List of bacterial strains used in this study.

<p>List of bacterial strains used in this study.</p

Biofilm values of <i>A</i>. <i>actinomycetemcomitans</i> strains with addition of probiotic bacteria on pre-formed biofilm.

<p>(A) represents Y4 strain (serotype b). Whereas (B and C) represents SUNY75 strain (serotype a) and OMZ534 strain (serotype e) respectively. Probiotic species and controls were labelled with number as follows; 1: <i>L</i>. <i>fermentum</i> JCM 1137, 2: <i>L</i>. <i>acidophilus</i> JCM 1021, 3: <i>L</i>. <i>fermentum</i> NBRC 15885, 4: <i>L</i>. <i>fructosum</i> NBRC 3516, 5: <i>L</i>. <i>plantarum</i> NBRC 15891, 6: <i>L</i>. <i>casei subsp</i>. <i>rhamnosus</i> NBRC 3831, 7: <i>L</i>. <i>johnsonii</i> NBRC 13952. Positive controls (8) are <i>A</i>. <i>actinomycetemcomitans</i> biofilm without probiotic addition, and <i>A</i>. <i>naeslundii</i> JCM 8349 was used as a negative control (9). Bars represent the mean, error bars represent standard deviation and significance was measured using paired T-test (* = P< 0.05, ** = P< 0.001).</p

Influence of probiotic bacteria supernatant and lactic acid on biofilm growth.

<p>Percent biofilm degradation following (A) the addition of an untreated cell-free supernatant (black column) or an adjusted pH (6.5) cell-free supernatant (gray column). (B) Biofilm growth of <i>A</i>. <i>actinomycetemcomitans</i> Y4 with lactic acid at various concentrations compared with the addition of probiotic cells. Biofilm of <i>A</i>. <i>actinomycetemcomitans</i> Y4 was allowed to pre-form under static anaerobic conditions for 72 h prior to the addition of lactic acid or probiotic cells. Bars represent the mean, error bars represent standard deviation and significance was measured using paired T-test (* = P< 0.05, ** = P< 0.001).</p

Percentage of biofilm degradation by probiotic cells or cell-free supernatant (concentrated) against <i>A</i>. <i>actinomycetemcomitans</i> Y4 biofilm.

<p>Black bars represent biofilm degradation by cells and white columns represent biofilm degradation by a cell-free supernatant. Bars represent the mean, error bars represent standard deviation.</p

Influence of culture medium nutrient and cell viability on biofilm degradation activity.

<p>Fig 3A represents a comparison of biofilm degradation by probiotic cells in a nutrient rich medium and a co-aggregation buffer. The nutrient rich medium contained a low density of probiotic cells (OD 0.05 at 600 nm absorbance). Washed cell pellets from an overnight culture were co-incubated with pre-formed <i>A</i>. <i>actinomycetemcomitans</i> Y4 biofilm to form the co-aggregation buffer. Fig 3B represents biofilm degradation by dead probiotic cells (autoclaved). Bars represent the mean, error bars represent standard deviation.</p

Co-incubation of pre-formed <i>A</i>. <i>actinomycetemcomitans</i> Y4 biofilms with probiotic cells leading to biofilm degradation.

<p><i>A</i>. <i>actinomycetemcomitans</i> Y4 biofilm was allowed to form under static anaerobic conditions for 72 h before a further 24 h co-culture with probiotic cells in a co-aggregation buffer. Biofilm with the addition of a co-aggregation buffer was used as a control. Grey columns represent biofilm formation and empty circles represent viable <i>A</i>. <i>actinomycetemcomitans</i> Y4 from the biofilm supernatant. Bars and circle represent the mean, error bars represent standard deviation.</p

Mature Biofilm Degradation by Potential Probiotics: <i>Aggregatibacter actinomycetemcomitans</i> versus <i>Lactobacillus</i> spp.

<div><p>The biofilm degradation of <i>Aggregatibacter actinomycetemcomitans</i> is essential as a complete periodontal disease therapy, and here we show the effects of potential probiotic bacteria such as <i>Lactobacillus</i> spp. for the biofilm of several serotypes of <i>A</i>. <i>actinomycetemcomitans</i> strains. Eight of the 13 species showed the competent biofilm degradation of ≥ 90% reduction in biofilm values in <i>A</i>. <i>actinomycetemcomitans</i> Y4 (serotype b) as well as four of the seven species for the biofilm of <i>A</i>. <i>actinomycetemcomitans</i> OMZ 534 (serotype e). In contrast, the probiotic bacteria did not have a big impact for the degradation of <i>A</i>. <i>actinomycetemcomitans</i> SUNY 75 (serotype a) biofilm. The dispersed <i>A</i>. <i>actinomycetemcomitans</i> Y4 cells through the biofilm detachment were still viable and plausible factors for the biofilm degradation were not due to the lactic acid and low pH conditions. The three enzymes, protease, lipase, and amylase may be responsible for the biofilm degradation; in particular, lipase was the most effective enzyme for the biofilm degradation of <i>A</i>. <i>actinomycetemcomitans</i> Y4 along with the protease activity which should be also important for the other serotypes. Remarkable lipase enzyme activities were detected from some of the potential probiotics and a supporting result using a lipase inhibitor presented corroborating evidence that lipase activity is one of the contributing factors for biofilm degradation outside of the protease which is also another possible factor for the biofilm of the other serotype of <i>A</i>. <i>actinomycetemcomitans</i> strains. On the other hand, the biofilm of <i>A</i>. <i>actinomycetemcomitans</i> SUNY 75 (serotype a) was not powerfully degraded by the lipase enzyme because the lipase inhibitor was slightly functional for only two of potential probiotics.</p></div

Specific binding of EGFR-MBs to EGFR on Ca9-22 cells.

<p>(A) Fluorescence intensity was measured by flow cytometry; untreated Ca9-22 cells (a), Ca9-22 cells treated with DIO-labeled MBs (b), DIO-labeled IgG-MBs (c), and DIO-labeled EGFR-MBs (d). (B) Ca9-22 cells were incubated with EGFR-MBs for 30 min at 37°C, fixed and stained as indicated. Stained cells were examined using a fluorescence microscope.</p

Apoptosis in Ca9-22 cells after BLM delivery <i>in vitro</i>.

<p>(A) Cell cycle distribution analyses by flow cytometry in the no treatment, BLM alone, BLM + sonoporation + MBs, or BLM + sonoporation + anti-EGFR antibody-conjugated MBs groups. The percentage of cells in the sub-G1 phase is indicated. (B) Apoptosis analysis by flow cytometry in the no treatment, BLM alone, BLM + sonoporation + MBs, or BLM + sonoporation + anti-EGFR antibody-conjugated MBs groups. (C) Percentages of apoptotic cells. *P<0.001 (D) Hoechst staining was performed to observe morphological changes in Ca9-22 cells after 48 h BLM delivery treatment. Cells were exposed to no treatment, BLM alone, BLM + sonoporation + MBs, or BLM + sonoporation + anti-EGFR antibody-conjugated MBs. Apoptotic cells (arrows) exhibited characteristic chromatin condensation under fluorescence microscopy. Bar: 20 μm. (E) Percentages of apoptotic cells. *P<0.01.</p

TUNEL analysis of xenografts after <i>in vivo</i> sonoporation with BLM.

<p>Ca9-22 xenograft–bearing mice were exposed to sonoporation <i>in vivo</i>. (a) control, (b) BLM injection, (c) sonoporation with MBs and BLM injection, (d) sonoporation with EGFR-MBs and BLM injection. TUNEL signal was visualized by diaminobezidine (DAB, brown) and cell nuclei were counterstained by methyl green. Magnification, 400×; bar, 100 μm.</p