Abstract / Summary
Abstract Background Mycobacterium wolinskyi is an emerging, rapidly growing non-tuberculous mycobacterium (NTM) increasingly associated with postoperative and device-related infections. While biofilm formation contributes to the survival of several NTM species, to our knowledge, surface-associated biomass formation by M. wolinskyi has not previously been assessed quantitatively. Objective To provide the first quantitative time-course assessment of surface-associated biomass formation by a clinical isolate of M. wolinskyi. Methods A wound-derived M. wolinskyi isolate was identified using MALDI-TOF MS, and this identification was confirmed via targeted Next Generation Sequencing (NGS). Surface-associated biomass was quantified in a time-course experiment using a CV-based static microtiter model, with early short-interval measurements (48 h and 96 h) to define the onset of adhesion, followed by extended time points (weeks 1, 3, and 6) to evaluate changes in CV-retained surface- associated biomass over time.Absorbance was measured at 570 nm after dye solubilization with 30% acetic acid. Experiments were performed in technical sextuplicate in five independent biological experiments. Temporal differences were evaluated using one-way ANOVA. The presence and structure of the biofilm were also assessed after one week using confocal fluorescence microscopy with a Zeiss LSM 900 microscope equipped with an Airyscan2 detector. Results No detectable surface-associated biomass was observed at 48 or 96 hours. Biomass became detectable after one week and increased significantly between week 1 (OD570 0.6 ± 0.2) and week 3 (OD570 1.9 ± 0.3; p < 0.001). Between weeks 3 and 6, mean OD₅₇₀ values remained similar (1.9 ± 0.3 versus 2.0 ± 0.2; p = 0.35), indicating a plateau in CV-retained biomass measurements under the experimental conditions tested. Confocal microscopy performed after one week confirmed the presence of an extracellular matrix associated with the bacterial aggregates. Conclusion To our knowledge, this is the first study to provide a quantitative, time-based assessment of the formation of surface-associated biomass by M. wolinskyi in CV under the static in vitro conditions tested. Biomass measurements increased between weeks 1 and 3, reaching a plateau by week 6. These results demonstrate M. wolinskyi's capacity to produce quantifiable surface-associated biomass, paving the way for future research into extracellular matrix composition, viability, and antimicrobial tolerance. Clinical trial number Not applicable.