Children with Autism spectrum disorder are associated with gut microbiota disorder

Purpose The aim of this study was to evaluate the occurrence and clinical characteristics of autism spectrum disorder (ASD) associated to the stable state of the gut microbiota. Methods A total of 9 children with ASD and 6 healthy children used as control were selected and feces samples were collected from all of them. The 16S gene ribosomal RNA sequencing was used to analyze the difference in gut microbiota between healthy control children and ASD patients. Results The results of 16S sequencing based on operational taxonomic units (OTUs) analysis showed that the ASD group and the healthy control (HC) group had a large difference in the abundance of microbiota at the level of family, genus and species. The abundance of Bacteroidales and Selenomonadales was significantly lower in the ASD group than in the HC group (p=0.0110 and p=0.0076, respectively). The abundance of Ruminococcaceae in the ASD group was higher than that in the HC group (p=0.0265), while the amount of Prevotellaceae was significantly lower in the ASD group than in the HC group (p=0.0265). The Tax4Fun analysis based on Kyoto Encyclopaedia of Genes and Genomes (KEGG) data indicated differentially expressed functional pathway between the ASD group and healthy control group associated to the nervous system, environmental information processing and cellular processing. Conclusions The abundance of gut microbiota in the ASD group is different from that in the healthy control children. These differences affect the biological function of the host. These results suggest that a disorder in the gut microbiota may be associated, at least in part, with ASD in children.


Background
In recent years, autism spectrum disorder (ASD) has been associated with the microbial gut composition and its different pathophysiological effects (such as toxic substances associated with intestinal immune responses, increased neuronal alpha-synuclein delivery or increased permeability of the systemic inflammation) [1][2][3]. Children with ASD are prone to dysregulation in the intestinal microbiota due to environmental and behavioral factors and because of the imperfect development of the immune system [4], especially the nervous immune system [5], thus, disorders of the gut microbiota may have a greater impact on children with ASD [6][7][8]. According to the above evidence, the gut microbiota from fecal samples of ASDs children and healthy control (HC) children was analyzed. Our results showed a significant difference in the microbial community composition between ASDs and HCs. Thus, children's targeted improvement of intestinal microbiota might be helpful in improving the health of ASD children.

Study design and patient selection
A total of 9 ASD patients aged 3-12 years, receiving rehabilitation in the rehabilitation center for children with autism at the Department of Autism Rehabilitation, Wuxi Huishan District Rehabilitation Hospital, China, from December 2018 to April 2019 were enrolled in our study and considered as the ASD group. In addition, 6 healthy children with an age matching the one of the patients were selected from kindergartens in the same area, and were considered as the HC group. 16S gene ribosomal RNA sequencing was performed on fecal samples of all the 15 children.

RNA sequencing and data analysis
Feces were collected from each subject, placed in a sterile stool container, frozen immediately in liquid nitrogen and stored at -80 °C. Since fecal samples differed in their collection dates, total bacterial DNA was extracted from fecal samples within one month using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, California) according to the manufacturer's protocol with minor modifications. The V3-V4 region of the 16S ribosomal RNA (rRNA) gene was amplified and sequenced on the Illumina MiSeq platform (Illumina, San Diego, California) in multiple runs, pooling together all 15 samples using a 2 × 250 bp paired-end protocol, according to the manufacturer's instructions. Raw reads from the microbiota sequencing were analyzed using Pandaseq, processed through the QIIME (version 1.8.0), clustered into operational taxonomic units (OTUs) at 97% identity level and taxonomically assigned via Ribosomal Database Project classifier against the Greengenes database (release 13.5; http://greengenes.secondgenome.com) [9]. The KEGG pathways database was used to predict differences in bacterial biochemical pathways between the ASD group and HC group. A p value < 0.001 was considered statistically significant.

Statistical analysis
Statistical evaluation of differences in alpha-diversity measures was performed by a nonparametric Monte Carloifferences in bacterial biochemical pathwaysadonis"donistical evaluation of di"vegan" (https://cran.r-project.org/web/packages/vegan/index.html) was used to determine statistical separation of the microbiota profiles in terms of beta diversity and predictive analysis. Other analyses were performed with the software SPSS 20.0 (Chicago), including two-tailed t-test and Pearson Chi-square test. P values < 0.05 were considered statistically significant.

Microbial Composition in ASD and HC
In our study, a total of 15 fecal samples from children were collected by us, 9 ASDs and 6 HCs. The basic features of the 15 children included in our study are shown in table 1. The microbial composition at the Phylum level (relative abundance > 5% in the 15 samples) is shown in Figure 1A Figure 1A-2. The level of Bacteroidales in the ASD group (29.291±8.689) was significantly less than that in the HC group (49.310±17.384) (t=2.963, p = 0.0110). The abundance of Selenomonadales in the ASD group (0.069±0.031) was significantly less than that in the HC group (7.423±3.186) (t= 3.157, p = 0.0076) ( Figure 1B-1). The microbiota at the Order level is shown in Figure 1A Table 2 shows the baseline data associated to the ASD and HC children enrolled in this study. The correlation between ASD and HC and gender, cesarean section and breastfeeding was further analyzed. A significant difference in gender, cesarean section and breastfeeding was not obtained between the two groups because of the small sample size. However, based on the differential analysis of gut microbiota of ASD and HC group, striking results were obtained. As regard the principal component analysis, ASD and HC children are distinguished in different quadrants, and they have distinct gut microbiota composition (Figure 2-A). The same result was obtained with the Tags Sampled analysis.

Microbiota and Clinical Features of ASD children
The Chao1 index of ASD and HC group also showed the characteristics of two different biomes (Figure 2-B). In other alpha diversity analyses, the analysis of the number of OTUs and the Shannon Index also reflected the differences between ASD and HC children (

Predictive Microbiota Functional Profiling
The functional contribution of the bacteria in the ASD and HC group was predicted based on OTUs using the Tax4Fun package in R software. A total of 53 KEGG orthology were found across all samples, mainly belonging to the pathway Organismal Systems, Environmental Information Processing, and Cellular processes. The most abundant functional pathways are presented in Figure 3. Cluster analysis of the KEGG pathway revealed that the gut microbes differentially expressed in the ASD group are mostly representing the Organismal Systems pathway and the difference between the two groups was mainly in the nervous system pathway. Similarly, the Environmental Information pathway was mostly represented in the ADS group and was also significantly lower in the ADS group than in the HC group.

Discussion
The alteration in the profile of gut microbiota was investigated in a population of patients with ASD and correspondent HCs by applying a robust statistical approach, adjusting for potential confounders. Although our results confirm that ASD was characterized by several changes in microbiota composition when compared with HCs, the results emphasized that this analysis is greatly influenced by several confounders, including the disease severity itself [10]. Our focus on de novo patients supports the hypothesis of a pathophysiological connection between ASD and gut microbiota, particularly with the reduced abundance of the Bacteroidales and Selenomonadales Class. In addition, the within-group analysis according to ASD clinical features suggests the potential role of microbial composition (in particular Ruminococcaceae and Prevotellaceae) and the differential effects of the gut microbiota that further affect the children.
In the intestinal microbiota biodiversity analysis, the concentration trend in HCs was significantly different from the one in the ADS children. In the intestinal microbiota biodiversity analysis in the ASD children, the trend of HC concentration is significantly different, and in the alpha diversity analysis based on the biological microbiota, the trend of intestinal microbiota diversity reflecting ASD children is different from HCs [11][12].
Because children's nervous system and immune system are not yet well developed, factors such as cesarean section and breastfeeding were consider because they have a significant impact on the microbial community of infants and young children, and these differences affect children's development [13][14]. However, the baseline parameters of the children included in this study are referred to a small sample size and, thus, the results were not significant. However, in combination with the biological function prediction of KEGG, it was remarkable to notice that the imbalance of intestinal microbiota led to the inhibition of ASD nervous system and environmental information processes, in addition to the changes of Cellular Processes, which explained the influence of microbiota imbalance to some extent. Since the gut microbiota is controlled by the brain -gut axis, it affects the cognitive abilities of children with ASD and their responses to environmental information.
Due to our small sample size, the relationship between ASD and HC baseline parameters and cesarean section, breastfeeding and other factors, as well as the occurrence of ASD and gut microbiota imbalance, did not significantly differ. Thus, further studies with a larger sample and prospective follow-up study is needed. However, our results demonstrated that it might be possible to improve the state of the nervous system development in patients with ASD by altering the food intake of children with ASD, thus interfering in a positive manner with the composition of the gut microbiota [15][16][17][18].

Conclusion 8
The gut microbiota composition between ASD and HC group was significantly different.

Funding
This research had no funding

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on a validated request.

Ethics approval and consent to participate
This study was performed in agreement with the principles of the Declaration of Helsinki and approved by the ethics committees of Wuxi Fifth People's Hospital and Rehabilitation Hospital of Huishan District Wuxi City . Written informed consent was obtained from the parent or guardian of each child included in this study.

Consent for publication
We have obtained a written authorization statement from the guardians of this study participants.

Declarations
The authors declare that they have no competing interests.   The predicted KEGG categories abundance of the expression in ASD group and HC group.