Cm1135

The bacterial strain used in this study was Staphylococcus aureus ATCC 25923, obtained from the culture collection of the Laboratory of Microbiology in the Interdisciplinary Research Platform within Banat's “King Michael I of Romania” University of Agricultural Science and Veterinary Medicine, Timisoara. In the laboratory, the ATCC strains are maintained at −50°C. The Staphylococcus aureus ATCC 25923 was revived by overnight growth in Brain Heart Infusion (BHI) broth (Oxoid, CM1135), at 37°C and, subsequently, passed on BHI Agar, for 24 hours at 37°C. The strain was then diluted with saline solution 4.5‰, at an optical density (OD) of 0.5 McFarland standard (1.5 × 10⁸ UFC/mL). 1 mL of this solution was then suspended in BHI broth 1 : 30.

Streptococcus oralis DSM 20627 (DSMZ, Braunschweig, Germany) was grown at 37 °C in tryptic soy broth (TSB, CM0129, Oxoid, Hampshire, UK), supplemented with 0.3% w/v yeast extract (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), for 18 hours under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂, Don Whitley Scientific Limited, Shipley, UK) and with stirring. The S. oralis biofilm was grown on supporting hydrophilic polyethersulfone membrane (Merck Millipore, Darmstadt, Germany). Sterile membranes with 13 mm diameter were placed into 24-well plates with the dull side up and washed in 1 mL PBS for 18 hours under anaerobic conditions with parallel shaking. The overnight bacterial culture was centrifuged at 4,000× g for 15 min at 4 °C and diluted in brain heart infusion broth (BHI, CM1135, Oxoid, Hampshire, UK), supplemented with 5% w/v D(+)-saccharose (4621.2, Carl Roth GmbH, Karlsruhe, Germany) to give an optical density of 0.06 at 600 nm, corresponding to 8.7 × 10⁷CFU/mL. This S. oralis suspension was added on top of the membranes (600 μL/well). For the control groups, the sterile culture medium was used. Membranes were incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 72 hours. The medium was replenished on alternate days.

The microplate method was used to examine the ability of E. coli to form biofilms with different media [33 (link)]. We studied the following six different media: Tryptone Soya broth (TSB), Tryptone Soya broth containing 1% (w/v) glucose, Yeast Extract, Brain Heart Infusion Broth (BHI; Oxoid CM 1135), Nutrient broth (NB; Oxoid CM0001), and LB broth (Miller) (LB; Merck 110285, Darmstadt, Germany).
E. coli isolates were incubated overnight (12–18 h) on TSB Agar at 37 °C. Then, the overnight cultures were adjusted to a density of 0.5 of McFarland and pipetted into microplates (3599 Corning Costar; Corning, NY, USA) with different media, followed by incubation at 37 °C for 24 h. After incubation, absorption was measured using a microplate reader (BioTek Epoch; Agilent, Santa Clara, USA) at 600 nm. The microplates were washed three times for biofilm detection as follows: 0.9% NaCl, methanol, crystal violet (Merck 1159400, Darmstadt, Germany), and 33% acetic acid (Merck 159166, Darmstadt, Germany) to remove the excess stain. After drying, the microplates were incubated with 5 mL of 96% ethanol for 15 min. The optical density (OD) at 595 nm was measured with a microplate reader (Bio Tek Epoch; Agilent, Santa Clara, USA) [34 (link)].

Each Salmonella strain was thawed from liquid nitrogen and cultured onto an XLDT4 agar plate. After overnight incubation at 37 °C, one colony was used to seed a Brain Heart Infusion broth (BHI) (Oxoid, CM1135), which was incubated overnight at 37 °C. Before the beginning of the trials, the virulence of the three Salmonella strains was enhanced as previously described [43 (link),44 (link)]. Briefly, each strain was inoculated by gavage into the crop using two Salmonella-free one-day-old chicks. After two days, these chicks were euthanized, and their livers and spleens were cultured onto XLDT4 plates that were processed as before. For standardization of the proceedings, the growth from the agar plates was collected, aliquoted, and kept frozen in liquid nitrogen until used.

Listeria monocytogenes EGD wild-type was used in the study (Table 1). Bacterial stock cultures were stored at -80°C and inoculated on Brain Heart Infusion (BHI; Oxoid CM 1135) agar and grown at 37°C overnight. An overnight culture was obtained by inoculating one colony in 5 ml BHI broth and incubating aerobically at 37°C with shaking (250 rpm).