Tryptic soy agar

The mastitis isolate S. aureus Newbould 305 (ATCC 29740) was grown in brain heart infusion (Thermo Scientific, Belgium) broth for 5 h from a frozen stock. The CFU/mL was determined after 24 h of incubation at 37°C from the 1/100 diluted bacterial preculture by spectrophotometric measurements at 600 nm (OD600) and were confirmed a posteriori by plating on tryptic soy agar (Thermo Scientific, Belgium) plates. The pathogen was suspended in PBS to achieve the required concentration (10³–10⁴ CFU/100μl). This 100 µl solution was then used to inoculate the mammary gland in the different mouse experiments (Figure 1). Ultrapure LPS 0111:B4 (InvivoGen, USA) and ultrapure LTA from S. aureus (InvivoGen, USA) were suspended in endotoxin-free water and diluted to the required concentration in PBS. These 100 µl LPS and LTA solutions were injected 24 h prior to the intramammary infection with S. aureus (Figure 1).

S. aureus strains PS80 [50 (link)], USA300 LAC::lux [51 (link)] and Newman [52 (link)], and E. coli strain EC958 [53 (link)] have been described previously. S aureus strains were cultured overnight on Columbia agar supplemented with 2% NaCl (PS80), or on tryptic soy agar (USA300 and Newman); E. coli was grown overnight on tryptic soy agar supplemented with 4% defibrinated sheep’s blood (ThermoFisher Scientific, Gloucester, UK). Bacteria were suspended in PBS, enumerated by optical spectrometry and diluted to 1 × 10⁸ CFU/mL. CFU counts were verified by plating on appropriate agar overnight.

The procedure for determining antibiotic activity followed a previous publication [27 (link)]. Escherichia coli ATCC 25922 (ATCC, Manassas, VA, USA) was sub-cultured in Tryptic soy broth (Thermo Scientific, Waltham, MA, USA) at 37 °C for 12 h. E. coli approximately at 2.4 × 10⁶ CFU, as determined by a spectrophotometer (ELx808 absorbance reader; BioTek, Shoreline, WA) at optical density 600 nm at 0.003 (OD 600 nm at 0.003), were diluted in Tryptic soy broth (Thermo Scientific) and added with AuNP in different concentrations: 12.5, 25 and 50 ppm, respectively. Then the solutions were incubated at 37 °C for 4 h. After that, the supernatant in serial dilution was plated in Tryptic soy agar (Thermo Scientific), kept at 37 °C overnight before bacterial colony enumeration. E. coli in Tryptic soy broth alone or in 100 μg/ml of gentamicin were used as the positive and negative control group, respectively.

Previous studies investigating the atmospheric microbiome have used culturing techniques or direct next-generation sequencing of air samples to analyze the atmospheric microbiome. Limitations of culturing techniques or direct next-generation sequencing are well-known (Stephens et al. 2015 (link)). We chose to investigate the culturable microorganisms only in order to eliminate the possibility of sequencing DNA artifacts carried by the air. Sampling was conducted using Petri plates containing potato dextrose agar (PDA) (Thermo-Fisher Scientific, Waltham, MA, USA) or tryptic soy agar (TSA) (Thermo-Fisher Scientific, Waltham, MA, USA). The plates were held open into the incoming air for 2 min to collect airborne fungi and bacteria. The plates were then incubated at room temperature for 48 h and stored at 4°C until sequencing. Originally, 47 samples for 22 days were collected using both PDA and TSA media. The number of replicates per day varied from 2 to 4. However, some samples were discarded because of their low-read quality after sequencing and that they would not represent the true microbial diversity in the environment. Therefore, we analyzed the culturable fungal microbiome using 47 replicates collected from 22 days, while for the culturable bacterial microbiome, 36 replicates collected from 17 days were used.

Delta-hemolysin activities were assessed by cross-streaking overnight cultures of the S. aureus isolates perpendicularly to the reference strain RN4220, which only produces beta-hemolysin, on tryptic soy agar containing sheep blood (Thermo Scientific). Agar plates were incubated at 37°C for 18 to 24 h. Given that beta- and delta-hemolysins act synergistically, enhanced hemolysis can be observed at the intersection of a strain that secretes delta-hemolysin and the reference strain (19 (link)). The wild-type strain HG003 and its agrC mutant (agrC::erm) were used as the positive and negative controls, respectively. Three independent cultures were examined to ensure reproducibility.