Cell-free culture media (CFCM) was obtained as previously described by allowing A. baumannii strains to grow for 48 h at 37°C under shaking conditions (200 rpm) in LB, PF, LB + 1% pyruvate (PYR), PF + 1% PYR, LB + 0.02% phenylalanine (PA), PF + 0.02% PA, LB + 0.02% PAA, PF + 0.02% PAA, LB + 0.02% phenylpyruvate (PP), PF + 0.02% PP, and culture (Ramsey et al., 2016 (link); Castellanos et al., 2019 (link)). At the end of the incubation period, bacterial cultures were centrifuged for 10 min at 7,000 × g. The supernatant was filtered through a Steriflip Vacuum Filtration System with Millipore Express PLUS Membrane (0.22 μm) (Millipore, #SCGP00525).
PYR (1%) was used, since it was previously shown to induce a significant increase in the exoproteome profile of S. aureus, enhancing its pathogenicity (Harper et al., 2018 (link)); 0.02% PA, PAA, and PP CFCM were used due to the minimal growth seen in A118 growth curves of PAA with or without PF in both minimal media growth curves and in LB. To be consistent, 0.02% was used in CFCM studies for all three conditions.
PYR (1%) was used, since it was previously shown to induce a significant increase in the exoproteome profile of S. aureus, enhancing its pathogenicity (Harper et al., 2018 (link)); 0.02% PA, PAA, and PP CFCM were used due to the minimal growth seen in A118 growth curves of PAA with or without PF in both minimal media growth curves and in LB. To be consistent, 0.02% was used in CFCM studies for all three conditions.