Mueller hinton broth (mhb)

All strains used in this work are described in Table 1. Bacteria were grown from stocks maintained at -80°C on Columbia Blood Agar (Lab M, Heywood, Lancashire, UK) supplemented with 5% defibrinated horse blood (Oxoid, Basingstoke, Hampshire, UK) for 48h in microaerobic conditions (80% N₂, 12% CO₂, 5% O₂ and 3% H₂) at 41.5°C. Liquid cultures were grown for 24h in 10 ml of Mueller-Hinton broth (MHB) (Lab M, Heywood, Lancashire, UK) in microaerobic conditions at 41.5°C.

Bacterial strains isolated from dosed bioreactor culture vessels were subjected to MIC determination according to the methods defined in the Clinical and Laboratory Standards Institute guidelines (CLSI) [22 ]. The MIC of tetracycline for isolates was determined in a 96-well microtiter plate. Serial dilutions of tetracycline in concentrations ranging from 0.25 to 256 µg/mL were prepared in 200 µl of Mueller-Hinton broth (MHB, LabM, Farmingdale, NY, USA). The wells were inoculated with 2 × 10⁵ CFU/mL, and the plates were incubated for 24 h at 37 °C. The MIC of tetracycline, in which no bacterial growth was observed, was then determined. E. coli ATCC25922 was used as quality control for MIC determination.

The strains used in the present study are listed in Table 1. The strains were stored at -80°C using Microbank vials (Prolab Diagnostics, Richmond Hill, ON, Canada) and subcultured at 37°C on Trypton Soy agar (TSA; Lab M, Lancashire, UK) or TSA supplemented with 800 μg/ml trimethoprim (Ludeco, Brussels, Belgium) for MDL2. Overnight cultures were grown aerobically in Mueller Hinton broth (MHB; Lab M) at 37°C. Except for cultures on which the H₂DCFDA assay was performed, Luria Bertoni agar (LBA; Lab M) and Luria Bertoni broth (LBB; Lab M) were used.

Tetraethyl orthosilicate (TEOS, ≥99.0%) and silver nitrate (≥99.0%) were obtained from Sigma-Aldrich (Steinheim, Germany and St. Louis, MO, USA, respectively). Ammonium hydroxide (25%) and ethanol (EtOH, 96.1 vol %) were purchased from JT Baker (Phillipsburg, NJ, USA) and Altia (Rajamäki, Finland), respectively. Cellulose acetate membranes (25 mm syringe filter w/0.2) were obtained from VWR International (Wallkill, NY, USA). Staphylococcus aureus subsp. aureus (MRSA, ATCC 43300, KWIK-STIK) was purchased from Microbiologics (St. Cloud, MN, USA), and E. coli (VTT E-94564) was provided by the culture collection of the Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University. Luria–Bertani (LB) broth and LB agar were purchased from BD Difco (Franklin Lakes, NJ, USA). Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) were purchased from Lab M Limited (Heywood, Lancashire UK). The pristine gauze (Mepore) and the CSD (Hansaplast, Sensitive MED XXL Antibacterial Plaster) were manufactured by Mölnlycke Health Care (Gothenburg, Sweden) and Beiersdorf AG (Hamburg, Germany), respectively. According to the manufacturer, the Hansaplast MED plasters are non-adhesive wound pads, containing Ag-coated polyethylene nets releasing Ag⁺ at contact with the wound fluid.