Oligonucleotide primers were obtained from Sigma-Genosys Ltd. (Cambridge, United Kingdom). The positive control strains for detection of potential virulence factors were the MCC950 cell line following: E. faecalis P4 for cylL L –cylL s , cylL L –cylL S –cylM, agg, gelE S3I-201 in vitro and efaAfs, E. faecalis P36 for esp[32], and E. faecium C68 for hyl[35]. PCR-amplifications were performed from total bacterial DNA obtained using the Wizard DNA Purification Kit (Promega, Madrid, Spain) in 25 μl reaction mixtures with 1 μl of purified DNA, 0.7 μM of each primer,

0.2 mM of each dNTP, buffer 1×, 1.5 mM MgCl2 and 0.75 U of Platinum Taq DNA polymerase (Invitrogen, Madrid, Spain). Samples were subjected to an initial cycle of denaturation (97°C for 2 min), followed by 35 cycles of denaturation (94°C for 45 s), annealing (48 to 64°C for 30 s) and elongation (72°C for 30 to 180 s), ending with a final extension step at 72°C for 7 min in an Eppendorf

Mastercycler thermal cycler (Eppendorf, Hamburg, Germany). PCR products were analyzed by electrophoresis on 1-2% (w/v) agarose (Pronadisa, Madrid, Spain) gels stained with Gel red (Biotium, California, USA), and visualized with the Gel Doc 1000 documentation system (Bio-Rad, Madrid, Spain). The molecular size markers used were HyperLadder II (Bioline GmbH, Germany) KPT-8602 and 1Kb Plus DNA ladder (Invitrogen). Production of gelatinase by enterococci Gelatinase production was determined using the method previously

described by Eaton and Gasson [32]. Briefly, enterococci were grown in MRS broth overnight at 32°C, and streaked onto Todd-Hewitt (Oxoid) agar plates (1.5%, w/v) containing 30 g of gelatine per litre. After incubation overnight incubation at 37°C, the plates were placed at 4°C for 5 h before examination for zones of turbidity (protein hydrolysis) around the colonies. E. faecalis P4 was used as positive control. Production of hemolysin To investigate hemolysin production by the 99 LAB, the strains grown in MRS broth were streaked onto layered check fresh horse blood agar plates (BioMérieux, Marcy l’Étoile, France) and grown at 37°C for 1–2 days [32]. β-hemolysis was revealed by the formation of clear zones surrounding the colonies on blood agar plates. E. faecalis P4 was used as positive control. Determination of antibiotic susceptibility Antibiotic susceptibility of the 59 enterococci was determined by overlaying antibiotic-containing disks (Oxoid) on Diagnostic Sensitivity Test Agar (Oxoid) previously seeded with approximately 1 × 105 CFU/ml of each enterococcal isolate. The antibiotics tested were ampicillin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), gentamicin (120 μg), nitrofurantoin (300 μg), norfloxacin (10 μg), penicillin G (10 IU), rifampicin (5 μg), teicoplanin (30 μg), tetracycline (30 μg), and vancomycin (30 μg).