Frailty is a common geriatric syndrome with multiple causes and is characterized by increased vulnerability and functional impairment that decreases the ability to deal with stress (Morley et al., 2013). This health status mainly includes the loss of muscle mass and strength, reduced endurance, decreased physiological reserves, and cognitive impairment (Morley et al., 2013; Wilson et al., 2017; Pansarasa et al., 2019). Older adults with frailty are more prone to experience a series of clinical adverse events such as falls, disability, depression, institutionalization, and mortality (Pansarasa et al., 2019). Frailty is associated with an increased demand for long-term care and high medical costs (Hoogendijk et al., 2019). The prevalence rate of frailty among older adults in the community is 4.0%–59.1%, increases with age, and affects women more commonly than men (Collard et al., 2012).

Age-related changes to the immune system, including immune senescence and “inflammaging,” are thought to play a role in the frailty and sarcopenia process (Wilson et al., 2017). Levels of circulating inflammatory mediators increase with age, and the primary cause of this age-related inflammation may be related to microbial dysbiosis. Animal experiments from Thevaranjan et al. had shown that age-associated microbial dysbiosis promotes intestinal permeability and systemic inflammation (Thevaranjan et al., 2017). The gut microbiota, which is also referred to as the “the second genome” influences the human health status and has characteristics of adaptability and plasticity (Kim and Jazwinski, 2018; Durack and Lynch, 2019). Machine learning algorithms can be used to establish microbiome profiles that may predict the age of healthy individuals, indicating that gut microbiota may serve as a marker for aging-related research (Galkin et al., 2018). However, there are relatively few studies on the relationship between intestinal dysbacteriosis and frailty.

Here, we analyzed the serum levels of the inflammatory cytokines IL-6 and HGMB1 and the intestinal permeability biomarker zonulin in older adults living in communities. We characterized and compared the microbial community composition between frail older adults and healthy controls using 16S rRNA gene sequencing and analyzed the relationships between fecal microbiota and clinical characteristics.

Soysal et al. analyzed the relationship between inflammation and frailty through a systematic review and meta-analysis and concluded that frailty and prefrailty are associated with higher inflammatory parameters, especially CRP and IL-6. This is in agreement with the results of our studies. The reason for frailty-related inflammation is not clear. Previous studies have shown that age-associated microbial dysbiosis can lead to aging-related inflammation and early mortality in mice. The process may involve increasing intestinal permeability and translocation of bacterial components, further causing a state of immune activation, systemic inflammation, and damage of cellular antibacterial function (Thevaranjan et al., 2017). The zonulin family is a group of proteins that correlates with increased epithelial permeability by regulating intercellular tight junctions (Barbaro et al., 2020). In this study, we show that the levels of serum inflammatory cytokines and intestinal permeability of community-dwelling older adults with frailty are higher than those of healthy controls.

A previous report investigated the gut microbiota changes associated with frailty in a younger community-dwelling population and found a negative correlation between early frailty and gut microbiota diversity (Jackson et al., 2016). Higher diversity is generally considered to be related to health, and reduced diversity is associated with health issues such as metabolic disease (Lim et al., 2017), Alzheimer’s disease (Calvani et al., 2018), and inflammatory bowel disease (Matsuoka and Kanai, 2015; Zuo et al., 2018). Our analysis suggests that the richness and uniformity of the gut microbiota in the two groups are not significantly different. Age- and gender-matched older adults were selected as controls in our study. Many observational studies have demonstrated that gut microbiota changes consist of reduced diversity and overexpression of pathobionts as people age (Kim and Jazwinski, 2018; Calvani et al., 2018). Aging-related diseases, including frailty, are based on the degeneration of body organs, and the gut microbiota is thought of as a forgotten organ in human health and disease. Physical frailty and sarcopenia (PF&S) show remarkable clinical overlap and share multisystem disorders (Wilson et al., 2017). No significant differences in alpha diversity between PF&S and age- and gender-matched non-PF&S older adults were noted, and these results are in line with our study (Picca et al., 2019).

The commensal genera such as Faecalibacterium, Prevotella, Roseburia, Megamonas, and Blautia were decreased in the frail group. Faecalibacterium prausnitzii, the only species of the genus Faecalibacterium, is a highly abundant gut microbiota with anti-inflammatory and immunoregulatory properties in healthy human individuals (Laursen et al., 2017). It is considered to be a marker of gut health. Prevotella is a dominant genus member of the Bacteroidetes and is more widespread in individuals with non-Western lifestyles (Ley, 2016; Tett et al., 2019). The role of Prevotella to the body has remained controversial and might be dependent on diet. Previous studies have shown that improved glucose metabolism and insulin tolerance induced by dietary fiber is associated with an increased abundance of Prevotella (Kovatcheva-Datchary et al., 2015). Roseburia is an anaerobic bacterium belonging to the Clostridium cluster XIVa (Seo et al., 2020), which has been shown to play a major role in maintaining the intestinal barrier function and immune defense (Kasahara et al., 2018; Seo et al., 2020). A reduced abundance of SCFA-producing bacteria Megamonas was also found in heart failure (Hayashi et al., 2018), depression (Cheung et al., 2019), multiple system atrophy (Wan et al., 2019), and Bechet’s disease (Shimizu et al., 2016). However, little research on the relevance of Megamonas in human health has been conducted. In addition, Blautia in the gut microbiota of obese children might help reduce intestinal inflammation (Benitez-Paez et al., 2020).

The frail group was significantly enriched for Parabacteroides and Klebsiella. Parabacteroides distasonis is one of the core bacteria in the human body. A correlation analysis showed that its content was negatively correlated with obesity, nonalcoholic fatty liver disease, diabetes, and other diseases, suggesting that it may play a positive regulatory role in glucose and lipid metabolism (Wang et al., 2019). Weight loss is a sign of frailty syndrome. Cancer cachexia is an extremely frail state involving multiple organs, including weight loss, muscle consumption, and systemic inflammation. Cachectic mice exhibit an increase in Enterobacteriaceae and Parabacteroides (Herremans et al., 2019). Pglyrp-regulated gut microflora P. distasonis can enhance DSS-induced colitis in mice, identifying P. distasonis as colitis-promoting species (Dziarski et al., 2016). Higher levels of stress are associated with increased Crohn’s disease activity. The relative abundances of Parabacteroides are significantly higher in patients with high perceived stress scale (PSS) scores compared with those with low PSS scores. However, whether the abundance variation of Parabacteroides is the cause or a consequence of increased intestinal inflammation remains to be further studied (Mackner et al., 2020). Nakamoto et al. identified Klebsiella pneumoniae in the gut microbiota of patients with primary sclerosing cholangitis that can disrupt the epithelial barrier to initiate bacterial translocation and promote liver inflammatory responses (Nakamoto et al., 2019). Oral pathobionts Klebsiella can also promote colitis by the colonization of the gut and activation of the inflammasome in colonic mononuclear phagocytes (Kitamoto et al., 2020).

Additionally, a higher level of the bacteria Akkermansia was found in the frail group. Akkermansia is a symbiotic bacterium colonizing the mucosal layer that uses mucin as carbon and nitrogen element sources and has been widely disputed in recent years (Zhang et al., 2019). Akkermansia muciniphila is one of the top 20 most abundant species detected in the human gut microbiota (Arumugam et al., 2011; Zhang et al., 2019). Its colonization starts in early life, but the concentration of A. muciniphila in fecal samples was decreased in older adults aged 80 to 82 (Collado et al., 2007). Many researchers also revealed a higher abundance of Akkermansia in centenarians aged 99 to 104 and in semi-supercentenarians aged 105 to 109 (Kong et al., 2016). However, the rate of frailty increases with age, and we should be more concerned about the beneficial effect of microbiota composition in nonfrailty older adults instead of in all older adults. Previous studies have shown that a low-fiber diet promotes the activity and expansion of colonic mucus-degrading bacteria A. muciniphila, as well as increases in the activity of mucus-targeting enzymes, which can damage the intestinal mucosal barrier function and enhance pathogen susceptibility (Desai et al., 2016). Considering that aging is accompanied by multiple organ functional aging, we suspect that a low abundance of Akkermansia may be more conducive to healthy aging. Alistipes are pathogenic in colitis and colorectal cancer (Moschen et al., 2016; Parker et al., 2020) and are related to liver fibrosis (Rau et al., 2018) and signs of depression (Bangsgaard Bendtsen et al., 2012). In hypertension, the increase in Alistipes finegoldii was also observed to be a potential driver for gut barrier dysfunction and inflammation (Kim et al., 2018).

Although our study has very strict inclusion and exclusion criteria to control confounding factors, we cannot completely exclude the possibility that some factors such as comorbidity and lifestyle including smoking and drinking may affect our results, which is a common difficulty in the study of gut microbiota. It should be noted that comorbidity was widespread among older adults with frailty and has been considered synonymous with frailty (Fried et al., 2001). However, many frailty-related diseases such as hypertension and diabetes have been reported to be associated with gut microbiota (Breuninger et al., 2021). We recorded the variables of drinking and smoking at present in the healthy control group and frail group. However, the amount of smoking and drinking and smoking cessation were not recorded. Some researchers have proposed that gut microbial biodiversity was higher in human drinkers than nondrinkers (Kosnicki et al., 2019). Smoking and smoking cessation led to changes in the gut microbiota (Sublette et al., 2020). Therefore, it is not clear how these factors affect our results, which is one of our main limitations. This problem needs to be clarified by matching these variables between the groups being compared.

There are some other general limitations to our study. Firstly, our work is based on a small sampling of older adults living in a community. Thus, the generalization of these findings must be carefully considered, and studies in different populations worldwide are required to confirm these data. Secondly, this study was cross-sectional based on community, and a longitudinal study might provide more robust evidence. Thirdly, fecal microbiota may not provide or reflect the whole bowel microbial environment, and the stool consistency, which is strongly associated with gut microbiota richness and composition, was not recorded in this study. Fourthly, the 16S rRNA gene sequencing method exhibits limited efficacy, as it cannot provide direct data of gene function and more in-depth analysis of species. Analyses of gut microbiota changes using metagenomics sequencing are therefore required. Due to the limited sample size, the predictive power of 20 genera random forests has not been evaluated. Fifthly, some gastrointestinal symptoms such as abdominal pain, constipation, and diarrhea that may be related to gut microbiota composition were not recorded in this study.

In conclusion, the results of the present study indicate that the inflammatory cytokines IL-6 and HGMB1 and the intestinal permeability biomarker zonulin were significantly increased in older adults with frailty. We provide a detailed comparison of gut microbiota in both frail participants and healthy controls using high-throughput sequencing and found that the bacterial community composition was significantly different from that of healthy controls. Some of the gut microbiota have a close association with inflammatory and intestinal mucosal permeability biomarkers. Focusing on a target for the treatment methods based on gut microbiota adjustment may represent an effective therapy for preventing and managing frailty. To develop more reliable microbiome biomarkers of frailty, large-scale prospective studies and rigorous animal experiments should be conducted.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: (https://www.ncbi.nlm.nih.gov/), PRJNA704230.

The studies involving human participants were reviewed and approved by Affiliated LiYuan Hospital of Tongji Medical College, Huazhong University of Science and Technology. The patients/participants provided their written informed consent to participate in this study.

Clinical analyses and manuscript writing: YX and DC. Sample collection and DNA extraction: HL, YD, ZH, HY, and JH. Statistical analyses: YW and YX. Sequencing analyses and management: MW and XJ. Project supervision and manuscript revision: ZX. Study design, project management, financial support, and manuscript revision: ZX. All authors contributed to the article and approved the submitted version.

This study was supported by the National Key Research and Development Program of China (2018YFC2002000).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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