We report a positive independent relationship between carotid IMT and cumulative periodontal bacterial burden. Furthsermore, we have shown that the observed relationship with carotid IMT reflects both the burden and dominance of those pathogens etiologically related to periodontal disease in the subgingival microbial niche. These findings strengthen the hypothesis that oral infections may contribute to cardiovascular disease morbidity and bolster the supposition that accelerated atherosclerotic development is a possible mechanism connecting chronic infections and cardiovascular disease.
In the present study, we collected and analyzed an average of 7 subgingival plaque samples per mouth and were careful not to simply select the most periodontally diseased sites for bacterial sampling. Although the latter might have increased our likelihood of positive findings, we opted instead for a protocol that would give us a representative picture of the subject’s periodontal microbiota, under the assumption that it would likely be most relevant to systemic health.
Furthermore, because we were concerned that overall pathogen burden might simply reflect poor oral and perhaps general health, we performed additional analyses focused on the burden of those specific bacteria admitted as causally related to periodontal disease.23,24
Both the validity and robustness of the present findings were enhanced, because these etiologic pathogens were the ones driving the relationship with increasing carotid IMT. This specificity argues against an overall health effect. Moreover, these findings could not be accounted for by lifestyle variables such as brushing, flossing, and physical activity, which adds further support to the concept of a relationship independent of healthy behaviors. Additionally, by design, subjects were enrolled from 5 specific zip codes in northern Manhattan, minimizing the possibility of socioeconomic bias. Interestingly, although these periodontally etiologic pathogens were not the largest contributors to the measured absolute bacterial burden (representing only 23% of bacterial distribution), only 1 additional bacterial species (M micros
) added to the relationship with carotid IMT.
Neither WBC nor CRP values increased with overall bacterial burden; however, WBC values, but not CRP values, tended to rise with both increasing levels of etiologic bacterial burden and carotid IMT, which suggests a possible direct role of certain, but not all, infections. Although these findings add to the evidence that microbiological burden may be important in atherosclerosis,5,6
they also demonstrate that a level of specificity exists. In the present study, the implicated bacteria share a common pathogenicity toward the periodontium, which may extend to the vasculature, via release of cytokines,25
repeated bacteremias, or a hyper-inflammatory response to a distant aggression.7–9
Because CRP values in this population were relatively elevated (mean 4.1±7.6 mg/L; median 1.9 mg/L), it is possible that the contributions of CRP were masked; CRP might discriminate better at lower values. Nevertheless, even after removal of the extreme CRP values or natural log transformation, we consistently found no relationship between CRP and either IMT or etiologic burden. This would be consistent with the findings by Folsom et al27
of no cross-sectional relationship between CRP and carotid IMT in the Atherosclerosis Risk In Communities (ARIC) study. This does not, however, exclude an indirect inflammatory role, because other studies have found positive associations between increasing extent of periodontal disease and systemic markers of inflammation.7,8
Slade et al7
noted that the relationship between periodontal disease and CRP was only present among individuals with low BMI, with the strongest findings among participants with BMI=20 kg/m2
. If BMI is truly a modifying factor, the higher average BMI in the INVEST population (>28 kg/m2
) might explain the differing findings.
DNA-DNA hybridization is a reliable method that has been directly compared with culture.18
It generally yields higher values than culture because, unlike culture, it does not require preservation of bacterial viability, thus allowing for identification of dead bacteria whose DNA was preserved in the subgingival plaque. Thus, in the context of the hypothesis of chronic infections influencing carotid IMT, we believe this method to be preferable, because it allows an investigation of a larger window of past exposure. It is also particularly suitable for epidemiological studies, because it allows the relatively rapid analysis of large numbers of plaque samples with respect to multiple species.
Direct assessment of bacterial load by means of DNA-DNA hybridization may be preferable over the serological study of antibody titers in dentate subjects in that it quantifies the microbial burden in a manner unregulated by the host immune status. Conversely, bacterial DNA recovery does not discriminate between states of colonization, with or without disease. Unlike the presence of bacteria in the bloodstream, which generally signifies pathology, the oral cavity naturally harbors multiple bacterial species, some of them associated with good health.23,24
This underscores the importance of taking into account the relative burden and dominance of specific bacteria.
Nevertheless, we make no assumptions about the precise number of microbes present but only as to the relative ranking of proportional burden between individuals. Thus, we refrained from reporting exact bacterial level values but rather ranked bacterial burden across tertiles. The present findings should be seen in the context of both the advantages and limitations of the hybridization method used.
The magnitude of carotid IMT changes reported here is in keeping with values that have been deemed significant in prior observational and interventional studies. The Etude du Vieillssement Arteriel (EVA) reported that a cross-sectional difference of 0.03 mm in carotid IMT was associated with a 15-mm Hg increase in systolic blood pressure,28
and the Longitudinal Study of Aging reported that a cross-sectional difference of 0.04 mm in carotid IMT thickness was equivalent to a 10-year age difference in subjects without bulbar plaque.29
In progression studies, Hodis et al30
reported that a 0.03-mm/y increase in carotid IMT is associated with a 2.3-increased relative risk for nonfatal myocardial infarction or coronary death. Interventional studies assessing the effect of statins report as clinically significant a difference of 0.0082 mm/y in mean maximum carotid IMT between annualized progression rates with placebo (0.067 mm) and pravastatin (0.059 mm).31
Thus, notwithstanding obvious design differences, the present finding of a 0.03- to 0.04-mm mean difference between the highest and lowest tertiles of bacterial burden/dominance appears to fall within the range of clinically relevant differences.
The present study shares with others the limitations of cross-sectional data. Because both carotid IMT and periodontal microbiology were measured concurrently, the time sequence cannot be established, and causal inferences cannot be made. We must await the prospective results of INVEST and other studies to make firmer conclusions. It is also possible that the bacterial changes noted here might reflect other risk factors not properly measured or identified; however, we adjusted extensively for confounders, and the relationship strengthened after statistical adjustment, which renders this possibility less likely.
Admittedly, there are hundreds of bacterial species that colonize the oral cavity.32
Although we opted for a selection of microorganisms originating from different bacterial complexes that are relevant to population studies,9,27
additional pathogens might have been of value and could conceivably modify the overall relationship.
Further studies are needed to confirm those findings. However, in INVEST, patients with a dominance of oral pathogens causally related to periodontal disease had thicker carotid IMT after adjustment for conventional risk factors. This study provides the first direct evidence of a possible role of periodontal bacteria in atherosclerosis. This relationship appears to be independent of CRP. If confirmed, these findings could be of public health importance because they raise the possibility that atherosclerotic damage possibly could be reduced and perhaps reversed through selective control of pathogenic periodontal bacteria by antibacterial or immunologic means.