Human microbiota and atherosclerosis

I've been wanting to post about this study for a while now. I think its a good update while I finish my first post on my nutritional immunotherapy protocol. This study was performed given the preliminary evidence linking infections and atherosclerosis, and the association of the human microbiota with the atherosclerotic plaque. For example, bacterial DNA has been observed in atherosclerotic plaques from young and old subjects (1, 2). This relationship has been investigated with more focus on oral bacteria, due to the association of periodontal disease and cardiovascular disease (CVD) (3, 4) and the presence of periodontal pathogens in  atherosclerotic plaques (5). 

The authors tried to answer the following questions:

Is there a core atherosclerotic plaque microbiota? 

Are bacteria present in the plaque also detectable in the oral cavities or guts of the same individuals?

Do the microbiotas of the oral cavity, gut, and atherosclerotic plaque relate to disease markers such as plasma levels of apolipoproteins and cholesterol? 

Is an altered oral or fecal microbiota associated with atherosclerosis?

Using 16S rRNA sequences (from patients with clinical atherosclerosis and controls) and the unweighted UniFrac distance metric (qualitative instead of quantitative), they found strong clustering of samples according to body site, suggesting that the oral, gut and atherosclerotic plaque (AP) sites have different microbial communities:

PC1 and PC2 refer to the first two principal coordinates from the principal coordinate analysis of unweighted UniFrac, plotted for each sample (See also Fig S1). Of these sites, bacterial diversity was higher for the gut microbiota. 

The analysis of the atherosclerotic plaque microbiota revealed that there was a positive correlation between the amount of bacterial 16S rRNA and the number of leukocytes present in the AP, and there was significantly higher levels of Proteobacteria and fewer Firmicutes compared with the oral and gut samples. Supporting the role for a "core" AP microbiota, several OTUs were present in all AP samples, which differentiated these samples from oral or fecal samples: Chryseomonas was detected at high levels in the AP samples, but not in gut or oral samples, being the most discriminative genus between sites and driving the differences between body sites. Other OTUs, three for the genus Staphylococcus, three classified as Propionibacterineae and one belonging to the genus Burkholderia, were specific for AP samples and were present in all AP samples analyzed.

There were no OTUs differentiating oral samples from healthy subjects and patients, but there were correlations between the abundances of OTUs in the oral cavity and CVD markers: the abundance of Fusobacterium was positively correlated with levels of cholesterol (P = 0.028) and LDL (P = 0.005), the abundance of Streptococcus was positively  correlated to HDL (P = 0.0001) and ApoAI (P = 0.01) levels and the abundance of Neisseria was negatively correlated to levels of these last two markers (P = 0.02 and 0.005, respectively). This is interesting, given that Fusobacterium has been associated with periodontal disease (6). As with oral samples, there were no differentiating OTUs between gut samples from controls and patients (in terms of OTU abundances). In gut samples, the abundance of two OTUs classified as uncharacterized members of Erysipelotrichaceae and Lachnospiraceae families were positively correlated with cholesterol (P = 0.009 and 0.001, respectively) and LDL (P = 0.012 and 0.007, respectively). 

Finally, inter-individual comparisons between sites showed that some OTUs were shared among sites. These included OTUs for Veillonella (in AP and oral samples in 11 of 13 patients, detected also in the gut sample of two patients) and Streptococcus (in AP and oral samples in 6 of 10 patients, detected also in the gut of four patients). Within patients, the AP samples contained OTUs shared with oral (Propionibacterium, Rothia, Burkholderia, Corynebacterium, Granulicatella, Staphylococcus) and gut (Bacteroides, Bryantella, Enterobacter, Ruminococcus) samples. 

Summary

• The study identified a "core" atherosclerotic plaque microbiota, comprising higher levels of Proteobacteria and fewer Firmicutes, compared with the gut and oral samples. 
• The AP microbiota contained specific OTUs not shared with the analyzed body sites.
• The abundance of some OTUs in the gut and oral cavity was correlated with CVD markers. 
• Shared OTUs among sites included Streptococcus and Veillonella, the correlation being stronger among the oral cavity and the AP, and these OTUs were also found in the gut samples from some patients. Across patients, the abundance of both were correlated in the oral cavity and the AP.

Commentary

I find this study very interesting because it supports the role of infection on the pathogenesis of atherosclerosis and CVD. The "infection hypothesis" of atherosclerosis has been proposed before (7). The fact that specific bacteria is present in AP and not in other body sites analyzed and that the amount of bacterial 16S rRNA was positively correlated with leukocyte counts, support the notion that these pathogens support directly atherosclerosis progression. However, the study only analyzed the oral cavity and the gut, so it is impossible to conclude that these pathogens couldnt have been derived from other body sites (for example, the skin). Moreover, primers commonly utilized to amplify 16S rRNA sequences are limited to some species, an inherent property of the method (8). Nevertheless, it seems more feasible to suppose that the origin of AP bacteria is the oral cavity because of the close proximity of the bacterial communities in the mouth to the highly vascularized gingival lining and because of the thickness of the subgingival epithelium, which differs from other protective layers such as the skin or the gut mucosa (9). Accordingly, any mechanical disruption of oral bacterial biofilms can trigger bacteremia, and these include oral procedures (periodontal probing, tooth extractions, etc), oral hygiene activities (such as brushing) and physiological phenomena (like chewing) (9). This, coupled with the findings that the abundance of OTUs in the AP were correlated with that of the oral cavity support this hypothesis. Gut bacterial origin is more complicated but feasible (as shown by the presence of gut bacteria in AP samples). The authors suggest that one possible way of this transfer is by phagocytosis of macrophages at epithelial linings. 

If indeed bacteria play a role in the formation and/or progression of atherosclerosis, the million dollar question is why do these specific pathogens adhere to the vascular endothelium? Moreover, is this colonization the initial trigger for the localized inflammatory response or just aggravates the condition? With the available evidence it is hard to answer these questions. It has been proposed that atheromas might act as mechanical sieves, collecting bacteria from the cirulation (10). This would have deleterious consequences, as bacterial accumulation in the AP would lead to an increased inflammatory response. It could also link the fact that endotoxemia increases the risk of CVD (11), for which periodontal pathogens seem to play an important role (12). Supporting the role of infection as secondary to atherosclerotic inflammation, fungal DNA has been observed in AP (13), with some species correlated with that found on human microbial communities. It is of worth noting that in this study, fungal richness was not associated with classical CVD risk factors. Because not all normal residing oral bacteria are found in AP samples, AP invasion might be related to the virulence properties of some species (9). This seems to be the case, as in the study reviewed here, there was a common abundance of Streptococcus and Veillonella in AP samples. Streptococcus is able to adhere to the endothelium, while Veillonella is able to change its adherence capacity in the presence of some factors from Streptococcus (14). In fact, there is a tight relationship between Streptococcus and Veillonella in the oral cavity as some strains co-aggregate, partially because Veillonella seems to be metabolically dependent on Streptococcus (15). This relationship is so important that Veillonella is unable to establish an infection without Streptococcus.

In conclusion, microbial accumulation in AP might contribute to the progression of atherosclerosis. Although the mechanism by which these microorganisms colonize this site is not defined, it is clear that several microbes found in other body sites are also found in AP, which suggests that the normal human microbial communities are an important source of pathogens contributing to atherosclerosis progression. Translocation from these sites, in turn, is controlled by the host inflammatory status. This seems to be relevant to translocation from the oral cavity: transient bacteremia is experienced by everyone because of mechanical disruption of microbial biofilms (for instance, when eating), but it is controlled quickly. However, there are always some persisters which resist by-standing immune mechanisms. Obviously, a higher microbial load facilitates dissemination into the bloodstream and could possible influence the degree of transient bacteremia. A higher bacterial load coupled with a compromised subgingival epithelium barrier increases the risk of bacteremia and secondary colonization. So, in order to reduce the risk of AP colonization by oral pathogens, it is wise to target these two factors. For reducing oral bacterial load (overgrowth), reducing sucrose intake might be of benefit, as bacterial glucosyltransferase (GTF) plays a crucial role in plaque formation (16) and S.mutans is only pathogenic in the presence of sucrose (17). Dietary sucrose has been shown to increase total viable microbial density and S.mutans population in human dental plaque (18). Sucrose alone seem to be more cariogenic than sucrose plus fructose (19, 20). Additionally, sucrose alters the ionic concentration in the biofilms' matrix, altering the normal de- and re-mineralization process of enamel and dentin  (21). The role of starches in dental plaque formation is controversial (22), although some authors are in agreement with the Cleave & Yudkin hypothesis, which states that an excess of fermentable carbohydrate intake (in the absence of dental interventions) promotes dental diseases and then systemic diseases (23). Nevertheless, starchy foods commonly ate might promote dental plaque formation and disease. Pollard (24) showed that cornflakes, branflakes and wholemeal bread produced the minimum dental plaque pH peak, while all foods tested promoted enamel demineralization*. This might be related to the fact that, although starches can reduce plaque pH and induce demineralization, sucrose accelerates this effects (25). This is probably mediated by the interaction between bacterial GTF and salivary amylase (26). In contrast to what some might expect, whole fruit and fruit juices induce enamel demineralization by the same magnitude (27). This has been also found in some observational studies, where high fruit consumption is associated with increased caries risk (28). On the contrary, cheese and nuts have shown a negative association (29). Finally, inflammation increases the risk of oral bacterial growth and translocation, which might induce and/or aggravate systemic diseases (30). Periodontal disease has been positively associated with obesity (31), metabolic syndrome (32), type 2 diabetes (33), Alzheimer's disease (34), among other. Thus, controlling inflammation is key to avoid secondary diseases caused by pathogenic oral bacteria. 

* "Test foods were oranges, apples, bananas, Cornflakes, Branflakes, Weetabix, Alpen (no added sugar), white bread, wholemeal bread, rice, and spaghetti, with positive and negative controls of sucrose and sorbitol."

Koren O, Spor A, Felin J, Fåk F, Stombaugh J, Tremaroli V, Behre CJ, Knight R, Fagerberg B, Ley RE, & Bäckhed F (2011). Human oral, gut, and plaque microbiota in patients with atherosclerosis. Proceedings of the National Academy of Sciences of the United States of America, 108 Suppl 1, 4592-8 PMID: 20937873