*2.4. Genomic DNA Extraction, RFLP and Amplification of (GTG)5/REP-PCR and ERIC Elements*

Genomic DNA extraction was performed on liquid cultures, which were previously incubated for 72 h at +28 ◦C. Flasks were vigorously shaken to remove the embedded cells from BC, the liquid medium was transferred to a sterile tube and centrifuged at 12,000× *g* for 5 min. gDNA was extracted using the method previously proposed by Gullo et al. [24]. gDNA was checked by 3% gel agarose in 1X TBE buffer and quantified by spectrophotometric measure (NanoDrop ND-1000). Band sizes were determined using a 100 bp DNA ladder (Invitrogen, Carlsbad, CA, USA).

RFLP analysis on the full-length 16S rRNA gene was performed using *Rsa*I and *Alu*I endonucleases (Fermentas, Hanover, ND, USA), incubating at +37 ◦C for 2 h. The restriction reaction was stopped by incubation at 80 ◦C for 20 min. The fragments were checked on 3% gel agarose in 1X TBE buffer and sizes were determined using 100 bp DNA ladder (Invitrogen).

(GTG)5 rep-PCR fingerprinting was carried out according to the method of Versalovic et al. [25], with some minor modifications. Isolates were subjected to REP-PCR with a single oligonucleotide GTG5 (5 - GTGGTGGTGGTGGTG-3 ). Samples were incubated for 5 min at 94 ◦C and then cycled 35 times at 94 ◦C for 30 s, 40 ◦C for 1 min, and 72 ◦C for 4 min. The samples were incubated for 7 min at 72 ◦C for final extension and kept at 4 ◦C. ERIC elements were initially amplified according to Versalovic et al. [26] using the primers ERIC 1R (5 -ATGTAAGCTCCTGGGGATTCAC-3 ) and ERIC 2 (5 -AAGTAAGTGGGGTGAGCG-3 ). Samples were incubated for 5 min at 94 ◦C and then cycled 30 times at 94 ◦C for 30 s, 57 ◦C for 30 s, and 65 ◦C for 4 min. The final extension was performed at 65 ◦C at 8 min and kept at 4 ◦C until tested. The reproducibility of ERIC/PCR was also tested by amplifying gDNA from randomly chosen strains several times. For both rep-PCR, pattern band lengths were determined by comparison against a 100 bp plus DNA ladder for the smallest bands and by 1 Kbp DNA ladder for the largest bands (Takara Bio, Inc., Otsu, Shiga, Japan).
