Nontuberculous mycobacteria (NTM) are a group of environmental bacteria that encompass nearly 200 described species, some of which can cause chronic pulmonary and extrapulmonary infections in humans. What makes these infections unique is that they are environmentally acquired, yet there remains a limited understanding of how different environments contribute to potential pathogen exposure. Here, we use new and existing marker gene data sets to compare the amounts and types of NTM across three environments known to harbor mycobacteria, surface waters, soil, and household plumbing biofilms, to better understand potential pathogen occurrence in each environment. We used 16S rRNA gene sequencing, in tandem with mycobacterial-specific marker gene sequencing, to characterize variation in the relative abundances of the genus Mycobacterium and specific mycobacterial taxa across the three environments, with a focus on a clinically significant NTM. We found that household plumbing biofilms contained both the highest relative abundance of the genus Mycobacterium (on average, 13.7% of bacteria were members of the genus), as well as the highest occurrence of clinically relevant species detected (Mycobacterium avium and Mycobacterium abscessus), compared to surface waters and soil. Although mycobacteria are ubiquitous across many different environments, mycobacterial diversity is highly variable between environments with clinically relevant species largely restricted to household plumbing biofilms, information that is critical for understanding the ecology and epidemiology of NTM disease.IMPORTANCENontuberculous mycobacteria, or NTM, are a diverse group of bacteria within the genus Mycobacterium that are common in many environments. While most members of the genus pose little threat to human health, a handful of species, namely the Mycobacterium avium complex, M. abscessus, and M. kansasii, can cause severe and prolonged lung infections. These environmentally acquired infections are on the rise in the United States and around the world, yet we still do not have a good understanding of which environment types pose the greatest risk of infection to susceptible populations. Our study used cultivation-independent approaches to identify the specific NTM taxa found in over 1,000 samples from three potentially important environmental reservoirs-surface waters, soils, and household plumbing systems, to determine which of these environments are most likely to harbor NTM of clinical significance. Our results highlight the high degree of variability in the types of NTM taxa detected in different environments (including extensive novel diversity within the genus) and show that household plumbing biofilms are likely the most important reservoir and subsequent route of transmission for clinically significant NTM.

Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms that can cause severe infections in vulnerable individuals. However, the environmental reservoirs and transmission routes of clinically relevant NTM remain insufficiently understood. Given the rising incidence of NTM infections, understanding where these bacteria thrive can inform public health measures and reduce infection risk. In this study, we analysed the prevalence and genetic diversity of mycobacteria in indoor water samples from private residences and a hospital in Frankfurt am Main, Germany, and characterised them using whole genome sequencing and phenotypic drug susceptibility testing. Core genome multi-locus sequence typing (cgMLST) was employed to compare environmental isolates with nearly 3,000 clinical and environmental NTM isolates from around the world, aiming to identify potential epidemiological connections and global lineages. NTM were isolated from 9 of 41 households (22.0%) and 15 of 102 water samples (14.7%). Eight different NTM-species were identified, including one novel species. For Mycobacterium abscessus and Mycobacterium chimaera, strains belonging to globally distributed lineages were detected. Notably, M. abscessus dominant circulating clone 3 (DCC3) was present in shower water and we report, for the first time, global lineages of M. chelonae in both environmental and clinical samples. While NTM from different households were genetically distinct, isolates from five households (M. chimaera, M. abscessus, and M. chelonae) were closely related to clinical isolates from Germany and abroad. In conclusion, our findings highlight indoor water systems as reservoirs for some clinically significant NTM, including globally relevant clones such as M. abscessus DCC3, and underscore the potential for targeted public health interventions to reduce exposure risks, especially for vulnerable populations.

Erector spinae muscle thickness predicts mortality in patients with pulmonary diseases.

Surgical site infection of orthopedic trauma patients is common and tends to be treated with optimal therapy after an organism is identified from intraoperative cultures. However, delayed presentation and multidrug resistance can lead to complications. In our case, we investigate a delayed presentation of Mycobacterium abscessus orthopedic hardware infection in a patient with a forearm fracture secondary to a motor vehicle accident. Six weeks after stabilization of the fracture, purulence from an iliac crest bone graft donor site was noted. Further investigation led to the diagnosis of M. abscessus infection of both the graft donor and recipient sites in the forearm. After a multitude of surgeries, including orthopedic hardware removal from the forearm, and concurrent antibiotic therapy of amikacin, imipenem, tedizolid, and omadacycline, the patient was deemed medically cleared of her infection. Rapid-growing nontuberculous mycobacterium (RGNTM) are rare infections of trauma patients that warrant further clinical investigation into best management practices. This has implications for both civilian and military battlefield-related trauma infections. This case serves as an example of successful multi-disciplinary management through appropriately aggressive surgery and tailored antibiotic therapy.

Mycobacteria possess a uniquely complex cell envelope and rely on a diverse array of secretion systems to interact with their environment, ensure survival, and modulate host immune responses. This review provides a comprehensive overview of these secretion pathways, from the universally conserved Sec and Tat systems to the specialized ESX/type VII secretion systems, as well as lipid transporters of the MmpL family, with particular emphasis on Mycobacterium tuberculosis and other clinically relevant members of the M. tuberculosis complex and non-tuberculous mycobacteria. By integrating findings from historical literature and the most recent experimental and bioinformatic studies, we outline the genetic organization, structure, regulation, and functional interplay of these pathways. Emphasis is placed on how these systems are not isolated entities but form a highly interconnected network that coordinates protein and lipid export essential for virulence, immune modulation, and cell wall integrity. We also explore the translational potential of secreted effectors and their transport machineries, discussing their relevance as targets for therapeutic interventions, including novel inhibitors, diagnostic biomarkers, and vaccine candidates. We highlight critical knowledge gaps and propose avenues for future research, particularly those that leverage multidisciplinary approaches. By drawing connections across secretion systems and emphasizing their shared and distinct roles, this work aims to provide an integrated framework that supports both fundamental understanding and biomedical innovation in mycobacterial pathogenesis.

Non-tuberculous mycobacteria are emerging respiratory pathogens that can persist in treated water systems. In 2018, a cluster of Mycobacterium intracellulare lung infections was linked to a pool facility in Australia, prompting an epidemiological and genomic investigation. M. intracellulare was isolated from five sputum samples across four clinical cases and from fourteen pool water samples across a total of five collection time points. All cases were resolved following exclusion from the pool facility, with only one patient requiring short-term steroids; none of the patients required anti-mycobacterial treatment. To test if this was a point-source outbreak, whole-genome sequencing of mycobacteria recovered from patients and the pool was implemented. Initial analysis confirmed all patient and water isolates were M. intracellulare with sequence type 210. A complete, circular genome was constructed from one of the isolates linked to this cluster and was used as a reference genome for high-resolution core genome SNP analysis. This analysis showed tight clustering of M. intracellulare genomes from patient and pool water isolates that were distinct from other M. intracellulare. Thus, epidemiological and comparative genome analysis strongly implicated the pool as the origin of these infections.

Infections in VEXAS syndrome: a systematic review of the literature.

Clinical characteristics analysis of anti-interferon-γ autoantibodies-associated adult-onset immunodeficiency with mycobacterium tuberculosis infection.

Enhanced targeted NGS assay for comprehensive diagnosis in tuberculosis and drug-resistant tuberculosis patients.

The impact of sodium-glucose cotransporter 2 inhibitors on the incidence of non-tuberculous mycobacteria infection in diabetes populations

Uncommon pulmonary mycobacterium wolinskyi infection in a hemodialysis patient.

The genetic basis underlying nontuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well‐established mycobacteria‐optimized dCas9 Sth1 ‐mediated gene knockdown system to develop a new subset of fluorescence‐based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use Mycobacterium smegmatis ( M. smeg ) and Mycobacterium abscessus ( M. abs ) as NTM model species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene silencing when targeting essential genes but also genes involved in smooth‐to‐rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.

Previous studies have not covered the distribution of non-tuberculous mycobacteria (NTM) in various regions of Hainan Province. This study provides the first comprehensive geographic map of NTM in Hainan, China's tropical island hotspot. By monitoring NTM across 14 cities over 4 years, we reveal key patterns in their habitat preferences and dominant species in infections. These findings establish a foundational framework for region-specific surveillance and treatment protocols in Hainan and offer a replicable model for global tropical regions combating NTM infections. Ultimately, this work empowers public health initiatives to reduce misdiagnosis, enhance antibiotic stewardship, and lower mortality rates among at-risk populations.

Introduction The growing prevalence of multidrug-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) highlights the urgent need for alternative therapeutic approaches. Mycobacteriophages, viruses that selectively infect mycobacteria, have emerged as promising tools. Here, we report the isolation and characterization of a new subcluster K4 phage, Yasnaya_Polyana, with a focus on its regulatory motifs and engineered lytic variant. Methods The phage was isolated by enrichment on Mycobacterium smegmatis mc(2)155, followed by genome sequencing and functional annotation. Start-Associated Sequences (SAS) and Extended SAS (ESAS) were analyzed in silico across 188 cluster K phages. A lytic derivative, YPΔ47, was engineered by deleting the repressor gene and characterized in terms of morphology, stability, infection dynamics, and host range. Results Yasnaya_Polyana exhibited siphovirus morphology and high genetic similarity with other subcluster K4 phages. Regulatory motif analysis revealed a reduced abundance of SAS and ESAS elements in subcluster K4 phages, including Yasnaya_Polyana, along with specific ESAS sequence deviations. The engineered YPΔ47 mutant retained morphology and infection parameters comparable to the wild-type phage but exhibited a decline in lysogeny frequency (from 18% to <0.01%), confirming a lytic phenotype. Host range analysis revealed limited activity of YPΔ47 against NTM, while the phage demonstrated a high efficiency of plating (EOP = 10 −1 ) on M. tuberculosis H37Rv and effectively lysed clinical isolates. Discussion These findings suggest that Yasnaya Polyana, and apparently other subcluster K4 phages, harbor distinct regulatory features that may reflect divergent transcriptional control strategies. Moreover, YPΔ47 shows potential as a candidate for phage therapy targeting mycobacterial infections.

Mycobacteroides abscessus is a rapidly growing non-tuberculous mycobacterium that causes chronic lung and soft tissue infections. Treatment options are severely limited. Amikacin has historically been a mainstay of combination treatment regimens. However, irreversible hearing loss and vestibular toxicity have led to a search for alternative agents. Apramycin is a novel aminoglycoside currently in Phase I clinical trials that may offer lower potential for ototoxic and renal toxic side effects. The goal of this study was to compare apramycin's in vitro activity with amikacin and other aminoglycosides against a large collection of M. abscessus clinical isolates, alone, and for apramycin and amikacin, in combination with clofazimine or linezolid. We also examined activity against a more limited collection of other rapidly growing mycobacteria. Analysis was performed using reference broth microdilution MIC testing, inkjet printer-assisted checkerboard assays, and time-kill assays. Against M. abscessus, the MIC50/90 for apramycin (2 mg/L) was one-eighth that of amikacin (16 mg/L). Plazomicin was inactive, and organisms were rarely susceptible to tobramycin. Synergy of either apramycin or amikacin with clofazimine or linezolid was not detected by checkerboard assay. In time-kill studies, clofazimine modestly increased activity of apramycin and, to a lesser extent, amikacin. Apramycin and amikacin showed delayed bacterial killing that either achieved or approached a bactericidal threshold. Apramycin was similarly potent against other rapidly growing mycobacteria tested. Apramycin exhibits more potent in vitro activity against a diverse set of M. abscessus and other rapidly growing mycobacteria than currently recommended aminoglycosides.

Effective monitoring of the treatment response is essential for successful tuberculosis (TB) management as prolonged therapies require accurate evaluation to prevent relapse, treatment failure, or drug resistance. This study highlights the diagnostic and treatment-monitoring potential of the PATHFAST TB LAM Ag assay, which quantifies lipoarabinomannan (LAM) concentrations in sputum samples and correlates with bacterial load. For the first time, we also demonstrate the assay's applicability for detecting and monitoring nontuberculous mycobacterial (NTM) pulmonary diseases, which are increasingly prevalent in industrialized countries. The semi-automated, rapid format (<17 minutes) of the PATHFAST TB LAM Ag assay provides a simple and reliable approach for assessing bacillary load during treatment, representing a promising tool for improving patient management and diagnostic efficiency in both TB and NTM-PD.

Metagenomic next-generation sequencing (mNGS) was suggested to potentially replace traditional microbiological methods because of its comprehensiveness. However, the diagnostic utility of mNGS for tissue hasn’t been fully explored, especially for patient with HIV infection. HIV-positive and negative patients with suspected infectious diseases who performed tissue mNGS and conventional microbiological tests (CMTs) were retrospectively enrolled between October, 2020 and May 2024. The microbial spectrum of tissue mNGS and CMTs was analyzed, and the diagnostic accuracy and consistency of mNGS and CMTs for tissue were compared. The related factors of positive rate of mNGS was analyzed. Of 70 patients with suspected infectious diseases, 44 cases were confirmed with the infectious diseases. Among 44 patients with infectious diseases, aerobic bacteria (36.4%) was the most common detected pathogen, followed by mycobacterium tuberculosis (MTB, 18.2%), non-tuberculous mycobacteria (NTM, 13.6%) and fungus (11.4%). The sensitivity of tissue mNGS (72.7%, 95%CI 56.9%-84.5%) was significantly higher than that that in tissue CMTs (29.5%, 95%CI 17.2%-45.4%) (p&amp;lt;0.001), but the specificity was not statistically significant(P = 0.656). mNGS demonstrated higher detection rates than CMTs in the case with single microbial infections (70.0% vs. 30.0%; p&amp;lt;0.01). For the case with multiple microbial infections, the detection rates of mNGS and CMTs was 100.0% and 25.5% (p=1.000), respectively. Both positive mNGS and CMTs were observed in 22.7% patients with infectious diseases, and sole positive mNGS and sole positive CMTs were observed in 50.0% and 6.8% patients, respectively. There were no statistically differences in age, gender, HIV infection, PCT levels, neutrophil counts, CD4 + lymphocyte count and antibiotic exposure between mNGS positive and mNGS negative groups (P &amp;gt; 0.05). Tissue mNGS could provide a higher sensitivity, more robust and broader method for pathogen identification by comparison with CMTs. However, CMTs shouldn’t be ignored since the low consistency between CMTs and mNGS.

Background Infections and exacerbations are common for people living with Cystic Fibrosis (pwCF). This project aimed to develop patient-focused diagnostic target product profiles (TPP) to address unmet diagnostic needs for pwCF with international relevance. Methods This project involved three phases, guided by an expert advisory group of key opinion leaders (KoLs) including methodologists, pwCF, and clinical staff, and including lived experience perspectives at every stage. Phase 1 involved a landscape analysis to identify and prioritise unmet diagnostic needs via focus groups with clinical experts and pwCF, as well as a scoping review of the diagnostic space and available diagnostic tests. Phase 2 involved drafting the TPPs using evidence from existing literature and regulatory documentation, focus groups, one-to-one interviews with KoLs, and web-based surveys, to define ‘minimal’ and ‘optimal’ characteristics for each TPP. Phase 3 refined and validated the TPP content through additional interviews, a two-round modified Delphi exercise, and a virtual symposium. Results A comprehensive document detailing four TPPs was drafted and released freely for use. The document includes aspirational TPPs covering diagnostics for managing acute pulmonary exacerbations, rapid pathogen identification, and antimicrobial susceptibility tests, as well as a detailed TPP defining in-vitro diagnostic tests for rapid detection of non-tuberculous mycobacteria (NTM) pulmonary infections. The document contains additional guidance for diagnostic development and research needs identified for pwCF. Conclusions The project consulted with over 150 individuals and experts in CF infection management, and diagnostic development. The TPP guidance document supports research and development of patient-focused diagnostics for the benefit of pwCF.

Investigate the increased incidence of Mycobacterium chelonae positive respiratory cultures in hospitalized patients. Apply the Healthcare-Associated Links in Transmission of Nontuberculous Mycobacteria (HALT NTM) toolkit to an outbreak investigation of M. chelonae. Quaternary-care pediatric hospital and medical center in the United States with a recently opened LEED-certified critical care tower. Adult and pediatric patients with M. chelonae positive respiratory cultures between June 2022 and January 2024. An epidemiological investigation involving clinical and laboratory practices, water management, building construction and renovation projects. Environmental sampling of air vents, water sources and endoscope reprocessing equipment was performed. M. chelonae isolates recovered from patients and the environment were analyzed using whole genome sequencing and compared for relatedness. Three clusters of matching environmental and patient isolates were identified. The most common environmental source of M. chelonae was ice/water dispensers with 40% positivity of sampled units. The critical care tower's water system performance and metrics were suboptimal, leading to four physical remediation activities along with a hyperchlorination treatment. Recent and ongoing construction along with the implementation of a LEED-certified, low-flow water system in a new critical care tower provided enhanced opportunities for M. chelonae exposure at point of use locations such as ice/water dispensers. More national infection prevention and control guidance is needed to address the infection risks from water sustainability efforts and construction activities in healthcare facilities.

Digestion and decontamination during acid-fast bacilli (AFB) culture processing are performed to suppress growth of normal microbiota in respiratory specimens; however, these steps may render AFB nonviable. This study aimed to evaluate the performance of NTM Elite agar, a decontamination-free selective medium, for the recovery of nontuberculous mycobacteria (NTM) compared to standard of care (SOC) processing and solid and liquid media inoculation for AFB culture in five hundred lower respiratory samples from non-cystic fibrosis patients. Concurrently, the same specimens were directly inoculated onto NTM Elite agar (NTM Elite-Direct Inoculation) or centrifuged and washed with saline prior to inoculation onto NTM Elite agar (NTM Elite-Concentrated) and incubated up to 28 days. The overall median time to positivity was 7.0 days (IQR = 7-18) for NTM Elite-Direct Inoculation and 14 days (IQR = 7-14) for NTM Elite-Concentrated, which were similar to SOC broth but shorter than SOC solid agar at 35 days (IQR = 2-41). Breakthrough non-AFB growth rate was 1.4% (7/500) NTM Elite-Direct Inoculation, which was less than NTM Elite-Concentrated at 9.0% (45/500) and SOC media at 10.0% (50/500). Forty-five unique isolates were included in the analysis for sensitivity of NTM detection. Sensitivity was 43.5% (95% CI = 30.2-57.8) for SOC media, 20.0% (95% CI = 10.9-33.8) for NTM Elite-Direct Inoculation, and 82.6% (95% CI = 69.3-90.9) for NTM Elite-Concentrated. Combined with SOC broth culture, sensitivity was 93.3% (95% CI = 82.1-97.7) for NTM Elite-Concentrated. The high sensitivity of the latter procedure indicates potential for NTM Elite agar to replace SOC solid agar for detection of NTM in areas of high NTM incidence but low incidence of tuberculosis.