肠道微生物如何影响自闭症？ | 知几文献汇编
孤独症（自闭症）是一组神经系统广泛性发育障碍。患者的两个基本特征是：交互性社交交流和社交互动的持续损害，以及受限的、重复的行为、兴趣或活动模式。
2016年，美国精神医学学会将“早期婴儿孤独症、儿童孤独症、卡纳氏（Kanner）孤独症、高功能孤独症、非典型孤独症、未特定的广泛发育障碍、儿童期瓦解障碍和艾斯伯格综合征”这些疾病，统一归类于孤独症谱系障碍（Autism Spectrum Disorder），简称为“ASD”。
美国疾控预防中心（CDC）的最新数据是，2014年，全美8岁儿童中，每59人中就有一例孤独症（自闭症）患者。
目前，中国暂未开展过全国性的流行病学调查。据推测，我国ASD患病人数或已超过千万。一份由专业机构发布的《中国自闭症儿童发展状况报告》估计，我国0-14岁ASD患儿的数量可能超过200万。
对于ASD儿童，往往有非常详细的个案描写，但鲜有整体群像——因为ASD本身具有高度个体特征，患者的能力和障碍呈光谱式分布，异质性极强。
ASD是医学界的著名难题之一。不仅没有确定的发病原因、机制，也没有对应的药物。
将ASD与肠道微生物联系在一起，是近十年才发生的事情。科学家们的研究起始点在于：他们发现，约四分之三的ASD患者同时伴随胃肠道症状。
据美国精神学会，回避性/限制性摄食障碍是ASD患者相当常见的特征，并且可能持续存在极端和有限的食物偏好。
一些ASD儿童只吃固定的几种食物（或某几种颜色的食物），并坚决拒绝饮食方式的改变，挑食严重。在出现胃肠道问题时，也往往因为不会表达而被忽视，或出现易激惹状态。相对于一般儿童，ASD患者感觉统合失调、胃肠道问题、睡眠障碍、饮食问题也更为多发。
随着研究的深入，研究者们还发现，肠道微生物可以影响血脑屏障的通透性。这说明，肠道微生物对人类大脑的影响可能远远超过我们的预期。
其影响机制，可能通过血液系统、内分泌系统和神经系统而作用，这一相互关系被称为“微生物-肠道-大脑轴”（microbiome-gutbrain axis，菌肠脑轴）。
十年间，对ASD的基础研究进入快速发展，聚焦于肠道微生物与ASD的研究正吸引着广泛注意。
为此，知几未来研究院出品了本篇《ASD与肠道微生物干预法》报告。本报告共包含了9篇ASD与肠道微生物的最新研究，截取部分原英文文献进行解读，重点关注饮食与益生菌对ASD患者肠道微生物的干预法。
您可以通过下方的文献目录快速了解文献内容👇👇👇
微生物重建逆转因母体饮食引起的后代社会和突触缺陷
中国ASD患儿肠道微生物群分布及相关性分析
嗜酸乳杆菌、乳杆菌和双歧杆菌粉剂改善孤独症的严重程度（由ATEC评估）及胃肠道症状
使用益生菌胶囊“Children Dophilus”干预ASD儿童肠道微生物
营养补充剂胶囊Delpro®改善ASD严重程度
益生菌有效降低ASD儿童尿液中DA水平和DA/LA比值，预防胃肠道念珠菌入侵
植物乳杆菌WCSF1显著增加ASD儿童Lab158数量，显著减少Erec482数量
粪菌移植疗法改变肠道生态系统并改善胃肠和ASD症状
微生物菌群调节神经发育障碍相关的行为和生理异常
为了方便大家学习，知几未来研究院也对每篇文献都做出了详尽解读。
本报告约2.1k字，全部读完大约需要50分钟。
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1.微生物重建逆转因母体饮食引起的后代社会和突触缺陷
Microbial Reconstitution Reverses Maternal Diet-Induced Social and Synaptic Deficits in Offspring（2016），Cell，IF: 31.398
Shelly A. Buffington, Gonzalo Viana Di Prisco, Thomas A. Auchtung, Nadim J. Ajami, Joseph F. Petrosino, Mauro Costa-Mattioli
In Brief：
A maternal high-fat diet leads to changes in the gut microbiome of offspring and induces behavioral alterations that can be restored via selective reintroduction of a commensal bacterial strain.
Highlights：
a. Maternal high-fat diet (MHFD) induces behavioral alterations in offspring
b. MHFD causes alterations in gut microbial ecology in offspring
c. MHFD offspring show deficient synaptic plasticity in the VTA and oxytocin production
d. L. reuteri treatment restores oxytocin levels, VTA plasticity and social behaviors
（1）实验方法：
孕鼠正常饮食孕育的后代与孕鼠高脂饮食孕育的后代分别在催产素水平、中脑腹侧被盖区（VTA）可塑性和社交行为等方面进行对比。并通过罗伊氏乳杆菌改善自闭症等症状。
（2）研究结论：
母体高脂肪饮食(MHFD)诱导后代行为改变
母体高脂肪饮食（MHFD）导致后代肠道微生物生态的改变
母体高脂肪饮食的后代在中脑腹侧被盖区（VTA）和催产素的产生过程中缺乏突触可塑性
罗伊氏乳杆菌（L. reuteri）治疗可恢复催产素水平、中脑腹侧被盖区（VTA）可塑性和社交行为
2.中国ASD患儿肠道微生物群分布及相关性分析
Analysis of gut microbiota profiles and microbe-disease associations in children with autism spectrum disorders in China（2018），Scientific Reports，IF: 4.122
Mengxiang Zhang1,2, Wei Ma3, Juan Zhang4, Yi He 1,2 & Juan Wang1,2
In Brief：
Autism spectrum disorder (ASD) is a set of complex neurodevelopmental disorders. Recent studies reported that children with ASD have altered gut microbiota profiles compared with typical development (TD) children. However, few studies on gut bacteria of children with ASD have been conducted in China. Here, in order to elucidate changes of fecal microbiota in children with ASD, 16S rRNA sequencing was conducted and the 16S rRNA (V3-V4) gene tags were amplified. We investigated differences in fecal microbiota between 35 children with ASD and 6 TD children. At the phylum level, the fecal microbiota of ASD group indicated a significant increase of the Bacteroidetes/Firmicutes ratio. At the genus level, we found that the relative abundance of Sutterella, Odoribacter and Butyricimonas was much more abundant in the ASD group whereas the abundance of Veillonella and Streptococcus was decreased significantly compared to the control group. Functional analysis demonstrated that butyrate and lactate producers were less abundant in the ASD group. In addition, we downloaded the association data set of microbe–disease from human microbe–disease association database and constructed a human disease network including ASD using our gut microbiome results. In this microbe– disease network based on microbe similarity of diseases, we found that ASD is positively correlated with periodontal, negatively related to type 1 diabetes. Therefore, these results suggest that microbe-based disease analysis is able to predict novel connection between ASD and other diseases and may play a role in revealing the pathogenesis of ASD.
（1）实验方法：
研究对35例（ASD）自闭症儿童和6例正常(TD)儿童相比，对其粪便微生物区系进行16S rRNA测序，扩增16S rRNA (V3-V4)区域，对其进行生物信息比对。
（2）研究结论：
ASD组的粪便菌群中，拟杆菌门/厚壁菌门的比值显著增加。
在属水平，与正常儿童相比，ASD组的萨特氏菌、臭气杆菌和丁酸亚胺的相对丰度更高，而韦荣氏球菌和链球菌的丰度显著降低。
功能分析显示ASD组的丁酸和乳酸产生的量减少。
在人类微生物疾病关联数据库中，发现ASD与牙周病正相关，与1型糖尿病负相关因此，这些结果表明，基于微生物的疾病分析能够预测ASD与其他疾病之间新的联系，并可能在揭示ASD的发病机制方面发挥作用。
3. 嗜酸乳杆菌、乳杆菌和双歧杆菌粉剂改善孤独症的严重程度（由ATEC评估）及胃肠道症状
The role of probiotics in children with autism spectrum disorder: A prospective, open-label study（2017），Nutritional Neuroscience，IF: 3.313
Sanaa Y. Shaaban, Yasmin G. El Gendy, Nayra S. Mehanna, Waled M. El- Senousy, Howaida S. A. El-Feki, Khaled Saad & Osama M. El-Asheer
Objective:
There are limited data on the efficacy of probiotics in children with ASD, therefore, this study aims to evaluate the efficacy and tolerability of probiotics in an Egyptian cohort of children with ASD.
Methods:
Gastrointestinal (GI) flora were assessed by quantitative real-time PCR of stool samples of 30 autistic children from 5 to 9 years old. GI symptoms of autistic children were assessed with a modified six- item Gastrointestinal Severity Index (6-GSI) questionnaire, and autistic symptoms were assessed with Autism Treatment Evaluation Checklist (ATEC) before and after 3 months of supplementation of probiotics nutritional supplement formula (each gram contains 100×106 colony forming units of three probiotic strains; Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacteria longum).
Results:
After probiotic supplementation, the stool PCR of autistic children showed increases in the colony counts of Bifidobacteria and Lactobacilli levels, with a significant reduction in their body weight as well as significant improvements in the severity of autism (assessed by the ATEC), and gastrointestinal symptoms (assessed by the 6-GSI) compared to the baseline evaluated at the start of the study.
Conclusions:
We concluded that probiotics have beneficial effects on both behavioral and GI manifestations of ASD. Probiotics (a non-pharmacological and relatively risk-free option) could be recommended for children with ASD as an adjuvant therapy. At this stage, this study is a single center with a small number of patients and a great deal of additional wide-scale randomized controlled trials are needed to critically confirm the efficacy of probiotics in ASD.
（1）实验方法：
选择人群：选取30例（19个男孩和11个女孩）5-9岁的自闭症儿童，另外从患者亲属中选取30例年龄、性别匹配的健康儿童作为对照组。所有患者接受问卷调查（年龄、性别、病史、饮食、行为史等），对所有患者进行常规检查以及神经系统检查。
入组条件：排除患有其他神经发育障碍或精神疾病的自闭症儿童。另外排除了任何临床重大慢性疾病患者，包括：贫血、大脑畸形、代谢疾病、癫痫、有机胃肠道疾病（如胃食管返流，食物过敏，肠易激综合症IBS）和腹腔疾病。此外，患者服用抗真菌药物、抗生素、特殊饮食（即无麸质饮食，无酪蛋白饮食，高蛋白饮食，生酮饮食）和当前使用精神类药物被排除在外。最后，所有患者在研究之前2周以及研究期间没有使用维生素治疗或其他替代治疗。
检测方法：收集干预之前的所有自闭症患儿以及健康组的粪便样本，通过实时定量PCR检测粪便样本中微生物组成（主要是双歧杆菌和乳酸菌），服用益生菌3个月之后，通过PCR来评估自闭症患儿粪便中益生菌的比例。通过六项胃肠道严重程度指数（6-GSI，(便秘、腹泻，粪便一致性、粪便的气味，肠胃气胀，腹痛)）问卷来评估自闭症儿童胃肠道症状。通过自闭症治疗评估检查表（ATEC，ATEC包含四个分量表：言语/语言通信（14个项目；分数范围从0到28），社交能力（20项；分数范围从0到40)、感觉/认知意识（18项；分数范围从0到36）和健康/身体/行为（25项；分数范围从0到75）来评估益生菌使用前和使用三个月之后的自闭症症状。
（2）干预方法：
所有自闭症儿童服用益生菌粉剂（包含有嗜酸乳杆菌、乳杆菌和双歧杆菌三种有益菌的益生菌粉剂），每天服用5克，每克粉剂含有100×10^6 CFUs，连续服用3个月。
（3）结论：
通过实时定量PCR对所有受检者粪便样本的有益菌（主要是双歧杆菌和乳酸菌）进行检测，结果显示，益生菌补充之前，自闭症儿童的双歧杆菌含量明显低于对照组（P值= 0.0001）；益生菌补充3个月后，自闭症患儿的双歧杆菌和乳酸杆菌水平明显增加。
益生菌摄入3个月后，患儿体重指数显著下降(P值< 0.01)，显著改善孤独症的严重程度（由ATEC评估）以及胃肠道症状。
结果表明益生菌对有ASD患儿的胃肠道症状和行为表现是有利的。益生菌（相对无风险的）可以推荐给患有自闭症的孩子，作为辅助治疗。
4.使用益生菌胶囊“Children Dophilus”干预ASD儿童肠道微生物
Gastrointestinal microbiota in children with autism in Slovakia（2015），Physiology & Behavior，IF: 2.517
Aleksandra Tomova a,⁎, Veronika Husarova a, Silvia Lakatosova a, Jan Bakos a, Barbora Vlkova b, Katarina Babinska a, Daniela Ostatnikova a
In Brief：
Development of Autism Spectrum Disorders (ASD), including autism, is based on a combination of genetic predisposition and environmental factors. Recent data propose the etiopathogenetic role of intestinal microflora in autism. The aim of this study was to elucidate changes in fecal microbiota in children with autism and determine its role in the development of often present gastrointestinal (GI) disorders and possibly other manifestations of autism in Slovakia. The fecal microflora of 10 children with autism, 9 siblings and 10 healthy children was investigated by real-time PCR. The fecal microbiota of autistic children showed a significant decrease of the Bacteroidetes/Firmicutes ratio and elevation of the amount of Lactobacillus spp. Our results also showed a trend in the incidence of elevated Desulfovibrio spp. in children with autism reaffirmed by a very strong association of the amount of Desulfovibrio spp. with the severity of autism in the Autism Diagnostic Interview (ADI) restricted/repetitive behavior subscale score. The participants in our study demonstrated strong positive correlation of autism severity with the severity of GI dysfunction. Probiotic diet supplementation normalized the Bacteroidetes/Firmicutes ratio, Desulfovibrio spp. and the amount of Bifidobacterium spp. in feces of autistic children. We did not find any correlation between plasma levels of oxytocin, testosterone, DHEA-S and fecal microbiota, which would suggest their combined influence on autism development. This pilot study suggests the role of gut microbiota in autism as a part of the “gut-brain”axis and it is a basis for further investigation of the combined effect of microbial, genetic, and hormonal changes for development and clinical manifestation of autism.
Highlights：
a. Fecal microbiota in autistic children in Slovakia differs from controls and siblings.
b. There is a correlation of the autism severity with the severity of GI dysfunction.
c. In our study Desulfovibrio spp. abundance is associated with the severity of autism.
d. Probiotic supplementation normalizes bacterial balance in fecal microbiota.
e. No joined influence of oxytocin, testosterone, DHEAS and fecal microbiota on autism.
（1）实验方法：
选择人群：选取三组人群，第一组为10例自闭症儿童，包括9个男孩和一个女孩，年龄从2到9岁，9例自闭症儿童接受了益生菌干预；第二组为自闭症儿童正常的兄弟姐妹9例，包含7个男孩和两个女孩，年龄从5到17岁；第三组为10例正常儿童作为对照组，包含10个男孩，年龄从2到11岁。
检测方法：通过实时定量PCR检测所有受试者（自闭症儿童、自闭症儿童益生菌干预4个月后、自闭症儿童正常的兄弟姐妹、对照儿童组）粪便样本中微生物丰度（主要是双歧杆菌、乳酸菌、脱硫孤菌、梭状芽胞杆菌），通过ELISA检测细胞因子TNF-α及等离子体激素，通过儿童自闭症评定量表（CARS）对自闭症儿童进行物理评估，用于自闭症儿童的识别和鉴别诊断；通过自闭症诊断访谈（ADI），了解自闭症儿童的行为。ADI用于研究4内容领域，包括社会/互惠的互动、沟通、演讲和语言、限制/重复行为。胃肠道情况评估是基于父母的调查问卷，根据症状的严重程度，范围从1到4。
（2）干预方法：
膳食补充益生菌胶囊的“Children Dophilus”，包含3种乳酸菌（60%），2种双歧杆菌 （25%）和1种链球菌（15%），每天口服3次，连续服用4个月。
（3）结论：
研究发现自闭症儿童粪便样本中肠道微生物群与健康儿童的肠道微生物群存在差异：拟杆菌门/厚壁菌门比例降低，乳酸菌丰度升高。在自闭症儿童中，梭状芽胞杆菌l和脱硫弧菌呈升高趋势。
自闭症儿童兄弟姐妹肠道菌群中拟杆菌门/厚壁菌门比例与对照组儿童有显著差异。
研究还发现，自闭症的严重程度（ADI）与胃肠功能障碍的严重程度相关，胃肠功能障碍的严重程度与促炎细胞因子TNFa的粪便水平相关。
补充益生菌后，自闭症患儿拟杆菌/厚壁菌门比例恢复正常、总乳酸菌水平上升，粪便中梭菌、脱硫弧菌以及肿瘤坏死因子（TNF）-的水平明显减少。
由于自闭症患者血浆催产素和脱氢表雄酮- s水平显著下降，睾酮或催产素水平与自闭症呈强正相关，研究未发现其血浆水平与粪便微生物群之间有任何强相关性，这表明它们在疾病发展中具有共同影响。
5.营养补充剂胶囊Delpro®改善ASD严重程度
Improvements in Gastrointestinal Symptoms among Children with Autism Spectrum Disorder Receiving the Delpro® Probiotic and Immunomodulator Formulation（2013），Probiotics & Health
Rachel West DO1, Emily Roberts2, Lubov S Sichel3 and John Sichel3*
In Brief：
Children with Autism Spectrum Disorder (ASD) frequently exhibit gastrointestinal (GI) distress. They often have deficiencies of beneficial intestinal microflora, which may lead to inflammation or immune dysfunction, malabsporption, food intolerance, failure to thrive, gas, bloating, constipation or diarrhea. We conducted a survey of caregivers for ASD children with GI distress receiving Delpro®, a nutritional supplement consisting of a mixture of five probiotic strains formulated with the immunomodulator Del-Immune V® (Lactobacillus rhamnosus V lysate). Caregivers assessed ASD signs and symptoms before and after 21 days of treatment using the autism treatment evaluation checklist (ATEC). Almost half of the respondents (48%) reported decreases in diarrhea severity and 52% reported decreases in constipation severity (n=25). Caregivers reported an increase in stool frequency, from one movement every 1.5 days to every 1.3 days, although this change was not statistically significant. Overall, 88% reported a decrease in total ATEC score, signifying an improvement of ASD symptoms. Mean ATEC values decreased from 72.8 prior to treatment to 58.3 following treatment initiation. Participants also had significant improvements in all ATEC domains (speech/language/communication, sociability, sensory/cognitive awareness, and health/physical/ behavior). Since this was a survey of caregivers for ASD children with GI distress receiving Delpro®, there was no control arm of no-treatment or placebo. Accordingly, these survey results need to be validated in controlled clinical trials. These data suggest that probiotic/immunomodulator Delpro® may have significant benefit in the treatment of GI distress and other ATEC signs and symptoms among this population.
（1）实验方法：
选择人群： 33例自闭症儿童，年龄3-16岁。
检测方法：没有检测肠道微生物，参与者需记录服用Delpro®之前以及服用21天后的大便频率，还要求提供干预之前呵和干预之后的ATEC成绩。ATEC是自闭症治疗研究所开发的工具用来评估自闭症严重程度在四类：1）言语/语言/通信，2）社交能力，3）感觉/认知意识，和4）健康/身体/行为。ATEC评分系统，值越高代表疾病越严重以及改善程度越小。ATEC分数报告两个时间点： 21天治疗前任何时间和21天治疗后任何时间。
（2）干预方法：
参与者接受Delpro® 营养补充剂胶囊（美国博尔德有限公司）。每个胶囊含有100亿CFUs不同的益生菌（嗜酸乳杆菌、干酪乳杆菌、乳酸菌、长双歧杆菌、两歧双歧杆菌，每种含有20亿CFUs）和8毫克Del-Immune V®粉。Del-Immune V®粉是一种细胞溶解的冻干粉，其中包含肽聚糖，胞壁肽和含有核酸化合物或DNA。推荐剂量为每日3次，每次1粒胶囊，补充21天。
（3）结论：
Delpro®治疗后， ATEC得分显著降低，自闭症障碍的严重程度降低。
48%的受检者报告腹泻严重程度减少和52%受检者便秘严重程度减少。
6.益生菌有效降低ASD儿童尿液中DA水平和DA/LA比值，预防胃肠道念珠菌入侵
The level of arabinitol in autistic children after probiotic therapy（2012），Nutrition，IF: 3.734
Joanna Ka1uz_na-Czaplin ska Ph.D.a,*, Sylwia B1aszczyk Ph.D.b
Objective:
The level of D-arabinitol (DA) and the ratio of D-/L-arabinitol (DA/LA) in the urine of children with autism were investigated. The changes in DA/LA after probiotic treatment in urine samples of children with autism were studied.
Methods:
DA and LA and the DA/LA ratio were identified by capillary gas chromatography/mass spectrometry in urine before and after the probiotic therapy.
Results:
The level of DA is significantly higher (P < 0.05) in the urine of autistic children before (A) and after probiotic supplementation (A1) (160.04 ! 22.88 mmol/mmol creatinine and 89.53 ! 37.41 mmol/mmol creatinine, respectively). Nonetheless, the probiotic supplementation let to a significant decrease in DA and DA/LA and to a significant improvement in ability of concentration and carrying out orders.
Conclusion:
The use of probiotics seems to be helpful in reducing the level of DA and the ratio of DA/LA in the urine of children with autism.
（1）实验方法：
选择人群： 22例自闭症儿童，年龄4-10岁。
检测方法： D-arabinitol （D-阿拉伯糖醇，DA）是许多致病性念珠菌属的代谢产物，其在自闭症患者中含量较高，通过毛细管气相色谱/质谱法检测益生菌治疗前、后尿液中DA（念珠菌的代谢产物）、LA以及 DA /LA比值（侵袭性念珠菌的一种生物标志物）。
（2）干预方法：
所有自闭症儿童口服益生菌胶囊（嗜酸乳杆菌, strain Rosell-11, 5×10^9 CFU / g），剂量为每日2次，服用2个月。
（3）结论：
补充益生菌后，自闭症儿童的尿液中DA水平显著降低（P < 0.05），DA /LA比值也降低，即益生菌疗法可以有效地降低自闭症儿童尿液中DA水平和DA/LA比值，预防胃肠道念珠菌的入侵。
7.植物乳杆菌WCSF1显著增加ASD儿童Lab158数量，显著减少Erec482数量
A double-blind, placebo-controlled, crossover-designed probiotic feeding study in children diagnosed with autistic spectrum disorders (2010), International Journal of Probiotics and Prebiotics
1*Helena M. R. T. Parracho, 1Glenn R. Gibson, 2Fiona Knott, 3Douwina Bosscher, 4Michiel Kleerebezem and 1Anne L. McCartney
In Brief：
There is growing interest in the role of gastrointestinal (GI) pathology and clinical expression of autism. Recent studies have demonstrated differences in the faecal clostridial populations harboured by autistic and non-autistic children. The potential of Lactobacillus plantarum WCSF1 (a probiotic) to modulate the gut microbiota of autistic subjects was investigated during a double- blind, placebo-controlled, crossover-designed feeding study. The faecal microbiota, gut function and behaviour scores of subjects were examined throughout the 12-week study. Lactobacillus plantarum WCFS1 feeding significantly increased Lab158 counts (lactobacilli and enterococci group) and significantly reduced Erec482 counts (Clostridium cluster XIVa) compared to placebo. Probiotic feeding also resulted in significant differences in the stool consistency compared to placebo and behaviour scores (total score and scores for some subscales) compared to baseline. The major finding of this work was the importance of study protocol in relation to the specific considerations of this subject population, with an extremely high dropout rate seen (predominantly during the baseline period). Furthermore, the relatively high inter-individual variability observed suggests that subsequent studies should use defined subgroups of autistic spectrum disorders, such as regressive or late-onset autism. In summary, the current study has highlighted the potential benefit of L. plantarum WCFS1 probiotic feeding in autistic individuals.
（1）实验方法：
选择人群： 22例自闭症儿童，年龄3-16岁。
入组条件：排除前一个月或整个研究期间补充益生菌、使用抗生素或活性药物治疗胃肠道以及参加另一项涉及实验性药物的研究。
检测方法：
进行一组随机、双盲、安慰剂对照实验，招募的受试者被随机分配到两个喂养组。第一组在第一次喂养期间（3周）服用安慰剂，第二次喂养期间（3周）服用益生菌，同时，第二组在第一次喂养期间（3周）服用益生菌，第二次喂养期间（3周）服用安慰剂。每个喂养期之后的3周是washout period（洗脱期）。
收集粪便样本的时间点为在开始服用益生菌前一周随机的两天（baseline samples；分别为:B1、B2）、益生菌干预期结束时（F1、F2）、washout period 结束时（W1、W2）。
使用荧光原位杂交法（FISH）检测粪便样本中细菌种群水平。
研究对象的父母/监护人被要求填写与胃肠道功能和症状有关的日记。每天记录粪便样本的数量和浓度（硬、成型、软），同时记录腹痛（无、中、重度）、肠胀气（有、无）、胃胀气（正常、多于正常)。伴随用药（药物、剂量、起始日期和治疗时间）和不良事件（事件、日期和持续时间的描述）也在整个研究过程中以不同的形式记录下来。
使用DBC-P（初级标准发展行为检查表）和 TBPS（行为问题总分）评分来评价安慰剂和益生菌治疗对行为的影响。
（2）干预方法：
所有自闭症儿童服用植物乳杆菌WCSF1，每日剂量4.5 × 10^10 CFUs，连服3周，对照组服用安慰剂（麦芽糖糊精，110毫克/胶囊）。
（3）结论:
研究人员对受试者的粪便微生物群、肠道功能和行为评分进行了评估。
研究发现，与安慰剂组相比，植物乳杆菌WCFS1喂养显著增加Lab158数量（乳酸菌和肠球菌组），显著减少Erec482数量（梭菌群XIVa）。
益生菌喂养后的实验组与安慰剂组相比，其粪便一致性存在差异，行为评分（总分和部分亚量表得分)与基线也存在显著差异。
8.粪菌移植疗法改变肠道生态系统并改善胃肠和ASD症状
Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study（2017），Microbiome，IF: 9.133
Dae-Wook Kang1†, James B. Adams2†, Ann C. Gregory3,15†, Thomas Borody4, Lauren Chittick5,15, Alessio Fasano6, Alexander Khoruts7,8,9, Elizabeth Geis2, Juan Maldonado1, Sharon McDonough-Means10, Elena L. Pollard2, Simon Roux5,15, Michael J. Sadowsky8,11, Karen Schwarzberg Lipson12, Matthew B. Sullivan3,5,15,16*, J. Gregory Caporaso12,13* and Rosa Krajmalnik-Brown1,14*
Background:
Autism spectrum disorders (ASD) are complex neurobiological disorders that impair social interactions and communication and lead to restricted, repetitive, and stereotyped patterns of behavior, interests, and activities. The causes of these disorders remain poorly understood, but gut microbiota, the 1013 bacteria in the human intestines, have been implicated because children with ASD often suffer gastrointestinal (GI) problems that correlate with ASD severity. Several previous studies have reported abnormal gut bacteria in children with ASD. The gut microbiome-ASD connection has been tested in a mouse model of ASD, where the microbiome was mechanistically linked to abnormal metabolites and behavior. Similarly, a study of children with ASD found that oral non-absorbable antibiotic treatment improved GI and ASD symptoms, albeit temporarily. Here, a small open-label clinical trial evaluated the impact of Microbiota Transfer Therapy (MTT) on gut microbiota composition and GI and ASD symptoms of 18 ASD-diagnosed children.
Results:
MTT involved a 2-week antibiotic treatment, a bowel cleanse, and then an extended fecal microbiota transplant (FMT) using a high initial dose followed by daily and lower maintenance doses for 7–8 weeks. The Gastrointestinal Symptom Rating Scale revealed an approximately 80% reduction of GI symptoms at the end of treatment, including significant improvements in symptoms of constipation, diarrhea, indigestion, and abdominal pain. Improvements persisted 8 weeks after treatment. Similarly, clinical assessments showed that behavioral ASD symptoms improved significantly and remained improved 8 weeks after treatment ended. Bacterial and phagedeep sequencing analyses revealed successful partial engraftment of donor microbiota and beneficial changes in the gut environment. Specifically, overall bacterial diversity and the abundance of Bifidobacterium, Prevotella, and Desulfovibrio among other taxa increased following MTT, and these changes persisted after treatment stopped (followed for 8 weeks).
Conclusions:
This exploratory, extended-duration treatment protocol thus appears to be a promising approach to alter the gut microbiome and virome and improve GI and behavioral symptoms of ASD. Improvements in GI symptoms, ASD symptoms, and the microbiome all persisted for at least 8 weeks after treatment ended, suggesting a long-term impact.
（1）实验方法：
观察18名ASD患儿肠道微生物群的纵向变化，评价粪菌移植对ASD患儿胃肠症状（主要预后）和ASD相关症状（次要结局）的治疗效果，以及粪菌移植对肠道微生物群的影响。
（2）研究结论：
胃肠症状评定量表显示，粪菌移植治疗结束时胃肠症状减少了大约80%，包括便秘、腹泻、消化不良和腹痛等症状的明显改善。治疗后8周持续改善。同样，临床评估显示， ASD症状明显改善，并在治疗结束后8周仍有改善。
测序分析显示，供体微生物区系部分植入成功，肠道环境发生有益变化。
粪菌移植后，微生物的总体多样性以及双歧杆菌、普氏菌和脱磷孤菌属等类群的丰度增加，治疗停止后，这些变化持续存在(8周后)。
9.微生物菌群调节神经发育障碍相关的行为和生理异常
Microbiota Modulate Behavioral and Physiological Abnormalities Associated with Neurodevelopmental Disorders（2013），Cell，IF: 31.398
Elaine Y. Hsiao, Sara W. McBride, Sophia Hsien, Gil Sharon, Embriette R. Hyde, Tyler McCue, Julian A. Codelli, Janet Chow, Sarah E. Reisman, Joseph F. Petrosino, Paul H. Patterson, and Sarkis K. Mazmanian
Summary：
Neurodevelopmental disorders, including autism spectrum disorder (ASD), are defined by core behavioral impairments; however, subsets of individuals display a spectrum of gastrointestinal (GI) abnormalities. We demonstrate GI barrier defects and microbiota alterations in the maternal immune activation (MIA) mouse model that is known to display features of ASD. Oral treatment of MIA offspring with the human commensal Bacteroides fragilis corrects gut permeability, alters microbial composition, and ameliorates defects in communicative, stereotypic, anxiety- like and sensorimotor behaviors. MIA offspring display an altered serum metabolomic profile, and B. fragilis modulates levels of several metabolites. Treating naive mice with a metabolite that is increased by MIA and restored by B. fragilis causes certain behavioral abnormalities, suggesting that gut bacterial effects on the host metabolome impact behavior. Taken together, these findings support a gut-microbiome-brain connection in a mouse model of ASD and identify a potential probiotic therapy for GI and particular behavioral symptoms in human neurodevelopmental disorders.
（1）实验方法：
建立母体免疫激活（MIA）小鼠模型，并对其后代进行脆弱拟杆菌（Bacteroides fragilis）干预，检测干预后MIA后代肠道屏障的完整性、特异微生物群的变化情况、ASD相关的行为异常，以及血清代谢组。
（2）研究结论：
MIA后代表现出人类ASD的胃肠道症状，并显示出肠道微生物菌群的失衡。
脆弱拟杆菌可以改善MIA后代的肠道屏障完整性、恢复MIA后代的特异性微生物群变化，并纠正与ASD相关的行为异常。
脆弱拟杆菌治疗可以调节血清代谢组，并纠正MIA诱导的血清代谢物水平。
血清代谢产物4-乙基苯基硫酸盐（4EPS）可以诱导焦虑样行为。
（脆弱拟杆菌治疗恢复MIA后代的特异性微生物群变化
B. fragilis Treatment Alters the Intestinal Microbiota and Corrects Species-Level Abnormalities in MIA Offspring）
近年来，益生菌干预作为ASD患者的辅助治疗方法，已经受到临床医生及科研人员的广泛关注。
然而，也有最新的文献研究指出，益生菌的使用并不具备普适性，益生菌的具体机制作用也尚需进一步验证。
因此，在选择益生菌或饮食干预治疗时，首先对患者进行相关检测显得尤为重要。
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