For the isolation of Bifidobacteria, fecal samples were collected from healthy Koreans (aged 20 to 30 years old) by BD BBLanaerobic sample collection and transport system (Becton Dickinson and Co, USA) to maintain anaerobic conditions. Fecal samples were serially diluted tenfold from 10^-1 to 10^-8, and 100 μl were spread into selective blood liver agar (Nissui Pharm, Japan) containing 5% sheep blood. After 48 h of incubation in anaerobic conditions (90% N₂, 5% H₂, 5% CO₂) (Bactron Anaerobic Chamber, USA) at 37°C, brown or reddish-brown colonies 2 mm to 3 mm in diameter were selected for further identification 18 . A fructose-6-phosphate phosphoketolase (F6PPK) test was performed to ensure that the colonies selected were Bifidobacteria 19 . To identify the isolated Bifidobacterium spp. at the species level, 16S rRNA sequencing was performed by Bio leaders (Daejeon, Korea). We established an N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutant of B. adolescentis SPM1005, which we named SPM1005-A. B. adolescentis SPM1005-A, was cultured at 37°C for 48 h on general anaerobic medium (GAM, Nissui Pharm, Japan) under anaerobic conditions and then centrifuged at 1,200 rpm for 15 minutes. The supernatant was separated from the bacterial cell pellet and filtered using an 0.2-μm syringe filter (Sartorius Stedim Biotech, Germany). The purified supernatant was used for further experiments. Written informed consents were obtained from all volunteer who provided samples and the protocol was approved by the Institution Review Board of Office of Research Development, Sahmyook University.

SiHa cervical cancer cells expressing HPV type 16 were obtained from the American Type Culture Collection http://www.atcc.org. The cell lines were grown in Minimum essential medium alpha (Gibco) containing 10% heat-inactivated fetal bovine serum (Sigma, USA), 10,000 U/ml penicillin and 10,000 μg/ml streptomycin in a humidified incubator at 37°C with 5% CO₂. SiHa cells (1.0 × 10⁵ cells per flask) were incubated for 0, 24, or 48 h in the presence or absence of B. adolescentis SPM1005-A at a concentration of 5.1 × 10⁷ cfu/ml. After incubation, the culture medium was removed and the monolayers were washed with phosphate-buffered saline (PBS) without phenol red and supplements; the cells were then immediately used for total protein and RNA extraction.

Cell viability was determined by a Trypan blue dye exclusion assay. Cells (3.6 × 10⁵) in culture plates were incubated overnight, and then the medium was changed to new medium either with or without B. adolescentis SPM1005-A at a concentration of 5.1 × 10⁷ cfu/ml, and incubated for 0, 24, 48, and 72 h. After incubation, each cell suspension was mixed with an equal volume of 0.3% Trypan blue solution (Samchun Chemical, Korea). Finally, cells were observed under a microscope, and living cells were counted on hemocytometer. Morphological changes were observed using an inverted microscope (Olympus, Japan) at × 400 magnification. Each assay was performed three times in triplicate and results presented as % of control.

Total RNA was extracted from the cell line using RNeasy mini Kit (Qiagen, USA) according to the manufacturer's recommendation. RNA concentration was quantified by measuring the absorbance at 260 nm. In order to analyze the same amount of cDNA in every sample, we performed reverse transcription as a separate step from PCR. The mRNA was reverse transcribed into cDNA using oligo-dT primer and Omniscript reverse transcription kit (Qiagen). The cDNA was stored at -20°C or directly used in qRT-PCR. As growth of HPV-transformed cervical cancer cells is dependent on sustained viral oncogene E6 and E7 expression, we investigated the inhibitory effects of B. adolescentis SPM1005-A on the cervical carcinoma cell line, SiHa, with quantitative real-time PCR (qRT-PCR), which is an excellent method for quantitation of viral DNA 20 21 .

qRT-PCR was conducted in a Light Cycler (Roche) system and the data were analyzed with Light Cycler software version 4.5. The quantity of HPV16-E6 and HPV16-E7 transcripts in each sample was standardized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript levels. For absolute quantification, cDNA constructed with specific binding sites for HPV16 E6 and E7 primers were used to derive the standard curves. Reactions contained 2 μl each of the cDNA solution, MgCl₂, primers and Fast Start DNA Master SYBR Green I (Qiagen). The primer sequences used for qRT-PCR of HPV16-E6 were 5'-GAC CCA GAA AGT TAC CAC AG-3' (nucleotide 44 to nucleotide 64) and 5'-CAT AAA TCC CGA AAA GCA AAG-3' (nucleotide 153 to nucleotide 173), and of E7 were 5'-GGA GGA GGA TGA AAT AGA TGG-3' (nucleotide 99 to nucleotide 199) and 5'-TGA GAA CAG ATG GGG CAC AC-3' (nucleotide 268 to nucleotide 287). GAPDH was used as internal control with primer sequences of 5'-CTG CAC CAC CAA CTG CTT AG-3' (forward) and 5'-TTC TGG GTG GCA GTG ATG-3' (reverse) 22 . The amplification conditions were initial incubation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s and 72°C for 10 s. Results were calculated using the ΔΔCT method with cDNA from all samples as a reference as described previously 23 . Positive and negative controls were included in each experiment to ensure reproducible results.

After 24 or 48 h of treatment, each sample was washed with PBS and treated with lysis buffer (Thermo, USA). The samples, containing 30 μg of protein, were suspended in a 2 × sample buffer (Sigma, USA), boiled for 4 minutes and resolved on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The proteins were transferred to nitrocellulose membrane (Whatman, USA), and equal loading was verified with Ponceau S staining (Sigma). Immunodetection was performed with HPV16-E6 antibody and HPV16-E7 antibody (Santa Cruz Biotechnology, USA), followed by incubation with donkey anti-goat horseradish-peroxidase-conjugated antibody and detection using enhanced chemiluminescence western blotting detection reagents (Abfrontier). β-Actin was used as a normalization standard. Each blot is representative of three experiments.

Results were expressed as mean ± SD. One-tailed Student's t tests were employed whenever individual data points were compared. Mean values of P < 0.05 were considered statistically significant.

Representative images of the morphological changes observed after 24, 48, or 72 h are shown in Figure 1. Compared to control PBS-treated cells, cells exposed to B. adolescentis SPM1005-A for 72 h slowly shrank in appearance, but there was no observed difference in 24-h to 72-h treated cells.

The cytotoxicity of B. adolescentis SPM1005-A was evaluated by Trypan blue dye exclusion assay. Based on the results of the assay, the cell survival rate decreased slightly (92, 95, 89%, respectively) with 24-h to 72-h exposure to 5.1 × 10⁷ cfu/ml B. adolescentis SPM1005-A (Figure 2). This indicates that B. adolescentis SPM1005-A does not significantly affect the cytotoxicity of SiHa cells. Consequently, experiments to assess the antiviral activity were carried out at this tested concentration in this study.

For this purpose, total RNA was converted to cDNAs for E6, E7 and GAPDH as a control by using oligo-dT primer reverse transcription. The qRT-PCR results showed a reduction of both genes mRNA transcript levels after incubating the cells with B. adolescentis SPM1005-A for 24 and 48 h (Figure 3A). PBS-treated cells were used as a control. A reduction in E6 mRNA expression was observed compared with that in untreated cells after 24 h. In particular, E6 and E7 expression levels were significantly inhibited by B. adolescentis SPM1005-A treatment for 48 h (*P < 0.05, **P < 0.01).

Western blot analysis revealed that the expression of both proteins decreased slightly after incubation with B. adolescentis SPM1005-A (Figure 3B). Inhibition of E6 protein levels was observed at both 24 and 48 h in comparison to β-actin. The qRT-PCR results revealed that E7 mRNA levels decreased after treatment in the SiHa cell line, whereas the level of E7 protein expression in cells treated with B. adolescentis SPM1005-A appeared to be unaffected compared with that in the control. In this respect, the protein change did not consistently occur in a time-dependent manner.