Ultrapur

The PFGE molecular fingerprints of LAB isolates were obtained using the method adapted from Smith and Cantor [17 (link)]. The culture and the agarose blocks were prepared as previously described [18 (link)]. The blocks were equilibrated for one hour in a restriction buffer at 4°C and transferred to 300 μL of fresh digestion buffer containing 15 U of SmaI or 25 U of AscI endonucleases (New England Biolabs, Hitchin, UK). The blocks were incubated for 4 h at 25°C for SmaI and at 37°C for AcsI. PFGE was carried out with a CHEF-DR II apparatus (Bio-Rad, Australia) in a 1% agarose gel (w/v) (Ultrapur, Gibco-BRL, Scotland) in 0.5 × TBE at 200 V and at 14°C with the following pulse times and total running time: SmaI (initial time—2 s; final time—20 s; total running time—20 h), AscI (2 s; 20 s; 21 h). After electrophoresis, gels were stained with GelRed and visualized under UV light. Photographs of PFGE gels were scanned and the band profiles were analyzed using BioNumerics, version 4.1 (Applied Maths, Kortrijk, Belgium). Comparisons between the normalized band profiles were made using the Dice similarity coefficient with an optimization of 1%. Clustering of strain profiles was accomplished using the unweighted pair group method with arithmetic averages (UPGMA).

The clonal diversity of Lactococcus lactis was determined by PFGE analysis of the strains after phenotypic sorting. Bacterial cultures and agarose plugs were prepared, as described previously by Lortal et al. [24 (link)]. The plugs were equilibrated for one hour in a restriction buffer (CutSmart, New England Biolabs, Beverly, MA, USA) at 4 $°$ C, and were then transferred to a fresh digestion buffer containing 15 units SmaI endonuclease (New England Biolabs, Beverly, MA, USA) for one hour. The plugs were then incubated at 25 $°$ C for 4 h. PFGE was performed in a Bio-Rad CHEF DRII electrophoretic cell on 1% (w/v) agarose gel (Ultrapur, Gibco-BRL, Inchinnan, Scotland) in 0.5x TBE buffer (45mM Tris, 45mM boric acid, 1mM EDTA, pH 8.0) at 200 V and 14 $°$ C, under the following conditions: initial time—2 s, final time—25 s, total running time—21 h. Strain CIRM-BIA 127 was used as a home-made ladder for this study. After migration, the gels were stained with gelRed, visualized using UV light and then analyzed with GelCompar software (BioNumerics, Applied Math, Austin, TX, USA). Conversion, normalization, and further analysis were performed using the Pearson coefficient and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis with BioNumerics software (Applied Math, Sint-Martens-Latem, Belgium).