Total Synthesis of a Trehalose-Containing Lipooligosaccharide Analogue from Mycobacterium linda.

A short and efficient methodology has been developed to synthesize an analogue of a lipooligosaccharide from Mycobacterium linda isolated from Crohn's disease. The total synthesis of the tetrasaccharide was achieved via a convergent [2 + 2] glycosylation approach. The key features of the synthesis involve the selective functionalization of a trehalose core via highly regioselective acylations and regioselective glycosylations. The synthesis was completed via a longest linear sequence of 14 steps in a 14.2% overall yield.

Inborn errors of human transcription factors governing IFN-γ antimycobacterial immunity.

Inborn errors of immunity (IEI) delineate redundant and essential defense mechanisms in humans. We review 15 autosomal-dominant (AD) or -recessive (AR) IEI involving 11 transcription factors (TFs) and impairing interferon-gamma (IFN-γ) immunity, conferring a predisposition to mycobacterial diseases. We consider three mechanism-based categories: 1) IEI mainly affecting myeloid compartment development (AD GATA2 and AR and AD IRF8 deficiencies), 2) IEI mainly affecting lymphoid compartment development (AR FOXN1, AR PAX1, AR RORγ/RORγT, AR T-bet, AR c-Rel, AD STAT3 gain-of-function (GOF), and loss-of-function (LOF) deficiencies), and 3) IEI mainly affecting myeloid and/or lymphoid function (AR and AD STAT1 LOF, AD STAT1 GOF, AR IRF1, and AD NFKB1 deficiencies). We discuss the contribution of the discovery and study of inborn errors of TFs essential for host defense against mycobacteria to molecular and cellular analyses of human IFN-γ immunity.

Application of chromogenic media for preliminary identification of acid-resistant bacteria.

Background: The variety of morphological and cultural characteristics of acid-resistant bacteria (ARB) makes it possible to use microscopy and estimate the growth rate and pigment formation when cultivating on solid egg media for preliminary identification only as additional indicative methods. It is necessary to develop new approaches for the cultivation and primary identification of ARB isolated from the biological material. It will allow to obtain data on the prevalence, structure, epidemiological, and clinical features of infectious processes caused by opportunistic ARB. Methods: Three hundred and sixty strains of ARB were isolated from the various biological materials obtained from the patients during the examination for tuberculosis. All biological material samples were negative on Mycobacteria tuberculosis complex. Species identification of all bacteria was performed by matrix-assisted lazer desorption/ion-ization time-of-flight mass spectrometry. The cultural characteristics of ARB were evaluated on a universal chromogenic media. As a selective additive, a mixture of bacitracin and polymyxin sulfate which had no effect on ARB was tested to suppress concomitant Gram-positive and Gram-negative microflora. Results: Cultural characteristics were identified and described for all tested representatives of fast-growing nontuberculous mycobacteria (NTM), as well as for all types of nocardia, gordonia, and streptomycetes. Representatives of other genera of ARB on a universal chromogenic media gave meager growth or did not show it at all. When inoculated on a universal chromogenic media with a selective addition, 100% of the strains from the ARB group showed abundant or moderate growth. Incubation time for fast-growing species was up to 7 days; for slow-growing species, it was up to 28 days. Concomitant control strains of Gram-positive and Gram-negative bacteria on universal chromogenic media with selective growth additive did not show the growth. Conclusions: The use of a universal chromogenic media allows to preliminarily identify NTM and other ARB by cultural characteristics. The addition of bacitracin and polymyxin sulfate does not reduce the growth properties of ARB, which can be used when working with both biological materials and for the isolation of pure ARB cultures from mixtures with other bacteria.

Nontuberculous mycobacteria isolation from sputum specimens: A retrospective analysis of 1061 cases.

Background: In recent years, with the development of laboratory methods, the frequency of nontuberculosis mycobacteria (NTM) infections has increased. The primary aim of this study was to evaluate the clinical significance of therapeutic drug monitoring (TDM) growths in respiratory samples, and the secondary aim was to evaluate the treatment regimens and treatment outcomes of treatment for TDM disease. Methods: This study was a retrospective cohort study. Persons with NTM growth in respiratory samples admitted to the reference hospital between 2009 and 2020 were included in this study. Samples detected as NTM by the immunochromatographic rapid diagnostic test, those requested by the clinicians, species were determined by the hsp65PCRREA method. The subjects were classified into 3 groups: patients with NTM infection who received treatment (135, 12.7%), those followed up without treatment (690, 65.1%), and a last group of patients with Mycobacterium tuberculosis (TB) complex strains were isolated and received TB treatment (236, 22.2%). Initiating NTM treatment was decided in accordance with the American Thoracic Society recommendations. Results: The mean ± standard deviation age of patients was 53.8 ± 16.5 years, and 749 (70.6%) were male. In total, 278 (26.2%) out of 1061 cases had identified, and the most frequent species were MAC (81; Mycobacterium avium: 39, Mycobacterium intracellulare: 39, and MAC: 3), Mycobacterium abscessus (67), Mycobacterium kansasii (48), Mycobacterium fortuitum (23), Mycobacterium chelonae (12), Mycobacterium gordonae (11), and Mycobacterium szulgai (11). In the NTM treatment group, 116 (85.9%) of 135 patients had multiple culture positivity. Previous TB treatment history had 51 (37.8%) of 135 patients, respiratory comorbidities were evident in 37 (27.4%) of 135 patients. Thorax computed tomography imaging in 84 patients revealed nodule 38 (45.2%), consolidation 46 (54.8%), cavity 52 (61.9%), and bronchiectasis 27 (32.1%). Treatment results in the NTM treatment group were as follows: ongoing treatment 14 (10.4%), cure 64 (47.4%), default 33 (24.4%), exitus 19 (14.1%), recurrence 3 (2.2%), and refractory disease 2 (1.5%). Conclusion: This is a large case series evaluating the clinical significance of NTM growths and NTM treatment in Turkey. The clinical significance of NTM growth in respiratory samples is low. Treatment success rates of NTM patients who are treated are low. Treatment defaults and mortality rates are high. New drugs and new regimens are needed.

Azido Inositol Probes Enable Metabolic Labeling of Inositol-Containing Glycans and Reveal an Inositol Importer in Mycobacteria.

Bacteria from the genus Mycobacterium include pathogens that cause serious diseases in humans and remain as difficult infectious agents to treat. Central to these challenges are the composition and organization of the mycobacterial cell envelope, which includes unique and complex glycans. Inositol is an essential metabolite for mycobacteria due to its presence in the structural core of the immunomodulatory cell envelope glycolipids phosphatidylinositol mannoside (PIM) and PIM-anchored lipomannan (LM) and lipoarabinomannan (LAM). Despite their importance to mycobacterial physiology and pathogenesis, many aspects of PIM, LM, and LAM construction and dynamics remain poorly understood. Recently, probes that allow metabolic labeling and detection of specific mycobacterial glycans have been developed to investigate cell envelope assembly and dynamics. However, these tools have been limited to peptidoglycan, arabinogalactan, and mycolic acid-containing glycolipids. Herein, we report the development of synthetic azido inositol (InoAz) analogues as probes that can metabolically label PIMs, LM, and LAM in intact mycobacteria. Additionally, we leverage an InoAz probe to discover an inositol importer and catabolic pathway in Mycobacterium smegmatis. We anticipate that in the future, InoAz probes, in combination with bioorthogonal chemistry, will provide a valuable tool for investigating PIM, LM, and LAM biosynthesis, transport, and dynamics in diverse mycobacterial organisms.

A One Health approach revealed the long-term role of Mycobacterium caprae as the hidden cause of human tuberculosis in a region of Spain, 2003 to 2022.

IntroductionMycobacterium caprae is a member of the Mycobacterium tuberculosis complex (MTBC) not routinely identified to species level. It lacks specific clinical features of presentation and may therefore not be identified as the causative agent of tuberculosis. Use of whole genome sequencing (WGS) in the investigation of a family microepidemic of tuberculosis in Almería, Spain, unexpectedly identified the involvement of M. caprae.AimWe aimed to evaluate the presence of additional unidentified M. caprae cases and to determine the magnitude of this occurrence.MethodsFirst-line characterisation of the MTBC isolates was done by MIRU-VNTR, followed by WGS. Human and animal M. caprae isolates were integrated in the analysis.ResultsA comprehensive One Health strategy allowed us to (i) detect other 11 M. caprae infections in humans in a period of 18 years, (ii) systematically analyse M. caprae infections on an epidemiologically related goat farm and (iii) geographically expand the study by including 16 M. caprae isolates from other provinces. Integrative genomic analysis of 41 human and animal M. caprae isolates showed a high diversity of strains. The animal isolates' diversity was compatible with long-term infection, and close genomic relationships existed between isolates from goats on the farm and recent cases of M. caprae infection in humans.DiscussionZoonotic circulation of M. caprae strains had gone unnoticed for 18 years. Systematic characterisation of MTBC at species level and/or extended investigation of the possible sources of exposure in all tuberculosis cases would minimise the risk of overlooking similar zoonotic events.

Mycobacteriophage Derived Lipoarabinomannan Binding Protein for Recognizing Non-Tuberculosis Mycobacteria.

Non-tuberculosis mycobacteria (NTM) is one family of pathogens usually leading to nosocomial infections. Exploration of high-performance biological recognition agent plays a pivotal role for the development of point-of-care testing device and kit for detecting NTM. Mycobacterium smegmatis (M. smegmatis) is a NTM which has been frequently applied as an alternative model for highly pathogenic mycobacteria. Herein, a recombinant tail protein derived from mycobacteriophage SWU1 infecting M. smegmatis was expressed with Escherichia coli system and noted as GP89. It shows a fist-like structure according to the results of homology modeling and ab initio modeling. It is confirmed as a lipoarabinomannan (LAM) binding protein, which can recognize studied NTM genus since abundant LAM constructed with d-mannan and d-arabinan is distributed over the mycobacterial surface. Meanwhile an enhanced green fluorescent protein (eGFP)-fused GP89 protein was acquired with a fusion expression technique. Then GP89 and eGFP-fused GP89 were applied to establish a sensitive and rapid method for fluorescent detection of M. smegmatis with a broad linear range of 1.0 × 102 to 1.0 × 106 CFU mL-1 and a low detection limit of 69 CFU mL-1. Rapid and reliable testing of antimicrobial susceptibility was achieved by the GP89-based fluorescent method. The present work provides a promising recognition agent for studied NTM and opens an avenue for clinical diagnosis of NTM-induced infections.

Mycobacterium indicus pranii therapy suppresses systemic dissemination of tumor cells in B16F10 murine model of melanoma.

Cancer associated morbidity is mostly attributed to the dissemination of tumor cells from their primary niche into the circulation known as "metastasis". Mycobacterium indicus pranii (MIP) an approved immunotherapeutic agent against lung cancer (NSCLC) has shown potent anti-tumor activity in prior studies. While evaluating anti-tumor activity of MIP in mouse model, MIP treated animals typically exhibited less metastatic lesions in their pulmonary compartment. To study the role of MIP in metastasis closely, B16F10 melanoma cells were implanted subcutaneously in the mice, and the dissemination of tumor cells from the solid tumor was evaluated over a period of time. When B16F10 melanoma cells were treated with MIP in vitro, downregulation of epithelial mesenchymal transition markers was observed in these cells, which in turn suppressed the invasion, migration and adhesion of tumor cells. Notably, MIP therapy was found to be effectively reducing the metastatic burden in murine model of melanoma. Molecular characterization of MIP treated tumor cells substantiated that MIP upregulates the PPARγ expression within the tumor cells, which attenuates the NFκB/p65 levels within the nucleus, resulting in the suppression of Mmp9 expression in tumor cells. Besides that, MIP also downregulated the surface expression of chemokine receptor CXCR4 in murine melanoma cells, where chromatin immunoprecipitation confirmed the impeded recruitment of p50 and c-Rel factors to the Cxcr4 promoter, resulting in its downregulation transcriptionally. Taken together, MIP suppressed the dissemination of tumor cells in vivo, by regulating the expression of MMP9 and CXCR4 on these cells.

Mycobacterium vaccae protects against glucocorticoid resistance resulting from combined physical and psychosocial trauma in mice.

Stress-related somatic and psychiatric disorders are often associated with a decline in regulatory T cell (Treg) counts and chronic low-grade inflammation. Recent preclinical evidence suggests that the latter is at least partly mediated by stress-induced upregulation of toll-like receptor (TLR)2 in newly generated neutrophils and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as glucocorticoid (GC) resistance in predominantly PMN-MDSCs following stress-induced upregulation of TLR4 expression. Here we show in mice exposed to the chronic subordinate colony housing (CSC) paradigm that repeated intragastric (i.g.) administrations of a heat-killed preparation of Mycobacterium vaccae NCTC 11659, a saprophytic microorganism with immunoregulatory properties, protected against the stress-induced reduction in systemic Tregs, increase in basal and LPS-induced in vitro splenocyte viability, as well as splenic in vitro GC resistance. Our findings further support the hypothesis that i.g. M. vaccae protects against CSC-associated splenic GC resistance via directly affecting the myeloid compartment, thereby preventing the CSC-induced upregulation of TLR4 in newly generated PMN-MDSCs. In contrast, the protective effects of i.g. M. vaccae on the CSC-induced upregulation of TLR2 in neutrophils and the subsequent increase in basal and LPS-induced in vitro splenocyte viability seems to be indirectly mediated via the Treg compartment. These data highlight the potential for use of oral administration of M. vaccae NCTC 11659 to prevent stress-induced exaggeration of inflammation, a risk factor for development of stress-related psychiatric disorders.

The synergetic effect of sitafloxacin-arbekacin combination in the Mycobacterium abscessus species.

Mycobacterium abscessus species (MABS) is the most commonly isolated rapidly growing mycobacteria (RGM) and is one of the most antibiotic-resistant RGM with rapid progression, therefore, treatment of MABS is still challenging. We here presented a new combination treatment with sitafloxacin that targeted rough morphotypes of MABS, causing aggressive infections. Thirty-four clinical strains of MABS were isolated from various clinical samples at the Juntendo university hospital from 2011 to 2020. The susceptibility to a combination of sitafloxacin and antimicrobial agents was compared to that of the antimicrobial agents alone. Out of 34 MABS, 8 strains treated with sitafloxacin-amikacin combination, 9 of sitafloxacin-imipenem combination, 19 of sitafloxacin-arbekacin combination, and 9 of sitafloxacin-clarithromycin combination showed synergistic effects, respectively. Sitafloxacin-arbekacin combination also exhibited the synergistic effects against 10 of 22 Mycobacterium abscessus subspecies massiliense (Mma) strains and 8 of 11 Mycobacterium abscessus subspecies abscessus (Mab) strains, a highly resistant subspecies of MABS. The sitafloxacin-arbekacin combination revealed more synergistic effects in rough morphotypes of MABS (p = 0.008). We demonstrated the synergistic effect of the sitafloxacin-arbekacin combination against MABS. Further, this combination regimen might be more effective against Mab or rough morphotypes of MABS.

Thermostability enhancement and insight of L-asparaginase from Mycobacterium sp. via consensus-guided engineering.

Acrylamide alleviation in food has represented as a critical issue due to its neurotoxic effect on human health. L-Asparaginase (ASNase, EC 3.5.1.1) is considered a potential additive for acrylamide alleviation in food. However, low thermal stability hinders the application of ASNase in thermal food processing. To obtain highly thermal stable ASNase for its industrial application, a consensus-guided approach combined with site-directed saturation mutation (SSM) was firstly reported to engineer the thermostability of Mycobacterium gordonae L-asparaginase (GmASNase). The key residues Gly97, Asn159, and Glu249 were identified for improving thermostability. The combinatorial triple mutant G97T/N159Y/E249Q (TYQ) displayed significantly superior thermostability with half-life values of 61.65 ± 8.69 min at 50 °C and 5.12 ± 1.66 min at 55 °C, whereas the wild-type was completely inactive at these conditions. Moreover, its Tm value increased by 8.59 °C from parent wild-type. Interestingly, TYQ still maintained excellent catalytic efficiency and specific activity. Further molecular dynamics and structure analysis revealed that the additional hydrogen bonds, increased hydrophobic interactions, and favorable electrostatic potential were essential for TYQ being in a more rigid state for thermostability enhancement. These results suggested that our strategy was an efficient engineering approach for improving fundamental properties of GmASNase and offering GmASNase as a potential agent for efficient acrylamide mitigation in food industry. KEY POINTS: • The thermostability of GmASNase was firstly improved by consensus-guided engineering. • The half-life and Tm value of triple mutant TYQ were significantly increased. • Insight on improved thermostability of TYQ was revealed by MD and structure analysis.

Human IL-23 is essential for IFN-γ-dependent immunity to mycobacteria.

Patients with autosomal recessive (AR) IL-12p40 or IL-12Rß1 deficiency display Mendelian susceptibility to mycobacterial disease (MSMD) due to impaired IFN-γ production and, less commonly, chronic mucocutaneous candidiasis (CMC) due to impaired IL-17A/F production. We report six patients from four kindreds with AR IL-23R deficiency. These patients are homozygous for one of four different loss-of-function IL23R variants. All six patients have a history of MSMD, but only two suffered from CMC. We show that IL-23 induces IL-17A only in MAIT cells, possibly contributing to the incomplete penetrance of CMC in patients unresponsive to IL-23. By contrast, IL-23 is required for both baseline and Mycobacterium-inducible IFN-γ immunity in both Vδ2+ γδ T and MAIT cells, probably contributing to the higher penetrance of MSMD in these patients. Human IL-23 appears to contribute to IL-17A/F-dependent immunity to Candida in a single lymphocyte subset but is required for IFN-γ-dependent immunity to Mycobacterium in at least two lymphocyte subsets.

Harnessing CRISRP-Cas9 as an anti-mycobacterial system.

Rapid emergence of drug resistance has posed new challenges to the treatment of mycobacterial infections. As the pace of development of new drugs is slow, alternate treatment approaches are required. Recently, CRISPR-Cas systems have emerged as potential antimicrobials. These sequence-specific nucleases introduce double strand cuts in the target DNA, which if left unrepaired, prove fatal to the host. For most bacteria, homologous recombination repair (HRR) is the only pathway for repair and survival. Mycobacteria is one of the few bacteria which possesses the non-homologous end joining (NHEJ) system in addition to HRR for double strand break repair. To assess the antimicrobial potential of CRISPR-system, Cas9-induced breaks were introduced in the genome of Mycobacterium smegmatis and the survival was studied. While the single strand breaks were efficiently repaired, the organism was unable to repair the double strand breaks efficiently. In a mixed population of antibiotic-resistant and sensitive mycobacterial cells, selectively targeting a factor that confers hygromycin resistance, turned the entire population sensitive to the drug. Further, we demonstrate that the sequence-specific targeting could also be used for curing plasmids from mycobacterium cells. Considering the growing interest in nucleic acid-based therapy to curtail infections and combat antimicrobial resistance, our data shows that CRISPR-systems hold promise for future use as an antimicrobial against drug-resistant mycobacterial infections.

Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium.

C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Δ1-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17ß-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Δ1-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry. KEY POINTS: • A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum. • An 1,4-HP producer was constructed by KshA and Hsd4A deficiency. • The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.

Differences in skin test reactions to official and defined antigens in guinea pigs exposed to non-tuberculous and tuberculous bacteria.

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/ß-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/ß immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/ß. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/ß-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/ß-dependent antiviral immunity.

MGIT Enriched Shotgun Metagenomics for Routine Identification of Nontuberculous Mycobacteria: a Route to Personalized Health Care.

Currently, nontuberculous mycobacteria (NTM) are identified using small genomic regions, and species-level identification is often not possible. We introduce a next-generation sequencing (NGS) workflow that identifies mycobacteria to (sub)species level on the basis of the whole genome extracted from enriched shotgun metagenomic data. This technique is used to study the association between genotypes and clinical manifestations to pave the way to more personalized health care. Two sets of clinical isolates (explorative set [n = 212] and validation set [n = 235]) were included. All data were analyzed using a custom pipeline called MyCodentifier. Sequences were matched against a custom hsp65 database (NGS-hsp65) and whole-genome database (NGS-WG) created based on the phylogeny presented by Tortoli et al. (E. Tortoli, T. Fedrizzi, C. J. Meehan, A. Trovato, et al., Infect Genet Evol 56:19-25, 2017, https://doi.org/10.1016/j.meegid.2017.10.013). Lastly, phylogenetic analysis was performed and correlated with clinical manifestation. In the explorative set, we observed 98.6% agreement between the line probe assay and the NGS-hsp65 database. In the validation set, 99.1% agreement between the NGS-WG and NGS-hsp65 databases was seen on the complex level. We identified a cluster of Mycobacterium marinum isolates not represented by the Tortoli et al. phylogeny. Phylogenetic analysis of M. avium complex isolates confirmed misclassification of M. timonense and M. bouchedurhonense and identified subclusters within M. avium although no correlation with clinical manifestation was observed. We performed routine NGS to identify NTM from MGIT enriched shotgun metagenomic data. Phylogenetic analyses identified subtypes of M. avium, but in our set of isolates no correlation with clinical manifestation was found. However, this NGS workflow paves a way for more personalized health care in the future.

Self-recycling and partially conservative replication of mycobacterial methylmannose polysaccharides.

The steep increase in nontuberculous mycobacteria (NTM) infections makes understanding their unique physiology an urgent health priority. NTM synthesize two polysaccharides proposed to modulate fatty acid metabolism: the ubiquitous 6-O-methylglucose lipopolysaccharide, and the 3-O-methylmannose polysaccharide (MMP) so far detected in rapidly growing mycobacteria. The recent identification of a unique MMP methyltransferase implicated the adjacent genes in MMP biosynthesis. We report a wide distribution of this gene cluster in NTM, including slowly growing mycobacteria such as Mycobacterium avium, which we reveal to produce MMP. Using a combination of MMP purification and chemoenzymatic syntheses of intermediates, we identified the biosynthetic mechanism of MMP, relying on two enzymes that we characterized biochemically and structurally: a previously undescribed α-endomannosidase that hydrolyses MMP into defined-sized mannoligosaccharides that prime the elongation of new daughter MMP chains by a rare α-(1→4)-mannosyltransferase. Therefore, MMP biogenesis occurs through a partially conservative replication mechanism, whose disruption affected mycobacterial growth rate at low temperature.