Dietary live microbes intake and its association with cardiovascular and all-cause mortality: Shreds of evidence from NHANES 2005-2018

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Dietary live microbes intake was classified into low, medium, and high categories based on 24-hour dietary recall. All-cause mortality was the primary outcome, with cardiovascular mortality as the secondary. Kaplan-Meier survival analysis and Cox proportional hazards models, adjusted for confounders, were applied in R software ( P < 0.05). Higher intake groups showed significantly lower all-cause and cardiovascular mortality ( P < 0.001). The Cox models confirmed reduced all-cause mortality risk in medium (HR = 0.89) and high intake groups (HR = 0.69) compared to low intake. Cardiovascular mortality was also lower in the high intake group (HR = 0.70). Subgroup analyses revealed consistent benefits, with a stronger effect in younger individuals. These findings highlight the potential of dietary live microbes in reducing mortality, warranting further prospective studies. Biological sciences/Microbiology Health sciences/Health care NHANES Cardiovascular Mortality All-cause mortality Dietary live microbes Figures Figure 1 Figure 2 Introduction The human gut microbiota comprises complex communities of bacteria, viruses, protozoa, and fungi, which interact with multiple human systems and play a critical role in regulating their functions. Recent animal and clinical studies have shown that imbalances in the gut microbiota may affect human health and vice versa. Consequently, there is growing recognition of the potential of the gut microbiota as a promising target for dietary and therapeutic interventions along the microbiota-gut-X axis 1 . Previous studies have demonstrated that live microbes in the diet, such as beneficial bacteria or yeasts, can be ingested to improve digestive health and enhance immunity. These microorganisms are naturally present in certain foods like yogurt, kefir, and sauerkraut and are also available in supplement form 2 . The 20th century's epidemiological shift saw a decline in deaths and disabilities caused by communicable diseases alongside a rise in non-communicable diseases (NCDs). Among NCDs, cardiovascular disease (CVD) has emerged as the primary cause of mortality and morbidity globally 3 . High mortality rates due to cardiovascular diseases and other causes place a considerable strain on healthcare systems and the economy 4 . Lowering cardiovascular and all-cause mortality aligns with global health goals, such as the United Nations' Sustainable Development Goals (SDGs), which aim to reduce premature mortality from non-communicable diseases by one-third by 2030 5 . It is imperative to seek methods to reduce cardiovascular and all-cause mortality urgently. A scoping review identified that a microbial dose expressed as CFU/day was linked to non-negative reported outcomes. Positive outcomes were mainly observed in older population groups, with a median age of 39 6 . Han et al. found that a high intake of dietary live microbes was associated with a lower prevalence of cardiovascular disease (CVD), with significant associations explicitly observed when the analysis focused on stroke and heart attack 7 . However, the exact relationship between dietary live microbes intake and all-cause or cardiovascular mortality remains uncertain 8 . This study aims to utilize a nationally representative sample from the National Health and Nutrition Examination Surveys (NHANES) and employ Marco's classification system to define and estimate dietary live microbes intake to investigate the association between dietary live microbes intake and all-cause mortality and cardiovascular mortality, as well as to analyze differences across various demographic characteristics and disease state 9 . Methods Study design and population NHANES is conducted by the Centers for Disease Control and Prevention (CDC), which systematically gathers, analyzes, and shares data that accurately represents the health and nutrition of the national population. It is a large-scale survey project aimed at assessing the health and nutritional status of the U.S. population 10 . Our analysis was based on data from participants in the NHANES 2005–2018 cycle. We applied the following exclusion criteria: missing follow-up time and unavailable data on dietary live microbes. After using these criteria, 36,720 participants were included in the final analysis (Fig. 1). Fig. 1. Flowchart of the sample selection from NHANES 2005–2018. NHANES, National health and nutrition examination surveys. Calculation of explanatory variable: dietary live microbes Energy and nutrient intake was estimated using 24-hour dietary recall data linked to the Food and Nutrient Database of the Department of Agriculture (USDA). Experts (Maria L Marco, Mary E Sanders, Robert Hutkins, and Colin Hill ) evaluated 9,388 food codes in the NHANES database to estimate live microbes content colony-forming units per gram (CFU/g) for foods, categorizing foods as low (10 7 CFU/g) in microbial count 11 . These classifications were designed to represent pasteurized foods or foods processed at high temperatures (low levels), unpeeled fresh fruits and vegetables (medium levels), and unpasteurized fermented foods and probiotic supplements (high levels) 12 . Based on these estimates, participants were grouped into low, medium, or high dietary live microbes intake categories, consistent with prior research 9,13,14 . Definition of outcome variables: all-cause mortality and cardiovascular mortality The study's primary outcome was all-cause mortality, encompassing deaths from any cause 8 . The secondary outcome explicitly focused on cardiovascular mortality, referring to deaths associated with CVD. Mortality data, including specific causes of death, were sourced from the publicly available NHANES public use linked mortality file. Participant mortality status was ascertained by linking survey data with death certificate records from the National Death Index (NDI). The causes of death were classified according to the International Classification of Diseases, 10th Revision (ICD-10) coding system 15 . Cardiovascular deaths were categorized by the National Center for Health Statistics (NCHS) as those resulting from heart disease (ICD-10 codes I00–I09, I11, I13, and I20–I51) or cerebrovascular disease (ICD-10 codes I60–I69), based on previous literature 8,10,16,17 . The Centers for Disease Control and Prevention (CDC) has validated this classification, commonly used in official reports 8 . In this study, participants were tracked from the baseline interview date until their date of death or December 31, 2019, whichever came first 8,10 . Assessment of covariates Covariate data were collected through baseline questionnaires, physical examinations, and laboratory tests 10 . Age, BMI, coffee intake, race /ethnicity, sex, marital status, poverty income ratio (PIR), education level, NHANES cycle, diabetes, hypertension, history of CVD, alcohol drinking, and smoking status were identified as potential confounders based on published research and clinical expertise 18 . Race/ethnicity data were self-reported and categorized into five groups: Mexican American, non-Hispanic White, non-Hispanic Black, other Hispanic, and others (including multiracial individuals) 10 . Educational attainment was stratified into three levels: less than high school, high school or equivalent, and beyond high school 18 . The PIR was determined by dividing the monthly family income by the poverty thresholds defined by the Department of Health and Human Services (HHS) guidelines. This ratio was then categorized into three groups: 3.5 (high income) 19 . Marital status was categorized into four groups: married, never married, living with a partner, and other (e.g., widowed, divorced, or separated). Self-reported CVD history included diagnoses of heart failure, coronary heart disease, angina, myocardial infarction, or stroke 8 . Diabetes was defined as a physician diagnosis, glycohemoglobin levels >6.5%, fasting glucose ≥7.0 mmol/L, random or 2-hour oral glucose tolerance test glucose levels ≥11.1 mmol/L, or current use of diabetes medication/insulin 10 . Hypertension was characterized by a systolic blood pressure of ≥140 mmHg or a diastolic pressure of ≥90 mmHg, based on ICD-10 codes 20 . Smoking status was classified into three categories: (i) never smoked (fewer than 100 cigarettes), (ii) former smoker (more than 100 cigarettes but has quit), and (iii) current smoker (more than 100 cigarettes and currently smoking). Caffeine intake over the 24-hour period was estimated using the USDA Food and Nutrient Databases for Dietary Studies (FNDDS) 5.0, as provided in the NHANES database 21 . Participants were divided into five categories based on their weekly alcohol consumption: non/former/mild drinker, moderate drinker, or heavy drinker, with classifications defined and adjusted based on sex 22 . Statistical analysis In line with NHANES analytical guidelines, the complex sampling design and mobile examination center sample weights were incorporated into the analysis 9,23 . Participant characteristics were summarized according to their pattern of live microbes intake. Categorical variables were presented as frequencies and percentages, while continuous variables were reported as means with standard errors (SE). A two-sample Student's t-test was used to compare normally distributed continuous variables between the two groups. At the same time, the Wilcoxon rank-sum test was applied for non-normally distributed continuous variables. Categorical variables were analyzed using the χ² or Fisher's exact test 8 .Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using the Cox proportional hazards model 20 . Model 1 was the unadjusted model. Model 2 was adjusted for age, gender, race/ethnicity, PIR, marital status, and education level. Model 3 included the same adjustments as Model 2, with additional adjustments for NHANES cycles, physical activity, smoking status, BMI, history of CVD, hypertension, and D.M. In subgroup analyses, stratified analyses were conducted by age group (60–69, 70–79, or ≥80 years), gender (female or male), race/ethnicity (Mexican American, non-Hispanic Black, non-Hispanic White, other Hispanic, or others), CVD history (yes or no), diabetes (yes or no), hypertension (yes or no), and prebiotic/probiotic intake (yes or no). Interaction effects were tested by adding an interaction term to the regression model to assess the influence of one variable on the outcome concerning another variable 9,24 .The Kaplan-Meier method was employed to estimate cumulative incidence and time-to-event for all-cause mortality in the overall population and the CVD subgroup. Survival curves between different groups were compared using the log-rank test. These models enable the estimation of cumulative incidence probabilities for cardiovascular mortality while accounting for competing events 8,25 .All analyses were conducted using R, version 4.4.0, with the survey package (version 4.1-1). Statistical significance was defined as a two-sided P -value of less than 0.05 26 . Results Baseline population characteristics 36,720 participants were involved, with an average age of 46.647 years and a gender split of 18,506 (50.960%) female patients to 18,506 (49.040%) male patients. Age, BMI, coffee take, race/ethnicity, sex, marital status, PIR, education levels, diabetes, hypertension, CVD, alcohol drinking, smoking, and mortality status were significantly different between the three groups (all P <0.05). Weighted analyses of the participants showed that those in the medium dietary live microbes group were more likely to be older, non-Hispanic White, have a lower BMI, have a higher education level, be married or living with a partner, have a higher PIR, and be non-smokers. The high dietary live microbes group also had better overall health, with lower prevalence rates of diabetes, hypertension, and CVD and lower mortality rates. Conversely, the low intake group had non-Hispanic Black participants, lower education levels, higher rates of smoking, and a higher prevalence of chronic conditions such as CVD, diabetes, and hypertension. These participants also exhibited poorer physical health and lower socioeconomic status ( Table 1 ). Associations of dietary live microbes' intake with all-cause and cardiovascular mortality K-M survival analyses showed a significant difference in the incidence of all-cause mortality and cardiovascular mortality between the three groups during follow-up, the lowest all-cause mortality being in the high dietary live microbes intake group (log-rank P < 0.001). The details of the K-M survival analyses are presented in Fig. 2 A and Fig. 2B . Fig. 2. Kaplan–Meier survival curves showing the association between dietary live microbe levels and (A) all-cause mortality and (B) cardiovascular mortality. Table 2 presents the results of the sample-weighted Cox regression models evaluating the association between dietary live microbes intake and mortality outcomes. In the crude model (Model 1), both the medium and high dietary live microbes intake groups showed significantly lower hazard ratios for all-cause mortality compared to the low dietary live microbes intake group (Medium: HR = 0.89, 95% CI, 0.81–0.98, P = 0.02; High: HR = 0.69, 95% CI, 0.61–0.77, P < 0.001). After adjusting for age, sex, and race/ethnicity in Model 2, the associations strengthened, particularly in the high dietary live microbes intake group (HR = 0.65, 95% CI, 0.58–0.73, P < 0.001). In the fully adjusted model (Model 3), which further accounted for marital status, PIR, education level, BMI, diabetes, hypertension, CVD history, alcohol consumption, coffee intake, and smoking status, the high dietary live microbes intake group continued to show a significantly reduced risk of all-cause mortality (HR = 0.85, 95% CI, 0.76–0.96, P = 0.01). Regarding cardiovascular mortality, the high dietary live microbes intake group exhibited a notable reduction in risk across all models, with the strongest association observed in Model 2 (HR = 0.70, 95% CI, 0.55–0.89, P = 0.003). The medium dietary live microbes intake group was also significantly associated with lower cardiovascular mortality in the fully adjusted model (HR = 0.82, 95% CI, 0.69–0.99, P = 0.04). Trend tests indicated a significant dose-response relationship for all-cause and cardiovascular mortality, with increasing dietary live microbes intake linked to progressively lower mortality risk ( P for trend < 0.05 in all models). These findings suggest that higher dietary live microbes intake is consistently associated with reduced mortality risk, independent of potential confounding factors. Subgroup analysis of the association between dietary live microbes intake with all-cause and cardiovascular mortality Table 3 and Table 4 present the results of subgroup analyses examining the association between dietary live microbes intake and all-cause mortality ( Table 3 ) and CVD mortality ( Table 4 ) across various demographic and health-related factors, including smoking, age, sex, diabetes, PIR, hypertension, marital status, education level, and race/ethnicity. Table 3 showed that participants in the medium and high dietary live microbes intake groups generally exhibited lower HR for all-cause mortality compared to the low intake group. This trend was consistent across most subgroups, including both smokers and non-smokers, males and females, and individuals with and without diabetes or hypertension. There is a significant difference in the impact of dietary live microbes on all-cause mortality between different age groups (≤60 years and >60 years, P for interaction = 0.001), which suggests that age is a vital interaction factor, with dietary live microbes potentially offering more potent protective effects for the younger group (≤60 years). Table 4 revealed that participants in the medium and high dietary live microbes intake groups generally exhibited lower HR for CVD mortality compared to the low intake group, particularly among females and those with a lower PIR. The reduction in CVD mortality risk was more pronounced in females across both medium (HR = 0.713, 95% CI, 0.557–0.914) and high intake groups (HR = 0.701, 95% CI, 0.518–0.948), while males showed a weaker association, especially in the medium intake group (HR = 1.027, 95% CI, 0.809–1.305). Additionally, those with a PIR below 1.3 experienced a significant reduction in risk in the medium intake group (HR = 0.724, 95% CI, 0.541–0.969), with a trend towards reduced risk in the high intake group, though it was not statistically significant. However, no significant interaction effects were observed across the stratified variables, indicating that the beneficial impact of higher dietary live microbes intake on CVD mortality is generally consistent across different population subgroups, regardless of variations in these factors. Discussion In this nationally representative cross-sectional study, we identified various vital factors influencing the intake of live bacteria in a reasonable diet. We found that participants in the medium and high dietary intake of live microbes groups had significantly lower all-cause mortality rates than those in the low dietary intake group. Notably, this association was more pronounced among elderly participants. Similarly, the prognosis for cardiovascular diseases was better in the medium and high dietary live microbes intake groups. Our findings provide compelling evidence that food, particularly the role of microbes in health, serves as medicine, offering noninvasive and easy ways to enhance health and extend lifespan. Despite the limited research on the overall dietary intake of live bacteria, the Mediterranean diet is widely acknowledged as a healthy eating pattern that has been demonstrated to promote beneficial alterations in gut microbiota. 27 .The NU-AGE 1-year dietary intervention across five European countries supports the feasibility of improving the habitual diet to modulate the gut microbiota, which in turn has the potential to promote healthier aging 28 . The diversity and abundance of the gut microbiota are among the factors influencing human health. In unhealthy populations, gut microbiota diversity is reduced, and its composition significantly differs from healthy individuals. The microorganisms found in fresh produce and fermented foods exhibit a wide range of diversity, influenced by factors such as food type, source, and degree of processing. This study demonstrates that dietary intake of medium or high levels of live microbes (sourced from unpeeled fresh fruits and vegetables, 10 4 –10 7 CFU/g or > 10 7 CFU/g)) is associated with improved survival, highlighting the importance of reasonable foods as a source of beneficial bacteria 29 . The mechanism of association between the medium and high dietary live microbes and cardiovascular and all-cause mortality may be as follows. (a) Probiotics are "live microorganisms that, when taken in sufficient quantities, provide health benefits to the host." Numerous fermented products act as sources of probiotic strains. The effectiveness of probiotics is influenced by various factors, including the interactions between probiotic bacteria and the host's microbiome 29 . In recent decades, probiotics have grown significantly, both for enhancing general gut health and as biotherapeutic agents to address clinical disorders linked to dysbiosis 30 , 31 . For example, the research indicates that after consuming fermented milk, the ileal microbiota in humans is temporarily dominated by probiotics for several hours, suggesting that the ingested probiotic strains have sufficient time to interact with and stimulate host cells [33] continuously. (b) Hill et al. found foods with higher microbial concentrations are associated with modest health improvements across a range of outcomes, such as blood pressure, C-reactive protein, plasma glucose, plasma insulin, triglyceride, waist circumference, BMI, high-density lipoprotein cholesterols 14 . (c) Wastyk et al. revealed diet-specific effects. A high-fiber diet boosted glycan-degrading enzymes without altering microbial diversity, while three distinct immune responses were linked to baseline microbiota diversity. In contrast, a high-fermented diet increased microbiota diversity and reduced inflammation. These findings suggest that fermented foods could help reduce microbiome diversity and increase inflammation in industrialized societies 32 . However, due to the limited sample size in this study and the potential impact of confounding factors, these findings should be interpreted with caution. Further research involving well-designed prospective studies is essential to explore this topic more thoroughly. Subgroup analyses showed that the association was stable across smoking, sex, diabetes, PIR, hypertension, marital status, education level, and race/ethnicity stratification. However, the interaction for age was statistically significant, which may be attributed to the unique physiological changes associated with aging in older individuals. Therefore, it is crucial to evaluate whether the benefits of consuming live microbes surpass any potential risks 9 . Nonetheless, the heterogeneity of the association observed in subgroup analyses warrants testing in a larger population 11 . Moreover, socioeconomic factors are crucial in the differences in live microbes intake in a balanced diet. Differences in access to healthcare, education, and socioeconomic resources can significantly influence individuals' acceptance of the concept of food-medicine homology, ultimately affecting the quality of their diet. Our study is the first to explore the association between dietary live microbes, distinct from specific fermented foods or probiotics, and cardiovascular and all-cause mortality in a broadly representative U.S. population. In this study, the stringent quality control procedures and the complex sampling design employed by NHANES enabled us to evaluate the association between the intake of live microbes and mortality. As a result, enhancing the intake of dietary live microbes could be viewed as a novel dietary approach to mitigating mortality. Furthermore, it may be crucial to evaluate the intake of live microbes. However, this observational study also had several limitations that should be pointed. First, due to this study's cross-sectional design, we could not identify the temporal and causal relationship between dietary live microbes and cardiovascular and all-cause mortality and the possibility for reverse effect bias should be considered. Second, the relatively simplistic categorization method for dietary live microbes may not accurately reflect the intake of various microorganisms. Third, since this study was conducted solely on a U.S. population, caution is needed when extrapolating the findings to other ethnic groups. Moreover, although we accounted for demographic characteristics, lifestyle factors, and certain medical conditions, residual mediators and modifiers cannot be entirely ruled out. Finally, considering individual differences within the population, assessing whether the benefits of dietary live microbes intake outweigh the potential drawbacks is necessary. Prospective studies are crucial to validate its effectiveness in reducing mortality. Mendelian randomization should be employed to obtain more robust and reliable results. Additionally, further research is necessary to establish the optimal dosage of dietary live microbes and to assess their impact on mortality across various diseases and health conditions 11 , 20 , 33 . Conclusions This study shows that eating more foods rich in live microbes is linked to lower risks of dying from any cause or from heart disease. The results were consistent across different groups of people, suggesting that consuming more live microbes could be a simple way to improve health. However, more studies are needed to confirm these findings and to explore the best ways to use live microbes to reduce the risk of death. Abbreviations BMI Body mass index CDC Centers for Disease Control and Prevention CFU Colony-forming units per gram CI Confidence interval CVD Cardiovascular disease FDA Food and Drug Administration FNDDS Food and Nutrient Databases for Dietary Studies HHS Department of Health and Human Services HR Hazard ratios ICD-10 International Classification of Diseases, 10th Edition PIR Pverty income ratio NCHS National Center for Health Statistics NDI National death index NHANES National Health and Nutrition Examination Surveys SDGs United Nations' Sustainable Development Goals SE Standard error USDA United States Department of Agriculture. Declarations Data availability The National Health and Nutrition Examination Survey data is accessible through the National Center for Health Statistics at the Centers for Disease Control and Prevention (https://www.cdc.gov/nchs/nhanes/index.htm). Declaration of competing interest The authors report no relationships that could be construed as a conflict of interest. Acknowledgments We thank the National Health and Nutrition Examination Survey participants and staff and the National Center for Health Statistics for their invaluable contributions. We also (granted publication licenses) acknowledge the BioRender (www.biorender.com). Authorship contributions J.W. conducted the study and wrote the manuscript. H.Z. analyzed the data. W.L. and X.Y. performed the literature search. W.F. and Q.T. critically revised the manuscript. Each author made a substantial contribution to the forthcoming work. Funding This work was supported by the National Science Foundation of China (Grant No.: 82070362) and the Jilin Province Special Project of Medical and Health Talents (Grant No.: JLSWSRCZX2023-9). Competing interests The authors declare no competing interests. Correspondence and requests for materials should be addressed to Q.T. 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Recent advances in probiotic breads; a market trend in the functional bakery products. Crit. Rev. Food Sci. Nutr. 1–12 10.1080/10408398.2023.2261056 (2023). Kothari, D., Patel, S. & Kim, S. K. Probiotic supplements might not be universally-effective and safe: A review. Biomed. Pharmacother . 111 , 537–547 (2019). Wastyk, H. C. et al. Gut-microbiota-targeted diets modulate human immune status. Cell . 184 , 4137–4153e14 (2021). Liao, W. Z. et al. Association between coffee and caffeine intake and risk of COPD: Findings based on NHANES 2007–2012. Heart Lung . 67 , 53–61 (2024). Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx Table4.docx Onlinefloatimage1.png Abstract graph NHANES findings on the association between dietary live microbes intake, cardiovascular and all-cause mortality. NHANES, National Health and Nutrition Examination Survey. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5333788","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":382403158,"identity":"55b24b17-67c1-434c-9379-cb26fc72535f","order_by":0,"name":"Jingyue Wang","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jingyue","middleName":"","lastName":"Wang","suffix":""},{"id":382403160,"identity":"79da367d-bb3b-4d56-baa3-a7ceba5baa20","order_by":1,"name":"Huicong Zhang","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Huicong","middleName":"","lastName":"Zhang","suffix":""},{"id":382403167,"identity":"d02d2bfc-64bf-46c0-ad14-65cfd792a871","order_by":2,"name":"Wenyun Liu","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Wenyun","middleName":"","lastName":"Liu","suffix":""},{"id":382403169,"identity":"624c724d-4b70-47dc-9800-c58d1640c21b","order_by":3,"name":"Xinyu Yang","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Yang","suffix":""},{"id":382403171,"identity":"3e0c0c8b-b198-4a99-bb6b-6bd01e23006d","order_by":4,"name":"Wenbin Fu","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Wenbin","middleName":"","lastName":"Fu","suffix":""},{"id":382403173,"identity":"7d68ec97-3ff5-402e-82e8-4d4a98eb39b4","order_by":5,"name":"Qian Tong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYNACAwYGfobDDUAWM2HFPDAtkg0HSdIC0nWAkUgt9uy9h1+8KbDLMz54sE2CocI6sYH97AH8tvCcS7OcY5BcbHYApOVMemIDT14Cfi0SOWbGPAbMidtAWhjbDic2SPAYEKOlPnFzA0jLP+K0GD/mMTicuIEBpKWBGC1nzpgxzjE4njjjwMFmi4Rj6cZtPDn4tbC39xh/ePOnOrF/xuGDNz7UWMv2s5/BrwUI2CTAkSNxgIEhAcQlpB4ImD+AtfA3EKF2FIyCUTAKRiQAANvFRgQW9JkzAAAAAElFTkSuQmCC","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Qian","middleName":"","lastName":"Tong","suffix":""}],"badges":[],"createdAt":"2024-10-25 16:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5333788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5333788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71003829,"identity":"dc5c0e54-5ffb-480f-92bb-905fb4463bb3","added_by":"auto","created_at":"2024-12-10 06:07:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96098,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of the sample selection from NHANES 2005–2018. NHANES, National health and nutrition examination surveys.\u003c/p\u003e","description":"","filename":"floatimage254.png","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/09496fa4b6ae3716286ea4c9.png"},{"id":71005117,"identity":"68cf437b-f244-4e45-a1f2-4c584aa7c83e","added_by":"auto","created_at":"2024-12-10 06:15:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57286,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan–Meier survival curves showing the association between dietary live microbe levels and (A) all-cause mortality and (B) cardiovascular mortality.\u003c/p\u003e","description":"","filename":"Onlinefloatimage313.png","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/0ed13705bf236431d0d68f7b.png"},{"id":71005121,"identity":"b0067974-f188-4059-af59-2a7b556bf1ab","added_by":"auto","created_at":"2024-12-10 06:16:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":668819,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/cccb193e-4d77-4aba-9084-b7db8b571e68.pdf"},{"id":71003830,"identity":"c48ebb34-5bb3-4b01-912a-c8fc28a60869","added_by":"auto","created_at":"2024-12-10 06:07:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":55447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/dd8644e22dee7870d0bae159.docx"},{"id":71003833,"identity":"533cf512-9765-4a39-9553-e1afe3f9f791","added_by":"auto","created_at":"2024-12-10 06:07:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33521,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/76858e0a2e6bee77716319e7.docx"},{"id":71003832,"identity":"15287ecb-2afc-4b2b-894e-a4506e5e2d91","added_by":"auto","created_at":"2024-12-10 06:07:55","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":45911,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/04c53248dcc369a5f1ed2598.docx"},{"id":71005118,"identity":"2c102e3c-5632-4c02-a5d6-45d4ee8cc942","added_by":"auto","created_at":"2024-12-10 06:15:55","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":45263,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/25456ed2003924417d520519.docx"},{"id":71003835,"identity":"dc29889b-7a67-4272-bf43-989ab78f3d9e","added_by":"auto","created_at":"2024-12-10 06:07:55","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":62398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbstract graph\u003c/strong\u003e NHANES findings on the association between dietary live microbes intake, cardiovascular and all-cause mortality. NHANES, National Health and Nutrition Examination Survey.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5333788/v1/3d5952a9ccbb364e57db7517.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dietary live microbes intake and its association with cardiovascular and all-cause mortality: Shreds of evidence from NHANES 2005-2018","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe human gut microbiota comprises complex communities of bacteria, viruses, protozoa, and fungi, which interact with multiple human systems and play a critical role in regulating their functions. Recent animal and clinical studies have shown that imbalances in the gut microbiota may affect human health and vice versa. Consequently, there is growing recognition of the potential of the gut microbiota as a promising target for dietary and therapeutic interventions along the microbiota-gut-X axis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Previous studies have demonstrated that live microbes in the diet, such as beneficial bacteria or yeasts, can be ingested to improve digestive health and enhance immunity. These microorganisms are naturally present in certain foods like yogurt, kefir, and sauerkraut and are also available in supplement form \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe 20th century's epidemiological shift saw a decline in deaths and disabilities caused by communicable diseases alongside a rise in non-communicable diseases (NCDs). Among NCDs, cardiovascular disease (CVD) has emerged as the primary cause of mortality and morbidity globally\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. High mortality rates due to cardiovascular diseases and other causes place a considerable strain on healthcare systems and the economy \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Lowering cardiovascular and all-cause mortality aligns with global health goals, such as the United Nations' Sustainable Development Goals (SDGs), which aim to reduce premature mortality from non-communicable diseases by one-third by 2030 \u003csup\u003e5\u003c/sup\u003e. It is imperative to seek methods to reduce cardiovascular and all-cause mortality urgently.\u003c/p\u003e\u003cp\u003eA scoping review identified that a microbial dose expressed as CFU/day was linked to non-negative reported outcomes. Positive outcomes were mainly observed in older population groups, with a median age of 39 \u003csup\u003e6\u003c/sup\u003e. Han et al. found that a high intake of dietary live microbes was associated with a lower prevalence of cardiovascular disease (CVD), with significant associations explicitly observed when the analysis focused on stroke and heart attack \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, the exact relationship between dietary live microbes intake and all-cause or cardiovascular mortality remains uncertain \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study aims to utilize a nationally representative sample from the National Health and Nutrition Examination Surveys (NHANES) and employ Marco's classification system to define and estimate dietary live microbes intake to investigate the association between dietary live microbes intake and all-cause mortality and cardiovascular mortality, as well as to analyze differences across various demographic characteristics and disease state \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eStudy\u0026nbsp;design and population\u003c/h2\u003e\n\u003cp\u003eNHANES is conducted by the Centers for Disease Control and Prevention (CDC), which \u0026nbsp; systematically gathers, analyzes, and shares data that accurately represents the health and nutrition of the national population. It is a large-scale survey project aimed at assessing the health and nutritional status of the U.S. population \u003csup\u003e10\u003c/sup\u003e. Our analysis was based on data from participants in the NHANES 2005\u0026ndash;2018 cycle. We applied the following exclusion criteria: missing follow-up time and unavailable data on dietary live microbes. After using these criteria, 36,720 participants were included in the final analysis (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1.\u0026nbsp;\u003c/strong\u003eFlowchart of the sample selection from NHANES 2005\u0026ndash;2018. NHANES, National health and nutrition examination surveys.\u003c/p\u003e\n\u003ch2\u003eCalculation\u0026nbsp;of explanatory variable: dietary live microbes\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eEnergy and nutrient intake was estimated using 24-hour dietary recall data linked to the Food and Nutrient Database\u0026nbsp;of the\u0026nbsp;Department of Agriculture\u0026nbsp;(USDA). Experts\u0026nbsp;(Maria L Marco, Mary E Sanders, Robert Hutkins, and Colin Hill )\u0026nbsp;evaluated 9,388 food codes\u0026nbsp;in the NHANES database\u0026nbsp;to estimate live microbes content\u0026nbsp;colony-forming units per gram (CFU/g) \u0026nbsp;for foods, categorizing foods as low\u0026nbsp;(\u0026lt;10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eCFU/g), medium\u0026nbsp;(10\u003csup\u003e4\u003c/sup\u003e\u0026ndash;10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003eCFU/g), or high\u0026nbsp;(\u0026gt;10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003eCFU/g)\u0026nbsp;in microbial count\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e. These classifications were designed to represent pasteurized foods\u0026nbsp;or foods processed at high temperatures\u0026nbsp;(low levels), unpeeled fresh fruits and vegetables (medium levels), and unpasteurized fermented foods and probiotic supplements (high levels)\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;Based on these estimates, participants were grouped into low, medium, or high dietary live microbes intake categories, consistent with prior research\u0026nbsp;\u003csup\u003e9,13,14\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDefinition of outcome variables: all-cause mortality and cardiovascular mortality\u003c/h2\u003e\n\u003cp\u003eThe study\u0026apos;s primary outcome was all-cause mortality, encompassing deaths from any cause\u003csup\u003e8\u003c/sup\u003e. The secondary outcome explicitly focused on cardiovascular mortality, referring to deaths associated with CVD. Mortality data, including specific causes of death, were sourced from the publicly available NHANES public use linked mortality file. Participant mortality status was ascertained by linking survey data with death certificate records from the National Death Index (NDI). The causes of death were classified according to the International Classification of Diseases, 10th Revision (ICD-10) coding system\u0026nbsp;\u003csup\u003e15\u003c/sup\u003e. Cardiovascular deaths were categorized by the National Center for Health Statistics (NCHS) as those resulting from heart disease (ICD-10 codes I00\u0026ndash;I09, I11, I13, and I20\u0026ndash;I51) or cerebrovascular disease (ICD-10 codes I60\u0026ndash;I69),\u0026nbsp;based on previous literature\u0026nbsp;\u003csup\u003e8,10,16,17\u003c/sup\u003e. The Centers for Disease Control and Prevention (CDC) has validated this classification, commonly used in official reports\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e. In this study, participants were tracked from the baseline interview date until their date of death or December 31, 2019, whichever came first\u0026nbsp;\u003csup\u003e8,10\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eAssessment of covariates\u003c/h2\u003e\n\u003cp\u003eCovariate data were collected through baseline questionnaires, physical examinations, and laboratory tests\u003csup\u003e10\u003c/sup\u003e.\u0026nbsp;Age,\u0026nbsp;BMI, coffee intake, race /ethnicity, sex, marital status, poverty income ratio (PIR), education level, NHANES cycle,\u0026nbsp;diabetes, hypertension, history of CVD,\u0026nbsp;alcohol drinking, and\u0026nbsp;smoking status\u0026nbsp;were identified as potential confounders based on published research and clinical expertise\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e. Race/ethnicity data were self-reported and categorized into five groups: Mexican American, non-Hispanic White, non-Hispanic Black, other Hispanic, and others (including multiracial individuals)\u003csup\u003e10\u003c/sup\u003e. Educational attainment was stratified into three levels: less than high school, high school or equivalent, and beyond high school\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e. The\u0026nbsp;\u0026nbsp;PIR\u0026nbsp;was determined by dividing the monthly family income by the poverty thresholds defined by the Department of Health and Human Services\u0026nbsp;(HHS)\u0026nbsp;guidelines. This ratio was then categorized into three groups: \u0026lt;1.3 (low income), 1.3\u0026ndash;3.5 (middle income), and \u0026gt;3.5 (high income)\u003csup\u003e19\u003c/sup\u003e.\u0026nbsp;Marital status was categorized into four groups: married, never married, living with a partner, and other (e.g., widowed, divorced, or separated). Self-reported CVD history included diagnoses of heart failure, coronary heart disease, angina, myocardial infarction, or stroke\u0026nbsp;\u003csup\u003e8\u003c/sup\u003e. Diabetes\u0026nbsp;was defined as a physician diagnosis, glycohemoglobin\u0026nbsp;levels \u0026gt;6.5%, fasting glucose \u0026ge;7.0 mmol/L, random\u0026nbsp;or\u0026nbsp;2-hour oral glucose tolerance test glucose levels \u0026ge;11.1 mmol/L, or current use of diabetes medication/insulin\u0026nbsp;\u003csup\u003e10\u003c/sup\u003e. Hypertension was characterized by a systolic blood pressure of \u0026ge;140 mmHg or a diastolic pressure of \u0026ge;90 mmHg,\u0026nbsp;based on ICD-10 codes\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e. Smoking status was classified into three categories:\u0026nbsp;(i)\u0026nbsp;never smoked (fewer than 100 cigarettes),\u0026nbsp;(ii)\u0026nbsp;former smoker (more than 100 cigarettes but has quit), and\u0026nbsp;(iii)\u0026nbsp;current smoker (more than 100 cigarettes and currently smoking). Caffeine intake over the 24-hour period was estimated using the USDA Food and Nutrient Databases for Dietary Studies (FNDDS) 5.0, as provided in the NHANES database\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e.\u0026nbsp;Participants were divided into\u0026nbsp;five\u0026nbsp;categories based on their weekly alcohol consumption: non/former/mild drinker, moderate drinker, or heavy drinker, with classifications defined and adjusted based on sex\u003csup\u003e22\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eIn line with NHANES analytical guidelines, the complex sampling design and mobile examination center sample weights were incorporated into the analysis\u0026nbsp;\u003csup\u003e9,23\u003c/sup\u003e. Participant characteristics were summarized according to their pattern of live microbes intake. Categorical variables were presented as frequencies and percentages, while continuous variables were reported as means with standard errors (SE). A two-sample Student\u0026apos;s t-test was used to compare normally distributed continuous variables between the two groups. At the same time, the Wilcoxon rank-sum test was applied for non-normally distributed continuous variables. Categorical variables were analyzed using the \u0026chi;\u0026sup2;\u0026nbsp;or Fisher\u0026apos;s exact\u0026nbsp;test\u003csup\u003e8\u003c/sup\u003e.Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using the Cox proportional hazards model\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;Model 1 was the unadjusted model. Model 2 was adjusted for age, gender, race/ethnicity, PIR, marital status, and education level. Model 3 included the same adjustments as Model 2, with additional adjustments for NHANES cycles, physical activity, smoking status, BMI, history of CVD, hypertension, and D.M. In\u0026nbsp;subgroup\u0026nbsp;analyses, stratified analyses were conducted by age group (60\u0026ndash;69, 70\u0026ndash;79, or \u0026ge;80 years), gender (female or male),\u0026nbsp;race/ethnicity (Mexican American, non-Hispanic Black, non-Hispanic White, other Hispanic, or others), CVD history (yes or no), diabetes (yes or no), hypertension (yes or no), and prebiotic/probiotic intake (yes or no). Interaction effects were tested by adding an interaction term to the regression model to assess the influence of one variable on the outcome concerning another variable\u0026nbsp;\u003csup\u003e9,24\u003c/sup\u003e.The Kaplan-Meier method was employed to estimate cumulative incidence and time-to-event for all-cause mortality in the overall population and the\u0026nbsp;CVD\u0026nbsp;subgroup. Survival curves between different groups were compared using the log-rank test. These models enable the estimation of cumulative incidence probabilities for cardiovascular mortality while accounting for competing events\u0026nbsp;\u003csup\u003e8,25\u003c/sup\u003e.All analyses were conducted using R, version 4.4.0, with the survey package (version 4.1-1). Statistical significance was defined as a two-sided\u0026nbsp;\u003cem\u003eP\u003c/em\u003e-value of less than 0.05\u0026nbsp;\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eBaseline population characteristics\u003c/h2\u003e\n\u003cp\u003e36,720 \u0026nbsp;participants were involved, with an average age of 46.647 years and a gender split of 18,506 (50.960%) female patients to 18,506 (49.040%) male patients.\u0026nbsp;Age, BMI, coffee take, race/ethnicity, \u0026nbsp;sex, marital status, PIR, education levels, diabetes, hypertension, CVD, alcohol drinking, smoking, and mortality status were significantly different between the three groups (all \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eWeighted analyses of the participants showed that those in the medium dietary live microbes group were more likely to be older, non-Hispanic White, have a lower BMI, have a higher education level, be married or living with a partner, have a higher PIR, and be non-smokers. The high dietary live microbes group also had better overall health, with lower prevalence rates of diabetes, hypertension, and CVD and lower mortality rates. Conversely, the low intake group had non-Hispanic Black participants, lower education levels, higher rates of smoking, and a higher prevalence of chronic conditions such as CVD, diabetes, and hypertension. These participants also exhibited poorer physical health and lower socioeconomic status (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003ch2\u003eAssociations of dietary live microbes\u0026apos; intake with all-cause and cardiovascular mortality\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eK-M survival analyses showed a significant difference in the incidence of all-cause mortality and cardiovascular mortality between the three groups during follow-up, the lowest all-cause mortality being in the high dietary live microbes intake group (log-rank \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). The details of the K-M survival analyses are presented in\u003cstrong\u003e\u0026nbsp;Fig. 2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;A\u003c/strong\u003e and \u003cstrong\u003eFig. 2B\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2.\u003c/strong\u003e Kaplan\u0026ndash;Meier survival curves showing the association between dietary live microbe levels and (A) all-cause mortality and (B) cardiovascular mortality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003epresents the results of the sample-weighted Cox regression models evaluating the association between dietary live microbes intake and mortality outcomes. In the crude model (Model 1), both the medium and high dietary live microbes intake groups showed significantly lower hazard ratios for all-cause mortality compared to the low dietary live microbes intake group (Medium: HR = 0.89, 95% CI, 0.81\u0026ndash;0.98,\u0026nbsp;\u003cem\u003eP\u003c/em\u003e = 0.02; High:\u0026nbsp;HR = 0.69, 95% CI, 0.61\u0026ndash;0.77,\u0026nbsp;\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). After adjusting for age, sex, and race/ethnicity in Model 2, the associations strengthened, particularly in the high dietary live microbes intake group (HR\u0026nbsp;= 0.65, 95% CI, 0.58\u0026ndash;0.73,\u0026nbsp;\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). In the fully adjusted model (Model 3), which further accounted for marital status,\u0026nbsp;PIR, education level, BMI, diabetes, hypertension, CVD history, alcohol consumption, coffee intake, and smoking status, the high\u0026nbsp;dietary live microbes\u0026nbsp;intake group continued to show a significantly reduced risk of all-cause mortality (HR\u0026nbsp;= 0.85, 95% CI, 0.76\u0026ndash;0.96,\u0026nbsp;\u003cem\u003eP\u003c/em\u003e = 0.01).\u003c/p\u003e\n\u003cp\u003eRegarding cardiovascular mortality, the high\u0026nbsp;dietary live microbes\u0026nbsp;intake group exhibited a notable reduction in risk across all models, with the strongest association observed in Model 2 (HR\u0026nbsp;= 0.70, 95% CI, 0.55\u0026ndash;0.89,\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eP\u003c/em\u003e = 0.003). The medium\u0026nbsp;dietary live microbes\u0026nbsp;intake group was also significantly associated with lower cardiovascular mortality in the fully adjusted model (HR\u0026nbsp;= 0.82, 95% CI, 0.69\u0026ndash;0.99,\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e= 0.04). Trend tests indicated a significant dose-response relationship for all-cause and cardiovascular mortality, with increasing dietary live microbes intake linked to progressively lower mortality risk (\u003cem\u003eP\u0026nbsp;\u003c/em\u003efor trend \u0026lt; 0.05 in all models). These findings suggest that higher dietary live microbes intake is consistently associated with reduced mortality risk, independent of potential confounding factors.\u003c/p\u003e\n\u003ch2\u003eSubgroup analysis of the association between dietary live microbes intake with all-cause and cardiovascular mortality\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Table 4\u003c/strong\u003e present the results of subgroup analyses examining the association between dietary live microbes intake and all-cause mortality (\u003cstrong\u003eTable 3\u003c/strong\u003e) and CVD mortality (\u003cstrong\u003eTable 4\u003c/strong\u003e) across various demographic and health-related factors, including smoking, age, sex, diabetes, PIR, hypertension, marital status, education level, and race/ethnicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eshowed that participants in the medium and high dietary live microbes intake groups generally exhibited lower HR for all-cause mortality compared to the low intake group. This trend was consistent across most subgroups, including both smokers and non-smokers, males and females, and individuals with and without diabetes or hypertension. There is a significant difference in the impact of dietary live microbes on all-cause mortality between different age groups (\u0026le;60 years and \u0026gt;60 years, \u003cem\u003eP\u003c/em\u003e for interaction = 0.001), which suggests that age is a vital interaction factor, with dietary live microbes potentially offering more potent protective effects for the younger group (\u0026le;60 years).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003e revealed that participants in the medium and high dietary live microbes intake groups generally exhibited lower HR for CVD mortality compared to the low intake group, particularly among females and those with a lower PIR. The reduction in CVD mortality risk was more pronounced in females across both medium (HR = 0.713, 95% CI, 0.557\u0026ndash;0.914) and high intake groups (HR = 0.701, 95% CI, 0.518\u0026ndash;0.948), while males showed a weaker association, especially in the medium intake group (HR = 1.027, 95% CI, 0.809\u0026ndash;1.305). Additionally, those with a PIR below 1.3 experienced a significant reduction in risk in the medium intake group (HR = 0.724, 95% CI, 0.541\u0026ndash;0.969), with a trend towards reduced risk in the high intake group, though it was not statistically significant. However, no significant interaction effects were observed across the stratified variables, indicating that the beneficial impact of higher dietary live microbes intake on CVD mortality is generally consistent across different population subgroups, regardless of variations in these factors.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this nationally representative cross-sectional study, we identified various vital factors influencing the intake of live bacteria in a reasonable diet. We found that participants in the medium and high dietary intake of live microbes groups had significantly lower all-cause mortality rates than those in the low dietary intake group. Notably, this association was more pronounced among elderly participants. Similarly, the prognosis for cardiovascular diseases was better in the medium and high dietary live microbes intake groups. Our findings provide compelling evidence that food, particularly the role of microbes in health, serves as medicine, offering noninvasive and easy ways to enhance health and extend lifespan.\u003c/p\u003e\u003cp\u003eDespite the limited research on the overall dietary intake of live bacteria, the Mediterranean diet is widely acknowledged as a healthy eating pattern that has been demonstrated to promote beneficial alterations in gut microbiota.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.The NU-AGE 1-year dietary intervention across five European countries supports the feasibility of improving the habitual diet to modulate the gut microbiota, which in turn has the potential to promote healthier aging \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The diversity and abundance of the gut microbiota are among the factors influencing human health. In unhealthy populations, gut microbiota diversity is reduced, and its composition significantly differs from healthy individuals. The microorganisms found in fresh produce and fermented foods exhibit a wide range of diversity, influenced by factors such as food type, source, and degree of processing. This study demonstrates that dietary intake of medium or high levels of live microbes (sourced from unpeeled fresh fruits and vegetables, 10\u003csup\u003e4\u003c/sup\u003e–10\u003csup\u003e7\u003c/sup\u003e CFU/g or \u0026gt; 10\u003csup\u003e7\u003c/sup\u003e CFU/g)) is associated with improved survival, highlighting the importance of reasonable foods as a source of beneficial bacteria\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe mechanism of association between the medium and high dietary live microbes and cardiovascular and all-cause mortality may be as follows. (a) Probiotics are \"live microorganisms that, when taken in sufficient quantities, provide health benefits to the host.\" Numerous fermented products act as sources of probiotic strains. The effectiveness of probiotics is influenced by various factors, including the interactions between probiotic bacteria and the host's microbiome\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In recent decades, probiotics have grown significantly, both for enhancing general gut health and as biotherapeutic agents to address clinical disorders linked to dysbiosis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. For example, the research indicates that after consuming fermented milk, the ileal microbiota in humans is temporarily dominated by probiotics for several hours, suggesting that the ingested probiotic strains have sufficient time to interact with and stimulate host cells [33] continuously. (b) Hill et al. found foods with higher microbial concentrations are associated with modest health improvements across a range of outcomes, such as blood pressure, C-reactive protein, plasma glucose, plasma insulin, triglyceride, waist circumference, BMI, high-density lipoprotein cholesterols\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. (c) Wastyk et al. revealed diet-specific effects. A high-fiber diet boosted glycan-degrading enzymes without altering microbial diversity, while three distinct immune responses were linked to baseline microbiota diversity. In contrast, a high-fermented diet increased microbiota diversity and reduced inflammation. These findings suggest that fermented foods could help reduce microbiome diversity and increase inflammation in industrialized societies \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, due to the limited sample size in this study and the potential impact of confounding factors, these findings should be interpreted with caution. Further research involving well-designed prospective studies is essential to explore this topic more thoroughly.\u003c/p\u003e\u003cp\u003eSubgroup analyses showed that the association was stable across smoking, sex, diabetes, PIR, hypertension, marital status, education level, and race/ethnicity stratification. However, the interaction for age was statistically significant, which may be attributed to the unique physiological changes associated with aging in older individuals. Therefore, it is crucial to evaluate whether the benefits of consuming live microbes surpass any potential risks \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Nonetheless, the heterogeneity of the association observed in subgroup analyses warrants testing in a larger population \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Moreover, socioeconomic factors are crucial in the differences in live microbes intake in a balanced diet. Differences in access to healthcare, education, and socioeconomic resources can significantly influence individuals' acceptance of the concept of food-medicine homology, ultimately affecting the quality of their diet.\u003c/p\u003e\u003cp\u003eOur study is the first to explore the association between dietary live microbes, distinct from specific fermented foods or probiotics, and cardiovascular and all-cause mortality in a broadly representative U.S. population. In this study, the stringent quality control procedures and the complex sampling design employed by NHANES enabled us to evaluate the association between the intake of live microbes and mortality. As a result, enhancing the intake of dietary live microbes could be viewed as a novel dietary approach to mitigating mortality. Furthermore, it may be crucial to evaluate the intake of live microbes. However, this observational study also had several limitations that should be pointed. First, due to this study's cross-sectional design, we could not identify the temporal and causal relationship between dietary live microbes and cardiovascular and all-cause mortality and the possibility for reverse effect bias should be considered. Second, the relatively simplistic categorization method for dietary live microbes may not accurately reflect the intake of various microorganisms. Third, since this study was conducted solely on a U.S. population, caution is needed when extrapolating the findings to other ethnic groups. Moreover, although we accounted for demographic characteristics, lifestyle factors, and certain medical conditions, residual mediators and modifiers cannot be entirely ruled out. Finally, considering individual differences within the population, assessing whether the benefits of dietary live microbes intake outweigh the potential drawbacks is necessary. Prospective studies are crucial to validate its effectiveness in reducing mortality. Mendelian randomization should be employed to obtain more robust and reliable results. Additionally, further research is necessary to establish the optimal dosage of dietary live microbes and to assess their impact on mortality across various diseases and health conditions \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study shows that eating more foods rich in live microbes is linked to lower risks of dying from any cause or from heart disease. The results were consistent across different groups of people, suggesting that consuming more live microbes could be a simple way to improve health. However, more studies are needed to confirm these findings and to explore the best ways to use live microbes to reduce the risk of death.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003e\u003cp\u003eBMI Body mass index\u003c/p\u003e\u003cp\u003eCDC Centers for Disease Control and Prevention\u003c/p\u003e\u003cp\u003eCFU Colony-forming units per gram\u003c/p\u003e\u003cp\u003eCI Confidence interval\u003c/p\u003e\u003cp\u003eCVD Cardiovascular disease\u003c/p\u003e\u003cp\u003eFDA Food and Drug Administration\u003c/p\u003e\u003cp\u003eFNDDS Food and Nutrient Databases for Dietary Studies\u003c/p\u003e\u003cp\u003eHHS Department of Health and Human Services\u003c/p\u003e\u003cp\u003eHR Hazard ratios\u003c/p\u003e\u003cp\u003eICD-10 International Classification of Diseases, 10th Edition\u003c/p\u003e\u003cp\u003ePIR Pverty income ratio\u003c/p\u003e\u003cp\u003eNCHS National Center for Health Statistics\u003c/p\u003e\u003cp\u003eNDI National death index\u003c/p\u003e\u003cp\u003eNHANES National Health and Nutrition Examination Surveys\u003c/p\u003e\u003cp\u003eSDGs United Nations' Sustainable Development Goals\u003c/p\u003e\u003cp\u003eSE Standard error\u003c/p\u003e\u003cp\u003eUSDA United States Department of Agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData availability\u003c/h1\u003e\n\u003cp\u003e\u0026nbsp;The National Health and Nutrition Examination Survey data is accessible through the National Center for Health Statistics at the Centers for Disease Control and Prevention (https://www.cdc.gov/nchs/nhanes/index.htm).\u003c/p\u003e\n\u003ch2\u003eDeclaration of competing interest \u0026nbsp;\u003c/strong\u003e\u003c/h1\u003e\n\u003cp\u003eThe authors report no relationships that could be construed as a conflict of interest.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h1\u003e\n\u003cp\u003eWe thank the National Health and Nutrition Examination Survey participants and staff and the National Center for Health Statistics for their invaluable contributions. We also (granted publication licenses) acknowledge the BioRender (www.biorender.com).\u003c/p\u003e\n\u003ch2\u003eAuthorship contributions\u003c/h1\u003e\n\u003cp\u003eJ.W. conducted the study and wrote the manuscript. H.Z. analyzed the data. W.L. and X.Y. performed the literature search. W.F. and Q.T. critically revised the manuscript. Each author made a substantial contribution to the forthcoming work.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h1\u003e\n\u003cp\u003eThis work was supported by the National Science Foundation of China (Grant No.: 82070362) and the Jilin Province Special Project of Medical and Health Talents (Grant No.: JLSWSRCZX2023-9).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h1\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to Q.T.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h1\u003e\n\u003cp\u003eThe study protocol received approval from the NCHS institutional review board. 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Association between coffee and caffeine intake and risk of COPD: Findings based on NHANES 2007\u0026ndash;2012. \u003cem\u003eHeart Lung\u003c/em\u003e. \u003cb\u003e67\u003c/b\u003e, 53\u0026ndash;61 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NHANES, Cardiovascular Mortality, All-cause mortality, Dietary live microbes","lastPublishedDoi":"10.21203/rs.3.rs-5333788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5333788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the relationship between dietary live microbes intake and the risk of cardiovascular and all-cause mortality using data from 36,720 participants in the NHANES 2005-2018. Dietary live microbes intake was classified into low, medium, and high categories based on 24-hour dietary recall. All-cause mortality was the primary outcome, with cardiovascular mortality as the secondary. Kaplan-Meier survival analysis and Cox proportional hazards models, adjusted for confounders, were applied in R software (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Higher intake groups showed significantly lower all-cause and cardiovascular mortality (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). The Cox models confirmed reduced all-cause mortality risk in medium (HR = 0.89) and high intake groups (HR = 0.69) compared to low intake. Cardiovascular mortality was also lower in the high intake group (HR = 0.70). Subgroup analyses revealed consistent benefits, with a stronger effect in younger individuals. These findings highlight the potential of dietary live microbes in reducing mortality, warranting further prospective studies.\u003c/p\u003e","manuscriptTitle":"Dietary live microbes intake and its association with cardiovascular and all-cause mortality: Shreds of evidence from NHANES 2005-2018","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 06:07:50","doi":"10.21203/rs.3.rs-5333788/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ccd84f7d-649f-4cc1-9fdc-49db3641f99f","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40725824,"name":"Biological sciences/Microbiology"},{"id":40725825,"name":"Health sciences/Health care"}],"tags":[],"updatedAt":"2024-12-10T06:07:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-10 06:07:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5333788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5333788","identity":"rs-5333788","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}