Hand, foot, and mouth disease (HFMD), a gastrointestinal contagious disease that mostly affects individuals under the age of 10 years, is one of the top three class C notifiable infectious diseases in China (Ying et al., 2019). Hence, HFMD poses a great threat to pediatric and neonatal populations in Asian countries.

The HFMD pathogens, mainly enteroviruses A71(EV71) and coxsackievirus A16 (CVA16), target the gastrointestinal (GI) epithelium and utilize intestinal bacteria to enhance multiplication, pathogenesis, and transmission and thereby lead to development of the disease. Additionally, the gut microbiota has been reported to work in conjunction with the intestinal barrier to orchestrate a defense network that impedes the invasion of pathogens and maintains the homeostasis and functionality of the gut (Sanz and De, 2009; Odenwald and Turner, 2017; Takiishi et al., 2017; Robinson, 2019). Dysbiosis of the gut microbiota could also lead to intestinal barrier disruption by influencing the self-renewal of epithelial cell and the secretion of the mucus layer and tight junctions of intestinal epithelial cells, which could increase the susceptibility of the host to immune or inflammatory disorders, including enterovirus infection (Wu et al., 2013; Winglee et al., 2014; Jakobsson et al., 2015; Schuijt et al., 2015; Sun et al., 2016; Heidrich et al., 2018; Alam and Neish, 2018). While majority studies had primarily focused on patients in epidemiological and virological studies, the role of the fecal bacterial microbiome in HFMD progression remains barely undocumented. Thus far, only limited studies with very small sample sizes have suggested the existence of gut microbiota dysbiosis in children with HFMD, and larger population studies are urgently needed to reveal the relationship and the underlying mechanisms. Probiotics and prebiotics are widely utilized as complements to nutrition strategies designed to reinforce intestinal immunity. Several studies have provided scientific evidence of the benefits resulting from the use of probiotics and prebiotics for intestinal diseases. Currently, researchers are considering the use of probiotics and prebiotics for the prevention of HFMD, but no related studies have been conducted to date. It is necessary to assess whether the use of probiotics and prebiotics exhibits potential preventive efficacy on intestinal flora in children with HFMD.

Here, we conducted a two-site recruitment and investigated 749 participants (including 262 HFMD patients and 487 healthy children) from two regions in Guangdong, China. By collecting their information and fecal samples, we aimed to explore the differences in gut microbiota communities between children with HFMD and healthy children and the potential effects of probiotics and prebiotics on HFMD infection.

HFMD has become an emerging public health problem in Asia, but there is currently no vaccine to protect against the responsible viruses. The present study aimed to seek answers to unanswered questions related to the gut microbiota. The differences in the gut microbiota patterns of patients with HFMD compared with those of the healthy group and the effect of synbiotics supplements on the intestinal flora of patients with HFMD were explored. We observed that the gut microbiota were altered in children with HFMD. Furthermore, children with HFMD who were administered synbiotics supplements exhibited reduced morbidity, and their gut microbiota showed potential resistance to HFMD-related gut flora dysbiosis.

First, the distribution characteristics of the gut microbiota of patients with HFMD differed from those of the gut microbiota of the healthy group. A reduction in bacterial diversity, which is considered to indicate an unhealthy state, has been observed in children with HFMD (Rivera-Chávez et al., 2016; Sassone-Corsi et al., 2016; Kriss et al., 2018). This result was consistent with previous research, which demonstrated that changes in the diversity of butyrate-producing bacteria and inflammation-inducing bacteria are associated with HFMD. In the present study, several bacteria were found to differ between the HFMD and control groups. Our results showed that Prevotella and Streptococcus were found at higher relative abundances in patients with HFMD than in the healthy group. Prevotella is widely considered a commensal genus, and a series of controversial reports on its pathogenicity have been published (Brook, 1998; Brook, 2004; Nagy, 2010). For example, Prevotella copri, a widely distributed species, has been linked to inflammatory diseases and intestinal barrier dysfunction by directly promoting the upregulation of inflammatory cytokines. Additionally, Prevotella is a lipopolysaccharide-producing anaerobic gram-negative bacterium that can cause inflammatory reactions and damage the gut barrier (Hofstad et al., 1993; Choi et al., 2011). Streptococcus, which is one of the most classic opportunistic pathogens, comprises a wide variety of pathogenic bacterial strains. Streptococcus is one of the most prevalent inhabitants of the respiratory tract (Engholm et al., 2017), skin surface (Ho et al., 2021) and gut (Sokol et al., 2017; Ooi et al., 2018) of adults and children. Studies have shown that influenza virus can interact with S. pneumoniae and induce susceptibility to viruses by activating cytokines and dendritic cells (Short et al., 2012). The enrichment of Streptococcus has also been observed in the gut microbiota of patients with viral hepatitis cirrhosis (Wang et al., 2019). The cause of HFMD is enteral virus infection, e.g., infection with EV71 and CVA16. A study found that EV71 is transported from the intestine to peripheral tissue and other organs, which suggests that the small intestine is a gateway for EV71 infection in vivo (Feng et al., 2020). EV71 infection could reduce the expression of goblet cell-derived mucins (Good et al., 2019), which indicates that EV71 infection could alter the luminal environment of the gut microbiota. Previous data have confirmed that EV71 infect and replicate in intestinal epithelial cells by activating the p38 mitogen-activated protein kinase (MAPK) and protein kinase signaling pathways. This inflammatory pathway overlaps with the inflammatory reactions caused by bacterial lipopolysaccharide or translocation. In addition, EV71 is most frequently associated with severe diseases (Shu et al., 2015). In our study, the gut microbiota of patients infected with EV71 were more dysbiotic than that of patients with CVA16. Hence, in HFMD pathogenesis, the observed gut dysbiosis might interact with enteroviruses.

In contrast, the relative abundances of Faecalibacterium and Bifidobacterium were decreased in the feces of the HFMD group. These two bacterial genera, which are generally considered producers of short-chain fatty acids, are reportedly capable of regulating intestinal epithelial nutrition, stabilizing immune homeostasis and reinforcing intestinal barrier functions (Hiippala et al., 2018; Routy et al., 2018). Bifidobacteria protect gut mucus against diet-induced microbiota-mediated deterioration, and gut barrier defects (Schroeder et al., 2018). Bifidobacteria have also been identified as health-promoting genera that increases butyrate production and thus contributes to improvements in the gut barrier and metabolic outcomes (Seganfredo et al., 2017). Similarly, Faecalibacterium exhibits an enriched polyphenol-rich dietary pattern, which could improve gut barrier function (Del Bo' et al., 2020), and the prophylactic use of Faecalibacterium could also prevent the acute breakdown of the colonic epithelial barrier (Lapiere et al., 2020). Thus, in both the research and healthcare product markets, Bifidobacterium is a very common commercialized probiotics, and Faecalibacterium shows strong promise as effective and safe probiotics.

In clinical practice, probiotics and prebiotics are widely used as supplements for healthy children or are included in therapeutic approaches for many diseases (Krumbeck et al., 2016). In this study, we found that children who were administered supplements exhibited a lower risk of HFMD-related infection, whereas the application of probiotics or prebiotics alone was not found to be significantly associated with HFMD risk. This very interesting finding indicates that synbiotics was related to an improved protective effect. A series of studies conducted by Professor D. A. Mills from the University of California Davis revealed that Bifidobacterium can process the prebiotics in human milk oligosaccharides and that the colonization of the probiotics Bifidobacterium breve could also be influenced by prebiotics (Underwood et al., 2017). These studies suggested a potential close association between probiotics and prebiotics, and this association is also being investigated by Professor Mills’s group. The intake of prebiotics may modulate the composition of the microbiota, be fermented by the beneficial gut microbiota and increase the abundance of probiotics (Gibson and Roberfroid, 1995; Pandey et al., 2015). Synbiotics, defined as combinations of probiotics and prebiotics, could benefit the host by modulating the gut microbiota (Gibson and Roberfroid, 1995). Previous studies have reported that synbiotics exert protective or preventive effects against gastrointestinal or other diseases by maintaining the gut flora structure (Shimizu et al., 2018; Tian et al., 2018). Similarly, in our study, the beta diversity analysis showed that the composition of the gut flora of patients applied synbiotics was closer to that of the control group than to those of the other patient groups. The analysis of the abundances of identified biomarkers indicated their relative restoration and depletion in patients applied synbiotics compared with those found in the other patient groups. These gut microbiota alterations suggested that the gut microbiota of the children who received synbiotics supplements could acquire resistance to HFMD-related gut flora changes, which might manifest as a reduction in HFMD susceptibility.

Our study has some limitations. In the present study, we only collected information regarding whether the children were administered supplements, including probiotics and prebiotics. However, the specific types and dose of probiotics or prebiotics are unknown. Besides, prebiotics are also found in food (Roberta, et al., 2020), especially those that contain complex carbohydrates, such as fiber and resistant starch. Diet information wasn’t collected and dietary prebiotics assessment were required in further study. Another major limitation is that all the samples from the HFMD group were collected after infection. Thus, the analysis could only suggest a potential bacterial resistance in the children who were administered synbiotics. However, the existence of this resistance in the gut microbiota should be validated in future follow-up studies with repeated and larger populations sampled before and after infection.

To our knowledge, the present study involves the largest HFMD cohort used for gut microbiota analyses thus far. We found that the gut microbiota of children with HFMD exhibits dysbiosis, and synbiotics supplements might provide a potential protective effect against HFMD. However, further studies with a larger population and detailed information are needed to validate our findings and explore the underlying mechanisms of HFMD. In conclusion, our study indicated the changes in the composition of the gut flora of patients with HFMD and identified potential bacterial biomarkers of HFMD. Additionally, we provided new evidence showing that synbiotics might lower the susceptibility to HFMD by modulating the gut microbiota.

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.ebi.ac.uk/ena/browser/view/PRJEB45719.

The studies involving human participants were reviewed and approved by Ethical Review Committee of Chinese Center for Disease Control and Prevention. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

WW, HZ, and XY designed the study. ZR, CZ, TL, YW, and SC processed the samples. XG and ZL searched literature, analyzed the data, and drafted the manuscript. WW, HZ, and XY reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was funded by the Science and Technology Planning Project Foundation of Guangzhou city (202102080584).

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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