Peptone water

Synthetic wound fluid (SWF) comprised equal volumes of foetal bovine serum (Gibco) and peptone water (Sigma-Aldrich)^{24 (link)}. Mueller–Hinton Broth (MHB) was obtained from VWR. HyClone water (GE Healthcare) was used throughout.

Silver nanoparticles solution (4000 µg/ mL with the particle size of 35 nm) was purchased from Pars Nano Nasb Co (Tehran, Iran) and sterilized by filtration through 0.22 µm filters before use. Peptone water, phosphate buffered saline (PBS), Luria-Bertani (LB) broth and agar, Agar-agar, and resazurin sodium were obtained from Sigma Chemical Co (St. Louis, MO., USA). All other chemicals were purchased from Merck (Darmstadt, Germany). The plant, Zataria multiflora Boiss, was purchased from local groceries. Staph. aureus ATCC 25923 and Salm. Typhimurium ATCC 14028 were obtained from the Department of Food Hygiene and Quality Control, Urmia University, Urmia, Iran.

The LAB strains were isolated from homemade pickled beetroots. Pickled foods are widely regarded in Poland as having health-promoting properties and a being a good source of natural probiotics. A series of 10-fold dilutions ranging from 10⁻¹ to 10⁻⁴ in Peptone Water (Peptone Water, Sigma-Aldrich, Germany) was prepared from the tested samples. The samples and all dilutions were then applied to 7 different culture media. A total of 100 μL of the appropriate dilution was applied to the medium and evenly distributed with a spreader. The prepared plates were incubated for 24–72 h in three conditions: aerobic conditions at 37 °C, aerobic conditions at 25 °C, and anaerobic conditions at 30 °C. Then, based on morphological differences, single colonies of bacteria were selected, from which reduction cultures were prepared on the same media to obtain pure cultures. The following culture media were used: M-17 Agar (Sigma Aldrich, Steinheim, Germany), MRS Agar (Sigma Aldrich, Steinheim, Germany), China Blue Lactose Agar (Sigma Aldrich, Steinheim, Germany), APT Agar (Merc, Darmstadt, Germany), and TOS (Merck, Darmstadt, Germany).

Each of the three Lactobacillus inoculums was adjusted to an OD (625 nm) of 0.1 and then mixed in a proportion of 1:1:1. This pool was allowed to grow in SVF for 16 h in anaerobiosis (selected time based on the results from Supplementary Data Figure S1) with the different extracts at a concentration of 2% (w/v). At the end of the 16 h, the C. albicans inoculum (OD of 0.1, at 625 nm) was added at 2% (v/v). Sampling points were carried out at 0 h and 16 h pre-infection, and then at 18 h, 20 h, 24 h, and 40 h after infection with C. albicans. Sequential tenfold dilutions were carried out in sterile peptone water (Sigma-Aldrich, St. Louis, USA) and plated (in quadruplicate), using the drop technique (Miles et al. 1938 (link)). MRS agar with 98 mg/L of fluconazole (to inhibit Candida sp. growth) was used for Lactobacillus sp. counting, while CHROMagar was used for C. albicans counting, incubated at 37 °C under anaerobic conditions and 30 °C under aerobic conditions, respectively.

Concurrent to the seawater sampling, mollusk samples were gathered in the summer (Jul–Aug 2015; 34.0 °C ± 2.0) and winter seasons (Dec–Feb 2016; 17.2 °C ± 2.0). Shells (n = 55–60) were hand-picked, sorted in autoclaved bags (4 °C), and transported to the College of Science, Kuwait University laboratories for further processing [28 (link)]. The mollusc shells were removed to collect the flesh and fluid in a pre-autoclaved beaker (Borosil, Poole, UK). Approximately 20 g was macerated with 0.1% peptone water (Sigma, Plymouth, UK) and serially diluted [28 (link)]. The dilutions were passed through the 0.45 µm nitrocellulose membrane filters and placed on TBX-agar for E. coli, as mentioned previously. In total, 247 mollusc strains were isolated.

Rocket leaf samples (0.1 g) were homogenized with 7.5 mL 1% (w/v) peptone water (Difco) with a mortar and pestle and the extracts filtered through Whatman filter paper. Serial dilutions were prepared with peptone water and 200 μL of extracts were plated in duplicate onto plate count agar (Sigma-Aldrich, UK) with appropriate serial dilutions to enable colony counting. All plates were incubated for 3 days at 25 °C. After incubation, colonies were counted and normalized to leaf fresh weight.
Chlorophyll content was assessed using a SPAD-502 Plus portable chlorophyll meter (Minolta, Osaka, Japan). Three measurements were taken on each leaf of each biological replicate. Chlorophyll a fluorescence-related parameters were measured using a MINI-PAM II (Pulse-Amplitude Modulation) portable chlorophyll fluorometer (Walz, Germany) where values were recorded for the parameters Fo (initial fluorescence), Fm (maximum fluorescence), and Fv/Fm (quantum maximum efficiency of photosystem II, PSII). Control leaves were dark acclimatized for at least an hour and readings were carried out in triplicate in the dark, avoiding veins. Care was taken not to wound or damage leaves during measurements.