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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 a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa , tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection. The pathogen Mycobacterium abscessus can switch from a smooth colony form (S) into a highly inflammatory, rough colony form (R) during infection. Here, Kam et al. use an adult zebrafish model of M. abscessus chronic infection to illustrate differences in the immunopathogenesis induced by R and S variants.

Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon (Tn) insertion library of an M. abscessus subspecies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB₄ that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.

Background. Nontuberculous mycobacteria (NTM) commonly colonize municipal water supplies and cause healthcare-associated outbreaks. We investigated a biphasic outbreak of Mycobacterium abscessus at a tertiary care hospital. Methods. Case patients had recent hospital exposure and laboratory-confirmed colonization or infection with M. abscessus from January 2013 through December 2015. We conducted a multidisciplinary epidemiologic, field, and laboratory investigation. Results. The incidence rate of M. abscessus increased from 0.7 cases per 10 000 patient-days during the baseline period (January 2013–July 2013) to 3.0 cases per 10 000 patient-days during phase 1 of the outbreak (August 2013–May 2014) (incidence rate ratio, 4.6 [95% confidence interval, 2.3–8.8]; P < .001). Thirty-six of 71 (51%) phase 1 cases were lung transplant patients with positive respiratory cultures. We eliminated tap water exposure to the aerodigestive tract among high-risk patients, and the incidence rate decreased to baseline. Twelve of 24 (50%) phase 2 (December 2014–June 2015) cases occurred in cardiac surgery patients with invasive infections. Phase 2 resolved after we implemented an intensified disinfection protocol and used sterile water for heater-cooler units of cardiopulmonary bypass machines. Molecular fingerprinting of clinical isolates identified 2 clonal strains of M. abscessus; 1 clone was isolated from water sources at a new hospital addition. We made several water engineering interventions to improve water flow and increase disinfectant levels. Conclusions. We investigated and mitigated a 2-phase clonal outbreak of M. abscessus linked to hospital tap water. Healthcare facilities with endemic NTM should consider similar tap water avoidance and engineering strategies to decrease risk of NTM infection.

Pathogenic mycobacteria produce a wide array of lipids which participate in host cell interactions and virulence. While some of these are conserved across all mycobacteria, others, like glycopeptidolipids (GPL), are restricted to a few species. Mycobacterium abscessus , an emerging rapid-growing pathogen, transitions from a smooth to a virulent rough variant upon the loss of surface GPL. Here, we discovered that M. abscessus and phylogenetically-close species harbor a second GPL-related locus, comprising two adjacent non-ribosomal peptide synthetase genes, MAB_4690c and MAB_4691c . A MAB_4690c deletion mutant (Δ MAB_4690c ) failed to produce a yet undescribed lipid, designated GL8P for glycosylated lipooctapeptide, sharing an acylated octapeptide core adorned by mono or di- O -rhamnosyl substituents. Δ MAB_4690c exhibited impaired uptake and survival in THP-1 cells and was attenuated in mice. Importantly, GL8P elicited a strong humoral response in patients infected with M. abscessus . These results highlight the role of GL8P in the pathophysiology of infection by rough M . abscessus and suggest its potential as a selective marker for M . abscessus infections.

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.

Introduction The incidence of Mycobacterium abscessus complex (MABC) infections is rising, becoming a major pathogen of nontuberculous mycobacteria responsible for pulmonary disease (PD) and extrapulmonary disease (ED). However, studies on the clinical characteristics of MABC-ED remain limited. Methods A 7-year retrospective analysis was conducted on MABC-ED cases at Zhongshan Hospital in Shanghai, China. We analyzed predisposing factors, clinical features, metagenomic sequencing (MS) results, drug susceptibility testing (DST), and genomic characteristics of MABC-ED patients, comparing the data with those of PD cases. Results Among 17 MABC-ED patients, 15 had predisposing risk factors and underlying conditions, with 2 of 3 patients with rheumatic disease showing poor prognosis. The diagnostic performance of metagenomic sequencing for MABC-ED was comparable to that for MABC-PD. However, MABC-ED samples exhibited distinct microbiome features and a more diverse mycobacterial community structure compared to PD. Resistance rates among extrapulmonary MABC isolates were observed as follows: 0% (amikacin), 20% (macrolides), 30% (linezolid), and 40% (cefoxitin). One case showed paradoxical results between erm (41) T28 sequevar and susceptibility phenotype. Genomic analysis revealed no specific dominant circulating clones (DCC) for MABC-ED isolates. Conclusion MABC-ED patients commonly present with risk factors and underlying diseases. Metagenomic sequencing diagnosis of MABC-ED poses challenges, and DST and whole genome sequencing data indicate diversity among MABC-ED isolates. Our study provides detailed data on MABC-ED, contributing to a better understanding of its disease characteristics.

Mycobacterium abscessus , a multidrug-resistant nontuberculous mycobacterium, has emerged as a major pathogen affecting people with cystic fibrosis (CF). Although originally thought to be acquired independently from the environment, most individuals are infected with one of several dominant circulating clones (DCCs), indicating the presence of global transmission networks of M. abscessus . How and when these clones emerged and spread globally is unclear. Here, we use evolutionary analyses of isolates from individuals both with and without CF to reconstruct the population history, spatiotemporal spread and recent transmission networks of the DCCs. We demonstrate synchronous expansion of six unrelated DCCs in the 1960s, a period associated with major changes in CF care and survival. Each of these clones has spread globally as a result of rare intercontinental transmission events. We show that the DCCs, but not environmentally acquired isolates, exhibit a specific smoking-associated mutational signature and that current transmission networks include individuals both with and without CF. We therefore propose that the DCCs initially emerged in non-CF populations but were then amplified and spread through the CF community. While individuals with CF are probably the most permissive host, non-CF individuals continue to play a key role in transmission networks and may facilitate long-distance transmission. In this Article, the authors perform evolutionary analyses of M. abscessus clinical isolates and report the emergence of dominant circulating clones (DCCs) in non-cystic fibrosis (CF) individuals followed by amplification in the CF community.

Background Cell division is essential for bacterial survival and represents a promising target for the development of novel antibiotics, particularly in mycobacteria. The role of the division protein FtsL in Mycobacterium abscessus remains poorly understood. This study investigated the effects of MAB_1999, a predicted homolog of FtsL, on the growth and cell division of M. abscessus . Method To investigate the function of mab_1999 , a knockdown mutant was generated via CRISPR interference (CRISPRi). The phenotypic impact of mab_1999 suppression was evaluated, with a focus on its effects on M. abscessus growth, cellular morphology, and antibiotic susceptibility. Results The putative homolog of FtsL in M. abscessus (MAB_1999) shares 54% amino acid sequence identity with FtsL from M. smegmatis (MSMEG_4234). CRISPRi-mediated repression of mab_1999 expression resulted in cell elongation and growth defects, although complete growth arrest was not observed. Furthermore, reduced mab_1999 expression increased the susceptibility of M. abscessus to β-lactam antibiotics, including ceftriaxone and imipenem. Conclusions Our findings suggest that mab_1999 is involved in cell division and cell wall integrity in M. abscessus . However, further investigation is necessary to confirm its identity as FtsL and to fully elucidate its role in the cell division process and cell wall synthesis.