Application of a new designed high resolution melting analysis for mycobacterial species identification.

The Non-tuberculous mycobacterial (NTM) isolates should be distinguished from tuberculosis and identified at the species level for choosing an appropriate treatment plan. In this study, two molecular methods were used to differentiate NTM species, including a new designed High Resolution Melting (HRM) and Multilocus Sequence Analysis (MLSA). Seventy-five mycobacterial isolates were evaluated by sequencing four genes ( MLSA) and a HRM assay specifically targeting atpE was designed to rapidly and accurately identify and differentiate mycobacterium species. Out of 70 NTM isolates, 66 (94.3%), 65 (92.9%), 65 (92.9%) and 64 (91.4%) isolates were identified to the species level by PCR of atpE, tuf, rpoB and dnaK genes. We could identify 100% of the isolates to the species level (14 different species) by MLSA. By using HRM assay, all NTM isolates were identified and classified into eight groups, in addition, Mycobacterium tuberculosis and Nocardia were also detected simultaneously. The MLSA technique was able to differentiate all 14 species of NTM isolates. According to the results, the HRM assay is a rapid and beneficial method for identifying NTM, M. tuberculosis (MTB), and Nocardia isolates without sequencing.

Orphan response regulator NnaR is critical for nitrate and nitrite assimilation in Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.

Gut microbiota dysbiosis-related susceptibility to nontuberculous mycobacterial lung disease.

The role of gut microbiota in host defense against nontuberculous mycobacterial lung disease (NTM-LD) was poorly understood. Here, we showed significant gut microbiota dysbiosis in patients with NTM-LD. Reduced abundance of Prevotella copri was significantly associated with NTM-LD and its disease severity. Compromised TLR2 activation activity in feces and plasma in the NTM-LD patients was highlighted. In the antibiotics-treated mice as a study model, gut microbiota dysbiosis with reduction of TLR2 activation activity in feces, sera, and lung tissue occurred. Transcriptomic analysis demonstrated immunocompromised in lung which were closely associated with increased NTM-LD susceptibility. Oral administration of P. copri or its capsular polysaccharides enhanced TLR2 signaling, restored immune response, and ameliorated NTM-LD susceptibility. Our data highlighted the association of gut microbiota dysbiosis, systematically compromised immunity and NTM-LD development. TLR2 activation by P. copri or its capsular polysaccharides might help prevent NTM-LD.

Differential radiological features of patients infected or colonised with slow-growing non-tuberculous mycobacteria.

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is considered a growing health concern. The majority of NTM-PD cases in Europe are caused by slow-growing mycobacteria (SGM). However, distinct radiological features of different SGM remain largely uninvestigated. We applied a previously described radiological score to a patient cohort consisting of individuals with isolation of different SGM. Correlations between clinical data, species and computed tomography (CT) features were examined by logistic and linear regression analyses, as well as over the course of time. Overall, 135 pulmonary CT scans from 84 patients were included. The isolated NTM-species were mainly Mycobacterium avium complex (MAC, n = 49), as well as 35 patients with non-MAC-species. Patients with isolation of M. intracellulare had more extensive CT findings compared to all other SGM species (coefficient 3.53, 95% Cl - 0.37 to 7.52, p = 0.075) while patients meeting the ATS criteria and not undergoing therapy exhibited an increase in CT scores over time. This study provides insights into differential radiological features of slow-growing NTM. While M. intracellulare exhibited a tendency towards higher overall CT scores, the radiological features were similar across different SGM. The applied CT score might be a useful instrument for monitoring patients and could help to guide antimycobacterial therapy.

Successful diagnosis of Mycobacterium marinum infection by mNGS in a patient with Human Immunodeficiency Virus: a case report.

INTRODUCTION: Mycobacterium marinum infection rarely occurs and has atypical symptoms. It is challenging to distinguish disseminated M. marinum infection from multifocal dermatosis caused by other factors clinically. CASE PRESENTATION: Herein, we reported a 68-year-old male patient with Human Immunodeficiency Virus (HIV) who presented redness and swelling in his left hand after being stabbed by marine fish for over 2 months. Mycobacterium tuberculosis infection was considered according to biochemical and pathological examinations, while empirical anti-infection treatment was ineffective. RESULTS: The metagenomic next-generation sequencing (mNGS) detected a large amount of M. marinum sequences, and the patient was finally diagnosed with M. marinum infection. After one month of combination therapy with ethambutol, rifabutin, moxifloxacin, and linezolid, the swelling disappeared significantly. In this case, the successful application of mNGS in diagnosing and treating M. marinum infection has improved the understanding of the microbe both in the laboratory and clinically, especially in patients with HIV. CONCLUSIONS: For diseases with atypical symptoms or difficulty in determining the pathogens, mNGS is suggested in clinical procedures for rapid and accurate diagnosis and treatment.

Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.

Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.

Comparison of the sputum microbiome between patients with stable nontuberculous mycobacterial pulmonary disease and patients requiring treatment.

BACKGROUND: We evaluated whether the sputum bacterial microbiome differs between nontuberculous mycobacteria pulmonary disease (NTM-PD) patients with stable disease not requiring antibiotic treatment and those requiring antibiotics. METHODS: We collected sputum samples from 21 clinically stable NTM-PD patients (stable group) and 14 NTM-PD patients needing antibiotic treatment (treatment group). We also obtained 13 follow-up samples from the stable group. We analyzed the 48 samples using 16S rRNA gene sequencing (V3-V4 region) and compared the groups. RESULTS: In the linear discriminant analysis effect size (LEfSe) analysis, the species Porphyromonas pasteri, Haemophilus parahaemolyticus, Prevotella nanceiensis, and Gemella haemolysans were significantly more prevalent in the sputum of the stable group compared to the treatment group. No taxa showed significant differences in alpha-/beta-diversity or LEfSe between the 21 baseline and 13 follow-up sputum samples in the stable group. In the stable group, the genus Bergeyella and species Prevotella oris were less common in patients who achieved spontaneous culture conversion (n = 9) compared to those with persistent NTM positivity (n = 12) (effect size 3.04, p = 0.039 for Bergeyella; effect size 3.64, p = 0.033 for P. oris). In the treatment group, H. parainfluenzae was more common in patients with treatment success (n = 7) than in treatment-refractory patients (n = 7) (effect size 4.74, p = 0.013). CONCLUSIONS: Our study identified distinct bacterial taxa in the sputum of NTM-PD patients based on disease status. These results suggest the presence of a microbial environment that helps maintain disease stability.

Identification of the Optimal Cultivation Period Required to Isolate Representatives of Mycobacterium abscessus Complex Isolated from Patients with Cystic Fibrosis.

BACKGROUND: In patients with cystic fibrosis (CF), representatives of the fast-growing Mycobacterium abscessus complex (MABSc) are often distinguished, but the culture of the material taken from such patients increases the growth time. We analyzed the terms of cultivation of MABSc representatives on dense nutrient media and also evaluated the productivity of a modified nutrient medium based on agar for the isolation of Burkholderia cepacia complex (BCC). METHODS: Sixty-four strains of MABSc isolated from patients with CF and suspected tuberculosis were analyzed. The material from the patients was cultured on a universal chromogenic medium, 5% blood agar, yolk-salt agar, selective medium for isolation of BCC, and Löwenstein-Jensen medium. The cultures were incubated for 5 days (37°C, aerobic conditions), after for 23 days (28°C, aerobic conditions). The productivity of the developed nutrient medium was evaluated by the number of cells that gave visible growth after culturing 0.1 mL of a bacterial suspension of 103 CFU/mL. RESULTS: 76.8% of the strains grew in a 2-week period, and 23.2% of the strains were obtained at a later date from 18 to 28 days (average: 21.23 days). The modified medium with a concentration of 240 mg of iron (III) polymaltose hydroxide proved to be the most optimal for the isolation of MABSc. CONCLUSION: When using a chromogenic medium for culture material from patients with CF, it is necessary to extend incubation up to 28 days to increase the probability of MABSc isolation. The modified BCC medium showed a good selectivity result but required further investigation.

Modulating mycobacterial envelope integrity for antibiotic synergy with benzothiazoles.

Developing effective tuberculosis drugs is hindered by mycobacteria's intrinsic antibiotic resistance because of their impermeable cell envelope. Using benzothiazole compounds, we aimed to increase mycobacterial cell envelope permeability and weaken the defenses of Mycobacterium marinum, serving as a model for Mycobacterium tuberculosis Initial hit, BT-08, significantly boosted ethidium bromide uptake, indicating enhanced membrane permeability. It also demonstrated efficacy in the M. marinum-zebrafish embryo infection model and M. tuberculosis-infected macrophages. Notably, BT-08 synergized with established antibiotics, including vancomycin and rifampicin. Subsequent medicinal chemistry optimization led to BT-37, a non-toxic and more potent derivative, also enhancing ethidium bromide uptake and maintaining synergy with rifampicin in infected zebrafish embryos. Mutants of M. marinum resistant to BT-37 revealed that MMAR_0407 (Rv0164) is the molecular target and that this target plays a role in the observed synergy and permeability. This study introduces novel compounds targeting a new mycobacterial vulnerability and highlights their cooperative and synergistic interactions with existing antibiotics.

Identification of Mycobacterium abscessus using the peaks of ribosomal protein L29, L30 and hemophore-related protein by MALDI-MS proteotyping.

Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.

Clinical Characteristics and Treatment Outcomes of Pulmonary Diseases Caused by Coinfections With Multiple Nontuberculous Mycobacterial Species.

BACKGROUND: Coinfections with multiple nontuberculous mycobacterial (NTM) species have not been widely studied. We aimed to evaluate the clinical characteristics and treatment outcomes in patients with NTM-pulmonary disease (PD) caused by coinfection with multiple NTM species. METHODS: We retrospectively reviewed patients with NTM-PD at a tertiary referral hospital in Korea between March 2012 and December 2018. Coinfection was defined as two or more species of NTM pathogens isolated from the same respiratory specimen or different specimens within three months. RESULTS: Among 1,009 patients with NTM-PD, 147 (14.6%) NTM coinfections were observed (average age 64.7 years, 69.4% women). NTM species were identified more frequently (median 6 vs. 3 times, P < 0.001) in the coinfection group than in the single species group, and follow-up duration was also longer in the coinfection group (median 44.9 vs. 27.1 months, P < 0.001). Mycobacterium avium complex (MAC) and M. abscessus and M. massiliense (MAB) were the dominant combinations (n = 71, 48.3%). For patients treated for over six months in the MAC plus MAB group (n = 31), sputum culture conversion and microbiological cure were achieved in 67.7% and 41.9% of patients, respectively. We divided the MAC plus MAB coinfection group into three subgroups according to the target mycobacteria; however, no statistical differences were found in the treatment outcomes. CONCLUSION: In NTM-PD cases, a significant number of multiple NTM species coinfections occurred. Proper identification of all cultured NTM species through follow-up is necessary to detect multispecies coinfections. Further research is needed to understand the nature of NTM-PD in such cases.

Genomic and phenotypic characterization of Mycobacterium tuberculosis' closest-related non-tuberculous mycobacteria.

Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

Vitamin B12 uptake across the mycobacterial outer membrane is influenced by membrane permeability in Mycobacterium marinum.

Vitamin B12 (B12) serves as a critical cofactor within mycobacterial metabolism. While some pathogenic strains can synthesize B12 de novo, others rely on host-acquired B12. In this investigation, we studied the transport of vitamin B12 in Mycobacterium marinum using B12-auxotrophic and B12-sensitive strains by deleting metH or metE, respectively. These two enzymes rely on B12 in different ways to function as methionine synthases. We used these strains to select mutants affecting B12 scavenging and confirmed their phenotypes during growth experiments in vitro. Our analysis of B12 uptake mechanisms revealed that membrane lipids and cell wall integrity play an essential role in cell envelope transport. Furthermore, we identified a potential transcription regulator that responds to B12. Our study demonstrates that M. marinum can take up exogenous B12 and that altering mycobacterial membrane integrity affects B12 uptake. Finally, during zebrafish infection using B12-auxotrophic and B12-sensitive strains, we found that B12 is available for virulent mycobacteria in vivo.IMPORTANCEOur study investigates how mycobacteria acquire essential vitamin B12. These microbes, including those causing tuberculosis, face challenges in nutrient uptake due to their strong outer layer. We focused on Mycobacterium marinum, similar to TB bacteria, to uncover its vitamin B12 absorption. We used modified strains unable to produce their own B12 and discovered that M. marinum can indeed absorb it from the environment, even during infections. Changes in the outer layer composition affect this process, and genes related to membrane integrity play key roles. These findings illuminate the interaction between mycobacteria and their environment, offering insights into combatting diseases like tuberculosis through innovative strategies. Our concise research underscores the pivotal role of vitamin B12 in microbial survival and its potential applications in disease control.

Mycobacterium immunogenum acquisition from hospital tap water: a genomic and epidemiologic analysis.

We identified 23 cases of Mycobacterium immunogenum respiratory acquisition linked to a colonized plumbing system at a new hospital addition. We conducted a genomic and epidemiologic investigation to assess for clonal acquisition of M. immunogenum from hospital water sources and improve understanding of genetic distances between M. immunogenum isolates. We performed whole-genome sequencing on 28 M. immunogenum isolates obtained from August 2013 to July 2021 from patients and water sources on four intensive care and intermediate units at an academic hospital. Study hospital isolates were recovered from 23 patients who experienced de novo respiratory isolation of M. immunogenum and from biofilms obtained from five tap water outlets. We also analyzed 10 M. immunogenum genomes from previously sequenced clinical (n = 7) and environmental (n = 3) external control isolates. The 38-isolate cohort clustered into three clades with pairwise single-nucleotide polymorphism (SNP) distances ranging from 0 to 106,697 SNPs. We identified two clusters of study hospital isolates in Clade 1 and one cluster in Clade 2 for which clinical and environmental isolates differed by fewer than 10 SNPs and had less than 0.5% accessory genome variation. A less restrictive combined threshold of 40 SNPs and 5% accessory genes reliably captured additional isolates that met clinical criteria for hospital acquisition, but 12 (4%) of 310 epidemiologically unrelated isolate pairs also met this threshold. Core and accessory genome analyses confirmed respiratory acquisition of multiple clones of M. immunogenum from hospital water sources to patients. When combined with epidemiologic investigation, genomic thresholds accurately distinguished hospital acquisition.

Synergistic effects of sulopenem in combination with cefuroxime or durlobactam against Mycobacterium abscessus.

Mycobacterium abscessus (Mab) affects patients with immunosuppression or underlying structural lung diseases such as cystic fibrosis (CF). Additionally, Mab poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual ß-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with ß-lactamase inhibitors [BLIs-avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration (MIC) values for 54 clinical Mab isolates and ATCC19977 (MIC50 and MIC90 ≤0.25 µg/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding ~1.5 Log10 CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in an additional reduction of CFUs (~3 Log10 for sulopenem-CXM and AVI and ~4 Log10 for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (Ldts; LdtMab1-LdtMab4), penicillin-binding-protein B (PBP B), and D,D-carboxypeptidase (DDC). Acyl complexes, identified via mass spectrometry analysis, demonstrated the binding of sulopenem with LdtMab2-LdtMab4, DDC, and PBP B and CXM with LdtMab2 and PBP B. Molecular docking and mass spectrometry data suggest the formation of a covalent adduct between sulopenem and LdtMab2 after the nucleophilic attack of the cysteine residue at the ß-lactam carbonyl carbon, leading to the cleavage of the ß-lactam ring and the establishment of a thioester bond linking the LdtMab2 with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLIs. These findings potentially broaden the selection of oral therapeutic agents to combat Mab. IMPORTANCE: Treating infections from Mycobacterium abscessus (Mab), particularly those resistant to common antibiotics like macrolides, is notoriously difficult, akin to a never-ending struggle for healthcare providers. The rate of treatment failure is even higher than that seen with multidrug-resistant tuberculosis. The role of combination ß-lactams in inhibiting L,D-transpeptidation, the major peptidoglycan crosslink reaction in Mab, is an area of intense investigation, and clinicians have utilized this approach in the treatment of macrolide-resistant Mab, with reports showing clinical success. In our study, we found that cefuroxime and sulopenem, when used together, display a significant synergistic effect. If this promising result seen in lab settings, translates well into real-world clinical effectiveness, it could revolutionize current treatment methods. This combination could either replace the need for more complex intravenous medications or serve as a "step down" to an oral medication regimen. Such a shift would be much easier for patients to manage, enhancing their comfort and likelihood of sticking to the treatment plan, which could lead to better outcomes in tackling these tough infections. Our research delved into how these drugs inhibit cell wall synthesis, examined time-kill data and binding studies, and provided a scientific basis for the observed synergy in cell-based assays.

Immunological and microbial shifts in the aging rhesus macaque lung during nontuberculous mycobacterial infection.

Nontuberculous mycobacteria (NTM) are environmentally ubiquitous organisms that predominately cause NTM pulmonary disease (NTMPD) in individuals over the age of 65. The incidence of NTMPD has increased in the U.S., exceeding that of Mycobacterium tuberculosis. However, the mechanisms leading to higher susceptibility and severity of NTMPD with aging are poorly defined in part due to the lack of animal models that accurately recapitulate human disease. Here, we compared bacterial load, microbial communities, and host responses longitudinally between three young (two female and one male) and two aged (two female) rhesus macaques inoculated with Mycobacterium avium subsp. hominissuis (MAH) in the right caudal lobe. Unilateral infection resulted in a low bacterial load in both young and aged animals confined to the infected side. Although a robust inflammatory response was only observed in the inoculated lung, immune cell infiltration and antigen-specific T cells were detected in both lungs. Computed tomography, gross pathology, and histopathology revealed increased disease severity and persistence of bacterial DNA in aged animals. Additional analyses showed the translocation of gut and oral-pharyngeal bacterial DNA into the lower respiratory microbiome. Finally, single-cell RNA sequencing revealed a heightened inflammatory response to MAH infection by alveolar macrophages in aged animals. These data are consistent with the model that increased disease severity in the aged is mediated by a dysregulated macrophage response that may be sustained through persistent antigen presence. IMPORTANCE: Nontuberculous mycobacteria (NTM) are emerging as pathogens of high consequence, as cases of NTM pulmonary disease (NTMPD) have exceeded those of Mycobacterium tuberculosis. NTMPD can be debilitating, particularly in patients over 65 years of age, as it causes chronic cough and fatigue requiring prolonged treatments with antibiotics. The underlying mechanisms of this increased disease severity with age are poorly understood, hampering the development of therapeutics and vaccines. Here, we use a rhesus macaque model to investigate the impact of age on host-NTM interactions. This work shows that aging is associated with increased disease severity and bacterial persistence in aged rhesus macaques, thus providing a preclinical model to develop and test novel therapeutics and interventions.

Disasters pile up on the rubbing heel: Sporothrix globosa as secondary infection to Mycobacterium chelonae infection.

Mycobacterium chelonae and Sporothrix globosa, both of which are opportunistic pathogens, have been proved to be possible multidrug resistant. However, are all recurring symptoms in chronic infections related to decreasing susceptibility? Here we report a case of sporotrichosis secondary to M. chelonae infection. In addition, we find that the blackish-red spots under the dermoscopic view can be employed as a signal for the early identification and regression of subcutaneous fungal infection.

Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

Disseminated mycobacterium genavense infection with central nervous system involvement in an HIV patient: a case report and literature review.

BACKGROUND: Immunodeficient patients, particularly HIV patients, are at risk of opportunistic infections. Nontuberculous mycobacteria can cause severe complications in immunodeficient patients. CASE PRESENTATION: We describe a 57-year-old HIV patient, primarily presented with coughs and constitutional symptoms, with a unique Mycobacterium genavense abdominal, pulmonary, and central nervous system infection, accompanied by intracranial masses. CONCLUSION: The diagnosis of NTM, including M. genavense, must always be considered by clinicians in immunodeficient patients, especially those with HIV, who have a compromised immune system.