Impact of Zinc Oxide Nanoparticles on the Composition of Gut Microbiota in Healthy and Autism Spectrum Disorder Children

Autism spectrum disorder (ASD) seriously affects children’s health, while the gut microbiome has been widely hypothesized to be involved in the regulation of ASD behavior. This study investigated and compared the number, diversity, and population structure of gut microbiota between healthy and ASD children and their susceptibility to zinc oxide nanoparticles (ZnONPs) based on the measurement of live cell number, living/dead bacterial staining test, flow cytometry observation and bacterial community analysis using 16S rRNA gene amplicon sequencing. The result of this present study revealed that ASD children not only significantly reduced the live cell number and the community diversity of gut bacteria, but also changed the gut bacterial community composition compared to the healthy children. In addition, this result revealed that ZnONPs significantly reduced the number of live bacterial cells in the gut of healthy children, but not in that of ASD children. In contrast, ZnONPs generally increased the gut bacterial community diversity in both ASD and healthy children, while a greater increase was found in ASD children than that of healthy children. Furthermore, this study successfully isolated and identified some representative nanoparticle-resistant bacteria based on the color, shape, and edge of colony as well as the 16S rDNA sequence analysis. The community of nanoparticle-resistant bacteria differed in between healthy and ASD children. Indeed, the representative strains 6-1, 6-2, 6-3 and 6-4 from healthy children were identified as Bacillus anthracis, Escherichia coli, Bacillus cereus and Escherichia coli with sequence similarity of 97.86%, 99.86%, 99.03% and 99.65%, respectively, while the representative strains 8-1, 8-2 and 8-3 from ASD children were identified as Bacillus cereus, with sequence similarities of 99.58%, 99.72% and 99.72%, respectively. Overall, this study demonstrated that ZnONPs caused a change in number, diversity, and species composition of gut bacteria, but differed in healthy and ASD children.