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Abstract
The objective of this study was to analyze quantitatively whether Weissella cibaria could affect the proliferation of five periodontopathic bacteria, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum, after incubation for 8~48 h. In addition, by using real-time PCR with a dual-labeled probe, each growth of bacteria was examined under different growth media conditions. The proliferation of periodontopathic bacteria was significantly inhibited by W. cibaria after incubation for 24~48 h (p < 0.05), whereas the growth of W. cibaria was not affected by these pathogenic bacteria. The growth of P. gingivalis, T. forsythia and T. denticola significantly increased in each growth media after incubation for 24 h (p < 0.05), as compared to the culture in mixed growth media. However, no differences in the growth of five periodontopathic bacteria were observed between each growth media and mixed media after incubation for 48 h. The growth and pH of W. cibaria culture significantly were changed in MRS after incubation for 24~48 h (p < 0.05), as compared to the bacterial culture in mixed growth media. The pH of P. gingivalis and F. nucleatum culture significantly was changed in both growth media and mixed media after incubation for 24~48 h (p < 0.05). Our data indicate that W. cibaria significantly inhibits the proliferation of five periodontopathic bacteria and each growth of bacteria is quantitatively analyzed under various media conditions by real-time PCR.
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Figure 1.
Amplification of genomic DNA from lysed cells. Serial dilutions of genomic DNA from F. nucleatum (A) or A. actinomycetemcomitans (B) were used as templates for real-time PCR. The threshold fluorescence, or the level at which the threshold cycle was determined, is shown. The standard curves were generated from the amplification plots in the insets (correlation coefficients, 0.996 for F. nucleatum and 0.997 for A. actinomycetemcomitans). CT is the cycle number at which the threshold fluorescence is reached. The linearity with R value is observed from 102 to 108 bacterial cells.
Figure 2.
Quantitative comparison of bacterial cell numbers of W. cibaria and periodontopathic bacteria in the mixed cultures by real-time PCR. Fn, F. nucleatum; Aa, A. actinomycetemcomitans; Tf, T. forsythia; Td, T. denticola; Pg, P. gingivalis; Wc, W. cibaria. ∗p < 0.05 for coculture versus monoculture. Values are means ± standard deviations of three independent experiments.
Figure 3.
Comparison of bacterial cell numbers of periodontopathic bacteria over time under different growth media conditions by real-time PCR. The growth change of periodontopathic bacteria compared to the zero time (0 h) was shown as log scale. F. nucleatum was grown in FN (F. nucleatum growth medium) or FN-M (a mixture of equal volume of FN and MRS). A. actinomycetemcomitans was grown in AA (A. actinomycetemcomitans growth medium) or AA-M (a mixture of equal volume of AA and MRS). T. forsythia was grown in TF (T. forsythia growth medium) or TF-M (a mixture of equal volume of TF and MRS). T. denticola was grown in TYGVS (T. denticola growth medium) or TYGVS-M (a mixture of equal volume of TYGVS and MRS). P. gingivalis was grown in PG (P. gingivalis growth medium) or PG-M (a mixture of equal volume of PG and MRS). ∗p < 0.05, T. forsythia culture in TF-M versus T. forsythia culture in TF; #p < 0.05, T. denticola culture in TYGVS-M versus T. denticola culture in TYGVS; +p < 0.05, P. gingivalis culture in PG-M versus P. gingivalis culture in PG. Values are means ± standard deviations of three independent experiments.
Figure 4.
Comparison of bacterial cell numbers (A) and pH changes (B) of W. cibaria over time under different growth media conditions by real-time PCR. The growth change of W. cibaria compared to the zero time (0 h) was shown as log scale. FN-M, a mixture of equal volume of F. nucleatum growth medium and MRS; AA-M, a mixture of equal volume of A. actinomycetemcomitans growth medium and MRS; TF-M, a mixture of equal volume of T. forsythia growth medium and MRS; TYGVS-M, a mixture of equal volume of T. denticola growth medium and MRS; PG-M, a mixture of equal volume of P. gingivalis growth medium and MRS. ∗p < 0.05, culture in FN-M versus culture in MRS; #p < 0.05, culture in AA-M versus culture in MRS. +p < 0.05, pH at over time versus pH at zero time.
Table 1.
Species-specific primers and probes used for real-time PCR
Table 2.
The pH changes of periodontopathic bacterial culture over time under different growth media conditions
| Incubation time (h) | 0 | 8 | 24 | 48 |
|---|---|---|---|---|
| F. nucleatum | ||||
| FN | 6.91±0.01 | 6.93±0.03 | 6.82±0.02∗ | 6.79±0.07∗ |
| FN-M | 6.81±0.01 | 6.80±0.01 | 6.74±0.04∗ | 6.56±0.06∗ |
| A. actinomycetemcomitans | ||||
| AA | 6.91±0.01 | 6.86±0.04 | 6.91±0.05 | 6.47±0.07∗ |
| AA-M | 6.79±0.09 | 6.78±0.03 | 6.74±0.04 | 6.07±0.07∗ |
| T. forsythia | ||||
| TF | 6.82±0.06 | 6.80±0.10 | 6.83±0.13 | 6.84±0.04 |
| TF-M | 6.74±0.04 | 6.76±0.06 | 6.80±0.10 | 6.78±0.08 |
| T. denticola | ||||
| TYGVS | 7.01±0.19 | 7.01±0.19 | 7.08±0.08 | 7.08±0.08 |
| TYGVS-M | 6.86±0.02 | 6.86±0.07 | 6.84±0.04 | 6.84±0.04 |
| P. gingivalis | ||||
| PG | 6.93±0.03 | 6.97±0.06 | 7.14±0.13∗ | 7.12±0.13∗ |
| PG-M | 6.78±0.03 | 6.81±0.01 | 6.82±0.02∗ | 6.82±0.02∗ |
FN, F. nucleatum growth medium; FN-M, a mixture of equal volume of FN and MRS; AA, A. actinomycetemcomitans growth medium; AA-M, a mixture of equal volume of AA and MRS; TF, T. forsythia growth medium; TF-M, a mixture of equal volume of TF and MRS; TYGVS, T. denticola growth medium; TYGVS-M, a mixture of equal volume of TYGVS and MRS; PG, P. gingivalis growth medium; PG-M, a mixture of equal volume of PG and MRS.
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