ABSTRACT
BACKGROUND: The Mycobacterium avium complex includes the commonest non-tuberculous mycobacteria associated with human infections. These infections have been associated with the production of biofilms in many cases, but there are only a few studies about biofilms produced by the species included in this group. METHODS: Three collection strains (M. avium ATCC25291, M. intracellulare ATCC13950, and M. chimaera DSM756), three clinically significant strains (647, 657, and 655), and three clinically non-significant ones (717, 505, and 575) of each species were included. The clinical significance of the clinical isolates was established according to the internationally accepted criteria. The biofilm ultrastructure was studied by Confocal-Laser Scanning Microscopy by using BacLight Live-Dead and Nile Red stains. The viability, covered surface, height, and relative autofluorescence were measured in several images/strain. The effect of clarithromycin was studied by using the technique described by Muñoz-Egea et al. with modifications regarding incubation time. The study included clarithromycin in the culture medium at a concentration achievable in the lungs (11.3 mg/L), using one row of wells as the control without antibiotics. The bacterial viability inside the biofilm is expressed as a percentage of viable cells. The differences between the different parameters of the biofilm ultrastructure were analyzed by using the Kruskal-Wallis test. The correlation between bacterial viability in the biofilm and treatment time was evaluated by using Spearman's rank correlation coefficient (ρ). RESULTS: The strains showed differences between them with all the studied parameters, but neither a species-specific pattern nor a clinical-significance-specific pattern were detected. For the effect of clarithromycin, the viability of the bacteria contained in the biofilm was inversely proportional to the exposure time of the biofilm (ρ > -0.3; p-value < 0.05), excluding two M. chimaera strains (M. chimaera DSM756 and 575), which showed a weak positive correlation with treatment time (0.2 < ρ < 0.39; p-value < 0.05). Curiously, despite a clarithromycin treatment of 216 h, the percentage of the biofilm viability of the strains evaluated here was not less than 40% at best (M. avium 717). CONCLUSIONS: All the M. avium complex strains studied can form biofilm in vitro, but the ultrastructural characteristics between them suggest that these are strain-specific characteristics unrelated to the species or the clinical significance. The clarithromycin effect on MAC species is biofilm-age/time-of-treatment-dependent and appears to be strain-specific while being independent of the clinical significance of the strain.
ABSTRACT
The genus Mycobacterium includes some of the deadliest pathogens of History (Mycobacterium tuberculosis, Mycobacterium leprae), but most of the species within the genus are environmental microorganisms. Because some of these nontuberculous mycobacteria (NTM) species can be human pathogens, the study of these mycobacterial biofilms has increased during the last decades, and the interest in this issue increased as well as the growing number of patients with diseases caused by NTM. Different molecular mechanisms have been described, being especially well known the importance of glycopeptidolipids. Moreover, the knowledge of the extracellular matrix has shown important differences with other microorganisms, especially because of the presence of lipidic molecules as a key component of this structure. The clinical importance of mycobacterial biofilms has been described for many chronic diseases, especially lung diseases and implant-related ones, both in vitro and in vivo, and even in patients. Moreover, the biofilm-producing capacity has been proven also in M. tuberculosis, while its importance is not well understood. Biofilm studies have also shown the increasing resistance of mycobacteria in sessile form, and the importance of this resistance in the management of the patients is beyond doubt, being surgery necessary in some cases to cure the patients. Diagnosis of mycobacterial diseases is still based on culture-based techniques designed for the detection of M. tuberculosis. Molecular biology-based methods are also broadly used but again designed for tuberculosis diagnosis. Antimicrobial susceptibility testing is also well developed for tuberculosis, but only some species of NTM have standardized techniques for this purpose. New tools or approaches are necessary to treat these patients, whose importance is increasing, as the number of potential hosts is also increasing throughout the world.
