mitis and S. salivarius K12. Genes responsible for bacteriocin production (salA, sboB, sivA, srtA, scnA, nisA, nisF, nsuB, mutII, mutIII, srtF, lanB, and lanC) were amplified by PCR using primers previously published (Hynes et al., 1993; Karaya et al.,

2001; Upton et al., 2001; Wescombe et al., 2006; Wirawan et al., 2006) and those designed for this study see Table 1. For mef(E) Ixazomib detection and PCR, we used previously published protocols (Santagati et al., 2009). To exclude the presence of potential virulence determinants, hemolytic activity and detection of virulence genes were assayed. The hemolytic ability of 24SMB was tested using: (1) horse blood in a base containing starch medium (Saunders Epigenetics inhibitor & Ball, 1980); (2) TSA with 5% defibrinated sheep blood; and (3) Columbia Agar with 5% defibrinated sheep blood. In S. salivarius 24SMB, the main streptococcal virulence genes, sagA (streptolysin S), smeZ-2 (mitogenic exotoxin Z), speB (pyrogenic exotoxin), speC, speG and speJ (exotoxin type C, G, J), prtF, (fibronectin-binding protein),

and sof (serum opacity factor) were detected by PCR using the primers described in Table 1 and by hybridization with specific probes. Streptococcus pyogenes SF370 and S. pyogenes 2812A were used as positive control. All amplification products were purified by the ‘QIAquick PCR gel extraction Kit’ (Qiagen) and sequenced with a LICOR DNA 4000L sequencer. The DNA sequence was analyzed by the Gapped blast software (Altschul et al., 1997). This method used the HEp-2 cell line (human, Caucasian,

larynx, carcinoma, squamous cell), ATCC CCL 23. The bacteria were grown from 16 to 18 h in 5 mL of Todd Hewitt broth. The density of all bacterial cultures was adjusted photometrically so that cultures contained approximately 105–106 CFU mL−1 prior to their use in the assay. HEp-2 (ATCCCCL23) cells were maintained in Eagle’s Minimal Essential Medium (EMEM; Invitrogen). The medium was supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU mL−1), and streptomycin (100 μg mL−1). HEp-2 adherence assays were conducted as previously described P-type ATPase (Benga et al., 2004). The number of adherent bacteria was obtained by subtraction from the total number of CFU. This is expressed as percentage adherence. All experiments were performed in duplicate wells and repeated at least three times. In each experiment, wells containing only cells were used as controls. Bacterial adhesion to the HEp-2 cell layer was also performed on microscope cover glasses as previously described (Guglielmetti et al., 2010). Briefly, approximately 2 × 108 cells resuspended in PBS were incubated with a monolayer of HEp-2 cells for 1 h at 37 °C. After washes with PBS, the cells were fixed with 3 mL of methanol and incubated for 8 min at room temperature.