Bhi broth

L. monocytogenes EGD was maintained as a frozen glycerol stock and cultured in brain heart infusion (BHI) broth (Becton Dickinson, Franklin Lakes, NJ, USA) or on BHI broth containing 1.5% agar.

Listeria monocytogenes strain EGD, an LLO deletion mutant (Δhly), and an LLO-complemented strain (Δhly::hly) were used in this study [15 ]. The L. monocytogenes strains were maintained in frozen glycerol stocks and cultured overnight in brain-heart infusion (BHI) broth (Becton Dickinson, Franklin Lakes, NJ, USA) at 37°C with shaking, or on BHI broth containing 1.5% agar.

Oral microorganism strains, E. faecalis (ATCC 19433) and P. gingivalis (ATCC 33277) were obtained from State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China. E. faecalis were cultivated in brain heart infusion broth (BHI broth, Becton, Dickinson and Company, US) and BHI agar plate, while P. gingivalis were cultivated in BHI broth and blood agar plate supplemented with 0.0005% hemin, 0.0001% vitamin K. Both strains were incubated anaerobically (N₂ 80%; H₂ 10%; CO₂ 10%) at 37 °C.

Sodium azide-resistant E. coli J53 was used as a receptor, and the prophage-containing K. pneumoniae strains 19051, 675920, 721005, and 911021 were used as donors for the conjugative transfer experiments. Small amounts (10–20 μl) of donor and recipient glycerol bacteria were inoculated in 3 ml of brain heart infusion (BHI) broth (BD Biosciences, San Jose, CA, United States). After culture overnight, the donor bacteria were mixed with the recipient bacteria, centrifuged, resuspended in 80 μl of BHI broth, supplemented with a resuspended bacterial solution at the surface of a filter (filter size 1 cm², pore size 0.45 μm), and the filter was adhered to BHI (BD Biosciences) agar plates. The cells were fully absorbed by the filter and cultured at 37°C for 12–18 h. The bacteria were then washed from the filter and spotted on to the BHI agar plates containing 200 μg/ml sodium azide and 50 μg/ml amikacin to select conjugons harboring the prophage.

Secretion of antimicrobials by P. xiamenensis was assessed by diffusion assay on agar. Soft agar (0.8%) was prepared using Brain Heart Infusion (BHI) broth (BD, Sparks, MD, USA) or Tryptic Soy Broth (TSB, BD). The bacterial strains E. piscicida, S. epidermidis, A. hydrophila, and P. aeruginosa were incorporated into 4 mL of soft agar and overlaid onto pre-solidified respective agar media. P. xiamenensis was grown either in marine agar or broth. E. piscicida was grown on BHI broth medium while other strains were propagated on Tryptic Soy Broth (TSB; BD, Reno-Sparks, NV, USA) medium. Briefly, overnight cultures were inoculated into fresh respective mediums, 1% v/v, and allowed to grow to 0.4 OD₅₉₅. At the appropriate density, 200 µL was retrieved and suspended into 4 mL of soft agar prepared by the respective media for each strain. The suspensions were overlaid onto pre-solidified agar and allowed to solidify. Then, complete round plugs were removed and filled with P. xiamenensis suspended in marine agar in a similar procedure. The P. xiamenensis suspension (1 mL) was poured into the well and allowed to solidify. Marine soft agar alone, or chloramphenicol-treated (50 µg/mL), were used as negative and positive controls, respectively. Plates were incubated> 24 h for pink color development, and the diffusion zones were compared.

A cocktail of three strains of S. aureus was used: CECT976 (SEA producer) and CECT4466 (SED producer), from the Spanish Type Culture Collection, and CTC1008, as a meat isolate from the IRTA culture collection. Each strain was independently grown in Brain Heart Infusion (BHI) broth (Becton Dickinson, Sparks, MD, USA) at 37 °C for 24 h. The cultures were cryopreserved at −80 °C with 20% glycerol until use. Thawed cultures of each strain were mixed at equal concentrations before being inoculated on DCH.