Characterization of changes in the intestinal microbiome following combination therapy with zinc preparation and conventional treatment for children with rotavirus enteritis

Abstract

Background: Rotavirus (RV) is one of the most common pathogens causing diarrhea in infants and young children worldwide. Routinely, antiviral therapy, intestinal mucosa protection, and fluid supplementation are used in clinic, however this is not efficacious in some severe cases. Zinc supplementation has previously been shown to improve resolution of symptoms from infectious diarrhea.

Methods: In this study differences in response rate, duration of hyperthermia, vomiting, and diarrhea, and the persistence time of cough and lung rales in groups were compared. 16SrDNA gene sequencing technology was used to analyze and compare changes in the intestinal microflora of children with RV enteritis who received the conventional treatment with or without the zinc preparation. In addition, the correlations between the differential bacterial species and the related inflammatory factors were determined.

Results: Conventional therapy combined with the zinc preparation significantly shortened the duration of hyperthermia, vomiting, and diarrhea compared with the conventional treatment alone. In addition, the time to symptom relief showed that the absorption time of cough and lung rales was significantly shorter in the combination treatment group than that in the conventional treatment group in the children with pneumonia. Further, compared with the conventional treatment, the combined treatment significantly increased the diversity and abundances of florae as compared with the conventional treatment. This combination therapy containing zinc preparation markedly increased the abundances of Faecalibacterium, Bacteroidales, Ruminoccoccoccus, and Lachnospiraceae at the genus level. The LEfSe analysis suggested that Clostridiumbolteae were most significantly altered after the combination therapy. In addition, a correlation analysis revealed significantly negative correlations between the inflammatory factors especially IL-6, TNF-a, CRP and some intestinal florae such as Bacteroides, Faecalibacterium, Blautia, Parabacteroides, Subdoligranulum, and Flavonifractor.

Conclusion: Compared with the conventional therapy alone, the combined therapy with the zinc preparation significantly improves symptoms caused by RV. The combination therapy containing the zinc preparation significantly increases the diversity and abundances of some beneficial groups of bacteria. Further, The presence of these groups was further negatively correlated with relevant inflammatory factors. More importantly, this combination therapy containing the zinc preparation provides a reference for the clinical management of children with RV enteritis.

Keywords: flora changes; inflammatory factors; intestinal microbiome; rotavirus enteritis; zinc preparation.

Figure 1

Rarefaction curves of OTUs. The x-axis represents the number of valid sequences per sample, and the y-axis represents the number of observed OTUs.

Figure 2

α diversity and β diversity of intestinal flora. (A-C) α diversity indices. ^* p<0.05 vs. untreated. The feces samples were collected and 16S rDNA sequencing was then performed. α diversity indices including Chao1, Shannon, and Simpson were detected to compare the differences among the groups; (D) Venn diagram assay. The number of intestinal flora was highest in untreated group, and more than that in Treated-2 group and Treated-1 group. 16 kinds of the intestinal flora were the same in the three groups; (E) PCoA analysis. PCoA assay suggested a good sample separation among the three groups.

Figure 3

Analyses of intestinal flora composition at phylum class, order, family, and species levels (top 10 bacteria). Changes in flora compositions at phylum, class, order, family, and species levels. The significantly changed flora were shown to reflect their proportions in the community at various levels. The top 10 bacteria were displayed to show the differences of the groups.

Figure 4

Analysis of species differences between groups. Each bar in the graph represents the mean of species with a significant difference in abundance between the two groups. The right figure shows the confidence level of inter group differences. The left-most point of each circle represents the lowest limit of the 95% confidence interval (95% CI), and the rightmost point of the circle represents the highest limit of the 95% CI. The center of the circle represents the difference in mean, and the color of the circle represents the significance for the inter group differences in the corresponding different species.

Figure 5

LEfSE analysis of groups. (A) tree diagram showing species with significant differences among the three groups; (B) evolutionary branch diagram showing the species that are differentially more abundantin the group. Classification is performed from phylum level to genus level. Blue column represents the untreated group, green column represents the zinc-containing treatment group, and red column represents the conventional treatment group.

Figure 6

Heatmap analysis of co-abundance network species. Different nodes represent different genera, node size represents the average relative abundance of genus, nodes at same phylum have the same color (shown in the legend), thickness of connecting lines between nodes is positively correlated with absolute value of correlation coefficient between species, and color of connecting lines is positively correlated (red represents a positive correlation, blue represents a negative correlation).

Figure 7

KEGG enrichment analysis. (A) the horizontal axis represents the number of differentially expressed genes annotated onto the KEGG pathway, and the vertical axis represents the KEGG pathway; (B) heatmap analysis of the functions of the differential expressed genes between the conventional treatment group and the zinc-containing combination treatment group. The red dot represents significantly upregulated gene function, the green dot represents significantly downregulated gene function, the two vertical dashed lines represent log2FC, and the dashed lines represent an adjusted p value of 0.05.

Figure 8

Correlation analysis of community compositions and environmental factors at the genus (A) and species (B) levels. The correlation is more positive when the correlation analysis is closer to 1, and vice versa. Horizontal axis represents inflammatory factors, and vertical coordinates represents genus names.