Streptococcus species abundance in the gut is linked to subclinical coronary atherosclerosis in 8,973 participants from the SCAPIS cohort

BACKGROUND The gut microbiota are implicated in atherosclerotic disease, but the relation to subclinical coronary atherosclerosis and to the oral microbiome is unclear. The aim of the study was to identify associations between gut microbial species and computed tomography-based measures of asymptomatic coronary atherosclerosis in a large population-based cohort and to explore relevant clinical correlates. METHODS We conducted a cross-sectional study of 8973 participants 50 to 65 years old without known cardiovascular disease from the Swedish Cardiopulmonary BioImage Study (SCAPIS). Coronary atherosclerosis was assessed by coronary artery calcium score (CACS) and coronary computed tomography angiography (CCTA). Gut microbiota were identified by deep shotgun metagenomics sequencing of stool samples. Multiple regression models adjusted for cardiovascular risk factors were applied, and findings were validated in a case-control study of patients (n=373) with symptomatic atherosclerotic cardiovascular disease. CACS-associated species were assessed for association with carotid plaques, corresponding species in saliva samples, inflammatory markers and infection, and enrichment of metabolic pathways. RESULTS Gut bacterial associations with CACS were enriched for Streptococcus and Oscillobacter genera and for bacteria carrying genes linked to amino acid and carbohydrate degradation functions. In the main model, 67 gut microbial species were associated with CACS; associations were strongest for Streptococcus anginosus and Streptococcus oralis subsp. oralis . These two and 13 other associations were replicated in the validation study. Associations were largely similar across CCTA-based measurements. Five CACS-associated species correlated with the same species colonizing the mouth in the Malmö Offspring Dental study (MODS), which were in turn associated with worse dental health. Nineteen CACS-associated species were associated with high-sensitivity C-reactive protein concentrations in the same direction as with CACS, and 16 were associated with neutrophil counts. CONCLUSIONS This study provides robust evidence of an association of a higher abundance in the gut microbiome of several Streptococcus spp. and other species commonly found in the mouth with subclinical atherosclerosis and biomarkers of systemic inflammation and infection. Our findings call for a re-evaluation of the hypothesis of an infectious component in atherogenesis with further longitudinal and experimental studies. WHAT IS NEW? WHAT ARE THE CLINICAL IMPLICATIONS? ### Competing Interest Statement The authors declare the following competing interests: Ms Pita, Ms Nielsen, Drs Eklund, Holm, and Nielsen are employees of Clinical Microbiomics A/S, where samples were processed and DNA extraction and estimations of relative abundance of the metagenomics species were done. Dr Ärnlöv has received lecture fees from Novartis and AstraZeneca and has served on advisory boards for AstraZeneca and Boerhinger Ingelheim, all unrelated to the present paper. Dr Nilsson has received lecture fees from Novartis, Novo Nordisk, Amgen, and Boerhinger Ingelheim. The other authors declare no competing interests. ### Funding Statement The main financial support for the study was from the European Research Council [RC-2018-STG-801965 (T.F.), ERC-CoG-2014-649021 (M.O.-M.), and ERC-STG-2015-679242 (J.G.S.)]. Funding was also provided by the Swedish Research Council [VR 2019-01471 (T.F.), 2018-02784 (M.O.-M.), 2018-02837 (M.O.-M.), 2019-01015 (J.Ä.), 2020-00243 (J.Ä.), 2019-01236 (G.E.), 2017-02554 (J.G.S.), and EXODIAB 2009-1039 (M.O.-M.)]; the Swedish Heart-Lung Foundation [Hjärt-Lungfonden 20190505 (T.F.), 20200711 (M.O.-M.), 20180343 (J.Ä.), 2019-0526 (J.G.S.), 20200173 (G.E.)]; the A.L.F. governmental grant [2018-0148 (M.O.-M.)]; the Novo Nordisk Foundation [NNF20OC0063886 (M.O.-M.)]; the Swedish Diabetes Foundation [(DIA 2018-375 (M.O.-M.)]; the Swedish Foundation for Strategic Research [LUDC-IRC 15-0067 (M.O.-M.)]; Formas [2020-00989 (S.A.)]; Göran Gustafsson Foundationfoundation [2016 (T.F.)]; and Axel and Signe Lagerman's Foundation (T.F.). The Swedish CArdioPulmonary bioImage Study (SCAPIS) was funded mainly by the Swedish Heart-Lung Foundation. The study was also funded by the Knut and Alice Wallenberg Foundation, the Swedish Research Council and VINNOVA (Sweden's innovation agency), the University of Gothenburg and Sahlgrenska University Hospital, Karolinska Institutet and Stockholm County Council, Linköping University and University Hospital, Lund University and Skåne University Hospital, Ume#x00E5; University and University Hospital, and Uppsala University and University Hospital. The Malmö Offspring Dental Study (MODS) was funded mainly by Oral Health Related Research by Region Skåne (OFRS 422361, OFRS 512951, OFRS 567711, OFRS 655561, OFRS 752071, OFRS 853031, OFRS 931171 and OFRS968144). The Malmö Offspring Study (MOS) was funded by the Research Council of Sweden [VR 521-2013-2756 (P.M.N.)], the Swedish Heart and Lung Foundation [Hjärt-Lungfonden 20150427 (P.M.N.)], and by funds (“ALF”) from the local Region Skåne County Council (P.M.N.). In addition, funding was obtained from Ernhold Lundströms Stiftelse (L.B.). The computations and data handling were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC) at Uppsala University (SNIC CENTRE), which is partially funded by the Swedish Research Council through grant agreement no. 2018-05973. The computations were done using resources provided by SNIC through the Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under Project SNIC sens2019512. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was conducted in accordance with the Declaration of Helsinki and was approved by the regional ethics committee (Ume#x00E5;: Dnr 2010-228-31M; Uppsala: Dnr 2018/315). The Malmö Offspring Dental Study (MODS) and the Malmö Offspring Study (MOS) were approved by the regional ethics committee in Lund (MODS: Dnr 2013/761) (MOS: Dnr 2012/594). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Deidentified metagenomic sequencing data can be accessed in the ENA under accession number PRJEB51353 (). Access to the remaining data requires ethical approval from the Swedish Ethical Review Board and approval from the SCAPIS Data access board (). * CACS : Coronary artery calcium score CCTA : Coronary computed tomography angiography CVD : Cardiovascular disease GMM : Gut metabolic module GSEA : Gene-set enrichment analysis hsCRP : High-sensitivity C-reactive protein ln : Natural logarithm MODS : Malmö Offspring Dental Study MOS : Malmö Offspring Study SCAPIS : Swedish CArdioPulmonary bioImage Study SIS : Segment involvement score TSEA : Taxon-set enrichment analysis VGS : Viridans streptococci group