Vaginal and fecal reads from healthy adult women
bacterium
Vaginal swabs were collected at visits V1, V2, V3 and V4, transferred into 750 µl of PowerBead solution (Qiagen GmbH, Hilden, Germany) and stored at -80°C until they were processed for DNA extraction. In addition, during visits V1-4, the volunteers delivered a fecal sample collected during the previous 24 h in a special container that was provided during visit V0. The fecal specimens were also stored at -80°C until they were processed for DNA extraction, as follows. Vaginal swabs in PowerBead Solution were thawed on ice. Swabs were pressed several times by rotating on the inner wall of the sampling tube to release the entire amount of buffer or vaginal mucus adhered on the swab in aseptic conditions (later swabs were used for isolation of the target probiotic strain). The samples were then processed using the DNeasy® PowerLyzer® PowerSoil® Kit (Qiagen GmbH) with a minor modification consisting of the incubation of samples in PowerBead solution at 65°C for 10 min after the addition of C1 solution. The mechanical lysis of cells was carried out using a Bead Beater Precellys 24 (Bertin Technologies, Montigny le Bretonneux, France). A similar procedure was followed for DNA extraction from fecal samples. Specifically, after thawing on ice, the samples were mixed vigorously for 2-3 min with a sterile spatula. The samples were weighed (250 mg) in a PowerBead tube, and DNA extraction was carried out as mentioned above. The DNA obtained either from vaginal or fecal samples was quantified using Take3 Micro-Volume analyzed in a microplate reader with the use of Gen5 software (BioTek Instruments, Inc., Winooski, VT, USA), diluted to 10 ng/µl and stored at -80°C until use.