Omnilog phenotype microarray system

The phenotypic analysis of the P. veronii SM-20 strain was carried out using the fully automated Omnilog Phenotype MicroArray (PM) System (Biolog, Inc., Hayward, CA, USA) following the manufacturer’s instructions [20 (link)]. The strain was screened using Gen III MicroPlate (Biolog, Inc., Hayward, CA, USA), enabling the analysis of 94 metabolic tests, including the utilization of 71 carbon sources and 23 chemical sensitivity assays (listed in Supplementary Table S1), and drawing a “phenotypic fingerprint” of the microorganism. After overnight growth, cells were collected from a single colony on the LB agar plate using a cotton swab and inoculated to achieve a bacterial cell suspension with a cell density of T (transmittance) = 90–98%. Then, 100 µL of the cell suspension was dispensed into each well. The PM, typically containing a tetrazolium redox dye, was incubated in the dark at 27 °C under aerobic conditions. Positive metabolic activity was indicated by the appearance of a purple color, resulting from the reduction of tetrazolium dye in the wells. The color change representing the level of substrate consumption was recorded by the automated Omnilog System every 15 min for 120 h, until the plateau phase was reached. Data analysis was performed using the DuctApe software (https://combogenomics.github.io/DuctApe/index.html, accessed on 15 November 2023) [21 (link)].

The metabolic activity of the strains was measured using the Omnilog^® phenotype microarray (PM) system (Biolog Inc., Hayward, CA, USA). At least three biological replicates of each strain were tested in a microtiter plate-based microarray for the metabolism of 190 different carbon (plates PM1 and PM2A), 95 nitrogen (plate PM3B), 59 phosphorus, and 35 sulfur (both plate PM4A) sources. For the assay, the strains were freshly grown on sheep-blood-agar (Sifin, Berlin, Germany; overnight, 37 °C). The PM plates were inoculated with a bacterial suspension containing approx. 10⁵ cells/100 µL/well prepared in IF-Oa medium containing Dye-Mix A and, in the case of plates PM3B and PM4A, 20 mM Na-succinate as recommended by the manufacturer. Plates were incubated (48 h, 37 °C) and bacterial respiration measured every 15 min by reduction of the tetrazolium violet dye.

The growth of each variant was determined using the OmniLog PhenoType MicroArray system (Biolog Inc., Hayward, CA). Ma^Sm or Ma^Rg was grown for as long as 48 h in the OmniLog incubator in 7H9 broth with OADC, with or without Tween 80, and in the presence of Biolog Redox Dye A at a density of 1 × 10⁷ bacteria/well. Readings were taken every 15 min.
The ability of M. abscessus to aggregate was assessed using an optical density aggregative index as described elsewhere (63 (link)) with modifications. Briefly, Ma^Sm or Ma^Rg was grown for 48 h in 7H9 broth with OADC, with or without Tween 80. After removal from the shaking incubator, cultures were gently agitated, and the OD₆₀₀ was taken at time 1 (T₁), and again after 15 min (T₂), for each variant. The aggregative index (AI) was calculated as OD_T1 − OD_T2/OD_T1 × 100.