Glass beads

L. plantarum colonization was determined by plating on MRS+CM agar. The combination of the MRS selectivity for lactobacilli and enterococci together with aerobe incubation and presence of antibiotics allowed growth repression of all other bacteria. Data were compared to the theoretical washout curve determined for absence of growth, from the following equation: c_t = c₀ × e^(t/RT) (53 (link)), where c₀ and c_t represent cell concentration at time point zero and t, respectively, and RT corresponds to the retention time. Colonies were randomly picked, incubated in MRS+CM overnight, mixed 1:1 with 60% (vol/vol) glycerol (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), and stored at −80°C.
Natural biofilm formed in TR2, used for long-term planktonic adaptation, and the repetition experiment in TR1 (period 1). To recover L. plantarum from biofilms, the vessels were emptied and washed twice with PBS. Remaining biofilm was removed and homogenized with glass beads (5 mm; VWR International AG, Dietikon, Switzerland) in dilution solution containing 0.85% (wt/vol) NaCl and 0.1% (wt/vol) peptone from casein (VWR International AG, Dietikon, Switzerland). Dilutions were plated with subsequent strain recovery and storage performed as described above.

Resistant isolates detected by screening and EUCAST microdilution broth reference methods were identified by sequencing a portion of the β-tubulin gene [31 (link)]. Complete genomic DNA was extracted from a Malt-extract agar fungal culture using QIAamp DNA blood minikit (Qiagen Sciences Ing, Courtaboeuf, France). Briefly, conidia and hyphae were disrupted with glass beads (VWR, ref: 432-0064) and lysis buffer on MagNA Lyser instrument (Roche Diagnostics, Meylan, France). The resulting suspension was then treated according to the manufacturer’s instructions. PCRs were performed in a 50 µL-final volume containing 1× HF buffer (ThermoFisher, Les Ulis France), 200 µM of deoxynucleoside triphosphates (dNTPs), 1 µM of each primer, 3% of DMSO, 1 unit of Phusion™ High-Fidelity DNA Polymerase (ThermoFisher), and 100 ng of genomic DNA. The primers used for β-tubulin gene amplification were Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3′) and Bt2b (5′-ACCCTC AGTGTAGTGACCCTTGGC-3′) as previously described [32 (link)]. Sanger sequencing was performed at the Genomic platform of Henri Mondor Hospital Biomedical Research Institute (IMRB). Sequences were analyzed using DNA Baser Assemble v5.15.0 and compared to GenBank and MycoBank databases sequences. Nucleotide identification was achieved at >99% match.

Capsular AM was isolated and purified from the M. tuberculosis strains as described (16 (link), 25 (link), 59 (link)). In brief, bacteria were incubated at 37°C for 21 days in horizontally placed roller bottles containing minimal media without any detergents to preserve the formation of the capsule. The capsule was then collected by physically disrupting bacterial cells using glass beads (VWR). The protein portion was removed from the capsular extract, and AM was separated from other glycans by column chromatography. LAM purified from H37Rv was obtained from the Biodefense and Emerging Infectious Research Resources Repository (BEI Resources; NR-14848). LAM purified from CDC1551 was obtained from Delphi Chatterjee at Colorado State University (Fort Collins, Colorado, USA) (60 (link)).

L. plantarum colonization of five independent in vitro colonic microbiota was monitored by daily plating reactor effluent on MRS+CM agar. Naturally formed biofilms on the reactor's walls were collected at the end of fermentation by emptying and washing the reactor vessel twice with PBS to remove non-adherent cells. The complete biofilm was removed using a spatula and homogenized in falcon tubes containing dilution solution (0.85% NaCl, 0.1% peptone from casein (w/v), VWR International AG, Dietikon, Switzerland) using glass beads (5 mm, VWR International AG, Dietikon, Switzerland). L. plantarum viable cell count was determined by plating as described earlier.

For the extraction of TTXs, glass beads (250 mg, 0.15–0.25 mm, VWR) and 1% aqueous acetic acid (0.5 mL) were added to 200 mg of shellfish (oyster or clam) or 50 mg of bacterial cell pellet. The sample was briefly vortexed and ground using a mixer mill (MM400, Retsch, Eragny, France) for 10 min at 30 Hz. Tubes were centrifuged (15,000× g, 10 min) and debris-free supernatants were ultra-filtered onto 3 kDa (Nanosep, Pall) for shellfish and 0.22 µm (Nanosep MF, Pall) for bacteria.

Capsular AM was isolated and purified from the M. tuberculosis strains as described (16 (link), 25 (link), 59 (link)). In brief, bacteria were incubated at 37°C for 21 days in horizontally placed roller bottles containing minimal media without any detergents to preserve the formation of the capsule. The capsule was then collected by physically disrupting bacterial cells using glass beads (VWR). The protein portion was removed from the capsular extract, and AM was separated from other glycans by column chromatography. LAM purified from H37Rv was obtained from the Biodefense and Emerging Infectious Research Resources Repository (BEI Resources; NR-14848). LAM purified from CDC1551 was obtained from Delphi Chatterjee at Colorado State University (Fort Collins, Colorado, USA) (60 (link)).