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The Making of a Miscreant: Tobacco Smoke and the Creation of Pathogen-Rich Biofilms

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The Making of a Miscreant: Tobacco Smoke and the Creation of Pathogen-Rich Biofilms

Samir A Shah et al. NPJ Biofilms Microbiomes.

Abstract

We have previously reported that oral biofilms in clinically healthy smokers are pathogen-rich, and that this enrichment occurs within 24 h of biofilm formation. The present investigation aimed to identify a mechanism by which smoking creates this altered community structure. By combining in vitro microbial-mucosal interface models of commensal (consisting of Streptococcus oralis, Streptococcus sanguis, Streptococcus mitis, Actinomyces naeslundii, Neisseria mucosa and Veillonella parvula) and pathogen-rich (comprising S.oralis, S.sanguis, S.mitis, A.naeslundii, N.mucosa and V.parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, Filifactor alocis, Dialister pneumosintes, Selenonomas sputigena, Selenominas noxia, Catonella morbi, Parvimonas micra and Tannerella forsythia) communities with metatranscriptomics, targeted proteomics and fluorescent microscopy, we demonstrate that smoke exposure significantly downregulates essential metabolic functions within commensal biofilms, while significantly increasing expression of virulence genes, notably lipopolysaccharide (LPS), flagella and capsule synthesis. By contrast, in pathogen-rich biofilms several metabolic pathways were over-expressed in response to smoke exposure. Under smoke-rich conditions, epithelial cells mounted an early and amplified pro-inflammatory and oxidative stress response to these virulence-enhanced commensal biofilms, and a muted early response to pathogen-rich biofilms. Commensal biofilms also demonstrated early and widespread cell death. Similar results were observed when smoke-free epithelial cells were challenged with smoke-conditioned biofilms, but not vice versa. In conclusion, our data suggest that smoke-induced transcriptional shifts in commensal biofilms triggers a florid pro-inflammatory response, leading to early commensal death, which may preclude niche saturation by these beneficial organisms. The cytokine-rich, pro-oxidant, anaerobic environment sustains inflammophilic bacteria, and, in the absence of commensal antagonism, may promote the creation of pathogen-rich biofilms in smokers.

Conflict of interest statement

The authors declare that they have no competing financial interests.

Figures

Fig. 1
Fig. 1
Hierarchical circle packing plot of transcriptional activity in commensal (a) and pathogen-rich (b) biofilms. Each circle represents a gene and is sized by log2 fold change. Genes are grouped based on their functional roles based on SEED classification. Genes that were significantly over-expressed (log2 fold change >2, p < 0.05, FDR-adjusted Wald test) in a smoke-rich environment when compared to controls are in red, while those that were under-expressed following smoke conditioning are shown in gray. White circles indicate genes whose change in expression did not meet the above criteria. The data used in creating this Figure are shown in Supplementary Table 1
Fig. 2
Fig. 2
Cytokine released by OKF6-TERT cells in response to biofilm challenge. Levels of selected cytokines in response to commensal (greens) and pathogen-rich (reds) biofilms in smoke-free and smoke-rich environments are shown. Data represent means of six replicates, with standard deviation bars. (* p < 0.05, **p < 0.01, ***p < 0.001, between-group differences, brackets represent p < 0.01, repeated measures ANOVA)
Fig. 3
Fig. 3
Cytokine released by smoke-conditioned OKF6-TERT cells in response to smoke-conditioned and smoke-free biofilm challenge. Levels of selected cytokines in response to commensal (greens) and pathogen-rich (reds) biofilms in smoke-free and smoke-rich environments are shown. Data represent means of six replicates, with standard deviation bars. (* p < 0.05, **p < 0.01, ***p < 0.001, between-group differences, brackets represent p < 0.01, repeated measures ANOVA)
Fig. 4
Fig. 4
Intracellular ROS production in response to commensal and pathogen-rich biofilms in smoke-free and smoke-rich environments. Figure 4a shows ROS production in response to commensal biofilm challenge, while Fig. 4b shows ROS production in response to pathogen biofilm challenge. Data represent means of six replicates, with standard deviation bars. i–v indicate sample micrographs at 0, 2, 4, 6 and 8 h respectively, while a–d indicates the environment at the same time points (*p < 0.05, **p < 0.01, ***p < 0.001, repeated measures ANOVA)

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