Yeast extract

Two concentrations of lactose and potassium nitrate were tested for a pool of strains in preliminary tests.
lactose fermentation was tested in a modified API 50CH medium containing the following: lactose 5 or 20 g.L⁻¹ (Panreac, Lyon, France), tryptone 10 g.L⁻¹, yeast extract 5 g.L⁻¹, K₂HPO₄, 0.25 g.L⁻¹, MnSO₄ 0.05 g.L⁻¹, and bromocresol purple 0.17 g.L⁻¹. The medium was inoculated using 1% (v/v) of 48-h cultures in YEL, and incubated at 30 °C under anaerobiosis (using the Anaerocult A system, Merck, Darmstadt, Germany). The production of acid from lactose was determined from the colour change of bromocresol purple from purple to yellow after 2, 5, and 7 days of incubation.
Nitrate reductase activity was detected by means of the Griess reagent (Biomérieux, Marcy l'Etoile, France) after incubation of cultures at 30 °C under microaerophilic conditions (air atmosphere without agitation) in a broth containing potassium nitrate, 0.5 or 1.5 g.L⁻¹ (VWR International, Fontenay-sous-Bois, France), tryptone (Biokar Diagnostics, Allone, France) 10 g.L⁻¹, yeast extract (Biokar Diagnostics) 5 g.L⁻¹, and glucose (Grosseron, Saint-Herblain, France) 1 g.L⁻¹, according to Dalmasso et al. (2011 (link)). The results of the tests were read after 2 and 5 days of incubation.
All the tests were carried out in triplicate.

S. epidermidis (10⁵ CFU/mL) was loaded by the siphon technique into PSF MTAM, as previously described [54 (link)]. S. epidermidis in PSF MTAM was incubated in 10 mL rich media (10 g/L yeast extract (Biokar Diagnostics, Beauvais, France), 3 g/L TSB, 2.5 g/L K₂HPO₄, and 1.5 g/L KH₂PO₄), 2% glycerol, and the 0.002% (w/v) phenol red (Sigma) as a fermentation indicator. S. epidermidis alone or PSF MTAM alone without S. epidermidis in the presence or absence (Figure S2) of 2% glycerol in phenol red-containing rich media was included as a control. A color change from red-orange to yellow indicated the occurrence of bacterial fermentation and it was detected by OD₅₆₀.

A total of five strains of S. cerevisiae were used in this study. Four commercial strains of S. cerevisiae (Lallemand Inc., Montreal, QC, Canada) were selected from among the strains considered in our previous work (Bordet et al., 2021 (link)). These commercial strains were coded S2, S3, S4 and S8, supplied as active dry yeast (ADY) and stored at 4°C once opened. The other strain, S. cerevisiae S3GFP was a modified S. cerevisiae strain supplied from the VAlMiS team, at the Burgundy University Vine and Wine Institute. S3GFP is a derivative of the commercial strain S3 modified by CRIPSR-Cas 9 to strongly express eEGFP (Bordet et al., 2022 (link)). This modified strain was stored at – 80°C in YPD liquid medium [0.5% w/v yeast extract (Biokar, Beauvais, France), 1% w/v bactopeptone (Biokar), 2% w/v D-glucose (Prolabo, Fontenay sous-Bois, France) and 0.02% w/v chloramphenicol (Sigma, St Louis, MI, United States)], containing 20% v/v glycerol.

Culture and identification of bacteria (USA300 [39 (link)], MRSA252, invasive methicillin-susceptible S. aureus (MSSA) (ATCC29213), or commensal S. aureus) are described in Supplementary Materials. To induce fermentation, bacteria (10⁵ colony-forming unit (CFU)/mL) were incubated in rich medium (10 g/L yeast extract (Biokar Diagnostics, Beauvais, France), 5 g/L tryptic soy broth (TSB), 2.5 g/L K₂HPO₄ and 1.5 g/L KH₂PO₄) in the absence and presence of 20 g/L (2%) glycerol under anaerobic conditions using Gas-Pak (BD) at 30 °C for ten days. Rich medium plus 20 g/L glycerol without bacteria was included as a control. The 0.001% (w/v) phenol red (Sigma, St. Louis, MO, USA) in rich medium with 20 g/L glycerol served as an indicator, converting from red-orange to yellow when fermentation occurred. SCFA identification by nuclear magnetic resonance (NMR) analysis and enumeration of USA300 by overlay assays and co-culture of USA300 with commensal S. aureus are described in detail in Supplementary Materials.

The pre-cultures for the inoculation of the bioreactors were cultivated in 500 mL shake flasks containing 100 mL xylose mineral medium (composition as indicated above) until exponential phase. The cells were harvested by centrifugation (4,500×g, Sorvall STR40) and used to inoculate 0.5 l bioreactors (MiniBio, Applikon) that contained 250 mL medium at an OD₆₀₀ of ~1.2. The composition of the fermentation medium was similar to the mineral medium used in the shake flask experiments, except that it contained 55 g/L (d)-xylose, 2 g/L Na₂HPO₄ · 12H₂O, 0.8 g/L KH₂PO₄, 6 g/L (NH₄)₂HPO₄, 0.4 g/L (NH₄)₂SO₄, 1 g/L tryptone (Biokar), 0.5 g/L yeast extract (Biokar), 0.4 mL/L polypropylene glycol as antifoaming agent, 1 mM IPTG, and no MOPS. The pH of the cultures was kept at 7.0 by the addition of 10 M KOH, and reactors were aerated with air at 1 vvm. Dissolved oxygen tension was regulated by adjusting the appropriate agitation speed (300–1,200 rpm, Rushton rotor, 28 mm diameter), and was either kept at 40 % to impose fully aerobic conditions, or at 2 % to impose micro-aerobic conditions.

S. epidermidis (10⁷ CFU/mL) were incubated in 10 mL rich media (10 g/L yeast extract (Biokar Diagnostics, Beauvais, France), 3 g/L TSB, 2.5 g/L K₂HPO₄, and 1.5 g/L KH₂PO₄) in the absence and presence of glycerol (2%) under aerobic conditions at 37 °C with shaking at 200 rpm. The rich media glycerol (2%) without bacteria were included as a control. The 0.002% (w/v) phenol red (Sigma) in rich media with glycerol (2%) acted as a fermentation indicator. A color change from red-orange to yellow indicated the occurrence of bacterial fermentation, which was detected as absorbance at 560 nm.