Pyrosequencing analysis For the 454 pyrosequencing analysis, 16 subjects were randomly selected among the test (n = 8) and control (n = 8) subjects (Figure S1). DNA was extracted from the baseline, 12-week exposure, and 1-month follow-up tooth biofilm samples as previously described [34]. The V3-V4 hypervariable region of the 16S rRNA gene was amplified via PCR using the forward primer 347F and reverse primer 803R [35]. For sample identification, fusion primers were created from these primers and unique barcode sequences according to the Roche guidelines for experimental amplicon design (www.454.com). DNA was amplified under the following running conditions: initial denaturation at 94°C for 3 min; 30 cycles of 94ºC for 15 s, 58ºC for 15 s, and 72ºC for 30 s; and a final extension at 72ºC for 8 min.