[A case of HIV combined with Mycobacterium celatum infection].

Here, we reported the diagnosis and treatment of a case of HIV infected person complicated by an extremely rare infection with Mycobacterium celatum. Due to the similarity of homologous sequence regions between Mycobacterium celatum and Mycobacterium tuberculosis complex, the identification of conventional Mycobacterium species was incorrect, which was corrected after first-generation 16S rRNA sequencing. This report aimed to improve the clinical understanding of Mycobacterium celatum infection and the level of differential diagnosis between non-tuberculous mycobacterial disease and tuberculosis.

Impact of the COVID-19 pandemic on the real-world diagnostic infrastructure for tuberculosis-An ESGMYC collaborative study.

We determined the impact of the COVID-19 pandemic on mycobacterial diagnostic services. 40 laboratories from 22 countries completed an online questionnaire covering the redeployment of the laboratory infrastructure and/or staff for SARS-CoV-2 testing, staff shortages and supply chain disruptions. 28 laboratories reported monthly numbers of samples processed for mycobacterial investigations and monthly numbers of M. tuberculosis complex (MTBC) PCRs performed between October 1st 2018 and October 31st 2020. More than half (23/40) of the participating TB laboratories reported having performed COVID-19 diagnostics in the early phase of the pandemic, in part with negative impact on the mycobacterial service activities. All participating laboratories reported shortages of consumables and laboratory equipment due to supply chain issues. Average monthly sample numbers decreased by 24% between January 2020 and October 2020 compared to pre-pandemic averages. At the end of the study period, most participating laboratories had not returned to pre-pandemic average MTBC PCR throughput.

Is the new tuberculous antigen-based skin test ready for use as an alternative to tuberculin skin test/interferon-gamma release assay for tuberculous diagnosis? A narrative review.

In recent years, novel specific Mycobacteria tuberculous (TB) antigen-based skin test (TBST) has become available for clinical use. The mechanism of TBST is similar to the interferon-gamma release assay (IGRA), making it a potential alternative for identifying latent tuberculous infection (LTBI), especially in subjects with history of bacille Calmette-Guérin vaccination. Three different commercial brands have been developed in Denmark, Russia, and China. Clinical studies in the respective countries have shown promising sensitivity, specificity, and safety profile. Some studies attempted to address the applicability of TBST in specific subject groups but the discrepancy in defining LTBI and problematic methodologies undermine the generalisation of the results to other communities across the world. Limited cost-effectiveness studies for TBST have been conducted without exploring the health economics for preventing development of LTBI into active TB. Unlike IGRA, no clinical studies have addressed the correlation of TBST results (magnitude of induration) with the likelihood of development of active TB. Moreover, the different TBSTs are not widely available for clinical use. While TBST is a promising test to overcome the shortcomings of tuberculin skin tests, more clinical data are needed to support its general application globally for the diagnosis of LTBI.

Beyond copper: examining the significance of His-mutations in mycobacterial GroEL1 HRCT for Ni(II) complex stability and formation.

Recently, we have studied the coordination chemistry of the Cu(II)-histidine-rich C-terminal tail (HRCT) complex of the mycobacterial GroEL1 protein. The structure of this domain differs significantly compared to the well-known methionine-glycine-rich GroEL chaperonin - it was predicted that mycobacterial GroEL1 could play a significant role in the metal homeostasis of Mycobacteria, especially copper. However, we found that this particular domain's pattern also repeats in a number of Ni(II)-binding proteins. Here, we present the studies concerning the properties of GroEL1 HRCT as a ligand for Ni(II) ions. For this purpose, we chose eight model peptides: L1 - Ac-DHDHHHGHAH, L2 - Ac-DKPAKAEDHDHHHGHAH, and 6 mutants of the latter in the pH range of 2-11. We examined the stoichiometry, stability, and spectroscopic features of copper complexes. We noticed that similar to the Cu(II)-complex, the presence of a Lys5 residue significantly increases the stability of the system. The impact of His mutations was also examined and carefully studied using NMR spectroscopy. His9 and His13 are the crucial residues for Ni(II) binding, whereas His12 has minimal relevance in complex formation.

Compartmentalization of galactan biosynthesis in mycobacteria.

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.

4-(Benzyloxy)phenol-induced p53 exhibits antimycobacterial response triggering phagosome-lysosome fusion through ROS-dependent intracellular Ca2+ pathway in THP-1 cells.

Drug-resistant tuberculosis (TB) outbreak has emerged as a global public health crisis. Therefore, new and innovative therapeutic options like host-directed therapies (HDTs) through novel modulators are urgently required to overcome the challenges associated with TB. In the present study, we have investigated the anti-mycobacterial effect of 4-(Benzyloxy)phenol. Cell-viability assay asserted that 50 µM of 4-(Benzyloxy)phenol was not cytotoxic to phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. It was observed that 4-(Benzyloxy)phenol activates p53 expression by hindering its association with KDM1A. Increased ROS, intracellular Ca2+ and phagosome-lysosome fusion, were also observed upon 4-(Benzyloxy)phenol treatment. 4-(Benzyloxy)phenol mediated killing of intracellular mycobacteria was abrogated in the presence of specific inhibitors of ROS, Ca2+ and phagosome-lysosome fusion like NAC, BAPTA-AM, and W7, respectively. We further demonstrate that 4-(Benzyloxy)phenol mediated enhanced ROS production is mediated by acetylation of p53. Blocking of p53 acetylation by Pifithrin-α (PFT- α) enhanced intracellular mycobacterial growth by blocking the mycobactericidal effect of 4-(Benzyloxy)phenol. Altogether, the results showed that 4-(Benzyloxy)phenol executed its anti-mycobacterial effect by modulating p53-mediated ROS production to regulate phagosome-lysosome fusion through Ca2+ production.

Retrospective validation of MetaSystems' deep-learning-based digital microscopy platform with assistance compared to manual fluorescence microscopy for detection of mycobacteria.

This study aimed to validate Metasystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module (Neon Metafer) with assistance on respiratory and pleural samples, compared to conventional manual fluorescence microscopy (MM). Analytical parameters were assessed first, followed by a retrospective validation study. In all, 320 archived auramine-O-stained slides selected non-consecutively [85 originally reported as AFB-smear-positive, 235 AFB-smear-negative slides; with an overall mycobacterial culture positivity rate of 24.1% (77/320)] underwent whole-slide imaging and were analyzed by the Metafer Neon AFB Module (version 4.3.130) using a predetermined probability threshold (PT) for AFB detection of 96%. Digital slides were then examined by a trained reviewer blinded to previous AFB smear and culture results, for the final interpretation of assisted digital microscopy (a-DM). Paired results from both microscopic methods were compared to mycobacterial culture. A scanning failure rate of 10.6% (34/320) was observed, leaving 286 slides for analysis. After discrepant analysis, concordance, positive and negative agreements were 95.5% (95%CI, 92.4%-97.6%), 96.2% (95%CI, 89.2%-99.2%), and 95.2% (95%CI, 91.3%-97.7%), respectively. Using mycobacterial culture as reference standard, a-DM and MM had comparable sensitivities: 90.7% (95%CI, 81.7%-96.2%) versus 92.0% (95%CI, 83.4%-97.0%) (P-value = 1.00); while their specificities differed 91.9% (95%CI, 87.4%-95.2%) versus 95.7% (95%CI, 92.1%-98.0%), respectively (P-value = 0.03). Using a PT of 96%, MetaSystems' platform shows acceptable performance. With a national laboratory staff shortage and a local low mycobacterial infection rate, this instrument when combined with culture, can reliably triage-negative AFB-smear respiratory slides and identify positive slides requiring manual confirmation and semi-quantification. IMPORTANCE: This manuscript presents a full validation of MetaSystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module using a probability threshold of 96% including accuracy, precision studies, and evaluation of limit of AFB detection on respiratory samples when the technology is used with assistance. This study is complementary to the conversation started by Tomasello et al. on the use of image analysis artificial intelligence software in routine mycobacterial diagnostic activities within the context of high-throughput laboratories with low incidence of tuberculosis.

Evaluation of nucleotide MALDI-TOF-MS for the identification of Mycobacterium species.

Background: The accurate identification of the Mycobacterium tuberculosis complex (MTBC) and different nontuberculous mycobacteria (NTM) species is crucial for the timely diagnosis of NTM infections and for reducing poor prognoses. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been extensively used for microbial identification with high accuracy and throughput. However, its efficacy for Mycobacterium species identification has been less studied. The objective of this study was to evaluate the performance of nucleotide MALDI-TOF-MS for Mycobacterium species identification. Methods: A total of 933 clinical Mycobacterium isolates were preliminarily identified as NTM by the MPB64 test. These isolates were identified by nucleotide MALDI-TOF-MS and Sanger sequencing. The performance of nucleotide MALDI-TOF MS for identifying various Mycobacterium species was analyzed based on Sanger sequencing as the gold standard. Results: The total correct detection rate of all 933 clinical Mycobacterium isolates using nucleotide MALDI-TOF-MS was 91.64% (855/933), and mixed infections were detected in 18.65% (174/933) of the samples. The correct detection rates for Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, MTBC, Mycobacterium gordonae, and Mycobacterium massiliense were 99.32% (585/589), 100% (86/86), 98.46% (64/65), 94.59% (35/37), 100.00% (34/34), 95.65% (22/23), and 100% (19/19), respectively. For the identification of the MTBC, M. intracellulare, M. abscessus, M. kansasii, M. avium, M. gordonae, and M. massiliense, nucleotide MALDI-TOF-MS and Sanger sequencing results were in good agreement (k > 0.7). Conclusion: In conclusion, nucleotide MALDI-TOF-MS is a promising approach for identifying MTBC and the most common clinical NTM species.

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

Newly Identified Mycobacterium africanum Lineage 10, Central Africa.

Analysis of genome sequencing data from >100,000 genomes of Mycobacterium tuberculosis complex using TB-Annotator software revealed a previously unknown lineage, proposed name L10, in central Africa. Phylogenetic reconstruction suggests L10 could represent a missing link in the evolutionary and geographic migration histories of M. africanum.

Outbreak of Mycobacterium orygis in a Shipment of Cynomolgus Macaques Imported from Southeast Asia - United States, February-May 2023.

Nonhuman primates (NHP) can become infected with the same species of Mycobacteria that cause human tuberculosis. All NHP imported into the United States are quarantined and screened for tuberculosis; no confirmed cases of tuberculosis were diagnosed among NHP during CDC-mandated quarantine during 2013-2020. In February 2023, an outbreak of tuberculosis caused by Mycobacterium orygis was detected in a group of 540 cynomolgus macaques (Macaca fascicularis) imported to the United States from Southeast Asia for research purposes. Although the initial exposure to M. orygis is believed to have occurred before the macaques arrived in the United States, infected macaques were first detected during CDC-mandated quarantine. CDC collaborated with the importer and U.S. Department of Agriculture's National Veterinary Services Laboratories in the investigation and public health response. A total of 26 macaques received positive test results for M. orygis by culture, but rigorous occupational safety protocols implemented during transport and at the quarantine facility prevented cases among caretakers in the United States. Although the zoonotic disease risk to the general population remains low, this outbreak underscores the importance of CDC's regulatory oversight of NHP importation and adherence to established biosafety protocols to protect the health of the United States research animal population and the persons who interact with them.

The C-terminus is essential for the stability of the mycobacterial channel protein MspA.

Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.

[Lsr2: A Nucleoid Associated Protein (NAP) and a transcription factor in mycobacteria]. / Lsr2 : protéine associée au nucléoïde (NAP) et facteur transcriptionnel chez les mycobactéries.

Lsr2, a small protein mainly found in actinobacteria, plays a crucial role in the virulence and adaptation of mycobacteria to environmental conditions. As a member of the nucleoid-associated protein (NAPs) superfamily, Lsr2 influences DNA organization by facilitating the formation of chromosomal loops in vitro and, therefore, may be a major player in the three-dimensional folding of the genome. Additionally, Lsr2 also acts as a transcription factor, regulating the expression of numerous genes responsible for coordinating a myriad of cellular and molecular processes essential for the actinobacteria. Similar to the H-NS protein, its ortholog in enterobacteria, its role in transcriptional repression likely relies on oligomerization, rigidifying, and bridging of DNA, thereby disrupting RNA polymerase recruitment as well as the elongation of RNA transcripts.

Title: Lsr2 : protéine associée au nucléoïde (NAP) et facteur transcriptionnel chez les mycobactéries. Abstract: Lsr2, une petite protéine conservée chez les actinobactéries, joue un rôle crucial dans la virulence et l'adaptation des mycobactéries aux conditions environnementales. Membre de la superfamille des protéines associées au nucléoïde (NAP), Lsr2 influence l'organisation de l'ADN en facilitant la formation de boucle chromosomique in vitro, ce qui suggère qu'elle pourrait être un acteur majeur du repliement tridimensionnel du génome. Lsr2 agit également comme un facteur de transcription, régulant l'expression de nombreux gènes responsables de la coordination d'une multitude de processus cellulaires et moléculaires essentiels chez les actinobactéries. Tout comme la protéine H-NS, son orthologue chez les entérobactéries, son rôle de répresseur transcriptionnel repose probablement sur son oligomérisation conduisant à la rigidification de l'ADN et, dans certaines situations, sur le pontage de fragments génomiques distants. Ces mécanismes pourraient perturber le recrutement de l'ARN polymérase sur les promoteurs ainsi que l'élongation des transcrits.

An anti-mycobacterial conjugated oligoelectrolyte effective against Mycobacterium abscessus.

Infections caused by nontuberculous mycobacteria have increased more than 50% in the past two decades and more than doubled in the elderly population. Mycobacterium abscessus (Mab), one of the most prevalent of these rapidly growing species, is intrinsically resistant to numerous antibiotics. Current standard-of-care treatments are not satisfactory, with high failure rate and notable adverse effects. We report here a potent anti-Mab compound from the flexible molecular framework afforded by conjugated oligoelectrolytes (COEs). A screen of structurally diverse, noncytotoxic COEs identified a lead compound, COE-PNH2, which was bactericidal against replicating, nonreplicating persisters and intracellular Mab.COE-PNH2 had low propensity for resistance development, with a frequency of resistance below 1.25 × 10-9 and showed no detectable resistance upon serial passaging. Mechanism of action studies were in line with COE-PNH2 affecting the physical and functional integrity of the bacterial envelope and disrupting the mycomembrane and associated essential bioenergetic pathways. Moreover, COE-PNH2 was well-tolerated and efficacious in a mouse model of Mab lung infection. This study highlights desirable in vitro and in vivo potency and safety index of this COE structure, which represents a promising anti-mycobacterial to tackle an unmet medical need.

Soft tissue infection and osteomyelitis caused by Mycobacterium farcinogenes after heart surgery: Case report and literature review of human cases.

Mycobacterium farcinogenes (M. farcinogenes) is rapidly growing mycobacterium, belonging to non-tuberculous mycobacterial (NTM). M. farcinogenes is an exceedingly rare causative agent of human infection. Only seven cases with M. farcinogenes infections in humans were reported. This is a case of soft tissue infection and osteomyelitis caused by M. farcinogenes after heart surgery. Microbial identification was achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The clinical outcome was favorable after surgical debridement and 4-month antibiotics treatment. We also provide a comprehensive literature review on this disease.

Identification, characterization and application of M16AT, a new organic solvent-tolerant (R)-enantio-selective type IV amine transaminase from Mycobacterium sp. ACS1612.

Biocatalysis has emerged as a powerful alternative to traditional chemical methods, especially for asymmetric synthesis. As biocatalysts usually exhibit excellent chemical, regio- and enantioselectivity, they facilitate and simplify many chemical processes for the production of a broad range of products. Here, a new biocatalyst called, R-selective amine transaminases (R-ATAs), was obtained from Mycobacterium sp. ACS1612 (M16AT) using in-silico prediction combined with a genome and protein database. A two-step simple purification process could yield a high concentration of pure enzyme, suggesting that industrial application would be inexpensive. Additionally, the newly identified and characterized R-ATAs displayed a broad substrate spectrum and strong tolerance to organic solvents. Moreover, the synthetic applicability of M16AT has been demonstrated by the asymmetric synthesis of (R)-fendiline from of (R)-1-phenylethan-1-amine.

Lsr2 acts as a cyclic di-GMP receptor that promotes keto-mycolic acid synthesis and biofilm formation in mycobacteria.

Cyclic di-GMP (c-di-GMP) is a second messenger that promotes biofilm formation in several bacterial species, but the mechanisms are often unclear. Here, we report that c-di-GMP promotes biofilm formation in mycobacteria in a manner dependent on the nucleoid-associated protein Lsr2. We show that c-di-GMP specifically binds to Lsr2 at a ratio of 1:1. Lsr2 upregulates the expression of HadD, a (3R)-hydroxyacyl-ACP dehydratase, thus promoting the synthesis of keto-mycolic acid and biofilm formation. Thus, Lsr2 acts as a c-di-GMP receptor that links the second messenger's function to lipid synthesis and biofilm formation in mycobacteria.

Mechanical morphotype switching as an adaptive response in mycobacteria.

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.