Intestinal microbiota profiles in infants with acute gastroenteritis caused by rotavirus and norovirus infection: a prospective cohort study

  • Author Footnotes
    † These authors contributed equally to this study as the first authors.
    Lijing Xiong
    Footnotes
    † These authors contributed equally to this study as the first authors.
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Author Footnotes
    † These authors contributed equally to this study as the first authors.
    Yang Li
    Footnotes
    † These authors contributed equally to this study as the first authors.
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Jing Li
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Jing Yang
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Lihong Shang
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Xiaoqing He
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Lirong Liu
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Yurong Luo
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Xiaoli Xie
    Correspondence
    Corresponding author. Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu 610091, People's Republic of China
    Affiliations
    Department of Paediatric Gastroenterology, Hepatology and Nutrition, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, People's Republic of China
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  • Author Footnotes
    † These authors contributed equally to this study as the first authors.
Open AccessPublished:August 15, 2021DOI:https://doi.org/10.1016/j.ijid.2021.08.024

      Highlights

      • Rotavirus and human norovirus had different effects on intestinal microbiota in infants with acute gastroenteritis.
      • Bacillus was detected as the characteristic genus in infected infants.
      • Disturbance of microbiota may increase the risk of potentially pathogenic bacteria.

      ABSTRACT

      Objective: To compare the intestinal microbiota profiles in infants following rotavirus (RV) and human norovirus (HNoV) infection.
      Methods: Faecal specimens from 18 infants {mean age 11.8 months [standard deviation (SD) 3.0] months} with acute gastroenteritis caused by RV (G9P8) and 24 infants [mean age 8.8 (SD 6.4) months] with acute gastroenteritis caused by HNoV (GII) infection were collected prospectively. The faecal microbiome was assessed by 16S rRNA amplicon pyrosequencing. Alpha diversity, beta diversity, deferentially abundant taxa and microbial functions were assessed by bioinformatic analysis.
      Results: The Chao1 index for the HNoV group was significantly higher compared with the control group (P=0.0003), and was lower for the RV group compared with the HNoV group (P=0.0078). No significant difference in beta diversity was observed between the RV and HNoV groups. The RV group showed greater abundance of Actinobacteria at phylum level and Bifidobacterium spp., Streptococcus spp., Enterococcus spp. and Lactobacillus spp. at genus level. The HNoV group showed richness in Fusobacteria and Cyanobacteria at phylum level, and Enterococcus spp. and Streptococcus spp. at genus level. Bacillus was the characteristic genus in infected infants. In comparison with the control group, the viral group (P≤0.01), the RV group (P=0.002) and the HNoV group (P≤0.01) showed significant differences in potentially pathogenic bacteria.
      Conclusions: Changes in microbiotic structure were observed in infants following RV and HNoV infection. The Chao 1 index of alpha diversity increased significantly in the HNoV group. Bacillus was the characteristic genus in infected infants. An increase in pathogenic bacteria, particularly Streptococcus spp. and Enterococcus spp., was detected in infected infants.

      Keywords

      Introduction

      Diarrhoea in children is a major global public health issue, especially in developing countries, with a high burden of disease. It is the fourth leading cause of mortality in children under 5 years of age. Rotavirus (RV) is the most common cause of diarrhoea-related death in children worldwide (
      • Tate JE
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      ;
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      • Cao S
      • Blacker BF
      • Ahmed T
      • et al.
      Rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5 years.
      ). Seven years of continuous surveillance at 213 sentinel sites in China from 2009 to 2015 indicated that the detection rate of RV in children aged <5 years with diarrhoea was 30%, accounting for 39.5% of inpatient diarrhoea cases and 28.1% of outpatient diarrhoea cases (
      • Yu J
      • Lai S
      • Geng Q
      • Ye C
      • Zhang Z
      • Zheng Y
      • et al.
      Prevalence of rotavirus and rapid changes in circulating rotavirus strains among children with acute diarrhea in China, 2009–2015.
      ). Another major pathogen in infantile viral gastroenteritis is human norovirus (HNoV), the prevalence of which has increased rapidly in recent years (
      • Kirk MD
      • Pires SM
      • Black RE
      • Caipo M
      • Crump JA
      • Devleesschauwer B
      • et al.
      World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis.
      ;
      • Bartnicki E
      • Cunha JB
      • Kolawole AO
      • Wobus CE
      Recent advances in understanding noroviruses.
      ). With the introduction of RV vaccines, the incidence of RV gastroenteritis has decreased, while HNoV has become one of the main causes of acute gastroenteritis (AGE) in children (
      • McAtee CL
      • Webman R
      • Gilman RH
      • Mejia C
      • Bern C
      • Apaza S
      • et al.
      Burden of norovirus and rotavirus in children after rotavirus vaccine introduction, Cochabamba, Bolivia.
      ;
      • Mattison CP
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      • Hall AJ.
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      ).
      Evidence suggests that diarrhoea in infants is closely correlated with the disturbance and imbalance of intestinal microbiota (
      • Becker-Dreps S
      • Allali I
      • Monteagudo A
      • Vilchez S
      • Hudgens MG
      • Rogawski ET
      • et al.
      Gut microbiome composition in young Nicaraguan children during diarrhea episodes and recovery.
      ;
      • The HC
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      • Jie S
      • Pham Thanh D
      • Thompson CN
      • Nguyen Ngoc Minh C
      • et al.
      Assessing gut microbiota perturbations during the early phase of infectious diarrhea in Vietnamese children.
      ). Clinical evidence suggests that oral administration of probiotics during the acute phase could shorten the disease course and reduce the severity of diarrhoea (
      • Allen SJ
      • Martinez EG
      • Gregorio GV
      • Dans LF
      Probiotics for treating acute infectious diarrhoea.
      ;
      • Szajewska H
      • Guarino A
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      • Kolacek S
      • Shamir R
      • et al.
      Use of probiotics for management of acute gastroenteritis: a position paper by the ESPGHAN Working Group for Probiotics and Prebiotics.
      ;
      • Guarino A
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      • Lo Vecchio A
      • Shamir R
      • Szajewska H
      • et al.
      European Society for Pediatric Gastroenterology, Hepatology, and Nutrition/European Society for Pediatric Infectious Diseases evidence-based guidelines for the management of acute gastroenteritis in children in Europe: update 2014.
      ;
      • Lai HH
      • Chiu CH
      • Kong MS
      • Chang CJ
      • Chen CC
      Probiotic Lactobacillus casei: effective for managing childhood diarrhea by altering gut microbiota and attenuating fecal inflammatory markers.
      ). The diversity and structure of intestinal microbiota is closely correlated with the response to the RV vaccine (
      • Harris VC
      • Armah G
      • Fuentes S
      • Korpela KE
      • Parashar U
      • Victor JC
      • et al.
      Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural Ghana.
      ), and the intestinal microbiota may mediate HNoV infection (
      • Baldridge MT
      • Turula H
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      Norovirus regulation by host and microbe.
      ). Therefore, more attention should be focused on the host–intestinal microbiota and pathogen–intestinal microbiota interactions.
      During exclusive breastfeeding, the infant microbiota is influenced by factors such as mode of delivery, type of feeding and antibiotic usage. This diversifies progressively until weaning, when it becomes more like the adult microbiota by becoming more stable and complex. After the complementary food introduction, alpha diversity increases, resulting in the replacement of Proteobacteria and Actinobacteria phyla by Firmicutes and Bacteroidetes phyla as the dominant members of the infant microbiota (
      • Fallani M
      • Amarri S
      • Uusijarvi A
      • Adam R
      • Khanna S
      • Aguilera M
      • et al.
      INFABIO Team. Determinants of the human infant intestinal microbiota after the introduction of first complementary foods in infant samples from five European centres.
      ;
      • Koenig JE
      • Spor A
      • Scalfone N
      • Fricker AD
      • Stombaugh J
      • Knight R
      • et al.
      Succession of microbial consortia in the developing infant gut microbiome.
      ). Meanwhile, due to increased activity, motor development and changes in eating habits, infants are also at the peak stage for intestinal viral infections.
      As such, studying the infant intestinal microbiota following intestinal viral infection and comparing the changes caused by RV and HNoV could facilitate our understanding of their different effects on the infant intestinal microbiota, and help identify potential rare or differentiated species for diagnosis and the development of probiotic therapy. Moreover, it could explain the resultant changes in mucosal immune development.
      To the authors’ knowledge, no studies to date have used the high throughput method to detect and elaborate the infant intestinal microbiota following RV and HNoV infection. As such, this study aimed to determine the intestinal microbiota profiles in infants following RV and HNoV infection, and estimate the potential distinctive species and functional changes after infection.

      Materials and methods

       Patients

      This prospective cohort study compared the intestinal microbiota of infants with AGE caused by RV and HNoV infection and healthy infants in Chengdu, Sichuan Province, a central city in west China. Faecal samples were collected from the subjects on the first day of hospital admission in July 2017 and March 2019. Faecal samples with the G9P8 genotype of RV or the GII genotype of HNoV were selected after initial viral typing. The severity of diarrhoea for each infant was scored using the Vesikari Clinical Severity Scoring System (
      • Ruuska T
      • Vesikari T.
      Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes.
      ). The clinical record of treatment and prognosis were followed-up and documented for further analysis. This study was approved by the Research Ethics Committee of Chengdu Women's and Children's Central Hospital.
      The inclusion criteria were: age 1–59 months; at least three episodes of diarrhoea in a 24-h period; and/or at least one episode of vomiting within a 24-h period with watery changes in stool. The exclusion criteria were: mucus, bloody or purulent stool; white blood cell count >0–5 per high-power field and/or red blood cell count >0–5 per high-power field as revealed by routine stool microscopy; given antibiotics, probiotic formulations or the RV vaccine within the preceding week; and consent to collect samples or clinical data not given by parents.
      In total, 59 stool samples were collected, with 31 faecal samples from AGE cases with RV (G9P8) and 28 faecal samples from AGE cases with HNoV (GII). Faecal samples of the included cases were collected during the acute phase of diarrhoea after a mean duration of 2.61 [standard deviation (SD) 1.91] days. Dimension reduction of Principal co-ordinates analysis (PcoA) based on age group revealed that samples from 5-16-month-olds in the RV group and samples from 2-16-month-olds in the HNoV group produced more reliable results. Therefore, 18 samples [mean age 11.8 (SD 3.0) months] from the RV group and 24 samples [mean age 8.8 (SD 6.4) months] from the HNoV group were selected for comparison with 25 healthy infants [mean age 6.7 (SD 4.3) months] matched by age and feeding mode.

       DNA extraction

      Total bacterial genomic DNA was extracted from samples using the PowerMax DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA), in accordance with the manufacturer's instructions, and stored at −20°C before further analysis. The quantity and quality of extracted DNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.

       16S rRNA amplicon pyrosequencing

      Polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene V4 region was performed using the forward primer 515F(5’-GTGCCAGCMGCCGCGGTAA-3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Sample-specific paired-end 6-bp barcodes were incorporated into the TruSeq adaptors for multiplex sequencing. The PCR mix contained 25 μL of Phusion High-Fidelity PCR Master Mix, 3 μL (10 µM) of each forward and reverse primer, 10 μL of DNA template, 3 μL of DMSO and 6 μL of ddH2O. Thermal cycling consisted of initial denaturation at 98°C for 30 s, 25 cycles of denaturation at 98°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 15 s, with a final extension of 1 min at 72°C. PCR amplicons were purified with Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN, USA) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and paired-end 2 × 150-bp sequencing was performed on the Illumina NovoSeq6000 platform by GUHE Info Technology Co., Ltd. (Hangzhou, China).

       Sequence analysis

      The Quantitative Insights Into Microbial Ecology (QIIME, v2.0) pipeline was used to process the sequencing data. Briefly, raw sequencing reads with exact matches to the barcodes were assigned to the respective samples, and were identified as valid sequences. The low-quality sequences were filtered out, and paired-end reads were assembled using VSEARCH v2.4.4. A representative sequence was selected from each operational taxonomic unit (OTU) using the default parameters. An OTU table was generated to record the abundance of each OTU in each sample and the taxonomy of these OTUs. OTUs representing <0.001% of the total sequences across all samples were discarded. To minimize the difference in sequencing depth across samples, an averaged, rounded rarefied OTU table was generated by averaging 100 evenly resampled OTU subsets that were <90% of the minimum sequencing depth for further analysis.

       Bioinformatic and statistical analysis

      Sequence data were analysed mainly with the QIIME and R packages (v3.2.0). OTU-level alpha diversity indices, such as the Chao1 richness estimator, the Shannon diversity index and the Simpson index, were calculated. Beta diversity analysis was performed to investigate variation in microbial communities across samples, visualized via principal coordinate analysis (PCoA) with non-metric multi-dimensional scaling. Differences in the UniFrac distances for pairwise comparisons between groups were determined using Student's t-test and the Monte Carlo permutation test with 1000 permutations. Principal component analysis (PCA) was also conducted based on the genus-level compositional profiles. Taxon abundance at phylum, class, order, family, genus and species levels was compared between samples or groups using the Kruskal test. Linear discriminant analysis of effect size was performed to detect deferentially abundant taxa across groups using the default parameters. Random Forest analysis was applied to discriminate the samples from different groups. Microbial functions were predicted using phylogenetic investigation of communities by reconstruction of unobserved states. The output file was further analysed using the Statistical Analysis of Metagenomic Profiles software package.
      Statistical analysis of clinical data was performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Statistical analysis of taxonomic data was performed using Chi-squared test or Fisher's exact test. Continuous variables with a normal distribution are presented as mean variance, and statistical analysis was performed using Student's t-test. Continuous variables with a non-normal distribution are presented as median variance, and statistical analysis was performed using the Mann–Whitney non-parametric test. When P<0.05, the difference between the groups was considered significant.

      Results

       Clinical characteristics

      Among the 59 subjects included in this study, 41 were breast-fed (69.49%), nine were artificially fed (12.25%), and nine were both breast-fed and artificially fed (15.25%). Forty-five cases (76.3%) lived in urban areas, and 14 cases (23.7%) lived in rural areas. Scoring using the Vesikari Clinical Severity Scoring System yielded 14 moderate cases and 45 severe cases. Twenty-eight of 31 (90.32%) cases in the RV group were severe, compared with 16 of 28 (57.14%) cases in the HNoV group. At hospital admission, there was no significant difference in the severity of diarrhoea between the RV and HNoV groups, whereas the differences in the extent of emesis and dehydration were significant (Table 1). Following 3–5 days of hospitalization, all the infants recovered within 1 week after disease onset.
      Table 1Severity of the clinical symptoms of infants with viral diarrhoea caused by rotavirus (RV) or human norovirus (HNoV) infection.
      RV (%)HNoV (%)χ2P-value
      Max. number of stools (times/day)1–33 (9.68)4 (14.29)0.3010.86
      4–58 (25.81)7 (25)
      ≥620 (64.52)17 (60.71)
      Diarrhoea duration (days)1–424 (77.42)20 (71.43)0.8560.652
      54 (16.13)3 (10.71)
      ≥63 (9.68)5 (17.86)
      Max number of vomiting episodes (times/day)02 (6.45)18 (64.29)22.1330.000
      113 (41.94)5 (17.86)
      28 (25.81)3 (10.71)
      38 (25.81)2 (7.14)
      Vomiting duration (days)02 (6.45)18 (64.29)24.0250.000
      112 (38.71)7 (25)
      28 (25.81)2 (7.14)
      ≥39 (29.03)1 (3.57)
      Temperature (°C)≤38.413 (41.94)14 (50)0.4190.811
      38.5–38.93 (9.68)2 (7.14)
      ≥3915 (48.39)12 (42.86)
      DehydrationNo7 (22.58)15 (53.57)6.6770.035
      Moderate21 (67.74)10 (35.71)
      Severe3 (9.68)3 (10.71)

       Differences in microbiota structure

      In total, 3,664,535 sequences of 16S rRNA gene amplicons passed the quality filters and were assigned to a taxonomy. Overall, 3949 OTUs were generated, with a mean of 306 (SD 117) OTUs [95% confidence interval (CI) 275–336] per sample overall, 268 (SD 116) OTUs (95% CI 225–310) per sample in the RV group, and 348 (SD 106) OTUs (95% CI 307–389) per sample in the HNoV group. PCoA analysis was performed on samples from the viral and control groups, and there were significant differences in overall microbiota structure between the two groups (Figure 1).
      Figure 1
      Figure 1Principal coordinate analysis plot based on unweighted UniFrac calculations. The abundance of operational taxonomic units in the intestinal microbiota in infants with gastroenteritis in the viral group was compared with that of infants in the healthy control group. PC2 (P=0.0021) and PC3 (P=0.0095) showed significant differences, indicating that intestinal viral (rotavirus and human norovirus) infections led to changes in the structure of intestinal microbiota in infants.

       Analysis of diversity of intestinal microbiota

      The comparison of alpha diversity between the viral group and the healthy group revealed differences in the Chao1 index between groups (Figure 2A). The Simpson indices of the RV and HNoV groups were lower compared with that of the control group, while there was no significant difference in the Shannon index although decreasing trends were observed in both viral groups, especially the HNoV group. The Chao1 index of the HNoV group was higher than that of the control group (P=0.0003). The RV group had lower alpha diversity than the HNoV group, and the difference in the Chao1 index was significant (P=0.0078). There was a significant difference in beta diversity between the viral group and the control group, while no significant difference in beta diversity was observed between the RV and HNoV groups (Figure 2B).
      Figure 2
      Figure 2Comparisons of intestinal microbiota diversity. (A) Comparison of infant intestinal microbiota alpha diversity as indicated by the Chao1 index. The differences between the three groups were significant (P<0.05). The Chao1 indices of the rotavirus (RV) and human norovirus (HNoV) groups were significantly higher than that of the healthy control group, with the HNoV group showing a greater difference than the RV group. (B) Comparison of infant microbiota beta diversity between the RV and HNoV groups. The principal coordinate analysis plot based on unweighted UniFrac calculations was drawn to observe the differences between the two groups. There was no significant difference in beta diversity between the RV and HNoV groups (P>0.05).

       Composition and abundance distribution of intestinal microbiota

      Infants with viral diarrhoea showed lower relative abundance of Proteobacteria and higher relative abundance of Actinobacteria, Fusobacteria, Verrucomicrobia and Cyanobacteria at phylum level; and lower relative abundance of Veillonella spp. and higher relative abundance of Streptococcus spp. and Enterococcus spp. at genus level. Compared with the healthy group, infants in the RV group had a higher abundance of Actinobacteria at phylum level, and higher abundance of Bifidobacterium spp., Streptococcus spp., Enterococcus spp. and Lactobacillus spp. at genus level. Compared with the healthy group, infants in the HNoV group had higher abundance of Fusobacteria and Cyanobacteria at phylum level, and higher abundance of Enterococcus spp. and Streptococcus spp. at genus level (Figure 3). Comparisons between the two viral groups showed that, at phylum level, the RV group exhibited higher abundance of Actinobacteria and Verrucomicrobia, while the HNoV group exhibited higher abundance of Fusobacteria. At genus level, the RV group showed higher abundance of Veillonella spp. and Bifidobacterium spp., while the HNoV group showed higher abundance of Enterococcus spp., Clostridium spp. and Fusobacterium spp.
      Figure 3
      Figure 3(A) Distribution and comparison of relative abundance of intestinal microbiota at phylum level between the three groups of infants. Firmicutes and Proteobacteria predominated in the intestinal microbiota of all three infant groups. The rotavirus (RV) group showed a more significant decrease in Proteobacteria, while the HNoV group showed a more significant decrease in Bacteroidetes. The RV group showed a significant increase in Actinobacteria. (B) Distribution and comparison of relative abundance of intestinal microbiota at genus level between the three groups of infants. Compared with the healthy control group, there were significant increases in Streptococcus spp. and Enterococcus spp. at genus level in infants with viral diarrhoea, especially in the human norovirus (HNoV) group. Infants in the rotavirus (RV) group showed a significant increase in Bifidobacterium spp., while infants in the HNoV group showed a decrease in Veillonella spp.

       Differential microbiota analysis

      Linear discriminant analysis of effect size revealed the characteristic appearance of Bacillus spp. among the intestinal microbiota of infants with viral diarrhoea, both RV and HNoV, followed by Streptococcus spp. and Enterococcus spp. (Figure 4). Random Forest analysis indicated that the genus Neisseria was effective in distinguishing infants with viral diarrhoea from healthy infants [error rate=9.76%, area under the curve (AUC)=0.98], while Streptococcus spp. (error rate=12.7%, AUC=0.98) and Pseudomonas spp. (error rate=6.17%, AUC=0.99) were effective in distinguishing healthy infants from those infected with RV and HNoV, respectively. Leptotrichia spp. could be used with certain degree of accuracy (error rate=20.91%, AUC=0.8) to distinguish between RV- and HNoV-infected infants. However, certain degree of accuracy no differentiating genus was found between the RV and HNoV groups.
      Figure 4
      Figure 4Linear discriminant analysis of effect size of intestinal microbiota of three infant groups based on linear discriminant analysis (LDA). The selection criteria for species showing significant intergroup differences was a logarithmic LDA score >2. The genus Bacillus exhibited a significant role in infants with viral diarrhoea (A), including both the rotavirus (RV) (B) and human norovirus (NV) (C) groups, followed by Streptococcus spp. and Enterococcus spp. Clostridia spp. remained a key characteristic bacterial genus during infancy.

       Differences in microbiota metabolism

      Results from BugBase indicated that there were significant differences in aerobic (P=0.027), Gram-negative (P=0.048), Gram-positive (P=0.048) and stress-tolerant (P=0.002) bacteria between the viral and healthy groups. The microbiota of the RV group showed no significant phenotypic difference from the control group, but the HNoV group exhibited phenotypic differences in aerobic (P=0.007), anaerobic (P=0.002), mobile-element-containing (P=0.008), Gram-negative (P=0.038), Gram-positive (P=0.038) and stress-tolerant (P=0.061) bacteria. Compared with the control group, the viral group (P≤0.01), the RV group (P=0.002) and the HNoV group (P≤0.01) showed significant differences in potentially pathogenic bacteria. There were significant phenotypic differences in anaerobic (P=0.002) and mobile-element-containing (P=0.004) bacteria between the RV and HNoV groups.
      Functional annotation of taxa analysis showed increased proportions of pathogenic micro-organisms causing septicaemia and meningitis, and of chloroplast-related bacteria in the viral groups. KEGG metabolic pathway analysis revealed upregulation of calcium signalling (P=6.51 × 10−9) and photosynthesis (P=6.51 × 10−9) in the viral group, as well as upregulation of key metabolic pathways of biosynthesis, such as steroid biosynthesis (P=1.03 × 10−7), indole alkaloid biosynthesis (P=4.00 × 10−7), various types of N-glycan biosynthesis (P=2.42 × 10−6), and cellular functional pathways such as the apoptosis (P=1.53 × 10−5), cytochrome P450 (P=5.35 × 10−5) and mRNA surveillance pathways (P=8.59 × 10−5). However, no significant difference in metabolic pathways was observed between the HNoV and RV groups.

      Discussion

      Among the cases included in this study, clinical severity was significantly higher in the RV group compared with the HNoV group due to the enrolment of inpatient cases. HNoV usually results in emesis, and the majority of these cases were treated in clinics or emergency rooms due to milder clinical symptoms or faster relief of the symptoms. In terms of diarrhoea, there was a higher proportion of HNoV infection among infants with moderate diarrhoea, while the proportion of RV infection was higher among infants with severe diarrhoea. As infants are in the developmental stage of intestinal microbiota, healthy infants matched by age and mode of feeding were used for comparison in this study. However, samples with local dominant strains were collected: G9P8 of RV and GII of HNoV. Therefore, the direction and magnitude of genotype variation bias were the sources of limitation in this study.
      Overall, changes in microbiota structure and diversity were observed in infants with viral AGS. Examination of the intestinal microbiota during the acute phase of AGS revealed a marked increase in the Chao1 index in both the RV (P=0.0078) and HNoV (P=0.0003) groups, indicating increased abundance of microbiota after infection; a decrease in the Simpson index, indicating reduced diversity of microbiota; and a more significant decrease in the Shannon index in the HNoV group, indicating the possible appearance of more rare colonies. Unfortunately, there was no significant difference in the Simpson index (P=0.8385) and the Shannon index (P=0.6045) between the groups. Although the HNoV group had less severe diarrhoea, more pronounced intestinal microbiota changes with a significant increase in overall microbiota abundance was detected, while the RV group showed less diversity, as reported previously (
      • Fei P
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      ).
      Although RV and HNoV have similar clinical manifestations, differences in species changes were observed between the RV and HNoV groups during the acute phase. These results are similar to those from a study by
      • Chen SY
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      , although they had fewer samples. At phylum level, the RV group had higher abundance of Actinobacteria and Verrucomicrobia, while the HNoV group had higher abundance of Fusobacteria. At genus level, the RV group had higher abundance of Veillonella spp. and Bifidobacterium spp., while the HNoV group had higher abundance of Enterococcus spp., Clostridium spp. and Fusobacterium spp. Further studies are needed to investigate whether these differences result in any difference in pathogenesis or host mucosal immunity. In contrast to a previous study which found increasing opportunistic pathogens such as Shigella spp. in children with AGE with enteric viruses (
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      • et al.
      Molecular characterization of fecal microbiota in patients with viral diarrhea.
      ), the present study found that Streptococcus spp. and Enterococcus spp. common pathogenic bacteria – were the dominant genera in infected infants. Disturbance of the intestinal microbiota in infants during the acute phase of diarrhoea may increase the risk of secondary infection by potentially pathogenic bacteria. Differential microbiota analysis uncovered the characteristic intestinal existence of the genera Bacillus. Bacillus spp. are important symbiotic organisms in the intestine, producing digestive enzymes and antioxidants, and maintaining the intestinal ecobalance. In recent years, some Bacillus spp. have been given as probiotics for their antimicrobial, anticancer, antioxidant and vitamin production properties (
      • Lee NK
      • Kim WS
      • Paik HD.
      Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier.
      ). Similarly, the RV group exhibited high abundance of Bifidobacterium spp. The increase in abundance of intestinal probiotics may reflect the self-regulation and repair abilities of the intestinal microbiota (
      • Dinleyici EC
      • Martínez-Martínez D
      • Kara A
      • Karbuz A
      • Dalgic N
      • Metin O
      • et al.
      Time series analysis of the microbiota of children suffering from acute infectious diarrhea and their recovery after treatment.
      ). However, longitudinal comparisons of microbiota before and after infection are needed to provide more strain-specific data on probiotic therapies.
      The interactions between HNoV, microbiota and the host are complicated, including proviral and antiviral effects of intestinal microbiota. HNoV can promote infection through interactions between its surface components and the microbiota (
      • Monedero V
      • Buesa J
      • Rodríguez-Díaz J.
      The Interactions between host glycobiology, bacterial microbiota, and viruses in the gut.
      ); alter the host gut microbial communities, resulting in enhanced bacterial diversity; and even participate in direct interactions with pathogenic bacterial species. However, consensus has not been reached on specific characteristic changes following HNoV infection. In adults, significant changes in intestinal microbiota have only been detected in a small subset of HNoV-induced patients with AGE (
      • Nelson AM
      • Walk ST
      • Taube S
      • Taniuchi M
      • Houpt ER
      • Wobus CE
      • et al.
      Disruption of the human gut microbiota following norovirus infection.
      ). The present study found that, unlike in adults, the overall microbiota abundance in infants showed a significant increase after HNoV infection, and increases were observed in some bacterial genera that bind to HNoV in vitro (
      • Almand EA
      • Moore MD
      • Outlaw J
      • Jaykus LA
      Human norovirus binding to select bacteria representative of the human gut microbiota.
      ) such as Enterococcus spp. and Bacillus spp. Phenotypic analysis of the microbiota also revealed increases in potentially pathogenic bacteria, indicating that the interactions between HNoV and intestinal microbes are more intimate and complicated in infants, and the microbiota may mediate the course of viral infection in a way that leads to potential bacterial infection. However, this study is not sufficient to elucidate the causal relationship between this change and HNoV infection.
      Current research on RV infection and intestinal microbiota in infants is focused on the role of intestinal microbes in the performance of oral RV vaccines (
      • Harris VC
      • Armah G
      • Fuentes S
      • Korpela KE
      • Parashar U
      • Victor JC
      • et al.
      Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural Ghana.
      ,
      • Harris VC
      • Ali A
      • Fuentes S
      • et al.
      Rotavirus vaccine response correlates with the infant gut microbiota composition in Pakistan.
      ,
      • Harris VC
      • Haak BW
      • Handley SA
      • et al.
      Effect of antibiotic-mediated microbiome modulation on rotavirus vaccine immunogenicity: a human, randomized-control proof-of-concept trial.
      ). Contrary to the high efficacy of RV vaccines against severe RV-related AGE in developed countries, the efficacy of RV vaccines in undeveloped and developing countries is lower (
      • Bhandari N
      • Rongsen-Chandola T
      • Bavdekar A
      • John J
      • Antony K
      • Taneja S
      • et al.
      Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian children in the second year of life.
      ;
      • Hoest C
      • Seidman JC
      • Pan W
      • Ambikapathi R
      • Kang G
      • Kosek M
      • et al.
      Evaluating associations between vaccine response and malnutrition, gut function, and enteric infections in the MAL-ED cohort study: methods and challenges.
      ;
      • Czerkinsky C
      • Holmgren J.
      Vaccines against enteric infections for the developing world.
      ;
      • Bar-Zeev N
      • Jere KC
      • Bennett A
      • Pollock L
      • Tate JE
      • Nakagomi O
      • et al.
      Population impact and effectiveness of monovalent rotavirus vaccination in urban Malawian children 3 years after vaccine introduction: ecological and case–control analyses.
      ;
      • Taniuchi M
      • Platts-Mills JA
      • Begum S
      • Uddin MJ
      • Sobuz SU
      • Liu J
      • et al.
      Impact of enterovirus and other enteric pathogens on oral polio and rotavirus vaccine performance in Bangladeshi infants.
      ). The important relationship between early-stage intestinal microbiota in children and the development of the mucosal immune system may affect the efficacy of RV vaccines. However, there are differences in the reported relationship between changes in intestinal microbiota and immune responses to RV vaccines in different regions (
      • Hoest C
      • Seidman JC
      • Pan W
      • Ambikapathi R
      • Kang G
      • Kosek M
      • et al.
      Evaluating associations between vaccine response and malnutrition, gut function, and enteric infections in the MAL-ED cohort study: methods and challenges.
      ;
      • Harris VC
      • Armah G
      • Fuentes S
      • Korpela KE
      • Parashar U
      • Victor JC
      • et al.
      Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural Ghana.
      ,
      • Harris VC
      • Ali A
      • Fuentes S
      • et al.
      Rotavirus vaccine response correlates with the infant gut microbiota composition in Pakistan.
      ,
      • Harris VC
      • Haak BW
      • Handley SA
      • et al.
      Effect of antibiotic-mediated microbiome modulation on rotavirus vaccine immunogenicity: a human, randomized-control proof-of-concept trial.
      ).
      • Harris VC
      • Armah G
      • Fuentes S
      • Korpela KE
      • Parashar U
      • Victor JC
      • et al.
      Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural Ghana.
      found that the vaccine response correlated with increased abundance of the class Bacilli and Streptococcus bovis, and decreased abundance of the Bacteroidetes phylum, in Ghanaian infants. Infants in the present study are naturally infected with RV of type G9P8, and the changes in intestinal microbiota observed were similar to those in responders to RV vaccines in the study by
      • Harris VC
      • Armah G
      • Fuentes S
      • Korpela KE
      • Parashar U
      • Victor JC
      • et al.
      Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural Ghana.
      . Although baseline data on the intestinal microbiota of the two paediatric populations are not available, relevant data may be used in the future to verify whether the response of local children to RV vaccines can be predicted based on similar changes in the microbiota.
      Changes in intestinal microbiota structure may lead to changes in its function. Compared with the healthy group, the viral group showed a significant overall increase in the proportions of chloroplasts and potentially pathogenic micro-organisms, along with corresponding upregulation of chloroplast and photosynthesis pathways. The increase in chloroplast-related microbiota may be due to incomplete digestion of plant food in the intestine during acute diarrhoea, and may indirectly reflect the decrease in intestinal digestive function in infants during AGS. However, no significant difference in intestinal microbiota metabolic pathways was observed between the RV and HNoV groups.
      Further studies need to be carried out based on these results to investigate the dynamic changes and subsequent repair of the intestinal microbiota by comparisons at different time points to obtain a more complete description of the overall changes in intestinal microbiota in infants with viral AGS. Moreover, further research is necessary regarding the impact of these changes in structure and diversity of intestinal microbiota on the development of mucosal immunity in infants.

      Conclusion

      This study provides preliminary data on the profile of intestinal microbiota during the acute phase of AGS caused by RV and HNoV infection in infants. There were significant changes in overall microbiota structure and diversity in infected infants. The Chao 1 index – an important parameter of alpha diversity – increased significantly in the HNoV group, while no significant difference in beta diversity was observed. Comparison of the viral group with the control group, and comparison of the RV and HNoV groups showed that the composition and abundance distribution of intestinal microbiota differed at phylum and genus levels. Linear discriminant analysis of effect size revealed the characteristic appearance of Bacillus spp. among the intestinal microbiota of infected infants. The viral group (P≤0.01), the RV group (P=0.002) and the HNoV group (P≤0.01) showed an increase in potentially pathogenic bacteria, particularly Streptococcus spp. and Enterococcus spp.

      Conflict of interest statement

      None declared.

      Funding

      This work was supported by the Science and Technology Project of the Health Planning Committee of Chengdu, China (Grant No. 2021186) and Chengdu High-level Key Clinical Specialty Construction Project.

      Ethical approval

      This work was approved by the Research Ethics Committee of Chengdu Women's and Children's Central Hospital.

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