Filter mating

Transfer of antibiotic-resistant genes from LAB isolates to pathogenic microorganisms was examined by the filter mating method [13]. For this, LAB isolate (donor) and intestinal pathogen (recipient) were grown separately in a non-selective medium up to the mid-exponential phase of growth (~ 4h). The donor cells and the recipient cells were mixed in 1:1 ratio, and the mixture was filtered through a sterile composite cellulose ester filter (0.45μm, HAWP-02500, Millipore) using a swinex filter holder (SX 02500, Millipore). After filtration, sterile peptone physiological saline solution (PPS- 8.5gm/L NaCl and1gm/L peptone) was passed through the filters to trap the cells more tightly into the membrane. The filters were placed on a non-selective agar medium and incubated at recipient bacterial growth conditions. After incubation, filters were washed with 2ml PPS solution, filtrates (mating mixture) were spread on selective antibiotic agar medium plates and incubated at 37°C for 24–48 h, to screen for antibiotic-resistant transconjugants.