# Endospore-forming Bacillus subtilis isolated from third molar exudates and its association with cardiovascular disease: a retrospective cohort study

## TL;DR

This study found that spore-forming Bacillus subtilis bacteria from third molars are more common in people with cardiovascular disease and may contribute to systemic inflammation.

## Contribution

The study introduces a novel approach to investigate the link between oral Bacillus subtilis and cardiovascular disease in regions with limited medical records.

## Key findings

- B. subtilis isolates were more prevalent in patients with cardiovascular disease compared to controls.
- Biofilm-forming B. subtilis was independently associated with cardiovascular disease (OR 2.91).
- B. subtilis presence correlated with higher systolic and diastolic blood pressure.

## Abstract

Various techniques have been previously modified to reduce early postoperative complications following third molar extraction. Given the influence of the oral microbiome, increasingly resistant bacteria have been linked to systemic diseases such as cardiovascular disease (CVD).

In this study, we aimed to identify the spore-forming bacterium Bacillus subtilis isolated from third molar exudates and to investigate its potential association with CVD.

In Iraq, dental hospitals don't keep thorough medical records for each patient. This lack of documentation makes it tough to carry out hospital-based research. Therefore, by collecting exudates from third molars, we conducted a retrospective cohort study of the population undergoing third molar exudate removal in a private dental clinic as an alternative setting to compare cardiovascular outcomes between individuals with cardiovascular disease and controls. Based on clinical assessments, body mass index, LDL cholesterol, C-reactive protein, systolic blood pressure, diastolic blood pressure, hypertension, and smoking status were measured. The study was conducted on 40 men, comprising 20 patients with diagnosed cardiovascular disease and 20 controls. Light and transmission electron microscopy were used to perform a phenotypic evaluation of the bacterial isolates (spore formation, biofilm production). Biofilm formation was assessed using Congo red agar, crystal violet staining, and scanning electron microscopy (SEM). In addition, systolic and diastolic blood pressure (SBP and DBP) values were obtained to further assess cardiovascular risk.

The number of B. subtilis isolates was higher in the CVD group than in the control group (non-CVD) and demonstrated significantly greater biofilm-forming ability (OD600 = 1.45 ± 0.22 vs 0.85 ± 0.19, p < 0.01). TEM confirmed dense endospore architecture from patients with CVD, while SEM revealed extensive extracellular matrix formation within CVD biofilms. Patients with oral colonization by B. subtilis showed a significantly higher prevalence of CVD (32.6%) compared to those without colonization (12.6%, p = 0.008). The presence of biofilm-positive B. subtilis strains was independently associated with CVD (OR 2.91; 95% CI, 1.23–6.83). Spore-forming B. subtilis isolates from third molars of patients with CVD demonstrated enhanced biofilm formation and sporulation phenotypes. A moderate positive correlation (r = 0.48) was also observed between B. subtilis presence and SBP and DBP. These findings suggest that these bacterial characteristics are potentially the cause of systemic inflammation and represent a potential microbial link to CVD.

In countries like Iraq, researchers run into real problems when they try to study links between oral health and other diseases. There's no NHS dental system, so they can't rely on existing records for data. Therefore, this study provides a protocol for conducting investigations related to oral health through collaboration with other institutions, such as universities. With respect to the main finding of this study, spore-forming B. subtilis isolated from third molar exudates demonstrated phenotypic characteristics that may contribute to persistent oral colonization and increase systemic inflammatory risk. The observed association with cardiovascular disease warrants further investigation into oral–systemic microbial pathways.