Smoking cessation induces profound changes in the composition of the intestinal microbiota in humans

Abstract

Background: The human intestinal microbiota is a crucial factor in the pathogenesis of various diseases, such as metabolic syndrome or inflammatory bowel disease (IBD). Yet, knowledge about the role of environmental factors such as smoking (which is known to influence theses aforementioned disease states) on the complex microbial composition is sparse. We aimed to investigate the role of smoking cessation on intestinal microbial composition in 10 healthy smoking subjects undergoing controlled smoking cessation.

Methods: During the observational period of 9 weeks repetitive stool samples were collected. Based on abundance of 16S rRNA genes bacterial composition was analysed and compared to 10 control subjects (5 continuing smokers and 5 non-smokers) by means of Terminal Restriction Fragment Length Polymorphism analysis and high-throughput sequencing.

Results: Profound shifts in the microbial composition after smoking cessation were observed with an increase of Firmicutes and Actinobacteria and a lower proportion of Bacteroidetes and Proteobacteria on the phylum level. In addition, after smoking cessation there was an increase in microbial diversity.

Conclusions: These results indicate that smoking is an environmental factor modulating the composition of human gut microbiota. The observed changes after smoking cessation revealed to be similar to the previously reported differences in obese compared to lean humans and mice respectively, suggesting a potential pathogenetic link between weight gain and smoking cessation. In addition they give rise to a potential association of smoking status and the course of IBD.

Figure 1. Comparison of the major phyla of the gut microbiota before and after smoking cessation.

(A) Phyla Composition. The results for the intervention group (I) and the control groups (non-smoking  = N; smoking  = S) are given for samples taken one week prior to smoking cessation (t1) as well as four weeks (t2) and eight weeks (t3) thereafter. Whereas the intervention group revealed a significant increase in fractions of Firmicutes and Actinobacteria and a decrease in fractions of Proteobacteria and Bacteroidetes, the microbiota of the control groups remained rather stable. The phyla Tenericutes, Verrucomicrobia, Synergistetes, Fusobacteria, Deinococcus-Thermus, TM7, Acidobacteria and OD1 are summarized under “other”. (B) Heat Map. The result of paired Student's t-test is shown on the phylum level with a color coded heat map. Significance levels are shown in different colors (shades of red, significant shifts in bacteria composition; shades of yellow, green and blue, non-significant shifts) and are indicated by the exact significance values within the colored squares of the graph. The major changes in the microbiota in the intervention group were observed between the time points before (t1) and after (t2, t3) smoking cessation. In contrast no significant changes were detected in the control groups and – with the exception of Bacteroidetes – after smoking cessation between t2 and t3 in the intervention group (an extended version of the heat map including all identified genera is shown in Figure S5, n/a =  not applicable).

Figure 2. Phylogeny-based Principal Component Analysis.

Bacterial communities of the three different treatment groups were clustered using PCA and the unweighted UniFrac distance matrix as an input (weighted PCA is shown in Figure S6). With PCA, a multivariate statistical analyses, axes that reflect the largest part of sample variation are identified (Percentage values at the axes reflect the level of variation explained by each principal coordinate; the first axis indicates the largest fraction of difference). Separation of the different sample collectives in 3 dimensions is visualized. A separation of the samples from the intervention group (I), that is most predominant 4 weeks after smoking cessation, was revealed. In contrast, the samples from the non-smoking (N) and smoking (S) control groups clustered together closely, thus reflecting their overall similar microbial composition.

Figure 3. UniFrac distance between samples and rarefaction curve of phylogenetic diversity.

(A) Unweighted UniFrac distance. The higher a UniFrac distance value between two samples the more different the bacterial composition. The highest distance values were determined for subjects undergoing smoking cessation between t1 and t2 as well as between t1 and t3. All other distance values were substantially smaller (error bars indicate SEM; *: p<0.05; ns: not significant). (B) Rarefaction curves. These curves express the accumulation of phylogenetic richness that would be obtained with continuous sampling effort and hence minimize potential differences that would be a result of the variable number of sequences obtained per sample. For the control groups the three sampling time points were combined in a single curve, while for the intervention group separated curves for t1, t2 and t3 were depicted to visualize the increased phlyogenetic diversity (PD) in the samples 4 weeks after smoking cessation (I2) compared to I1 and I3 (additional indices of α-diversity are depicted in Figure S9).