Autophagy and Host Defense in Nontuberculous Mycobacterial Infection.

Autophagy is critically involved in host defense pathways through targeting and elimination of numerous pathogens via autophagic machinery. Nontuberculous mycobacteria (NTMs) are ubiquitous microbes, have become increasingly prevalent, and are emerging as clinically important strains due to drug-resistant issues. Compared to Mycobacterium tuberculosis (Mtb), the causal pathogen for human tuberculosis, the roles of autophagy remain largely uncharacterized in the context of a variety of NTM infections. Compelling evidence suggests that host autophagy activation plays an essential role in the enhancement of antimicrobial immune responses and controlling pathological inflammation against various NTM infections. As similar to Mtb, it is believed that NTM bacteria evolve multiple strategies to manipulate and hijack host autophagy pathways. Despite this, we are just beginning to understand the molecular mechanisms underlying the crosstalk between pathogen and the host autophagy system in a battle with NTM bacteria. In this review, we will explore the function of autophagy, which is involved in shaping host-pathogen interaction and disease outcomes during NTM infections. These efforts will lead to the development of autophagy-based host-directed therapeutics against NTM infection.

Challenges in the management of slowly growing non-tuberculous mycobacteria causing pulmonary disease: Perspectives from a high burden country.

PURPOSE: There is a dearth of data on epidemiology, diagnosis and management of slow growing non tuberculous mycobacteria(NTM) in India, despite being a TB endemic country. This study aims to describe the geographic distribution, risk factors, and the challenges in management of slow growing NTM causing pulmonary infections. METHODS: Over a period of 3 years, all slow growing NTM received from pulmonary specimens at a tertiary care centre were further studied from electronic hospital records to correlate non tuberculous mycobacteria species with demographics, geographic location, describe comorbidities including immunosuppression, radiologic findings and treatment regimes. RESULTS: M.intracellullare was found in the majority of isolates with significant geographic variation and M.simiae the second commonest found exclusively in southern states. Common comorbidities included a previous history of treatment for tuberculosis, structural lung disease as well as systemic risk factors. Disseminated disease only occurred in immunocompromised hosts as was expected, but at a high rate of 44%. Treatment completion and outcomes were difficult to attain in our population. CONCLUSION: The burden of NTM infection and its management in India remain a challenge. Ensuring it is made a notifiable disease may improve the current situation.

Conserved and specialized functions of Type VII secretion systems in non-tuberculous mycobacteria.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis, to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis. In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

Disseminated disease due to non-tuberculous mycobacteria in HIV positive patients: A retrospective case control study.

INTRODUCTION: Disseminated infection due to non-tuberculous mycobacteria has been a major factor of mortality and comorbidity in HIV patients. Until 2018, U.S. American guidelines have recommended antimycobacterial prophylaxis in patients with low CD4 cell counts, a practice that has not been adopted in Europe. This study aimed at examining the impact of disseminated NTM disease on clinical outcome in German HIV patients with a severe immunodeficiency. MATERIALS AND METHODS: In this retrospective case control study, HIV patients with disseminated NTM disease were identified by retrospective chart review and matched by their CD4 cell counts to HIV patients without NTM infection in a 1:1 alocation. Primary endpoints were mortality and time to first rehospitalisation. In addition, other opportunistic diseases, as well as antimycobacterial and antiretroviral treatments were examined. RESULTS: Between 2006 and 2016, we identified 37 HIV patients with disseminated NTM disease. Most of them were suffering from infections due to M. avium complex (n = 31, 77.5%). Time to event analysis showed a non-significant trend to higher mortality in patients with disseminated NTM disease (p = 0.24). Rehospitalisation took place significantly earlier in patients with disseminated NTM infections (median 40.5 days vs. 109 days, p<0.0001). CONCLUSION: In this retrospective case control study, we could demonstrate that mortality is not significantly higher in HIV patients with disseminated NTM disease in the ART era, but that they require specialised medical attention in the first months following discharge.

Direct Molecular Characterization of Acid-Fast Bacilli Smear of Nontuberculosis Mycobacterium Species Causing Pulmonary Tuberculosis in Guna Yala Region, Panama.

Mycobacterium tuberculosis (MTB) stands out as the main causative agent of pulmonary tuberculosis (TB). However, nontuberculous mycobacteria (NTM) species also have the potential to infect and cause TB in susceptible individuals. The objective of this study was to identify NTM species that cause public health problems in remote areas. The study was carried out using 105 sputum smears obtained from patients from the Guna Yala Region of Panama with clinical signs suggestive of TB. DNA was extracted from sputum smears. Nontuberculous mycobacteria and MTB were characterized using polymerase chain reaction restriction analysis (hsp65, rpob) and an evaluation of 24-mycobacterial interspersed repetitive units-variable number of tandem repeats loci. Twenty-six Mycobacterium species were characterized; 19 (18%) were identified as MTB, and 7 (6.7%) were identified as NTM (four M. avium complex, two M. haemophilum, one M. tusciae). These results suggest that at least one in five cases of pulmonary TB among this population is caused by an NTM. Thus, identifying the bacteria causing pulmonary disease is key even in remote regions of the world where standard diagnosis and culture are not available. Strengthening the laboratory capacity within the Guna Yala Region is needed to identify NTM infections promptly.

Cobalamin is present in cells of non-tuberculous mycobacteria, but not in Mycobacterium tuberculosis.

Cobalamin (vitamin B12) is a structurally complex molecule that acts as a cofactor for enzymes and regulates gene expression through so-called riboswitches. The existing literature on the vitamin B12 synthesis capacity in Mycobacterium tuberculosis is ambiguous, while in non-tuberculous mycobacteria (NTM) is rather marginal. Here we present the results of our investigation into the occurrence of vitamin B12 in mycobacteria. For detection purposes, immunoassay methods were applied to cell lysates of NTM and M. tuberculosis clinical and laboratory strains grown under different conditions. We show that whereas vitamin B12 is present in cells of various NTM species, it cannot be evidenced in strains of differently cultured M. tuberculosis, even though the genes responsible for vitamin B12 synthesis are actively expressed based on RNA-Seq data. In summary, we conclude that the production of vitamin B12 does occur in mycobacteria, with the likely exception of M. tuberculosis. Our results provide direct evidence of vitamin B12 synthesis in a clinically important group of bacteria.

Nucleic acid amplification techniques for rapid diagnosis of nontuberculous mycobacteria: A protocol of systematic review and meta-analysis.

BACKGROUND: Nontuberculous mycobacteria (NTM) infection is similar to Mycobacterium tuberculosis (MTB) infection. Early clinical identification and differentiation of NTM and MTB infections continues to be a major challenge. Nucleic acid amplification tests (NAATs) have the ability to efficiently and rapidly detect pathogens and are widely used in mycobacterial infections. The objective of this study is to estimate the diagnostic accuracy of NAATs for NTM. METHODS: We will search candidate studies that assessing the accuracy of NAATs for diagnosis of NTM through PubMed, Embase and the Cochrane Library until May 2021. Studies with full text that meet the inclusion criteria will be included. Following a revised tool for Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2), two researchers will independently evaluate the study quality. The STATA software (version 15.0) will be used to carry out meta-analyses. When heterogeneity is observed, subgroup analyses and meta-regression analyses will be used to explore sources of heterogeneity. Sensitivity analyses will be used to check the robustness of analyses. CONCLUSION: We hope that this study will provide meaningful evidence for the early and rapid diagnosis of NAATs for NTM, which will help to guide the treatment of NTM and improve the prognosis of patients.

Prevalence of Nontuberculous Mycobacterium Infections versus Tuberculosis among Autopsied HIV Patients in Sub-Saharan Africa: A Systematic Review and Meta-Analysis.

In industrialized countries, Mycobacterium avium complex and other nontuberculous mycobacteria (NTM) are major causes of opportunistic infection-related deaths in HIV patients. However, in resource-limited regions, data on NTM are scarce, and tuberculosis (TB) was often assumed to be the cause of death in HIV patients with a positive acid-fast smear. We searched MEDLINE and Embase databases for studies on autopsied HIV patients in sub-Saharan Africa published between January 1997 and April 2020. We included studies that reported histopathological or microbiological evidences for diagnosis of TB and NTM infection. We excluded articles without mycobacterial evidence from culture or molecular testing, such as those that used verbal autopsy, death certificates, or national registry data (systematic review registration number: CRD42019129836 at PROSPERO). We included six eligible studies that reported 391 autopsies in sub-Saharan African HIV patients. The prevalence of NTM and TB at autopsy ranged from 1.3% to 27.3% and 11.8% to 48.7%, respectively. The weighted prevalence ratio of NTM versus TB was 0.16 indicating that for every seven HIV patients died with mycobacterial infections, there was one died with NTM infection. Of the 13 NTM infections, six were caused by M. avium complex. Mycobacterium avium complex and other NTM infections are important differential diagnoses of TB at the time of death among HIV patients in sub-Saharan Africa. Our findings highlight the need to systematically survey the prevalence of NTM infections among HIV patients seeking medical care in resource-limited regions.

Genomic analysis of cardiac surgery-associated Mycobacterium chimaera infections in Italy.

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".

Detection of Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus) using an interferon gamma release assay in a captive elephant herd.

Tuberculosis is highly contagious disease that can be transmitted between humans and animals. Asian elephants (Elephas maximus) in captivity live in close contact with humans in many Asian countries. In this study, we developed an interferon gamma release assay (IGRA) for elephant TB detection using antigens from the MTB complex (MTBC) and nontuberculous mycobacteria (NTM) as stimulating antigens (PPD, ESAT6, CFP10) to elicit a cell-mediated immune response (CMIR). The developed assay was applied to an elephant herd of more than 60 animals in Thailand, and the results were compared with those obtained through serological detection. IGRA has sufficient sensitivity for detecting elephant interferon gamma (eIFNγ) from specific antigen-stimulated PBMCs. Among 60 animals tested, 20 samples (33.3%) showed negative results for both MTBC and NTM infection. Eighteen samples (30%) showed positive responses against PPD from M. bovis and/or ESAT6 and CFP10, indicating MTBC infection. In contrast, only 15.6% showed seropositivity in a commercial serological test kit for elephant TB. The discrepancies between serological and CMIR highlight that the two methods may detect different stages of elephant TB. Therefore, employing both tests may enable them to complement each other in correctly identifying elephants that have been exposed to MTBC.

Characterization of integrated prophages within diverse species of clinical nontuberculous mycobacteria.

BACKGROUND: Nontuberculous mycobacterial (NTM) infections are increasing in prevalence, with current estimates suggesting that over 100,000 people in the United States are affected each year. It is unclear how certain species of mycobacteria transition from environmental bacteria to clinical pathogens, or what genetic elements influence the differences in virulence among strains of the same species. A potential mechanism of genetic evolution and diversity within mycobacteria is the presence of integrated viruses called prophages in the host genome. Prophages may act as carriers of bacterial genes, with the potential of altering bacterial fitness through horizontal gene transfer. In this study, we quantify the frequency and composition of prophages within mycobacteria isolated from clinical samples and compare them against the composition of PhagesDB, an environmental mycobacteriophage database. METHODS: Prophages were predicted by agreement between two discovery tools, VirSorter and Phaster, and the frequencies of integrated prophages were compared by growth rate. Prophages were assigned to PhagesDB lettered clusters. Bacterial virulence gene frequency was calculated using a combination of the Virulence Factor Database (VFDB) and the Pathosystems Resource Integration Center virulence database (Patric-VF) within the gene annotation software Prokka. CRISPR elements were discovered using CRT. ARAGORN was used to quantify tRNAs. RESULTS: Rapidly growing mycobacteria (RGM) were more likely to contain prophage than slowly growing mycobacteria (SGM). CRISPR elements were not associated with prophage abundance in mycobacteria. The abundance of tRNAs was enriched in SGM compared to RGM. We compared the abundance of bacterial virulence genes within prophage genomes from clinical isolates to mycobacteriophages from PhagesDB. Our data suggests that prophages from clinical mycobacteria are enriched for bacterial virulence genes relative to environmental mycobacteriophage from PhagesDB. CONCLUSION: Prophages are present in clinical NTM isolates. Prophages are more likely to be present in RGM compared to SGM genomes. The mechanism and selective advantage of this enrichment by growth rate remain unclear. In addition, the frequency of bacterial virulence genes in prophages from clinical NTM is enriched relative to the PhagesDB environmental proxy. This suggests prophages may act as a reservoir of genetic elements bacteria could use to thrive within a clinical environment.

Port-site infection due to nontuberculous mycobacteria following laparoscopic surgery.

Nontuberculous mycobacteria (NTM) are commonly found in soil and water and can cause nosocomial infections by contaminating equipment and disinfectants solution used in hospitals. NTM port-site infection after laparoscopic surgery is increasingly observed, but its clinical features, management, and prevention have not been reviewed adequately. We performed a comprehensive literature review of reports that described the clinical manifestation and management of NTM port-site infections following laparoscopic surgery. The perceived increase in NTM port-site infections is likely multifactorial, influenced by greater awareness, better diagnostics, changes in medical practice, increased prevalence of immunosuppression, and potential pathogen spread. Widespread resistance to common disinfectants is a major concern. Patients with NTM port-site infections typically present 1-3 months after the laparoscopic intervention with chronic local and minimal systemic symptoms. Surgical excision plays an important role in localized or refractory cases. Medical treatment should be guided by species identification and in vitro drug-susceptibility testing (DST) of the infecting NTM strain, with a combination of second-line antituberculosis agents, given for a prolonged duration. NTM port site infection is best prevented by meticulous skin preparation and infection control, using only sterilized supplies for laparoscopic surgery.

Clinical-epidemiological profile and factors related to the mortality of patients with nontuberculous mycobacteria isolated at a reference hospital in Ceará, Northeastern Brazil.

Background: There is a significant shortage of official records that enable estimating the real prevalence of nontuberculous mycobacteria (NTM) infections in Brazil. The study aims to investigate the clinical, laboratory, and epidemiological aspects of patients with NTM isolation at an infectious diseases reference hospital, and to identify factors associated with mortality. Methods: This was an observational study in which clinical, epidemiological, and laboratory aspects were evaluated in patients with NTM isolated at care in Hospital São José, located in Northeastern Brazil, from 2005 to 2016. The records of the reference laboratory for NTM isolates were searched from the culture results of patients. Afterward, the medical records of the patients were reviewed. The analytical assessment was conducted by the Mann-Whitney and Fisher's exact test. The adopted level of significance was 5%. Results: A total of 69 patients were described, with a predominance of males (73.9%). The main clinical forms identified were: pulmonary (60.9%) and disseminated (27.5%). The most frequently NTM identified were Mycobacterium avium (24.6%) and Mycobacterium fortuitum (10.1%). Forty-eight (69.6%) patients had HIV infection. The mortality was 24.6%, and the risk factors for deaths identified were: origin from outside the metropolitan region; weight loss; HIV infection; anemia; hyperbilirubinemia; increased serum glutamic-oxaloacetic transaminase, alkaline phosphatase, lactate dehydrogenase; and impaired renal function. Among the patients with HIV, the main changes related to death were: lower counts of CD4+ and CD8+ T lymphocytes. Conclusion: Maintaining constant vigilance regarding the possibility of NTM infection is required, namely in patients co-infected with HIV/AIDS.

The Epidemiology of Extrapulmonary Non-tuberculous Mycobacterial Infection in a Pediatric Population.

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and a well-known cause of lymphadenitis, skin and soft tissue infections. The aim of this study was to evaluate the epidemiology of extrapulmonary pediatric NTM infections occurring from 2000 to 2017 in Queensland, Australia. METHODS: All cases of NTM and TB are notifiable under the Queensland Public Health Act (2005) and associated regulations (2005). Data from 2000 to 2017 inclusive was collected from the Notifiable Conditions Database, a laboratory based notification system that covers private and public laboratory systems. Pediatric population demographic data were obtained from the Australian Bureau of Statistics by researching the number of children 0-14 years of age in Queensland from 2000 to 2017; both annual and averaged population was determined. The statistical software SPSS and Tableau was used for analysis. RESULTS: The mean age of diagnosis was 2.5 years with a majority of the cohort being women. Mycobacterium avium was the most commonly diagnosed pathogen. Geographic regions with the highest numbers of cases were predominantly tropical and coastal areas. M. haemophilum emerged as a more common pathogen from 2011 following a period of major flooding. CONCLUSION: Characteristics of the cohort of children susceptible to NTM disease appears consistent with previous reports. However, changes in the epidemiology of NTM infection (such as incidence, species, and geographic distribution) in children may be linked to environmental and weather factors.

State-of-the-art treatment strategies for nontuberculous mycobacteria infections.

INTRODUCTION: Non-tuberculous Mycobacteria (NTM) are a group of organisms whose importance in medicine seems to be increasing in recent times. The increasing number of patients susceptible to these diseases make it necessary to expand our knowledge of therapeutic options and to explore future possibilities for the development of a therapeutic arsenal. AREAS COVERED: In this review, the authors provide a brief introduction about the present importance of NTM and describe the present recommendations of the available guidelines for their treatment. They include a description of the future options for the management of these patients, especially focusing on new antibiotics. The authors also look at possibilities for future therapeutic options, such as antibiofilm strategies. EXPERT OPINION: No actual changes have been made to the current recommendations for the management of most NTM infections (except perhaps the availability of nebulized amikacin). However, it is also true that we have increased the number of available antibiotic treatment options with good in vitro activity against NTM. The use of these drugs in selected cases could increase the therapeutic possibilities. However, some problems are still present, such as the knowledge of the actual meaning of a NTM isolate, and will probably be a key part of future research.

Cutaneous nontuberculous mycobacterial infection in Thailand: A 7-year retrospective review.

A remarkable increase in the prevalence of cutaneous nontuberculous mycobacterial (NTM) infection has occurred worldwide. However, updated data regarding cutaneous NTM infection in Thailand is limited.This study aim to describe the clinical manifestations, pathogenic organism, and prognostic factors of cutaneous NTM infections among patients living in Thailand.The electronic medical records of all patients with confirmatory diagnosis of cutaneous NTM infection from either positive cultures or polymerase chain reaction were retrospectively reviewed at a university-based hospital.From 2011 to 2017, a total of 88 patients with a confirmed diagnosis of cutaneous NTM infection were included. Mycobacterium abscessus was the most common pathogens followed by M haemophilum and M marinum (61.4%, 10.2%, and 8.1%, respectively). Nodule and plaque were 2 most common lesions (26.4% and 25.5%, respectively) and lower leg is the most common site of involvement (50.9%). The majority of patients presented with single lesion (67%). Seven patients (7.9%) had history of surgical procedure and/or cosmetic injection before the development of lesion and all pathogenic organisms in this group were rapidly growing mycobacteria. Sweet's syndrome and erythema nodosum were the 2 most common reactive dermatoses, presented in 3.4% and 2.3%, respectively. The majority of patients infected with cutaneous M haemophilum infections were immunocompromised and lacked history of preceding trauma (77.8%). Patients with cutaneous NTM that receiving less than 3 medications was associated with higher disease relapse (odds ratio 65.86; P = .02).M abscessus is the most common pathogen of cutaneous NTM infection in Thailand. The prevalence of M haemophilum is increasing and should be particularly cautious in immunocompromised patients. Rapidly growing mycobacteria should be suspected in all cases of procedure-related cutaneous NTM. We recommend at least 3 antibiotics should be considered for cutaneous NTM infection to reduce the rate of relapse.

Macrophage Signaling Pathways in Pulmonary Nontuberculous Mycobacteria Infections.

The incidence and prevalence of nontuberculous mycobacteria (NTM) lung disease is rising worldwide and accounts for most clinical cases of NTM disease. NTM infections occur in both immunocompetent and immunocompromised hosts. Macrophages are the primary host cells that initiate an immune response to NTM. Defining the molecular events that govern the control of infection within macrophages is fundamental to understanding the pathogenesis of NTM disease. Here, we review key macrophage host signaling pathways that contribute to the host immune response to pulmonary NTM infections. In this review, we focus primarily on NTM that are known to cause lung disease, including Mycobacterium avium intracellulare, M. abscessus, and M. kansasii.

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.