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Infections caused by non-tuberculous mycobacteria (NTM) are increasing globally and are notoriously difficult to treat due to intrinsic resistance of these bacteria to many common antibiotics. NTM are diverse and ubiquitous in the environment, with only a few species causing serious and often opportunistic infections in humans, including Mycobacterium abscessus. This rapidly growing mycobacterium is one of the most commonly identified NTM species responsible for severe respiratory, skin and mucosal infections in humans. It is often regarded as one of the most antibiotic-resistant mycobacteria, leaving us with few therapeutic options. In this Review, we cover the proposed infection process of M. abscessus, its virulence factors and host interactions and highlight the commonalities and differences of M. abscessus with other NTM species. Finally, we discuss drug resistance mechanisms and future therapeutic options. Taken together, this knowledge is essential to further our understanding of this overlooked and neglected global threat.Non-tuberculous mycobacteria, such as Mycobacterium abscessus, are an increasing global health burden, in part due their extensive drug resistance. In this Review, Johansen, Herrmann and Kremer discuss the infection process, host interactions, mechanisms of drug resistance and drug development.

Mycobacterium abscessus is the most frequently isolated rapidly growing mycobacterium in human disease and recently has emerged as responsible for severe pulmonary infections in cystic fibrosis patients. However, little is known about the virulence mechanisms of this human pathogen. We adapted the zebrafish embryo as a tractable infection model to study, at a spatiotemporal level, the physiopathology of M. abscessus infection. We describe the high propensity of virulent rough variant M. abscessus to produce serpentine cords in vivo , which are not observed with the less virulent smooth variant. We demonstrate that extracellular cording allows the bacterium to withstand phagocytosis, leading to uncontrolled growth and establishment of an acute and lethal infection, thus constituting a determinant of virulence. Mycobacterium abscessus is a rapidly growing Mycobacterium causing a wide spectrum of clinical syndromes. It now is recognized as a pulmonary pathogen to which cystic fibrosis patients have a particular susceptibility. The M. abscessus rough (R) variant, devoid of cell-surface glycopeptidolipids (GPLs), causes more severe clinical disease than the smooth (S) variant, but the underlying mechanisms of R-variant virulence remain obscure. Exploiting the optical transparency of zebrafish embryos, we observed that the increased virulence of the M. abscessus R variant compared with the S variant correlated with the loss of GPL production. The virulence of the R variant involved the massive production of serpentine cords, absent during S-variant infection, and the cords initiated abscess formation leading to rapid larval death. Cording occurred within the vasculature and was highly pronounced in the central nervous system (CNS). It appears that M. abscessus is transported to the CNS within macrophages. The release of M. abscessus from apoptotic macrophages initiated the formation of cords that grew too large to be phagocytized by macrophages or neutrophils. This study is a description of the crucial role of cording in the in vivo physiopathology of M. abscessus infection and emphasizes cording as a mechanism of immune evasion.

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.

Mycobacterium abscessus is the most pathogenic species among the predominantly saprophytic fast-growing mycobacteria. This opportunistic human pathogen causes severe infections that are difficult to eradicate. Its ability to survive within the host was described mainly with the rough (R) form of M . abscessus , which is lethal in several animal models. This R form is not present at the very beginning of the disease but appears during the progression and the exacerbation of the mycobacterial infection, by transition from a smooth (S) form. However, we do not know how the S form of M . abscessus colonizes and infects the host to then multiply and cause the disease. In this work, we were able to show the hypersensitivity of fruit flies, Drosophila melanogaster , to intrathoracic infections by the S and R forms of M . abscessus . This allowed us to unravel how the S form resists the innate immune response developed by the fly, both the antimicrobial peptides- and cellular-dependent immune responses. We demonstrate that intracellular M . abscessus was not killed within the infected phagocytic cells, by resisting lysis and caspase-dependent apoptotic cell death of Drosophila infected phagocytes. In mice, in a similar manner, intra-macrophage M . abscessus was not killed when M . abscessus -infected macrophages were lysed by autologous natural killer cells. These results demonstrate the propensity of the S form of M . abscessus to resist the host’s innate responses to colonize and multiply within the host.

ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M . abscessus EsxU and EsxT in vitro and in vivo . Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M . abscessus -infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M . abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M . abscessus during infection.

Mycobacterium abscessus (Mabs), an intracellular and opportunistic pathogen, is considered the most pathogenic fast-growing mycobacterium, and causes severe pulmonary infections in patients with cystic fibrosis. While bacterial factors contributing to its pathogenicity are well studied, the host factors and responses that worsen Mabs infection are not fully understood. Here, we report that Mabs systemic infection alters Drosophila melanogaster intestinal homeostasis. Mechanistically, Mabs remotely induces a self-damaging oxidative burst, leading to excessive differentiation of intestinal stem cells into enterocytes. We demonstrated that the subsequent increased intestinal renewal is mediated by both the Notch and JAK/STAT pathways and is deleterious to Drosophila survival. In conclusion, this work highlights that the ability of Mabs to induce an exacerbated and self-damaging response in the host contributes to its pathogenesis.

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark TM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of Biomark TM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

represents an important respiratory pathogen among the rapidly-growing non-tuberculous mycobacteria. Infections caused by are increasingly found in cystic fibrosis (CF) patients and are often refractory to antibiotic therapy. The underlying immunopathological mechanisms of pathogenesis remain largely unknown. A major reason for the poor advances in research has been a lack of adequate models to study the acute and chronic stages of the disease leading to delayed progress of evaluation of therapeutic efficacy of potentially active antibiotics. However, the recent development of cellular models led to new insights in the interplay between with host macrophages as well as with amoebae, proposed to represent the environmental host and reservoir for non-tuberculous mycobacteria. The zebrafish embryo has also appeared as a useful alternative to more traditional models as it recapitulates the vertebrate immune system and, due to its optical transparency, allows a spatio-temporal visualization of the infection process in a living animal. More sophisticated immunocompromised mice have also been exploited recently to dissect the immune and inflammatory responses to . Herein, we will discuss the limitations, advantages and potential offered by these various models to study the pathophysiology of infection and to assess the preclinical efficacy of compounds active against this emerging human pathogen.

Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections, particularly in individuals with underlying conditions, such as cystic fibrosis or chronic obstructive pulmonary disease. Macrolides, such as clarithromycin (CLR) or azithromycin (AZM), represent the cornerstone of antibiotherapy against the M. abscessus species. However, prolonged exposure to these macrolides can induce of Erm(41)-mediated resistance, limiting their spectrum of activity and leading to therapeutic failure. Therefore, inhibiting Erm(41) could thwart this resistance mechanism to maintain macrolide susceptibility, thus increasing the rate of treatment success. In our previous study, the Erm(41) methyltransferase was identified as a possible target enzyme of Cyclipostins and Cyclophostin compounds (CyC). Herein, we exploited this feature to evaluate the in vitro activity of CLR and AZM in combination with different CyC via the checkerboard assay on macrolide-susceptible and induced macrolide-resistant M. abscessus strains selected in vitro following exposure CLR and AZM. Our results emphasize the use of the CyC to prevent/overcome Erm(41)‑induced resistance and to restore macrolide susceptibility. This work should expand our therapeutic arsenal in the fight against a antibioticresistant mycobacterial species and could provide the opportunity to revisit the therapeutic regimen for combating M. abscessus pulmonary infections in patients, and particularly in erm(41)-positive strains.

Background In mycobacteria, conjugation differs from the canonical Hfr model, but is still poorly understood. Here, we quantified this evolutionary processe in a natural mycobacterial population, taking advantage of a large clinical strain collection of the emerging pathogen Mycobacterium abscessus (MAB). Results Multilocus sequence typing confirmed the existence of three M. abscessus subspecies, and unravelled extensive allelic exchange between them. Furthermore, an asymmetrical gene flow occurring between these main lineages was detected, resulting in highly admixed strains. Intriguingly, these mosaic strains were significantly associated with cystic fibrosis patients with lung infections or chronic colonization. Genome sequencing of those hybrid strains confirmed that half of their genomic content was remodelled in large genomic blocks, leading to original tri-modal ‘patchwork’ architecture. One of these hybrid strains acquired a locus conferring inducible macrolide resistance, and a large genomic insertion from a slowly growing pathogenic mycobacteria, suggesting an adaptive gene transfer. This atypical genomic architecture of the highly recombinogenic strains is consistent with the distributive conjugal transfer (DCT) observed in M. smegmatis . Intriguingly, no known DCT function was found in M. abscessus chromosome, however, a p-RAW-like genetic element was detected in one of the highly admixed strains. Conclusion Taken together, our results strongly suggest that MAB evolution is sporadically punctuated by dramatic genome wide remodelling events. These findings might have far reaching epidemiological consequences for emerging mycobacterial pathogens survey in the context of increasing numbers of rapidly growing mycobacteria and M. tuberculosis co-infections.