Mueller hinton medium

The bacterial strains Bacillus subtilis ATCC 6637, Bacteroides fragilis ATCC 25285, Clostridium bifermentans ATCC 638, Escherichia coli ATCC 35218, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 43300, and Staphylococcus epidermidis ATCC 12228 were cultured in Mueller–Hinton medium (Oxoid, Basingstoke, UK) at 37 °C for 24 h.

The disc diffusion method was used to assess the antimicrobial activity of OBEO. The inoculum of bacteria was grown at 37 °C for 24 h on tryptone soy agar (Oxoid, Basingstoke, UK), whereas the yeast inoculum was grown at 25 °C on Sabouraud dextrose agar (Oxoid, Basingstoke, UK). The obtained inoculum was then adjusted to the optical density of a 0.5 McFarland standard (1.5 × 10⁸ CFU/mL) then 100 µL was placed in a Petri dish (PD) with Mueller Hinton medium (Oxoid, Basingstoke, UK) for bacteria and Sabouraud dextrose agar for yeast. Then, sterile disks with a diameter of 6 mm were placed on the solidified medium, onto which 10 µL of OBEO was applied. The PD prepared in this way was incubated in the above-mentioned conditions for the next 24 h. Antibiotics were used as a positive control for bacteria: cefoxitin for Gram-negative and gentamicin for Gram-positive, and for yeasts, an antifungal agent was used—fluconazole. All substances were obtained from Oxoid (Basingstoke, UK). As a negative control, discs with 10 µL of 0.1% DMSO applied on them (Centralchem, Bratislava, SK) were used. After incubation, the zones of growth inhibition were determined: above 10 mm antimicrobial activity was determined as very strong, above 5 mm as mild and above 1 mm as weak [63 (link)].

The strains and plasmids used are listed in Table 1. E. coli strains were grown aerobically at 37°C in Luria-Bertani (LB) broth or agar plates. Mueller-Hinton (MH) medium (Oxoid, UK) was used for drug susceptibility testing. The strains for pKD46 or pCP20 temperature-sensitive plasmid maintenance were incubated at 30°C. When needed, appropriate antibiotics for plasmid selection and maintenance were used at the following concentrations: ampicillin at 100 µg/mL and kanamycin at 50 µg/mL.

Five phages (JEP1, 4, 6, 7, and 8) were reported in our previous study to inhibit ESBL-producing E. coli isolates from chicken carcasses (18 (link)). All cesium chloride (CsCl)-purified phages (≥10¹⁰ plaque forming units per milliliter [PFU/mL]) were used in this experiment. ESBL-producing E. coli strains E20, E41, E55, and E59 belonging to phylogroups A, B1, B2, and D, respectively, were isolated from retail raw chicken in our previous study (18 (link)) and routinely cultured at 37°C in Luria Bertani (LB) medium (Difco, NJ, USA). Campylobacter was cultured at 42°C in Mueller-Hinton (MH) medium (Oxoid, Hampshire, UK) under microaerobic conditions (5% O₂, 10% CO₂, 85% N₂). All bacterial strains were stored at −80°C in LB or MH broth with 15% glycerol. The sodium chloride-magnesium sulfate (SM) buffer (100 mM NaCl, 8 mM MgSO₄·7H₂O, and 50 mM Tris·HCl, pH 7.5) was used as the phage buffer.

Five phages (JEP1, 4, 6, 7, and 8) were reported in our previous study to inhibit ESBL-producing E. coli isolates from chicken carcasses (18 (link)). All cesium chloride (CsCl)-purified phages (≥10¹⁰ plaque forming units per milliliter [PFU/mL]) were used in this experiment. ESBL-producing E. coli strains E20, E41, E55, and E59 belonging to phylogroups A, B1, B2, and D, respectively, were isolated from retail raw chicken in our previous study (18 (link)) and routinely cultured at 37°C in Luria Bertani (LB) medium (Difco, NJ, USA). Campylobacter was cultured at 42°C in Mueller-Hinton (MH) medium (Oxoid, Hampshire, UK) under microaerobic conditions (5% O₂, 10% CO₂, 85% N₂). All bacterial strains were stored at −80°C in LB or MH broth with 15% glycerol. The sodium chloride-magnesium sulfate (SM) buffer (100 mM NaCl, 8 mM MgSO₄·7H₂O, and 50 mM Tris·HCl, pH 7.5) was used as the phage buffer.