Tryptic soy broth tsb

Two Gram-positive bacteria frequently found in the food industry, especially in the milk or meat industry were selected because of their strong biofilm forming ability [25 (link)]. Microbacterium lacticum (M. lacticum) D84 (EF 204392) was isolated from extended shelf-life (ESL) milk, and Staphylococcus capitis subsp. capitis (S. capitis) was isolated from an air decontamination step prior to packaging at a meat production facility. Bacterial stock and working cultures were maintained and prepared according to Zand et al. [25 (link)]. The isolates were preserved in a 50% (v/v) glycerol stock at −80 °C. To obtain a stock culture, bacteria were sub-cultured overnight in tryptic soy broth (TSB; Carl Roth, Karlsruhe, Germany) at 37 °C and in 0.8% (v/v) skimmed milk broth (MB; Carl Roth) at 30 °C, for S. capitis and M. lacticum, respectively. Subsequently, stock cultures were then streaked onto either tryptic soy agar (TSA) or milk agar (MA) (Carl Roth) and incubated at 37 °C or 30 °C overnight and stored at 4 °C. Before each experiment, one colony was inoculated in 10 mL fresh TSB or MB, and the optical density₆₀₀ (OD) was standardized to 0.1 to obtain a working culture.

Staphylococcus aureus 04-02981 (Nuebel et al., 2010 (link)) was grown at 37°C in Tryptic Soy Broth [TSB] (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) with constant agitation at 150 rpm or on Tryptic Soy Agar [TSA] (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Growth inhibition tests on agar surface were performed according to CLSI guidelines for disk diffusion test (Naas et al., 2006 (link)). For this assay, 0.25 cm² sheets of Ag and AGXX^® were used.
For generation of growth curves, bacteria were pre-cultured overnight, diluted in TSB to an optical density at 600 nm (OD₆₀₀) of 0.05 and incubated for further 8 h either in presence of AGXX^® or in the presence of silver (Ag), 24 cm² each in 30 mL medium to obtain a sheet surface to medium volume ratio (A: V) of 0.8. Cultures grown in the absence of a metal sheet served as controls. OD₆₀₀ of the cultures was measured using the Genesys 10S UV-Vis spectrophotometer (Thermo Scientific, China). Colony forming units (CFU) per mL were determined hourly from 0 to 8 h post inoculation. Growth experiments were performed in triplicate with independent biological replicates.

The peptides were added to the culture at different concentrations ranging from 0.015 to 250 μM (final concentrations) and the antimicrobial activity was determined against the Gram-positive bacteria Listeria monocytogenes (DSM 20600), L. fleischmannii (DSM 24998), L. grayi (DSM 20601), L. marthii (DSM 23813), L. innocua (DSM 20649), L. welshimeri (DSM 20650), L. seeligeri (DSM 20751), Staphylococcus aureus (DSM 2569) and S. epidermidis (DSM 3269), and the Gram-negative species Escherichia coli (D31) and Pseudomonas aeruginosa (DSM 50071). The assays were carried out in 384-well plates (Griener Bio One, Frickenhausen, Germany) using Brain Heart Infusion Broth (BHIB) medium for Listeria spp. or Tryptic Soy Broth (TSB; Roth, Karlsruhe, Germany) for the other bacteria. Cultures in the mid-logarithmic phase were used for growth inhibition assays. The initial optical density (OD), measured at a wavelength of 600 nm (OD₆₀₀), for Listeria spp. was set to 0.01 and for the rest of the bacteria to 0.001. Changes in OD₆₀₀ values were monitored every 20 min for 24 h using an Eon Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA). Each assay also included a medium-only control. Antimicrobial activity was tested against all bacteria and the assays were carried out at least twice with comparable results.