Mycobacterium abscessus biofilms have viscoelastic properties which may contribute to their recalcitrance in chronic pulmonary infections.

Mycobacterium abscessus is emerging as a cause of recalcitrant chronic pulmonary infections, particularly in people with cystic fibrosis (CF). Biofilm formation has been implicated in the pathology of this organism, however the role of biofilm formation in infection is unclear. Two colony-variants of M. abscessus are routinely isolated from CF samples, smooth (MaSm) and rough (MaRg). These two variants display distinct colony morphologies due to the presence (MaSm) or absence (MaRg) of cell wall glycopeptidolipids (GPLs). We hypothesized that MaSm and MaRg variant biofilms might have different mechanical properties. To test this hypothesis, we performed uniaxial mechanical indentation, and shear rheometry on MaSm and MaRg colony-biofilms. We identified that MaRg biofilms were significantly stiffer than MaSm under a normal force, while MaSm biofilms were more pliant compared to MaRg, under both normal and shear forces. Furthermore, using theoretical indices of mucociliary and cough clearance, we identified that M. abscessus biofilms may be more resistant to mechanical forms of clearance from the lung, compared to another common pulmonary pathogen, Pseudomonas aeruginosa. Thus, the mechanical properties of M. abscessus biofilms may contribute to the persistent nature of pulmonary infections caused by this organism.

Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus.

Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors.

Biofilms of Mycobacterium abscessus Complex Can Be Sensitized to Antibiotics by Disaggregation and Oxygenation.

Pulmonary infection with the multidrug-resistant Mycobacterium abscessus complex (MABSC) is difficult to treat in individuals with cystic fibrosis (CF). MABSC grows as biofilm aggregates in CF patient lungs, which are known to have anaerobic niches. How aggregation and anoxic conditions affect antibiotic tolerance is not well understood. We sought to determine whether disaggregation and oxygen availability sensitize MABSC isolates to recommended antibiotics. We tested the susceptibilities of 33 isolates from 22 CF patients with MABSC infection and a reference strain to the following antibiotics: amikacin, azithromycin, cefoxitin, ciprofloxacin, clarithromycin, imipenem, kanamycin, linezolid, moxifloxacin, rifampin, tigecycline, and sulfamethoxazole-trimethoprim. Isolates were grown in Mueller-Hinton broth with and without the disaggregating detergent Tween 80 (5%). Time-kill curves at days 1 and 3 were generated for oxic and anoxic amikacin treatment in 4-fold dilutions ranging from 2 to 512 mg liter-1 Scanning electron microscopy was used to visualize the aggregation patterns, while confocal laser scanning microscopy and microrespirometry were used to visualize biofilm growth patterns. Disruption of MABSC aggregates increased susceptibility to amikacin, tigecycline, kanamycin, azithromycin, imipenem, cefoxitin, and clarithromycin (P < 0.05, n = 29 to 31). Oxygenation enhanced the killing of disaggregated MABSC isolates by amikacin (P < 0.05) by 1 to 6 log units when 2 to 512 mg liter-1 of amikacin was used. This study explains why current drug susceptibility testing results correlate poorly with treatment outcomes. The conditions achieved by oxic culturing of planktonic isolates in vitro do not resemble the hypoxic conditions in CF patient lungs. Biofilm disruption and increased O2 availability during antibiotic therapy may be new therapeutic strategies for chronic MABSC infection.

Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus.

Mycobacterium abscessus is an emerging human pathogen that is notorious for being one of the most drug-resistant species of Mycobacterium. It has developed numerous strategies to overcome the antibiotic stress response, limiting treatment options and leading to frequent therapeutic failure. The panel of aminoglycosides (AG) usually used in the treatment of M. abscessus pulmonary infections is restricted by chemical modification of the drugs by the N-acetyltransferase Eis2 protein (Mabs_Eis2). This enzyme acetylates the primary amine of AGs, preventing these antibiotics from binding ribosomal RNA and thereby impairing their activity. In this study, the high-resolution crystal structures of Mabs_Eis2 in its apo- and cofactor-bound forms were solved. The structural analysis of Mabs_Eis2, supported by the kinetic characterization of the enzyme, highlights the large substrate specificity of the enzyme. Furthermore, in silico docking and biochemical approaches attest that Mabs_Eis2 modifies clinically relevant drugs such as kanamycin and amikacin, with a better efficacy for the latter. In line with previous biochemical and in vivo studies, our work suggests that Mabs_Eis2 represents an attractive pharmacological target to be further explored. The high-resolution crystal structures presented here may pave the way to the design of Eis2-specific inhibitors with the potential to counteract the intrinsic resistance levels of M. abscessus to an important class of clinically important antibiotics. DATABASE: Structural data are available in the PDB database under the accession numbers: 6RFY, 6RFX and 6RFT.

Non-Tuberculous Mycobacteria multispecies biofilms in cystic fibrosis: development of an in vitro Mycobacterium abscessus and Pseudomonas aeruginosa dual species biofilm model.

Lung disease in cystic fibrosis (CF) is characterized by the progressive colonization of the respiratory tract by different bacteria, which develop polymicrobial biofilms. In the past decades, there has been an increase in the number of CF patients infected with Non-Tuberculous Mycobacteria (NTM). Although Mycobacterium abscessus is the main NTM isolated globally, little is known about M. abscessus multispecies biofilm formation. In the present study we developed an in vitro model to study the phenotypic characteristics of biofilms formed by M. abscessus and Pseudomonas aeruginosa, a major pathogen in CF. For that purpose, dual species biofilms were grown on polycarbonate membranes with a fixed concentration of P. aeruginosa and different inoculums of M. abscessus. The biofilms were sampled at 24, 48, and 72 h and bacteria were quantified in specific media. The results revealed that the increasing initial concentration of M. abscessus in dual species biofilms had an effect on its population only at 24 and 48 h, whereas P. aeruginosa was not affected by the different concentrations used of M. abscessus. Time elapsed increased biofilm formation of both species, specially between 24 and 48 h. According to the results, the conditions to produce a mature dual species biofilm in which the relative species distribution remained stable were 72 h growth of the mixed microbial culture at a 1:1 ratio. A significant decrease in mycobacterial population in dual compared to single species biofilms was found, suggesting that P. aeruginosa has a negative influence on M. abscessus. Finally, in a proof of concept experiment, young and mature dual species biofilms were exposed to clarithromycin.