculosis. M. shigaense activated NF-κB, TNF, cytokines and chemokines in the host innate immune-related signaling pathways and elicited an early response shared with pathogenic bacilli except M. tuberculosis. M. shigaense upregulated specific host response genes such as TLR7, CCL4 and CXCL5. We performed an integrated and comparative analysis of M. shigaense. Multigroup comparison indicated certain differences with typical pathogenic bacilli in terms of gene features and the macrophage response.

There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.

Pulmonary infection due to Mycobacterium heckeshornense (M. heckeshornense) in healthy adults without underlying diseases is very rare and optimal treatments have not yet been established. A 39-year-old woman was admitted to our hospital for further examinations following the identification of a pulmonary cavitary nodule. Acid-fast bacilli were cultured from specimens obtained by bronchofiberscopy, and identified with M. heckeshornense using nucleotide sequencing. Antimycobacterial chemotherapy was effective temporarily, while the nodular lesion subsequently worsened. The patient underwent lobectomy and has not relapsed thus far. A lung specimen showed marked granulomatous inflammation with extensive caseous necrosis and the preservation of some parts of alveolar septa within caseous necrosis, indicating an exudative process and resistance to chemotherapy. M. heckeshornense is strongly pathogenic and switching to surgical intervention needs to be considered when chemotherapy is insufficient., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis (MTB) is a successful pathogen which increases persistence inside the host macrophage by subverting its defence mechanism. Mycobacteria regulate the pathogenesis and intracellular survival by controlling its interaction with host protein(s). Galectin 3 is a member of the β-galactoside binding gene family which is involved in several biological functions. In the present study, we have expressed the mycobacterial protein tyrosine kinase (PtkA) in the cytosol of host macrophages through a eukaryotic promoter vector and found that it down-regulates Galectin 3. Infection by ptkA knocked-out (KO) mycobacterial strain shows increased level of Galectin 3 in the cytosol of macrophages. PtkA regulates Galectin 3 level and stimulates host macrophage through MEK-JNK-cJUN pathway and initiates early apoptosis in H 37 Ra infected macrophage. The ptkA KO strain showed decreased progression of apoptosis confirming Galectin 3 as anti-apoptotic molecule. The intracellular survival was also found to be impaired in the mice infected with ptkA KO mycobacteria. The hypothesis was also confirmed by looking at the intracellular survival of mycobacteria in Galectin 3 silenced macrophages. The overall findings suggest the significance of Galectin 3 and PtkA interaction in intracellular persistence of mycobacteria., (Copyright © 2018 Elsevier Inc. All rights reserved.)