Abstract
Background:
Increasing studies suggest that the microbiome-gut-brain axis plays a fundamental role in developing autism spectrum disorder (ASD).Objectives:
We aimed to explore biomarkers from gut bacteria in ASD and the dietary vitamin A (VitA) relationship with intestinal bacteria of autistic children and provide a theoretical basis for dietary regulation of ASD.Methods:
Ten autistic children aged 2 to 6 from special training facilities were enrolled from 2017 to 2018. At the same time, 10 healthy children aged 2 to 6 from a kindergarten were collected as a control. All participants were from Chongqing, China. The 16sRNA amplicon sequencing was used to analyze children's intestinal bacteria. The serum retinol level was detected by high-performance liquid chromatography (HPLC), and children's dietary intake was analyzed using three-day 24-hour dietary recalls.Results:
There were significant differences in alpha diversity between the groups. Also, a higher relative abundance of Prevotellaceae, Prevotella 9, and Roseburia was observed among ASD children. We also found decreases in 9 bacteria (Enterobacteriales, Gammaproteobacteria, Enterobacteriaceae, Clostridiaceae 1, Clostridium sensu stricto 1, Escherichia-Shigella, Bacteroides fragilis, Escherichia coli, and Clostridium neonatale). Dietary VitA intake and serum retinol concentration were lower in the ASD group than in the control group. Meanwhile, serum VitA had a significantly negative correlation with the abundance of intestinal Prevotella 9.Conclusions:
Our study helps identify some bacterial biomarkers for ASD, as in previous reports. Meanwhile, the study suggests that dietary VitA may be involved in the clinical symptoms of ASD by regulating the intestinal bacteria Prevotella. It may provide a new way to treat ASD in the future. Further studies are needed to identify the results by expanding the sample size and developing animal experiments.Keywords
1. Background
Many studies have shown a significant relationship between intestinal bacteria and autism spectrum disorder (ASD) (1, 2). The microbiome-gut-brain axis is suggested to have a vital role in the development of ASD (3-5). Dysbiotic gut microbiota of children with ASD may be associated with brain dysfunction, but the underlying mechanisms are unclear. Furthermore, no specific gut bacteria are considered a biomarker for ASD so far. However, the symptom of some ASD cases has been improved statistically by therapies targeting intestinal bacteria (3, 4). Differences in biomarkers from various research studies might be linked to demographic characteristics of subjects such as region and race. Alcaligenaceae, Enterobacteriaceae, and Clostridium were considered candidate bacterial biomarkers by Li et al. (6). Another study showed gut bacterial biomarkers, such as Eubacterium, Bifidobacterium, Blautia, and Dialister (7). There are few reports about the intestinal microbiome of ASD in China. Furthermore, ASD initiates in the early life of children (8), while very little literature focuses on the early life of autistic children in China.
Vitamin A (VitA) is an important micronutrient for people. Researchers have recently reported that VitA plays a role in intestinal bacteriomes (9). Amit-Romach et al. suggested that VitA also regulates intestinal bacillus (10). Another study revealed that neonatal vitamin A supplementation and vitamin A status were associated with the composition of the intestinal microbiome among infants at 2 years old (11). Some studies have shown the relationship between vitamin A and ASD (12-14). Studies also showed that individuals with ASD have lower VitA concentrations than typically developing (TD) children. Recent studies have reported that serum VA level was negatively correlated with the severity of ASD (15). However, little is reported on how vitamin A regulates specific gut microbiota to affect ASD.
2. Objectives
This study further explored the characteristics of intestinal bacteria in ASD in China. In addition, the effect of VitA on the gut bacteria of ASD was investigated to find a new possible treatment for ASD.
3. Methods
Ten children with ASD aged 2 to 6 from special training facilities in Chongqing from 2017 to 2018 were enrolled in this study. According to the 5th edition of the diagnostic and statistical manual of mental disorders (DSM-5), the inclusion criteria included children diagnosed by more than two child behavioral health practitioners. The exclusion criteria included children diagnosed with other congenital disorders. Those with acute and chronic infectious diseases in the past month were also excluded. The other exclusion criteria were dietary supplements taken by children during the past six months and probiotics and antibiotics taken for almost a month. At the same time, 10 healthy children aged 2 to 6 years were enrolled as controls from a kindergarten between 2017 and 2018. The exclusion criteria were dietary supplements taken by children during the past six months and probiotics and antibiotics taken for almost a month.
A demographic questionnaire was used to obtain information, including gender, age, nationality, and living place. The study was consented to by parents (guardians) of all children, and we obtained the approval of the Committee of Chongqing Medical and Pharmaceutical College.
The dietary VitA intake was investigated using three-day 24-hour dietary recalls (validity, 0.73; reliability, 0.69) (16, 17), and the data were analyzed by a nutrition Software program (FeiHua V2.5, Beijing, China). Serum retinal analysis was detected by high-performance liquid chromatography (HPLC) (18).
The fresh and pollution-free stool was collected and temporarily stored in a refrigerator at -20°C and was transferred to the refrigerator at -80°C within 7 days.
The 16S rRNA genes of distinct regions (16SV4) were amplified using a specific primer (16S V4: 515F - 806R) with the barcode. Sequencing was performed by Uparse software (Uparse v70.1001) (19). Qiime software (version 1.9.1) was used to calculate the observed species, Chao1, Shannon, Simpson, and abundance-based coverage estimator (ACE) (20). The linear discriminant (LDA) analysis effect size (LEfSe) software was used for LEfSe analysis, and the filter value of the LDA score was set at the default value of 4.
Data were analyzed by SPSS 17.0 software package. The p values below 0.05 were significant.
4. Results
4.1. Demographic Characteristics
All children with ASD were 2 to 6 years old in the ASD group (8 males and 2 females; 57.50 ± 11.38 months on average). The control group contained 10 healthy children aged 2 to 6 (8 males and 2 females; 50.50 ± 11.22 months on average). There was no statistical difference between the two groups concerning gender and age (P > 0.05). All the children were of Han nationality and lived in Chongqing City.
4.2. Vitamin A in Diet and Serum
Vitamin A dietary intakes were 185.00 ± 38.12 and 261.70 ± 79.70 μgRE in the ASD and control groups, respectively. There were 5 cases (50%) in the ASD group that had an intake of lower than dietary nutrient reference intake (DRIS), according to the Chinese Academy of Nutrition, while there were two such cases (30%) in the control group. In addition, the retinol level in serum was 0.78 ± 0.24 μmol/L in the ASD group and 1.01 ± 0.24 μmol/L in the control group. VitA deficiency (VAD) was observed in 3 cases (70%) in the ASD group and 5 cases (50%) in the control group. The two groups significantly differed in dietary VitA intake and serum VitA levels (P < 0.05).
4.3. Characteristics of Gut Bacteria
4.3.1. Alpha Diversity Analysis
We obtained an average of 82,261 sequences and 619 operational taxonomic units (OUTs) per sample (Figure 1). The alpha diversity analysis revealed no significant differences using the Simpson. However, the scores of observed species (reflecting species richness), Chao, and ACE (reflecting species abundance) (Table 1) regarding the OTU levels in autistic samples decreased compared to those in controls, suggesting that autistic children had less species diversity. The findings revealed that the overall composition of the ASD gut microbiota differed from that of the control gut microbiota.
Operational taxonomic units of all samples. RS1.1-RS1.10 represented autism samples, and RS2.1-RS2.10 represented control samples.
Alpha Diversity Analysis in Two Groups
| Control Group | ASD Group | P Value | |
|---|---|---|---|
| Observed species | 595 | 515 | 0.0008 |
| Shannon | 5.534 | 4.913 | 0.0292 |
| Simpson | 0.918 | 0.895 | 0.3363 |
| Chao1 | 641.163 | 565.026 | 0.0011 |
| ACE | 655.215 | 582.327 | 0.0017 |
4.3.2. Beta Diversity Analysis
The LEFSe analysis can identify biomarkers with statistical differences between the groups. This study found increasing relative abundance in Prevotellaceae (family level), Prevotella 9, and Roseburia at the genus level in the ASD group. However, significantly lower abundances were observed in 9 bacteria (Enterobacteriales (order level), Gammaproteobacteria (class level), Enterobacteriaceae (family level), Clostridiaceae 1 (family level), Clostridium sensu stricto 1 (genus level), Escherichia-Shigella (genus level), Bacteroides fragilis (species level), Escherichia coli (species level), and Clostridium neonatale (species level) (Figure 2).
The histogram of linear discriminant (LDA) values. The histogram of LDA values shows the species with an LDA score greater than 4. RS1 refers to the autism group, and RS2 shows the control group.
4.3.3. Vitamin A is Associated with Intestinal Bacteria
The analysis found that the level of serum retinol was associated negatively with the abundance of Prevotella 9 (r = -0.708, P = 0.022), while there was no relationship between retinol level and the abundance of other bacteria which were considered biomarkers in this study (Table 2).
Correlation Between Serum Retinol and Gut Bacteria in Children with Autism Spectrum Disorder
| R | P | |
|---|---|---|
| f_Prevotellaceae | -0.48 | 0.165 |
| g_Prevotella 9 | -0.71 | 0.022 |
| g_Roseburia | 0.35 | 0.327 |
5. Discussion
In the present study, less species diversity and altered relative abundance of gut bacteria were observed in children with ASD from Chongqing, China, consistent with previous reports (21) to some extent.
There was an obvious increase in Prevotella at the family level, Prevotella 9, and Roseburia at the genus level in autistic children in our research. Several studies have demonstrated significant enrichment of Prevotella and Roseburia in the intestinal microbiome of children with ASD (22, 23). Studies also suggested the potential role of Prevotella as intestinal pathobionts (24). A report showed that some Prevotella strains may participate in human disease by promoting chronic inflammation (25). However, some reported that children with ASD showed a depletion of Prevotella, and microbial metastasis therapy improved autism-related symptoms and the abundance of Prevotella (26, 27). The different properties of Prevotella in the gut microbiome between these studies might be due to the high genetic diversity within and between the species (24). Further study is needed to explore the differences between their results and ours.
Moreover, autistic children displayed a reduction in nine bacteria, which is somewhat consistent with some studies, but different from others (23, 28, 29). Decreased Bacteroides fragilis was found in our study, consistent with previous reports (30, 31). Meanwhile, there was a lower abundance of Escherichia coli and Escherichia-Shigella in the intestinal microbiome of children with ASD in this study, similar to the literature (32, 33). The Clostridium sensu stricto 1 (genus level) and Clostridium neonatale (species level) were found to be lower in the intestinal bacteria of children with ASD in this study, while Clostridia spp. increased in ASD children (2). Further studies are needed to investigate the role of Clostridia in ASD. All studies strongly indicate that the microbiome-gut-brain axis is important in health and disease. Intestinal bacteria have been found to play a major role in the development of autism, although there have been no specific biomarkers yet, and the real causes leading to the development of autism are still unclear.
Furthermore, this study found a negative correlation between serum VitA and intestinal Prevotella in children with ASD, suggesting that vitamin A may regulate intestinal bacteria. Huda et al. reported that early infant supplementation of vitamin A could increase the abundance of Bifidobacterium in the intestines of boys (11). However, few reports have shown the relationship between VitA and Prevotella gut bacteria in autistic children. Our study proposes that VitA may affect the development of ASD by regulating the Prevotella of gut bacteria.
5.1. Limitations
The sample size was insufficient, which may lead to difficulty distinguishing true differences from noise in this study. Nevertheless, based on the existing methodology, we will be able to enlarge the sample for further study in the future. Meanwhile, this study was limited to children aged 3 to 6. Whether the results apply to autistic children of other age groups will be confirmed by expanding age groups in the future. In fact, this experiment selecting 3 to 6 years old children aimed to eliminate age interference on the results and make the results more reliable.
5.2. Conclusions
Our study helps identify some bacterial biomarkers for ASD, as in previous reports. Differing from some other research, increased Prevotella abundance was found in this study, which might be due to some reasons, such as the high genetic diversity within and between the species. Further study about the different results between those and ours is needed.
Meanwhile, the study suggests that dietary VitA may be involved in the clinical symptoms of ASD by regulating the intestinal bacteria Prevotella. It may provide a new way to treat ASD in the future. Further studies will be needed to identify the results by expanding the sample size and developing animal experiments.
Acknowledgements
References
-
1.
Cryan JF, O'Riordan KJ, Cowan CSM, Sandhu KV, Bastiaanssen TFS, Boehme M, et al. The Microbiota-Gut-Brain Axis. Physiol Rev. 2019;99(4):1877-2013. [PubMed ID: 31460832]. https://doi.org/10.1152/physrev.00018.2018.
-
2.
Srikantha P, Mohajeri MH. The Possible Role of the Microbiota-Gut-Brain-Axis in Autism Spectrum Disorder. Int J Mol Sci. 2019;20(9):2115. [PubMed ID: 31035684]. [PubMed Central ID: PMC6539237]. https://doi.org/10.3390/ijms20092115.
-
3.
Chernikova MA, Flores GD, Kilroy E, Labus JS, Mayer EA, Aziz-Zadeh L. The Brain-Gut-Microbiome System: Pathways and Implications for Autism Spectrum Disorder. Nutrients. 2021;13(12):4497. [PubMed ID: 34960049]. [PubMed Central ID: PMC8704412]. https://doi.org/10.3390/nu13124497.
-
4.
Alharthi A, Alhazmi S, Alburae N, Bahieldin A. The Human Gut Microbiome as a Potential Factor in Autism Spectrum Disorder. Int J Mol Sci. 2022;23(3):1363. [PubMed ID: 35163286]. [PubMed Central ID: PMC8835713]. https://doi.org/10.3390/ijms23031363.
-
5.
Settanni CR, Bibbo S, Ianiro G, Rinninella E, Cintoni M, Mele MC, et al. Gastrointestinal involvement of autism spectrum disorder: focus on gut microbiota. Expert Rev Gastroenterol Hepatol. 2021;15(6):599-622. [PubMed ID: 33356668]. https://doi.org/10.1080/17474124.2021.1869938.
-
6.
Li N, Yang J, Zhang J, Liang C, Wang Y, Chen B, et al. Correlation of Gut Microbiome Between ASD Children and Mothers and Potential Biomarkers for Risk Assessment. Genomics Proteomics Bioinformatics. 2019;17(1):26-38. [PubMed ID: 31026579]. [PubMed Central ID: PMC6520911]. https://doi.org/10.1016/j.gpb.2019.01.002.
-
7.
Xu YS, Wang YH, Xu JS, Song Y, Liu BQ, Xiong ZF, et al. Leveraging Existing 16SrRNA Microbial Data to Define a Composite Biomarker for Autism Spectrum Disorder. Microbiol Spectr. 2022;10(4):e00331-22. https://doi.org/10.1128/spectrum.00331-22.
-
8.
Dean DD, Agarwal S, Muthuswamy S, Asim A. Brain exosomes as minuscule information hub for Autism Spectrum Disorder. Expert Rev Mol Diagn. 2021;21(12):1323-31. [PubMed ID: 34720032]. https://doi.org/10.1080/14737159.2021.2000395.
-
9.
Cantorna MT, Snyder L, Arora J. Vitamin A and vitamin D regulate the microbial complexity, barrier function, and the mucosal immune responses to ensure intestinal homeostasis. Crit Rev Biochem Mol Biol. 2019;54(2):184-92. [PubMed ID: 31084433]. [PubMed Central ID: PMC6629036]. https://doi.org/10.1080/10409238.2019.1611734.
-
10.
Amit-Romach E, Uni Z, Cheled S, Berkovich Z, Reifen R. Bacterial population and innate immunity-related genes in rat gastrointestinal tract are altered by vitamin A-deficient diet. J Nutr Biochem. 2009;20(1):70-7. [PubMed ID: 18495461]. https://doi.org/10.1016/j.jnutbio.2008.01.002.
-
11.
Huda MN, Ahmad SM, Kalanetra KM, Taft DH, Alam MJ, Khanam A, et al. Neonatal Vitamin A Supplementation and Vitamin A Status Are Associated with Gut Microbiome Composition in Bangladeshi Infants in Early Infancy and at 2 Years of Age. J Nutr. 2019;149(6):1075-88. [PubMed ID: 31006815]. [PubMed Central ID: PMC6543205]. https://doi.org/10.1093/jn/nxz034.
-
12.
Robea MA, Luca AC, Ciobica A. Relationship between Vitamin Deficiencies and Co-Occurring Symptoms in Autism Spectrum Disorder. Medicina (Kaunas). 2020;56(5):245. [PubMed ID: 32443822]. [PubMed Central ID: PMC7279218]. https://doi.org/10.3390/medicina56050245.
-
13.
Guo M, Li L, Zhang Q, Chen L, Dai Y, Liu L, et al. Vitamin and mineral status of children with autism spectrum disorder in Hainan Province of China: associations with symptoms. Nutr Neurosci. 2020;23(10):803-10. [PubMed ID: 30570388]. https://doi.org/10.1080/1028415X.2018.1558762.
-
14.
Cheng B, Zhu J, Yang T, Guo M, Lai X, Li Q, et al. Vitamin A deficiency increases the risk of gastrointestinal comorbidity and exacerbates core symptoms in children with autism spectrum disorder. Pediatr Res. 2021;89(1):211-6. [PubMed ID: 32225174]. https://doi.org/10.1038/s41390-020-0865-y.
-
15.
Liu Z, Wang J, Xu Q, Hong Q, Zhu J, Chi X. Research Progress in Vitamin A and Autism Spectrum Disorder. Behav Neurol. 2021;2021:5417497. [PubMed ID: 34917197]. [PubMed Central ID: PMC8670912]. https://doi.org/10.1155/2021/5417497.
-
16.
Delgado C, Ward P, Chertow GM, Storer L, Dalrymple L, Block T, et al. Calibration of the brief food frequency questionnaire among patients on dialysis. J Ren Nutr. 2014;24(3):151-6-e1. [PubMed ID: 24613023]. [PubMed Central ID: PMC4145671]. https://doi.org/10.1053/j.jrn.2013.12.004.
-
17.
Mei CF, Faller EM, Chuan LX, Gabriel JS. Household Income, Food Insecurity and Nutritional Status of Migrant Workers in Klang Valley, Malaysia. Ann Glob Health. 2020;86(1):90. [PubMed ID: 32832384]. [PubMed Central ID: PMC7413208]. https://doi.org/10.5334/aogh.2859.
-
18.
Yang C, Chen J, Guo N, Liu Z, Yun C, Li Y, et al. Comparison on the status of vitamin A in 6- to 13- year-old children between 2002 and 2012 in China. Nutr J. 2016;15(1):50. [PubMed ID: 27146897]. [PubMed Central ID: PMC4857281]. https://doi.org/10.1186/s12937-016-0170-0.
-
19.
Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10(10):996-8. [PubMed ID: 23955772]. https://doi.org/10.1038/nmeth.2604.
-
20.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7(5):335-6. [PubMed ID: 20383131]. [PubMed Central ID: PMC3156573]. https://doi.org/10.1038/nmeth.f.303.
-
21.
Fu SC, Lee CH, Wang H. Exploring the Association of Autism Spectrum Disorders and Constipation through Analysis of the Gut Microbiome. Int J Environ Res Public Health. 2021;18(2):667. [PubMed ID: 33466802]. [PubMed Central ID: PMC7830459]. https://doi.org/10.3390/ijerph18020667.
-
22.
Li J, Ma Y, Bao Z, Gui X, Li AN, Yang Z, et al. Clostridiales are predominant microbes that mediate psychiatric disorders. J Psychiatr Res. 2020;130:48-56. [PubMed ID: 32781373]. https://doi.org/10.1016/j.jpsychires.2020.07.018.
-
23.
Zou R, Xu F, Wang Y, Duan M, Guo M, Zhang Q, et al. Changes in the Gut Microbiota of Children with Autism Spectrum Disorder. Autism Res. 2020;13(9):1614-25. [PubMed ID: 32830918]. https://doi.org/10.1002/aur.2358.
-
24.
Precup G, Vodnar DC. Gut Prevotella as a possible biomarker of diet and its eubiotic versus dysbiotic roles: a comprehensive literature review. Br J Nutr. 2019;122(2):131-40. [PubMed ID: 30924428]. https://doi.org/10.1017/S0007114519000680.
-
25.
Larsen JM. The immune response to Prevotella bacteria in chronic inflammatory disease. Immunology. 2017;151(4):363-74. [PubMed ID: 28542929]. [PubMed Central ID: PMC5506432]. https://doi.org/10.1111/imm.12760.
-
26.
Kang DW, Adams JB, Coleman DM, Pollard EL, Maldonado J, McDonough-Means S, et al. Long-term benefit of Microbiota Transfer Therapy on autism symptoms and gut microbiota. Sci Rep. 2019;9(1):5821. [PubMed ID: 30967657]. [PubMed Central ID: PMC6456593]. https://doi.org/10.1038/s41598-019-42183-0.
-
27.
Kang DW, Park JG, Ilhan ZE, Wallstrom G, Labaer J, Adams JB, et al. Reduced incidence of Prevotella and other fermenters in intestinal microflora of autistic children. PLoS One. 2013;8(7):e68322. [PubMed ID: 23844187]. [PubMed Central ID: PMC3700858]. https://doi.org/10.1371/journal.pone.0068322.
-
28.
Pulikkan J, Maji A, Dhakan DB, Saxena R, Mohan B, Anto MM, et al. Gut Microbial Dysbiosis in Indian Children with Autism Spectrum Disorders. Microb Ecol. 2018;76(4):1102-14. [PubMed ID: 29564487]. https://doi.org/10.1007/s00248-018-1176-2.
-
29.
Strati F, Cavalieri D, Albanese D, De Felice C, Donati C, Hayek J, et al. New evidences on the altered gut microbiota in autism spectrum disorders. Microbiome. 2017;5(1):24. [PubMed ID: 28222761]. [PubMed Central ID: PMC5320696]. https://doi.org/10.1186/s40168-017-0242-1.
-
30.
Gilbert JA, Krajmalnik-Brown R, Porazinska DL, Weiss SJ, Knight R. Toward effective probiotics for autism and other neurodevelopmental disorders. Cell. 2013;155(7):1446-8. [PubMed ID: 24360269]. [PubMed Central ID: PMC4166551]. https://doi.org/10.1016/j.cell.2013.11.035.
-
31.
Hsiao EY, McBride SW, Hsien S, Sharon G, Hyde ER, McCue T, et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell. 2013;155(7):1451-63. [PubMed ID: 24315484]. [PubMed Central ID: PMC3897394]. https://doi.org/10.1016/j.cell.2013.11.024.
-
32.
Xu M, Xu X, Li J, Li F. Association Between Gut Microbiota and Autism Spectrum Disorder: A Systematic Review and Meta-Analysis. Front Psychiatry. 2019;10:473. [PubMed ID: 31404299]. [PubMed Central ID: PMC6673757]. https://doi.org/10.3389/fpsyt.2019.00473.
-
33.
Ye F, Gao X, Wang Z, Cao S, Liang G, He D, et al. Comparison of gut microbiota in autism spectrum disorders and neurotypical boys in China: A case-control study. Synth Syst Biotechnol. 2021;6(2):120-6. [PubMed ID: 34095558]. [PubMed Central ID: PMC8163862]. https://doi.org/10.1016/j.synbio.2021.03.003.