Tryptic soy agar plates

Cryo-conserved cultures of selected bacterial strains (Table 4) were transferred to Tryptic Soy Agar (TSA) plates (Carl Roth) with a sterile inoculation loop, sealed with Parafilm (BEMIS, Neenah, WI, USA), and incubated for 1–2 days at 37 °C depending on growth. Single colonies were picked and transferred into 2–3 mL of the Mueller–Hinton II (MH II) broth (BD, Heidelberg, Germany). The bacteria were cultivated overnight, subcultured on fresh medium and grown for 3–4 h before testing at 37 °C, with shaking at 180 rpm. We measured the optical density at 600 nm (OD₆₀₀) of 1 mL of the bacterial suspensions in polystyrene cuvettes (SARSTED, Nümbrecht, Germany) using an Ultrospec 10 spectrophotometer (Biochrome, Cambridge, UK). We then diluted them with the MH II medium to the preferred OD₆₀₀ (Table 2). The peptides were dissolved in DMSO to generate 10 mmol/L stocks and diluted with the MH II medium to 200 µM before the assay. We used 10 µg/mL (~40 µg/mL for S. epidermidis) gentamicin and colistin (Sigma-Aldrich, Taufkirchen, Germany), DMSO and the medium only as controls. The bacteria were transferred to 96-multiwell plates with a final volume of 100 µL per well (50 µL bacterial suspension, and 50 µL test peptide). The OD₆₀₀ was measured at 0, 24 and 48 h using a BioTek Eon microplate reader followed by analysis with Gen5 v2.09. Raw data are given in Supplementary File S5.

Effects of Macrovipera venoms against Gram-positive and negative bacteria were determined on six strains (Table 2). Cryo-conserved cultures were transferred to tryptic soy agar (TSA) plates (Carl Roth) and incubated at 37°C. Single colonies were picked and transferred into either 4 mL Mueller-Hinton II (MH II) medium (BD, Heidelberg, Germany) or in the case of Listeria monocytogenes into 4 ml of Brain Heart Infusion (BHI) medium (Carl Roth) and cultivated for 24 h at 37°C and 180 rpm. 4 ml of fresh culture medium were inoculated with each bacterial suspension (L. monocytogenes 60 μl, other strains 30 µl) prior incubation for 4 h at the conditions outlined above. A five-step venom dilution series from 2–0.125 mg/ml was created for each venom and tested against all strains using gentamicin (0.02 μg/μl, Sigma-Aldrich, Taufkirchen, Germany) as a positive control. Assays were performed as biological triplicates in 96-multiwell plates with a final volume of 100 µl per well (50 µl of bacterial culture; 50 µl venom solution) after incubation for 48 h at 37°C and 140 rpm. The OD₆₀₀ was measured after 24 h on a BioTek Eon microplate reader and Gen5 v2.09. Normalized raw-data of the antibacterial assay is given in Supplementary Table S8.

We assessed the in vitro bacterial killing activity (BKA) of the plasma against Escherichia coli following the method of Tieleman et al. [49 ]. This assay has been used previously on different free-ranging and captive small mammal species, including several rodents [50 (link)–52 ] and is useful as it measures the functionality of the innate immunity [46 (link)]. After diluting the plasma samples 1:20 in PBS, to each diluted sample (140 μl) we added 10 μl of a suspension of live E. coli (ATCC #8739). The bacterial suspension was adjusted to a concentration of 200 colonies per 50 μl of diluted plasma-bacteria mixture. After incubation for 30 min at 37°C (mammalian body temperature), aliquots of the 50 μl of the plasma-bacteria mixture was spread onto Tryptic Soy Agar plates (#CP70.1, Carl Roth GmbH) in duplicate, and the plates were incubated overnight at 37°C. To obtain the initial number of bacteria that we had before starting to interact with the plasma, we diluted 140 μl media alone with bacterial suspension and plated immediately. On the following day the colony-forming units were counted and the bacterial killing activity (BKA%) was defined as the percent of the killed bacteria, which was calculated as 1– (average of the viable bacteria after incubation/the initial number of bacteria). The average was calculated from two plates per sample.