Shaking incubator

All assays were conducted with S. aureus Xen 31 MRSA strain (Perkin Elmer, Waltham, MA, USA). Bacterial colonies were maintained on LB plates containing 17 g/L agar (Fisher Scientific, Waltham, MA, USA). Bacteria were grown in Luria Bertani Miller (LB-Miller; Fisher Scientific) broth at 37°C until they reached exponential phase (optical density of 0.4–0.7 at 600 nm) in a shaking incubator (New Brunswick Scientific, Edison, NJ, USA) at 250 rpm. Bacterial growth was measured using the DU 730 spectrophotometer (Beckman Coulter, Inc., Chaska, MN, USA).

SA was GFP-tagged strain USA300 LAC (SA^GFP). Bacteria were stored at –80°C in 25% glycerol in autoclaved Luria-Bertani (LB) broth media (MP Biomedicals) and propagated on LB-agar plates containing chloramphenicol (10 μg/mL; MilliporeSigma). Plates were refreshed from frozen stock every 1–2 weeks. For experiments, single bacterial colonies were propagated in autoclaved LB media containing chloramphenicol (10 μg/mL) in a shaking incubator at 37°C and 200 rpm (New Brunswick Scientific) for 18 hours (stationary growth phase) or, for selected experiments, to OD_600nm = 1 (exponential growth phase). Bacteria were prepared for alveolar microinstillation or intranasal instillation, respectively, by dilution of 1 mL of culture in 500 μL or 1.3 mL in 300 μL of DPBS containing Ca²⁺ and Mg²⁺. Within 40 minutes of bacterial removal from the incubator, we instilled the bacteria-containing solution into lung alveolar airspaces by alveolar micropuncture or mice by intranasal instillation (30 μL to deliver 1 × 10⁸ CFU per mouse). For intranasal instillations, mice were rapidly anesthetized and instilled in pairs to ensure similarity of inocula across animals.

The bacteria were grown until the logarithmic phase in 7H9 broth supplemented with 10% ADS, 0.2% glycerol, and 0.05% tyloxapol or Sauton’s medium supplemented with 2% glycerol and 0.05% tyloxapol and diluted to an OD₆₀₀ of 0.05 before being incubated in a shaking incubator (New Brunswick Scientific; 175 rpm) at 37°C. Growth of the strains was evaluated every 24 h by measuring the OD₆₀₀ with a cell density meter (Biochrom WPA Biowave). In parallel, the same experiment was conducted for 2 days in 7H9 broth and 3 days in Sauton’s medium with growth measured by both OD₆₀₀ and CFU count to determine the CFU-OD₆₀₀ proportion of each strain.

Mab strains were grown in 7H9 supplemented with 10% ADS, 0.2% glycerol, and 0.05% tyloxapol until reaching their logarithmic phase and diluted to an OD₆₀₀ of 0.1 in the same medium. After 48 h of growth in a shaking incubator (New Brunswick Scientific; 175 rpm) at 37°C, the pellets of the strains were harvested and incubated in TRIzol reagent (Invitrogen) for 5 min at room temperature (RT). Next, the bacteria were lysed with BeadBug beads (Sigma; 0.1 mm Zirconium beads) by shaking at a speed of 6 m/second twice for 45 seconds using the FastPrep 24 Classic (MP biomedicals) followed by overnight incubation at −80°C. In order to separate the samples from the TRIzol reagent, Phasemaker tubes (Invitrogen) were used together with the addition of chloroform to the sample. Once separated, RNA isolation of the samples was completed using the RNeasy Plus Mini Kit (Qiagen) followed by a DNase treatment completed with TURBO DNAase (Qiagen) and ezDNase (Invitrogen). The final RNA concentration was measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific), and the RNA samples were stored at −80°C until further use.

Prior to use, acid whey was clarified by centrifugation to remove sediments and heated at 65°C for 30 min to decontaminate the product; no microorganisms were recoverable on microbiological media after heating. The acid whey was modified as follows: (a) pH adjustment to 6 or 7 with 5 N NaOH (Fisher Scientific, Fair Lawn, NJ, United States), (b) addition of ultra-high temperature (UHT) skim milk (1% w/v), and (c) supplementation with yeast extract (YE; Becton, Dickinson and Company) at 1% w/v level. Yeast extract was dissolved in water and autoclaved prior to addition to the whey.
The four selected antimicrobial producers were cultured at 1% (v/v) in 20 ml of the modified acid whey as shown in Table 2. The incubation was completed in 125–ml baffled Erlenmeyer flasks agitated at 150 revolutions/min (RPM) in a shaking incubator (New Brunswick Scientific, Edison, NJ, United States) at 30°C for 24 or 48 h. After incubation, cells of the producer bacteria were removed by centrifugation at 7,000 × g and 4°C for 20 min and the supernatant was collected and stored at 4°C until being used for antimicrobial testing, using the antimicrobial bioassay technique described later. Based on the antimicrobial activity assessment (Table 2), the highest antimicrobial producer in modified acid whey, under given bioreaction conditions, was selected for scaling up the antimicrobial production.

The biosynthesis of γ-PGA was carried out by growing Bacillus licheniformis ATCC-9945a using “E” medium adjusted to pH 7, as previously described in the literature [26 (link)]. The microorganisms were incubated at 37 °C in a shaking incubator at 250 rpm for 24–48 h (New Brunswick scientific, Enfield, CT, USA). γ-PGA was purified by centrifugation, and the supernatant was washed two times with a mixture of acetone and deionized water (1:1 v/v). Then, the precipitate was collected, lyophilized, and stored in a refrigerator.