Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 7 (9)

Nitric Oxide Donor Modulates a Multispecies Oral Bacterial Community-An In Vitro Study

Affiliations

Nitric Oxide Donor Modulates a Multispecies Oral Bacterial Community-An In Vitro Study

Takayuki Nambu et al. Microorganisms.

Abstract

The deterioration of human oral microbiota is known to not only cause oral diseases but also to affect systemic health. Various environmental factors are thought to influence the disruption and restoration of the oral ecosystem. In this study, we focused on the effect of nitric oxide (NO) produced by denitrification and NO synthase enzymes on dental plaque microbiota. Interdental plaques collected from 10 subjects were exposed to NO donor sodium nitroprusside (SNP) and then cultured in a specialized growth medium. Depending on the concentration of exposed SNP, a decrease in α-diversity and a continuous change in β-diversity in the dental plaque community were shown by sequencing bacterial 16S rRNA genes. We also identified eight operational taxonomic units that were significantly altered by NO exposure. Among them, the exposure of NO donors to Fusobacterium nucleatum cells showed a decrease in survival rate consistent with the results of microbiota analysis. Meanwhile, in addition to NO tolerance, an increase in the tetrazolium salt-reducing activity of Campylobacter concisus cells was confirmed by exposure to SNP. This study provides an overview of how oral plaque microbiota shifts with exposure to NO and may contribute to the development of a method for adjusting the balance of the oral microbiome.

Keywords: 16S rRNA gene; high-throughput sequencing; nitric oxide; oral microbiota; sodium nitroprusside.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Total bacterial density in the initial plaque samples and in vitro cultures. For qPCR quantification, the universal bacterial 16S rRNA primers were used to estimate the concentration of total bacterial DNA. Horizontal lines indicate statistical significance according to the Wilcoxon rank-sum test (* p < 0.05, ** p < 0.01).
Figure 2
Figure 2
Bacterial community shift under sodium nitroprusside (SNP) exposure and in vitro culture. Stacked bar charts show taxonomic distribution at the order level. Each bar represents a single sample, with each color representing different bacterial taxa; sorted by individuals. The detailed information for each operational taxonomic unit (OTU) can be found in Table S2.
Figure 3
Figure 3
Alpha diversity indices in dental plaque and cultured samples. The R-based Rhea pipeline [41] was used for data analysis. The indices are plotted with three alpha diversity indicators: (a) Shannon effective; (b) Simpson effective; (c) species richness. p-values determined by paired Wilcoxon signed rank-sum test (* p < 0.05, ** p < 0.01).
Figure 4
Figure 4
The effect of sodium nitroprusside (SNP) on beta diversity. Overall plaque microbiome composition was depicted by non-metric multidimensional scaling (NMDS) analysis. The plaque samples were treated with NO donor, SNP, at final concentrations in the range of 0–100 mM for 1 h. (a) All microbial communities were clustered according to treatment conditions. The relationships between untreated and 10 mM SNP-treated samples (b) and untreated and 20 mM SNP-treated samples (c) are extracted and depicted. The numbers in the squares indicate the final concentration (mM) of the added SNP. Samples on the first and second principal coordinates are plotted by nodes. Lines connect samples in the same groups, and colored circles cover the samples near the center of gravity for each group.
Figure 5
Figure 5
Relative abundance of the eight OTUs altered by SNP treatment. Differential abundance analysis was performed using the Wilcoxon rank-sum test at the OTU level (* p < 0.05, ** p < 0.01). Oral taxon IDs in HOMD are given in parentheses following bacterial names.
Figure 6
Figure 6
Effect of NO donor on the cell viability of bacterial strains. (a) Viability of F. nucleatum subsp. polymorphum growing in the presence or absence of NO donor SNP or S-nitrosoglutathione (GSNO). (b) Viability of C. concisus growing in the presence or absence of SNP. Values are expressed as the mean ± standard deviation, calculated from quadruplicate assays. p-values were calculated by Student’s t-test (* p < 0.05).
Figure 7
Figure 7
Relative WST-8-reducing activity of SNP-treated cells as determined by WST-8 reduction assay. Reduction of WST-8 to formazan by NADH in F. nucleatum subsp. polymorphum (a) and C. concisus (b) was measured at 450 nm using a microplate reader. Values are expressed as the mean and standard deviation, calculated from triplicate assays. p-values were calculated by Student’s t-test (** p < 0.01).

Similar articles

See all similar articles

References

    1. Aas J.A., Paster B.J., Stokes L.N., Olsen I., Dewhirst F.E. Defining the normal bacterial flora of the oral cavity. J. Clin. Microbiol. 2005;43:5721–5732. doi: 10.1128/JCM.43.11.5721-5732.2005. - DOI - PMC - PubMed
    1. Kuramitsu H.K., He X., Lux R., Anderson M.H., Shi W. Interspecies interactions within oral microbial communities. Microbiol. Mol. Biol. Rev. 2007;71:653–670. doi: 10.1128/MMBR.00024-07. - DOI - PMC - PubMed
    1. Dewhirst F.E., Chen T., Izard J., Paster B.J., Tanner A.C.R., Yu W.H., Lakshmanan A., Wade W.G. The human oral microbiome. J. Bacteriol. 2010;192:5002–5017. doi: 10.1128/JB.00542-10. - DOI - PMC - PubMed
    1. Zhou Y., Gao H., Mihindukulasuriya K.A., La Rosa P.S., Wylie K.M., Vishnivetskaya T., Podar M., Warner B., Tarr P.I., Nelson D.E., et al. Biogeography of the ecosystems of the healthy human body. Genome Biol. 2013;14:R1. doi: 10.1186/gb-2013-14-1-r1. - DOI - PMC - PubMed
    1. Kilian M., Chapple I.L.C., Hannig M., Marsh P.D., Meuric V., Pedersen A.M.L., Tonetti M.S., Wade W.G., Zaura E. The oral microbiome—An update for oral healthcare professionals. Br. Dent. J. 2016;221:657–666. doi: 10.1038/sj.bdj.2016.865. - DOI - PubMed
Feedback