S pyogenes

B. subtilis, S. aureus USA300 #417 and S. aureus USA300 #2690 tested in this assay were already available in our in-house collection, S. pyogenes (ID:0716100032) and S. agalactiae (ID:0216100214) are clinical isolated strains in the First Affiliated Hospital of Guangzhou Medical University, other strains were purchased from American Type Culture Collection (ATCC, USA). Antimicrobial susceptibility tests were conducted in 96-well microplates using the broth microdilution procedure described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (Wikler et al., 2009 ). Cation-adjusted Mueller Hinton broth for all the S. aureus strains, or Brain Heart Infusion broth for antibiotic-susceptible E. faecium strain ATCC 49624, vancomycin-resistant E. faecium strain ATCC 700221, S. agalactiae ATCC 13813, S. pyogenes ATCC 12344, S. pyogenes ATCC 19615 and M. catarrhalis ATCC 25240, or Mueller Hinton broth for the other strains were used in the assays. After incubation for 18 h at 37°C, the absorbance at 600 nm (A₆₀₀) was recorded using a microplate reader (Bio-Rad laboratory Ltd., UK) and the percentage of bacterial cell inhibition with respect to vehicles (1% DMSO) was calculated. The MIC was defined as the lowest compound concentration at which the growth of bacteria was inhibited by ≥90%. Three independent assays were performed for each test.

Hi (strain 51907), S. pyogenes (strain 700294), and S. pneumoniae (strain 700674) were purchased from ATCC (US). B. parapertussis bacteria (strain 529), influenza A virus subtype H1N1, and S. aureus bacteria were provided from the Polish Collection of Microorganisms. A single colony of each bacterium was inoculated in BHI broth (Sigma-Aldrich), and cultured overnight at a temperature of 37 °C, and next, the culture was centrifuged and the cell pellet was resuspended in 1 mL of PBS buffer.

The following four bacterial standard strains from the American Type Culture Collection (ATCC, Manassas, VA, USA) were used: H. influenzae ATCC 49247, S. aureus ATCC 29213, S. pneumoniae ATCC 49619, and S. pyogenes ATCC 19615. Cultivation and assay media (broth/agar) were Mueller–Hinton broth (MHB) complemented by Haemophilus test medium (H. influenzae), MHB (S. aureus), and brain–heart infusion (S. pneumoniae and S. pyogenes). The pH of the broths was adjusted to a final value of 7.6 using Trizma base (Sigma-Aldrich). All microbial strains and cultivation media were purchased from Oxoid (Basingstoke, UK). Stock cultures of bacterial strains were cultivated in broth medium at 37 °C for 24 h prior to testing in the incubator (Memmert GmbH & Co. KG, Buchenbach, Germany). For the preparation of inoculum, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard using a Densi-La-Meter II (Lachema, Brno, Czech Republic) to obtain a final concentration of 10⁸ colony-forming units per mL.