With the rapidly expanding global aged population, the proportion of psychiatric disorders in middle-aged and elderly individuals has dramatically increased. According to the Global Burden of Disease Study 2017 there were 1.13 million incident schizophrenia (SZ) cases in 2017 globally, while the number of newly diagnosed SZ cases was 0.29 million in China (He et al., 2020), ranking the first in the world. In 2010, there were 7.16 (6.57-7.75) million people in China affected by SZ during their lifetime (Chan et al., 2015). SZ is considered a severe and complex mental illness characterized by positive and negative symptoms, including delusions, hallucinations, thought disorder, apathy, and avolition as well as cognitive and functional impairment. Reports have shown that SZ prevalence changes with age in an inverted U-shape, with the highest value at around 40 years and nearly 71% of total cases being aged 25-54 years (Zhou et al., 2021). Due to SZ epidemiological characteristics, aging SZ patients are easy to ignore. With high stress and psychosocial burden for caregivers and a high suicide rate, SZ has become a major public problem, especially for affected elderly patients (Yu et al., 2021). Despite its low prevalence, the burden of SZ remains large and continues to increase, thereby increasing the burden on healthcare systems.

SZ is a complex psychiatric neurodevelopmental disorder with uncertain etiology. During the past decades of research, genetic, environmental, and behavioral risk factors, including development, industrialization, and urbanization have been proposed to be associated with the onset of SZ (Ma et al., 2020a). Recently, the role of non-human genetic factors such as gut microbiota has been identified in the pathophysiology of SZ (Nguyen et al., 2019; Pełka-Wysiecka et al., 2019; Li et al., 2020; Ma et al., 2020b; Pan et al., 2020; Zhu et al., 2020a; Zhu et al., 2020b; Li et al., 2021a; Yuan et al., 2021; Fan et al., 2022; Munawar et al., 2022). Well-balanced gut microbiota is critical to maintaining host general health. Mounting evidence suggests that the gut microbiota can modulate brain function and behavior via the “microbiota-gut-brain” axis via multiple routes including the vagus nerve, immune system, short-chain fatty acids and tryptophan, while its alterations may actively contribute to the SZ pathogenesis and development. Hsiao et al. have demonstrated that the gut microbiota can affect various complex behaviors, including social, emotional, and anxiety-like behaviors, and modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders (Hsiao et al., 2013). Zhu et al. have also found that transplantation of gut microbiota from drug-free SZ patients causes SZ-like abnormal behaviors and dysregulated kynurenine metabolism in mice (Zhu et al., 2020a), which provided direct evidence that the dysbiosis of the gut microbiome may have a causal role in the development of SZ. Although most of these causal studies are conducted in animal models, they also provide valuable clues for investigating the roles and underlying mechanisms of the gut microbiota in the development of SZ. Additionally, the gut microbiota undergoes a progressive age-related physiological succession of species across the life-cycle, and the aging gut microbiota is distinct from other healthy age groups (Ling et al., 2020b). Our previous study has established the structural and functional bacterial and fungal dysbiosis of fecal microbiota in elderly patients with Alzheimer’s disease (Ling et al., 2020c; Ling et al., 2020d). Several above-mentioned studies have shown gut microbiota alterations at various stages of SZ; however, most of these participants were non-elderly SZ patients. Thus, it is important to explore the alterations of the gut microbiota in Chinese elderly SZ patients, which can elucidate the non-invasive SZ diagnosis and novel gut microbiota-based alternative therapy.

This study aimed to explore the overall structure and composition of the fecal microbiota in the elderly SZ patients without gastrointestinal diseases hospitalized in a large Chinese geriatric ward from Lishui by using the 16S rRNA gene high-throughput MiSeq platform. Additionally, we also investigated the correlations between SZ-associated key functional bacteria and clinical indicators, which would provide novel targets for the early, non-invasive diagnosis and personalized treatment of SZ and the development of tailor-made psychobiotics designed for Chinese elderly SZ patients.

During the past decade, the new concept “microbiota-gut-brain axis” strongly connected the gut microbiota and the brain function emerging as a new focal point of biomedical research. A growing body of evidence indicates that the gut microbiota plays a crucial role in the complex bidirectional communication between the gut and the brain, which profoundly influences neural development, neuroinflammation, activation of the stress response, and neurotransmission, in addition to modulating complex behavior, such as sociability and anxiety. Since 2006, multi-omics sequencing and analytical technologies have provided valuable insights regarding the taxonomic diversity of the gut microbiota on a much deeper level and demonstrated its novel associations with human diseases. Recent advances in gut microbiota studies have expanded beyond simply profiling microbiota compositions and have been increasingly characterizing microbial functions by using functional meta-omics approaches and deciphering the cross-talk between the gut microbiota and host in detail Ling et al., 2022. With these developed omics techniques, our previous studies have found that the gut microbiota dysbiosis leading to changes in the bidirectional relationship between gut microbiota and the nervous system is linked to the pathogenesis of several neuropsychiatric diseases, such as major depressive disorder, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis and so on (Jiang et al., 2015; Ling et al., 2020a; Ling et al., 2020c; Ling et al., 2020d; Sun et al., 2020; Sun et al., 2021; Wu et al., 2021). Different from previous genetic and environmental findings, the alterations of the gut microbiota provide novel insights into the pathogenesis of these neuropsychiatric disorders. The dysfunctional gut microbiota can translate into brain phenotypes including psychiatric symptoms and cognitive deficits via the microbiota-gut-brain axis. A previous study has shown that the gut microbiota can control the expression of various neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), which can affect neural development and brain plasticity (Sudo et al., 2004). Recently, Li et al. have also observed a correlation between the gut microbiota and the brain structure and function in SZ (Li et al., 2021a). Since the gut microbiota is an ecosystem adjustable by genetic and environmental factors, these altered gut key functional bacteria can be used as novel targets for the diagnosis and treatment of psychiatric disorders and clarify their pathogeneses.

Being different from most previous microbiome studies on young and adult SZ patients, this study enrolled elderly SZ patients to explore the alterations of the gut microbiota and the host immune response. During the aging process, human gut microbiota underwent physiological succession and changed gradually across the life cycle. Compared with the age-matched elderly healthy controls, we found that the overall structure of the fecal microbiota (β-diversity) was different between SZ patients and healthy controls, although the α-diversity indices were not significantly different between the two groups. However, Yuan et al. have demonstrated that the first-episode unmedicated young SZ patients showed lower α-diversity (the Shannon and Simpson’s indices) compared to healthy controls (Yuan et al., 2021). The disparity between the two Chinese studies might have been caused by age-related differences in the gut microbiota baseline. Similar to our findings, Nguyen et al. have also found that the α-diversity indices were not significantly changed in the adult SZ Americans (Nguyen et al., 2021). Antipsychotics can influence the gastrointestinal hypomotility of SZ patients, leading to antipsychotic-induced constipation. Xu et al. have found that the antipsychotic-induced constipation group had a significantly increased α-diversity in observed species, Chao 1, and ACE as compared to the non-constipation group (Xu et al., 2021). The altered microbiota diversity might affect the gut-brain axis and influence the etiology and clinical manifestation of SZ.

Additionally, our results from 16S rRNA sequencing demonstrated significant changes in microbial composition in elderly SZ patients. SZ was associated with profound changes in the fecal microbiota, with an increase in Bacteroidetes and Actinobacteria and a decrease in Firmicutes on the phylum level. Although the changing patterns were not always similar to our findings, other previous studies have also shown that the compositional changes of the human gut microbiota were closely linked to SZ (Schwarz et al., 2018; Shen et al., 2018; Zheng et al., 2019; Zhu et al., 2020b). Indeed, the relative abundances and differences in taxa have shown extensive variation from study to study. In elderly SZ patients, we found that the abundance of several representative butyrate-producing genera, such as Roseburia, Faecalibacterium, Coprococcus and Butyricicoccus, were significantly reduced. Interestingly, our previous microbiome studies in multiple sclerosis and AD have also demonstrated similar changing microbiota profiles (Ling et al., 2020a; Ling et al., 2020d). Our correlation analyses also found that these butyrate-producing bacteria were positively and negatively correlated with the pro- and anti-inflammatory cytokines, respectively, indicating that these bacteria can modulate host immune response. Our findings suggested that these representative butyrate-producing genera might play crucial roles in preventing these neuropsychiatric diseases, although the biological role of these key functional bacteria still needs to be clarified deeply. In fact, the PiCRUSt analysis also revealed the altered functional pathways, such as fatty acid biosynthesis, in the SZ-associated microbiota. Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, were produced by various commensal bacteria in the human gut. SCFAs production and regulation have been proposed as one of the mechanisms by which probiotics promote health outcomes (Gargari et al., 2016). The imbalance of the gut microbiota in elderly SZ patients, especially the reduction of butyrate-producing bacteria, was accompanied by a decrease in the synthesis of butyrate subsequently. Among these three major SCFAs, butyrate has received particular attention for its beneficial effects on gut homeostasis and energy metabolism. Numerous in vitro and in vivo studies have shown that butyrate plays a vital role in modulating immune and inflammatory responses and gut mucosal barrier function (Tan et al., 2014). Recently, butyrate has been demonstrated to act as a signaling molecule to communicate between the gut, gut microbiota, and the brain, which can directly or indirectly modulate brain functions through immune, endocrine, vagal, and other humoral pathways. There is ample evidence showing that butyrate can cross the blood-brain barrier, and its concentrations in wet brain samples are about an order of magnitude higher than those in the peripheral blood, which will help to shape the brain immune milieu (Kim et al., 2013; Sun et al., 2016). Greenhill et al. have found that butyrate plays an essential role in regulating synaptic plasticity and cortical development (Greenhill et al., 2015). Animal studies have also shown that modulation of butyrate can improve cognitive performance and influence hippocampal neurogenesis (Liu et al., 2015; Val-Laillet et al., 2018; Yang et al., 2018). The impact of gut-derived butyrate on the brain has reinforced the notion of the existence of the microbiota-gut-brain axis. The level of butyrate in the gut and brain may be critical in determining the effects on behavioral and psychophysiological processes. Stilling et al. have found that supraphysiological doses of butyrate exert potent neuropharmacological effects, facilitating synaptic tagging and capturing (Stilling et al., 2016). A previous study has demonstrated that butyrate can attenuate microglia-mediated neuroinflammation in Alzheimer’s disease by regulating the microbiota-gut-brain axis (Sun et al., 2020). Li et al. have observed that increased serum levels of butyrate after 24-week risperidone treatment might be associated with a favorable treatment response (the reduction in the PANSS score) in drug-naïve, first-episode SZ (Li et al., 2021b). With risperidone monotherapy, the increased butyrate in the serum might be associated with the increased relative abundance of butyrate-producing bacteria in the gut in SZ patients. Our previous study has identified that administration of Clostridium butyricum can increase butyrate levels in feces and in the brain in a mouse model of vascular dementia, resulting in significant attenuation of cognitive impairment and histopathological changes in the hippocampus (Liu et al., 2015). An open-label inpatient pilot clinical trial has found that oligofructose-enriched inulin can increase serum butyrate concentrations in SZ patients and modulate cognitive function in visual learning, processing speed, and attention positively (Kelly et al., 2021). The butyrogenic prebiotic fibers can promote the increase in gut-derived butyrate by increasing the relative abundance of butyrate-producing bacteria. In addition, diet, especially the healthiest Mediterranean diet, in which a person consumes mainly plant-based food, can increase the butyrate-producing bacteria in the gut, thereby modulating butyrate production (Nagpal et al., 2019). These studies have suggested that butyrate supplementation directly or increased butyrate-producing bacteria via manipulating the gut microbiota indirectly can be utilized as a promising and convenient approach to treat SZ by targeting microbiota-gut-brain interactions in the elderly.

Other differential functional bacteria, such as Prevotella, Akkermansia, Streptococcus, Romboutsia and so on, were increased in elderly SZ patients. These genera, such as Prevotella and Actinomyces, were negatively and positively correlated with anti-inflammatory and pro-inflammatory cytokines, respectively, suggesting that these genera might actively participate in the pathogenesis of SZ. These correlation analyses are almost opposite to the findings in butyrate-producing bacteria. These significant increments in SZ genera might promote neuroinflammation and then contribute to cognitive dysfunction, a decline in motivation, and psychosis. Prevotella, a genus with high genetic diversity within and between species, has been considered commensal bacteria due to its extensive presence in the healthy microbiota. Our previous study has also found a decreased proportion of Prevotella in patients with multiple sclerosis (Ling et al., 2020a), whereas its abundance increased after successful treatment. However, increasing evidence has indicated that Prevotella (at least some strains) has been considered clinically important pathobionts that can participate in human disease by promoting chronic inflammation (Ley, 2016). Prevotella exhibits increased inflammatory properties, as demonstrated by the augmented release of inflammatory mediators from immune cells and various stromal cells (Larsen, 2017). There has been compelling mechanistic and causal evidence in mice that Prevotella can promote inflammatory disease features. By activating toll-like receptor 2, Prevotella can lead to the production of Th17-polarizing cytokines by antigen-presenting cells, including IL-23 and IL-1. Recently, Agarwala et al. have also shown a five-fold increase in Prevotella in autistic children (Agarwala et al., 2021). Prevotella-mediated local inflammation leads to systemic dissemination of inflammatory mediators, bacteria, and bacterial products, which in turn may affect gut-brain disorder outcomes. There is still a need for more studies to ascertain a causal and potential SZ-triggering role of Prevotella, which can help to open up novel therapeutic targets for SZ. Another SZ-associated genus, intestinal mucin layer-degrading Akkermansia, was significantly increased in elderly SZ patients. Similar to our previous study, we have also found increased Akkermansia in other gut-brain disorders, such as AD (Ling et al., 2020d). Akkermansia, an acetate producer, has been considered a next-generation probiotic candidate with significant prospects for its application. The beneficial roles of Akkermansia (typical strain is Akkermansia muciniphila) in metabolic diseases, inflammatory bowel disease and tumor immunotherapy has been widely recognized. However, this study found that Akkermansia should not always be considered a potentially beneficial bacterium since it might be harmful for the gut-brain axis in the context of SZ development in the elderly. Our previous study has also found that the traditional probiotic Bifidobacteria, another acetate producer, also participated in the development of AD (Ling et al., 2020d). Nishiwaki et al. have demonstrated that Akkermansia is significantly increased in patients with PD across countries (Nishiwaki et al., 2020). Akkermansia can degrade the mucus layer of the gut, leading to increased intestinal permeability (gut leakiness) in these elderly patients (Forsyth et al., 2011).

Previous studies have shown that increased bacterial translocation due to the increased intestinal permeability and innate immune imbalance are both presented in patients with SZ (Severance et al., 2013; Maes et al., 2019). As aforementioned, the different SCFAs profiles produced by these key functional bacteria might play distinct roles in the pathophysiology of various neurological conditions. Specifically, butyrate, but not acetate, can improve gut integrity, attenuate pro-inflammatory cytokine expression in microglia, and reduce peripheral inflammation concurrent to decreased brain neuroinflammation (Matt et al., 2018). A previous study has identified that increased acetate mediates the microbiome-brain-β-cell axis to promote metabolic syndrome (Perry et al., 2016). Pérez-Pérez et al. have also found that plasma acetate correlates with disability and immune response in multiple sclerosis (Pérez-Pérez et al., 2020). In combination with our previous microbiome studies in gut-brain disorders, the discrepancy in SCFAs profiles provided clues to how the gut microbiota might modulate the pathophysiology of SZ. Further studies are warranted to elucidate the relationships between gut-derived SCFAs and neuroinflammation in these elderly SZ patients. These new findings will elucidate the novel therapeutic options for these elderly SZ patients targeting the regulation of the SCFAs profiles.

The current diagnostic criteria of SZ emphasize mainly clinical symptoms and signs. However, the absence of pathognomonic clinical features or specific laboratory tests has made SZ diagnosis more subjective. Thus, it is urgent to find biomarkers for SZ diagnosis. This study found that bacterial dysbiosis was involved in the development of SZ in the elderly. The characteristics of the gut microbiota could be used as a clinical biomarker for SZ. These differential key functional genera, such as Faecalibacterium, Roseburia, Actinomyces, Butyricicoccus, and Prevotella, and the Faecalibacterium/Bifidobacterium ratio could be used as biomarkers to distinguish between SZ and healthy controls. Interestingly, the five genera in combination could significantly improve predictive performance when compared with those single genera. In another SZ metagenomic study, Zhu et al. have constructed and validated a discriminatory model using the deferential microbial species, which can clearly distinguish SZ patients from healthy controls (Zhu et al., 2020b). This discriminatory model provides an accurate and objective method for the diagnosis of SZ. Our previous studies have also shown that those deferential key functional genera could be used as diagnostic biomarkers for the diagnosis of neuropsychiatric diseases (Ling et al., 2020a; Ling et al., 2020d). In combination with other indicators, the gut microbiota, especially these key functional bacteria, could help to improve the accuracy of SZ diagnosis.

However, several limitations of this study should be mentioned. First, analysis of the bacterial 16S rRNA gene V3-V4 regions had a limited resolution in terms of bacterial species identification. The 16S rRNA gene amplicon sequencing used in this study generally captured reliable taxonomic classification at the genus level. Further metagenomic studies in elderly SZ patients would provide more information on the alterations of the gut microbiota. Second, this was a single-center study with a small sample size. Future multicenter microbiota studies should include larger cohorts to verify our conclusions. Third, this study only identified several SZ-associated key functional bacteria and predicted their roles in the development of SZ. However, their biological role requires further investigation in future in vitro and in vivo studies. Furthermore, clinical studies with fecal microbiota transplantation or specific psychobiotic strains administration might help to clarify the relationship between the gut microbiota and SZ more clearly.

In summary, the gut microbiota in elderly SZ patients significantly changed when compared with healthy controls. Although the α-diversity indices did not alter significantly, the β-diversity analysis, such as PCoA, could divide the SZ patients and healthy controls into two different clusters, suggesting structural dysbiosis in SZ patients. The LEfSe identified several changing genera, such as Faecalibacterium, Roseburia, Actinomyces, Butyricicoccus, Prevotella, and so on, demonstrating that the differences in the overall composition of the fecal microbiota between SZ patients and healthy controls were evident. These altered key functional bacteria could be used as non-invasive biomarkers to discriminate SZ patients from healthy controls. Targeting on these altered key functional bacteria through a personalized diet or intervention from beneficial microbiota may be useful for patient-tailored early intervention in SZ cases. In addition, the functional dysbiosis in SZ-associated fecal microbiota also suggests that the changed fecal microbiota is associated with decreased fatty acid biosynthesis and bile acid biosynthesis, and is correlated with the altered host immunity, which might play vital roles in the pathogenesis and development of SZ. Thus, our investigation of the fecal microbiota using a large and confirmed SZ cohort provides novel insights into disease pathogenesis, which could provide new approaches to managing neurodevelopmental disorders by modulating the gut microbiome.

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 in the article/ Supplementary Material .

The studies involving human participants were reviewed and approved by the Ethics Committee of Lishui Second People’s Hospital (Zhejiang, China). The patients/participants provided their written informed consent to participate in this study.

ZL, XL and LZ conceived and designed the experiments. ZL, GJ, XY, YC, LS, XL and QS performed the experiments. ZL, LS and XL analyzed the data. ZL, LS and XL wrote the paper and edited the manuscript. The final manuscript was read and approved by all authors. All authors contributed to the article and approved the submitted version.

This present work was funded by the grants of Key R&D Program of Zhejiang (2022C03060), the Research Fund for Lin He Academician New Medicine (17332005), the S&T Major Project of Lishui (2017ZDYF15), the National Natural Science Foundation of China (81771724, 31700800, 81790631), Zhejiang Basic Public Welfare Research Project (LGF20H090016), the Research Project of Jinan Microecological Biomedicine Shandong Laboratory (JNL-2022033C), the Taishan Scholar Foundation of Shandong Province (tsqn202103119), the Nutrition and Care of Maternal & Child Research Fund Project of Guangzhou Biostime Institute of Nutrition & Care (2019BINCMCF045), the National S&T Major Project of China (2018YFC2000500), and the Foundation of China’s State Key Laboratory for Diagnosis and Treatment of Infectious Diseases.

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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