Harboring Cnm-expressing Streptococcus mutans in the oral cavity relates to both deep and lobar cerebral microbleeds

Background: Cerebral microbleeds (CMBs) influence long-term prognoses of stroke patients. Streptococcus mutans expressing the collagen-binding protein Cnm induces cerebrovascular inflammation, impairing blood brain barrier integrity and causing cerebral bleeding. Here, we examine the association of Cnm-positive S. mutans with CMBs. Methods: Acute stroke patients were selected from a single-center registry database. Oral carriage of Cnm-positive or Cnm-negative S. mutans was determined using polymerase chain reaction assays. The associations of Cnm-positive S. mutans with CMB number and specifically the presence of > 10 CMBs were examined using quasi-Poisson and logistic regression models, respectively. Results: This study included 3154 stroke patients, of which 428 patients (median [inter-quartile range] age, 73.0 [63.0– 81.0] years; 269 men [62.9%]) underwent oral bacterial examinations. In total, 326 patients harbored S. mutans . After excluding four patients without imaging data, we compared patients with Cnm-positive ( n = 72) and Cnm-negative ( n = 250) S. mutans . Harboring Cnm-positive S. mutans was independently associated with

Hypertensive arteriopathy and cerebral amyloid angiopathy (CAA) are the two most common causes of CMBs.Previous studies have suggested that deep CMBs are mostly associated with hypertensive arteriopathy, whereas lobar CMBs reflect CAA [9].However, growing evidence has shown that hypertensive arteriopathy induces both deep and lobar CMBs [9].One pathological investigation showed that lobar CMBs were related to hypertensive arteriopathy in the absence of CAA [8].
We previously reported that oral carriage of Streptococcus mutans that expresses Cnm protein is related to an increased risk of deep CMBs [10][11][12].Streptococcus mutans is a major cariogenic bacterium that is detected in the oral cavity of approximately 90% of the general population [13].Cnm, encoded by the cnm gene, is a cell surface 120-kDa collagen-binding protein of S. mutans [14].Intravenous administration of Cnm-expressing S. mutans (Cnm-positive S. mutans) aggravates cerebral bleeding in both cortical and deep gray matter in stroke-prone spontaneously hypertensive rats (SHRs) [15].Nevertheless, the contribution of Cnm-positive S. mutans to lobar CMBs in humans remains unclear [10,13,16].Infective endocarditis is a critical consequence of dental bacteremia, including that caused by Cnm-positive S. mutans [17], and lobar CMBs precede intracerebral hemorrhage (ICH) in infective endocarditis [18].We therefore hypothesized that Cnm-positive S. mutans is associated with the development of lobar and deep CMBs.This cross-sectional study investigated the involvement of Cnm-positive S. mutans in deep and lobar CMBs in stroke patients.

Study design
The significance of oral carriage of Cnm-positive S. mutans with re-

Clinical characteristics
Clinical information, except for MRI findings, was obtained from the National Cerebral and Cardiovascular Center Stroke Registry database.Hypertension was defined as systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg, or a history of antihypertensive medication use.Diabetes mellitus was considered present when a patient used antidiabetic drugs or insulin, the fasting plasma glucose level was ≥126 mg/dL, or the glycated hemoglobin A1c level was ≥6.5%.The definition of dyslipidemia the presence of >10 CMBs (adjusted odds ratio 2.20 [1.18-4.10])and higher numbers of deep and lobar CMBs (adjusted risk ratio 1.61 [1.14-2.27]for deep; 5.14 [2.78-9.51]for lobar), but not infratentorial CMBs, after adjusting for age, sex, hypertension, stroke type, National Institutes of Health Stroke Scale score, and cerebral amyloid angiopathy.

Conclusions:
Harboring Cnm-positive S. mutans was independently associated with a higher number of CMBs in deep and lobar locations.Reducing Cnm-positive S. mutans in the oral cavity may serve as a novel therapeutic approach for stroke.
dental caries, microbleeds, risk factor, Streptococcus mutans, stroke was a low-density lipoprotein cholesterol level of ≥140 mg/dL, a high-density lipoprotein cholesterol level of ≤40 mg/dL, a triglyceride level of ≥150 mg/dL, or use of lipid-lowering drugs.National Institutes of Health Stroke Scale (NIHSS) scores were recorded upon admission.The diagnosis of possible or probable CAA was based on the modified Boston criteria [19].

Detection of Cnm-positive S. mutans
Dental plaque specimens were collected, inoculated in Mitis-Salivarius medium with bacitracin (Sigma-Aldrich) and on 15% sucrose agar plates, and anaerobically incubated at 37°C for 48 h.
Streptococcus mutans strains were identified and isolated based on rough morphological features on the agar plates.The strains were then cultured in brain-heart infusion broth (Becton, Dickinson and Company) at 37°C for 24 h.Bacterial genomic DNA was extracted from each strain.A polymerase chain reaction assay was performed using an S. mutans-specific primer set (forward, 5′-GGCAC CAC AAC ATT GGG AAG CTCAGTT-3′; reverse, 5′-GGAAT GCC GAT CAG TCA ACAGGAT-3′) following a method described previously [20].The presence or absence of the cnm gene was determined using primers designed to amplify the entire length of the cnm gene (forward, 5′-GACAA AGA AAT GAA AGATGT-3′; reverse, 5′-GCAAA GAC TCT TGT CCCTGC-3′) [20].Experiments were conducted by researchers who were blinded to the clinical information.

Evaluation of cerebral microbleeds
In this study, CMBs were illustrated on T2*-weighted MRI.We evaluated the "certain" brain microbleeds as CMBs according to the Brain Observer MicroBleed Scale [6].The MRI parameters are summarized in Table S1.We defined deep CMBs as hypointensities on T2*weighted MRI located in the deep gray matter of the basal ganglia or thalamus or the white matter of the corpus callosum or internal, external, or extreme capsule.Lobar CMBs were defined as those in the cortical gray or subcortical white matter.Infratentorial CMBs were defined as those in the brainstem or cerebellum.Strictly lobar CMBs were defined as CMBs restricted to a lobar region.We also classified deep and/or infratentorial and mixed CMBs [21].Mixed CMBs included those located in both lobar and deep and/or infratentorial regions [21].The term "all CMBs" encompasses CMBs in any brain region.The number of CMBs was independently determined by two trained neurologists blinded to the clinical data and hypothesis of this study.In the case of disagreement, the opinion of a third neurologist was sought.

Statistical analyses
Variables are presented as medians and interquartile ranges or numbers and percentages.The Mann-Whitney U test was used to analyze continuous data, and the χ 2 or Fisher exact test was used for categorical data.The numbers of all, deep, lobar, and infratentorial CMBs were stratified into six categories: 0, 1, 2-4, 5-10, 11-20, and ≥21 [1,2].We examined the linear trend between Cnm-positive S. mutans and each CMB category using the Cochran-Armitage test.
In addition, univariate and multivariate logistic regression models were applied to examine the association of Cnm-positive S. mutans with the presence of >10 CMBs, as harboring >10 CMBs predicts future ICH [1], ischemic stroke [2], and death [4].The association of Cnm-positive S. mutans with the number of CMBs was analyzed using quasi-Poisson regression models.These models were adjusted for age, sex, hypertension, stroke type (ischemic stroke/ICH), and CAA (none/possible or probable CAA) [22].A p value < 0.05 (twotailed) was considered statistically significant.Statistical analyses were conducted using SPSS version 27 (IBM Corp.) and SAS version 9.4 (SAS Institute).
The oral bacterial examination revealed that S. mutans was present in 326 patients (76.2%) and absent in 102 patients (23.8%).The clinical profiles were similar between patients with and without S. mutans in the oral cavity (Table S3).We identified 72 patients with Cnm-positive S. mutans and 254 with Cnm-negative S. mutans.Four patients were excluded from the analyses in the Cnm (−) group because no MRI data were available.

Demographics and clinical characteristics
The clinical profiles of the Cnm (+) and Cnm (−) groups are described in Table 1.The age, sex, blood pressure, vascular risk factors, and medication histories were similar between the two groups.The Cnm (+) group showed slightly lower NIHSS scores than the Cnm (−) group ( 2

Cnm-positive S. mutans and cerebral microbleeds
Increasing numbers of CMBs are predictive of poor prognoses [1][2][3][4].When the numbers of CMBs were stratified into six categories, the Cnm (+) group was significantly distributed in the "higher CMB" categories for all and lobar CMBs but not for deep or infratentorial CMBs (all CMBs, p = 0.030; deep, p = 0.178; lobar, p = 0.009; infratentorial, p = 0.721, Figure 2).Representative images showing two patients with a substantial number of deep and lobar CMBs are illustrated in Figure 3.

DISCUSS ION
The current study demonstrated that oral carriage of Cnm-positive S. mutans was independently associated with a greater number of all, deep, and lobar, but not infratentorial, CMBs.Cnm-positive S. mutans was significantly related to the presence of >10 CMBs, a predictor of future ICH [1], ischemic stroke [2], and mortality [4].
Cnm-positive S. mutans has been reported in many countries, including Japan, the United States [24], Canada [25], and Finland [20].We previously reported a high prevalence [10] and high incidence [12] of deep CMBs in stroke patients harboring Cnm-positive S. mutans.
The cross-sectional [10] and retrospective longitudinal [12] studies showed a strong association between Cnm-positive S. mutans and deep, but not lobar, CMBs.The present study showed that harboring Cnm-positive S. mutans was independently associated with the number of lobar CMBs after adjusting for several vascular risk factors and stroke severity.The seemingly different results regarding lobar CMBs may stem from the different sample sizes between the previous and current studies [10,12].In addition, the frequency of hypertension (93.1%) in patients with Cnm-positive S. mutans in the present study was also slightly higher than that in the previous studies (85.7-90.9%),which might have contributed to the higher incidence of lobar CMBs.Bacteremia caused by S. mutans is almost inevitable in daily life because of toothbrushing, flossing, or tooth extraction [11].TA B L E 1 Clinical profiles of Cnmpositive and Cnm-negative groups.
The major sources of S. mutans are mothers or caregivers [20].S.
mutans is vertically transmitted, colonizes the mouths of infants [20], and is rarely implanted during adulthood [26].However, it frequently disappears from the oral cavity of edentulous individuals because S. mutans resides on the tooth surface [27].
We therefore excluded patients without S. mutans and only compared patients with Cnm-positive and Cnm-negative S. mutans in this study.

Note:
The unadjusted and adjusted odds ratio was estimated by logistic regression analysis.
b Adjusted for Model 1 plus stroke type (ischemic stroke or intracerebral hemorrhage) and National Institutes of Health Stroke Scale score.
c Adjusted for Model 2 plus cerebral amyloid angiopathy.

CNM-POSITIVES. MUTANSINCREASESCMBS
Cnm-positive S. mutans is characterized by its binding to components of the vascular basement membrane, such as collagen-IV and laminin [14,17,28], whereas Cnm-negative S. mutans cannot attach to soft tissues [17,28].Aging and vascular risk factors, including hypertension, induce endothelial injury and increase the thickness of basement membranes, resulting in collagen-IV and laminin exposure in small cerebral arteries [29,30].Once Cnmpositive S. mutans adheres to the basement membrane [28], neutrophil infiltration may aggravate local inflammation, resulting in increased permeability of the blood-brain barrier and increased production of enzymes (e.g., matrix metalloproteinase-9 [15]) that accelerate endothelial damage, leading to CMBs (Figure 4) The "vascular centrencephalon" is a phylogenetically ancient part of the brain that is perfused by short straight arteries with few branches, transmitting pressure directly from large arteries to small arterioles [31,32].However, the cortex is supplied by long arteries TA B L E 3 Risk ratios and 95% confidence intervals for associations between Cnm-positive Streptococcus mutans and the number of CMBs.with many branches, resulting in a large blood pressure decrement in the brain [33].The marked differences in the arterial pressures between the deep and cortical regions could explain why lacunar infarcts related to high blood pressure-induced vasculopathies preferentially occur in the vascular centrencephalon rather than in the cortex [32].Conversely, stroke-prone SHRs (i.e., a rat model of systemic hypertension) exhibit endothelial damage not only in deep but also in cortical arteries [30].Furthermore, cerebrovascular integrity is more severely damaged in stroke-prone SHRs than SHRs, even though stroke-prone SHRs and SHRs show similar degrees of hypertension [34].These findings suggest that factors other than high blood pressure also contribute to endothelial injury in patients with systemic hypertension [11], which may explain the increased numbers of lobar and deep CMBs in patients with Cnm-positive S. mutans.
This study had certain limitations.First, only 428 patients However, the association between Cnm-positive S. mutans and CAA should be further clarified by newly proposed criteria such as the Edinburgh criteria [36] and the Boston criteria version 2.0 [37].
Fourth, no country other than Japan has reported the effects of Cnm-positive S. mutans on stroke and CMBs.Thus, we cannot extrapolate our findings to other countries, and further multinational validation studies are necessary.
In conclusion, we found that Cnm-positive S. mutans was associated with a higher number of both lobar and deep CMBs.
Transmission of Cnm-positive S. mutans can be prevented by improving oral hygiene in early childhood.In addition, several strategies to target Cnm-positive S. mutans, such as immunotherapy, probiotics, and prebiotics, are being innovated [38].Reducing Cnm-positive S.
mutans in the oral cavity may serve as a novel therapeutic approach to improve the long-term prognoses of stroke patients.

AUTH O R CO
gard to CMBs was evaluated in a cross-sectional study in accordance with the Declaration of Helsinki standards and the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects.Approval was obtained from the Ethics Committee of the National Cerebral and Cardiovascular Center (M23-073-8, M27-015-5).Acute stroke patients were selected from the National Cerebral and Cardiovascular Center Stroke Registry database (https://www.clini caltr ials.gov;unique identifier: NCT02251665) who fully satisfied the following criteria: (1) patients who underwent oral bacterial assessments from February 2014 to May 2016 or from May 2017 to October 2018; (2) patients who developed ischemic stroke or ICH during the above-mentioned period; (3) patients older than 40 years old; and (4) patients or their legal representatives who provided written informed consent for the current study.We did not perform oral bacterial assessments for any patients between June 2016 and April 2017.Patients without MRI data were excluded from the analyses.Patients with Cnm-positive and Cnm-negative S. mutans (the Cnm [+] and Cnm [−] groups, respectively) were compared.Acute stroke patients that did or did not undergo an oral bacterial examination were comrpaed to uncover potential sources of bias.

F I G U R E 1
Flow diagram of patient selection.Patients with Cnm-positive and Cnm-negative Streptococcus mutans (Cnm [+] and Cnm [−] groups, respectively) were compared.MRI, magnetic resonance imaging; NCVC, National Cerebral and Cardiovascular Center.

[ 12 ]
. However, no clinical studies have shown the effects of Cnm-positive S. mutans on the blood-brain barrier or neuroinflammation.Gadolinium-enhanced MRI and positron emission tomography are warranted in future studies.

Note:F I G U R E 3
Abbreviations: CI, confidence interval; CMB, cerebral microbleed; RR, risk ratio.a Adjusted for age, sex, and hypertension.b Adjusted for Model 1 plus stroke type (ischemic stroke or intracerebral hemorrhage) and National Institutes of Health Stroke Scale score.c Adjusted for Model 2 plus cerebral amyloid angiopathy.

( 13 .
6%) of the total 3154 stroke patients underwent an oral bacterial evaluation.We attempted to recruit a wide range of stroke patients; however, older and more severe stroke patients tended not to participate in the study, largely because of difficulties in explaining the research to those with impaired consciousness or other disabilities, including dementia and advanced frailty.As a result, the patients that underwent bacterial assessments were younger and had lower NIHSS scores.Most of the enrolled participants were therefore mild-to-moderate stroke patients, raising a potential risk of selection bias, and limiting the ability to generalize our results to more severely affected patients.Additionally, this cross-sectional study was performed retrospectively, and outcome data were not obtained.To this end, we are currently performing a prospective multicenter observational study to evaluate the effects of Cnm-positive S. mutans[35].Second, the proportion of ICH patients and the number of patients with a history of hypertension or atrial fibrillation differed between those who did and did not undergo oral bacterial examination.We previously reported that Cnm-positive S. mutans is more closely associated with hypertensive ICH than ischemic stroke[10], which might have influenced participation in the study or the success rate of informed consent acquisition.Third, we found no evidence that Cnm-positive S. mutans accelerates the pathophysiology of CAA, a strong risk factor for lobar CMBs.Considering the close association of Cnm-positive S. mutans with deep CMBs, it is challenging to diagnose CAA in a patient harboring Cnm-positive S. mutans.The sensitivity of the modified Boston criteria for the diagnosis of CAA was 94.7%[19].

F I G U R E 4
NTR I B UTI O N S Study conception: SS, RN, KN, and MI.Data acquisition: SI, SS, SH, ST, Hajime I, and Hiroyuki I. Analysis and interpretation of data: SI, SS, Yumi Y, RN, MT, and KN.Drafting the manuscript: SI, SS, and MI.Revising the manuscript critically for intellectual content: TT, YH, RPF, ROC, NK, Yusuke Y, HH, MK, and KT.Supervision of the study: SS, HH, and MI.Hypothetical model of Cnm-induced development of cerebral microbleeds.Cerebral bleeding may occur at the level of arterioles and capillaries.Several factors, including aging and hypertension, result in endothelial damage and thickening of the basement membrane (BM).Cnm-positive Streptococcus mutans that enters the bloodstream after toothbrushing, flossing, or tooth extraction can attach to the exposed BM.Here, neutrophil infiltration results in local inflammation, leading to cerebral microbleeds.[Colour figure can be viewed at wileyonlinelibrary.com]