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. 2021 Jan 15;11(1):1631.
doi: 10.1038/s41598-020-80921-x.

Lactobacillus reuteri AN417 cell-free culture supernatant as a novel antibacterial agent targeting oral pathogenic bacteria

Kyung Mi Yang #  1 , Ji-Sun Kim #  2 , Hye-Sung Kim  1 , Young-Youn Kim  1 , Jeong-Kyu Oh  1 , Hye-Won Jung  1 , Doo-Sang Park  3 , Kwang-Hak Bae  4
Affiliations

Lactobacillus reuteri AN417 cell-free culture supernatant as a novel antibacterial agent targeting oral pathogenic bacteria

Kyung Mi Yang et al. Sci Rep. .

Abstract

Lactobacillus reuteri AN417 is a newly characterized probiotic strain. The activity of AN417 against oral pathogenic bacteria is unknown. We investigated the antibacterial activity of cell-free L. reuteri AN417 culture supernatant (LRS) against three oral pathogens: Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans. P. gingivalis and F. nucleatum have been implicated in periodontal disease, whereas S. mutans causes dental caries. Exposing these oral pathogenic bacteria to LRS significantly reduced their growth rates, intracellular ATP levels, cell viability, and time-to-kill. The minimal inhibitory volume of LRS was 10% (v/v) against P. gingivalis, 20% (v/v) for F. nucleatum, and 30% (v/v) for S. mutans. LRS significantly reduced the integrity of biofilms and significantly suppressed the expression of various genes involved in P. gingivalis biofilm formation. The L. reuteri AN417 genome lacked genes encoding reuterin, reuteran, and reutericyclin, which are major antibacterial compounds produced in L. reuteri strains. LRS treated with lipase and α-amylase displayed decreased antibacterial activity against oral pathogens. These data suggest that the antibacterial substances in LRS are carbohydrates and/or fatty acid metabolites. Our results demonstrate that LRS has antimicrobial activity against dental pathogenic bacteria, highlighting its potential utility for the prevention and treatment of P. gingivalis periodontal disease.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Evaluation of the antimicrobial activity of culture supernatants of L. reuteri strains against E. coli, P. aeruginosa, S. mutans, and P. gingivalis. (A) The antimicrobial activity of culture supernatants from newly isolated and reference L. reuteri strains against E. coli, P. aeruginosa and S. mutans was analyzed through disk diffusion assays following 24 h incubation at 37 °C. (B) Growth curve of P. gingivalis at OD600 measured using a disposable curvet 48 h after treatment with the supernatants of each strains. Significant differences from the control (p < 0.01) are indicated by **. (C) The effect of various concentrations of LRS and BE on P. gingivalis growth, measured by OD600 (left panel) and intracellular ATP levels (right panel) following a 24 h incubation at 37 °C. MRS medium was used as the negative control. Significant differences from the control (p < 0.001) are indicated by ***. (D) The effect of 20% (v/v) and 40% (v/v) LRS on P. gingivalis growth (left panel) and intracellular ATP levels (right panel) over time. MRS medium was used as the negative control. Significant differences from the control (p < 0.001) are indicated by ***.
Figure 2
Figure 2
Effect of LRS on the growth of P. gingivalis, F. nucleatum, and S. mutans. (A) The effect of 0% (v/v), 10% (v/v), 20% (v/v), and 40% (v/v) LRS on the growth of pathogenic oral bacteria. Pure MRS medium was used as the control. Data presented are OD600 values after incubation for 24 h. Significant differences from the control are indicated by (**) for p < 0.01 and (***) for p < 0.001. (B) The effect of 40% (v/v) LRS on the growth of pathogenic oral bacteria over time. Pure MRS medium was used as the control. Data presented are culture OD600 values. Significant differences from the control are indicated by (*) for p < 0.05, (**) for p < 0.01, and (***) p < 0.001.
Figure 3
Figure 3
Analysis of the MIV of LRS and the effect of LRS on P. gingivalis, F. nucleatum, and S. mutans cell viability. (A) Representative fluorescence images of LIVE/DEAD BacLight viability assay of pathogenic bacteria exposed to 20% (v/v) LRS or MRS broth (control) for 24 h at 37 °C. Red fluorescence indicates dead or membrane-damaged bacterial cells and green fluorescence indicates live/healthy bacteria. Original magnification, ×400. (B) Quantification of P. gingivalis cell concentrations (CFU/mL) following treatment with 20% (v/v) MRS (control) or LRS. Black colonies developed after 6 days growth in a 37 °C anaerobic chamber. The time required to kill P. gingivalis treated with MRS or LRS is shown graphically. Significant differences from the control (p < 0.001) are indicated by ***. (C) MIV of LRS for pathogenic bacteria, determined based on OD600 values after 48 h incubation in an aerobic or aerobic chamber maintained at 37 °C. Significant differences from the control (p < 0.001) are indicated by ***.
Figure 4
Figure 4
Activity of LRS against P. gingivalis and S. mutans biofilm formation (A) Visualization using fluorescence microscopy (left panel) of the antibiofilm effect of LRS. Non-adherent bacteria were removed, and the biofilms stained using the LIVE/DEAD BacLight Bacterial Viability Kit. Green and red fluorescence indicates live and dead bacteria, respectively. The fluorescence intensity ratio of live/dead cells (right panel) was analyzed using ImageJ software (IJ version 1.46r; https://imagej.nih.gov/ij/download.html). Significant differences from the control (p < 0.001) are indicated by ***. (B) Images showing crystal violet-stained biofilms of P. gingivalis following treatment with various concentrations of MRS (control) and LRS. Significant differences from the control (p < 0.001) are indicated by ***. (C,D) The effect of LRS on the expression of genes involved in biofilm formation. Bacteria were treated with MRS broth (control) or LRS for 48 h following (C) initial biofilm colonization or (D) following established biofilm formation (5 days). The mRNA levels of hagA, hagB, rgpA, rgpB, and kgp genes were quantitatively measured using RT-qPCR. The data are expressed as the relative level of 16S rRNA. Significant differences from the control are indicated by (**) p < 0.01 and (***) p < 0.001.
Figure 5
Figure 5
ANI values between genomes of L. reuteri strains and their phylogenetic position. (A) ANI values between genomes of L. reuteri strains. (B) Phylogenetic tree reconstructed using the amino acid alignments of 766 core genes using the maximum likelihood approach. Numbers above branches show maximum likelihood bootstrap supports from 500 non-parametric replicates. The tree was rooted using L. gastricus LG045 and L. secaliphilus DSM 17896 as outgroups. The scale bar represents the number of substitutions per site. (C) Genes encoding proteins related to environmental adaptation and antimicrobial compounds.
Figure 6
Figure 6
Exploring antibacterial substances in LRS. (A–C) Antibacterial effects of LRS and enzyme-treated (α-amylase, lipase, and proteinase) LRS against P. gingivalis after a 24 h incubation. Significant differences from control and LRS treatments (p < 0.001) are indicated by (***) and (†††), respectively.
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