Patient enrollment and 16S ribosomal RNA (16S rRNA) sequencing

A total of 40 stool samples were collected, comprising 20 fecal samples from pediatric patients with AGE (mean age, 16.9 months; range, 0–91 months) and 20 stool samples from disease-free infants or children (negative controls; mean age, 17.4 months; range, 0–54 months). Of the 20 patients, 15 patients had a norovirus infection and 5 had a rotavirus infection, including 15 with severe AGE (Vesikari score >10)15 and 5 with mild-to-moderate AGE (Vesikari score ≦ 10). The 15 complication presentations comprised 10 AGE with convulsion (7 norovirus, 3 rotavirus infections), 2 necrotizing enterocolitis (norovirus infections), 2 gastroenteritis with renal failure or severe electrolyte imbalance (norovirus infections), and 1 gastroenteritis with chronic diarrhea of prolonged duration (>3 weeks) and malnutrition (norovirus infection). Antibiotics were administered in 2 infants with necrotizing enterocolitis before surgery and a further 1 infant with renal failure. All fecal samples were collected at least 1 week after discontinuation of the antibiotics. Enteric bacteria in the 40 fecal samples were classified into 9 phyla, 62 families, and 159 genera based on 16S rRNA sequences and metagenomic analysis of all selected reads.

Diversity of the intestinal microbiota in children with severe gastroenteritis

The Shannon diversity indices of the fecal samples were calculated and reported as entropy scores16. These are indicative of the intestinal microbiota composition and disruption of the microbiota, as scores are lower in the presence of overgrowth of a single bacterial species. The entropy score of the intestinal microbiota of the normal controls, and patients with mild-to-moderate and severe AGE were 0.56 (0.47–0.73), 0.7 (0.56–0.93), and 0.3 (0.05–0.6), respectively (Fig. 1A). The Shannon diversity index (entropy score) of the intestinal microbiota was significantly lower in patients with severe AGE than in normal controls (P = 0.017) and patients with mild-to-moderate AGE (P = 0.011).

Figure 1: Diversity of microbiota among healthy controls and AGE patients with different severity. Statistical analysis showed significantly decreased Shannon diversity (entropy score) of intestinal microbiota in severe viral AGE, when compared to normal controls (P = 0.017) and to patients with mild to moderate severity (P = 0.011) (A). The entropy score of the rotavirus infection was 0.05 (0.02–0.35), that was significantly lower than norovirus infection group (P = 0.048) (B). Full size image

The average severity score of the 5 patients with rotavirus infection was 13.8 (range, 11–15), which is significantly higher than that of the 15 patients with norovirus infection (mean, 10.3; range, 8–11) (P = 0.03). The entropy score in patients with norovirus infection was 0.65 (0.2–0.75), and was not significantly different compared with that of the normal controls. The entropy score of patients with rotavirus infection was 0.05 (0.02–0.35), which was significantly lower than that of patients with norovirus infection (P = 0.048) (Fig. 1B).

The intestinal microbiota composition at the phylum level of normal controls, and patients with mild-to-moderate and severe AGE is shown in Fig. 2A. The microbiota composition in patients with rotavirus or norovirus infection and the associated disease severity classification are shown in Fig. 2B. There was no significant difference in microbiota composition between the normal controls and patients with AGE at the phylum level. The feces of normal controls exhibited significant abundance of Rikenellaceae (P = 0.043) and Porphyromonadaceae (P = 0.02) at the family level, and Alistipes (P = 0.038) and Parabacteroides (P = 0.019) at the genus level compared with any severity of AGE patients.

Figure 2: Intestinal microbiota in family level in healthy controls and AGE patients with various severity and caused by rotavirus or norovirus. The intestinal microbiota distrubtion in phylum level of normal control, mild, and servere disease groups of acute viral gastroenteritis (A). The microbiota distribution in patients with rotavirus or norovirus infections and their disease severity classifications (B). Full size image

Clinical correlation

The correlations of the intestinal microbiota composition with clinical manifestations were evaluated (Table 1). At the family level, patients with abdominal pain exhibited greater abundance of Prevotellaceae (P = 0.013), Staphylococcaceae (P = 0.013), and Coriobacteriaceae (P = 0.015). Veillonellaceae (P = 0.048) abundance was lower in patients hospitalized for longer than 7 days. The abundance of Micrococcaceae (P = 0.011) and Campylobacteraceae (P = 0.05) was lower in patients with extraintestinal manifestations of viral infection. At the genus level, patients with abdominal pain had greater richness in Prevotella (P = 0.013), TM7 (P = 0.013), Staphylococcus (P = 0.013), and Atopobium (P = 0.017). Patients with convulsion showed absence of Haemophilus (P = 0.045) and with substantially decreased genus of Faecalibacterium (P = 0.059).

Table 1 Different Intestinal Microbiota at Family and Genus Level Assignments by Clinical Symptoms. Full size table

We also analyzed the intestinal microbiota composition of 3 patients with complicated AGE, comprising 1 patient with necrotizing enterocolitis after norovirus infection, 1 with rotavirus infection with convulsions, and 1 with norovirus infection and renal failure (Supplementary Fig. 1). In the norovirus infection patient with necrotizing enterocolitis, the entropy score of the intestinal microbiota was 0.008833, and Streptococcaceae (63%), Enterobacteriaceae (23%), and Pasteurellaceae (6%) comprised >90% of the microbiota. The entropy score of patient with rotavirus infection and convulsions was 0.02779, and Bacteroidaceae comprised almost 100% of the intestinal microbiota in this patient. The entropy score of norovirus infection patient with renal failure was 0.005949, and Enterococcaceae, Clostridiaceae, and Streptococcaceae comprised >95% of the microbiota.

Microbiota of viral AGE patients with and without complications

There were 15 children that experienced complications after viral AGE and 5 without complications. The intestinal microbiota of the children with viral AGE differed from that of healthy controls at the family level (Table 2). The overall differences in healthy children, and those with non-complicated and complicated AGE were in the richness of Porphyromonadaceae in healthy children (P = 0.035) and Streptococcaceae in uncomplicated AGE (P = 0.035), shown by the composition of fecal bacteria. Patients with complicated AGE had greater abundance of Campylobacteraceae (P = 0.0003), Neisseriaceae (P = 0.0115), Methylobacteriaceae (P = 0.0004), Sphingomonadaceae (P = 0.0221), and Enterobacteriaceae (P = 0.0451) than healthy children. Uncomplicated AGE was associated with a decrease in the abundance of Desulfovibrionaceae (P = 0.0198), Ruminococcaceae (P = 0.0125), Veillonellaceae (P = 0.0199) and increase in Carnobacteriaceae (P = 0.0124). Finally, patients with complicated AGE had greater Pasteurellaceae richness (P = 0.0071) than patients with AGE without complications.