TEA e Microbiota

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Microbiota and gut-brain axis dysfunction in Autism Spectrum Disorder:

Evidence for Functional Gastrointestinal disorders
I. Lasheras, P. Seral, E. Latorre, E. Barroso, P. Gracia-Garcı́a, J.

Santabárbara
PII: S1876-2018(19)30747-6
DOI: https://doi.org/10.1016/j.ajp.2019.101874
Reference: AJP 101874
To appear in: Asian Journal of Psychiatry
Received Date: 12 August 2019

Revised Date: 4 November 2019
Accepted Date: 4 November 2019
Please cite this article as: Lasheras I, Seral P, Latorre E, Barroso E, Gracia-Garcı́a P,
Santabárbara J, Microbiota and gut-brain axis dysfunction in Autism Spectrum Disorder:
Evidence for Functional Gastrointestinal disorders, Asian Journal of Psychiatry (2019),
doi: https://doi.org/10.1016/j.ajp.2019.101874
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Microbiota and gut-brain axis dysfunction in Autism Spectrum
Disorder: Evidence for Functional Gastrointestinal disorders
Lasheras I a, Seral P a, Latorre E b,c,d*

, Barroso E e, Gracia-García P d,f,g
,
Santabárbara J a,d,g
a
Department of Preventive Medicine and Public Health, Universidad de Zaragoza,
Zaragoza, Spain.
b
Department of Biochemistry and Molecular and Cell Biology, Universidad de Zaragoza,
Zaragoza, Spain
of
c
Instituto Agroalimentario de Aragón – IA2- (Universidad de Zaragoza – CITA),
Zaragoza, Spain
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d
Instituto de Investigación Sanitaria de Aragón (IIS Aragón), Zaragoza, Spain
e
Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), Madrid,
Spain
f
-p
Psychiatry Service. Hospital Clínico Universitario Miguel Servet, Zaragoza, Spain
re
g
Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Ministry of
Science and Innovation, Madrid, Spain.
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*Corresponding and reprint author:

Dr. Eva Latorre
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Department of Biochemistry and Molecular and Cell Biology

University of Zaragoza
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Pedro Cerbuna 12, 50009, Zaragoza, Spain

evalatorre@unizar.es
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1
Highlights
 Autism spectrum disorders (ASD) are associated with functional gastrointestinal
disorders
 Gut microbiota dysbiosis could be a potential factor for ASD pathogenesis
 Microbiome may be an interface between genetic and environmental risk factors
in ASD
 We review evidence of gut microbial shifts in ASD and gastrointestinal
disorders
 Gut microbiota-brain axis studies could identify novel targets for ASD
ABSTRACT
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INTRODUCTION: The high frequency of functional gastrointestinal disorders (FGIDs)
in autism spectrum disorders (ASD) has drawn attention to the composition of gut
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microbiota as a possible factor in ASD pathogenesis. However, characterization of a
distinctive ASD microbial pattern is still unclear.

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OBJECTIVE: To conduct a narrative review on ASD microbial profile and diversity
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changes relative to NT children and FGID comorbidity and ASD pathogenesis.
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METHODOLOGY: First, we searched the PubMed database in peer-reviewed journals
for evidence regarding the current epidemiological evidence on FGID comorbidity. For
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the identification of a microbial profile in ASD children, only original studies examining
gut bacterial and fungal abundances and diversity in ASD children and adolescents were
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included. Lastly, research on the role of microbial dysbiosis as an interface between
genetic and environmental risk factors in the pathogenesis of neuropsychiatric disorders,

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and specifically ASD, was examined.
RESULTS: Prevalence and risk of FGIDs is significantly higher in ASD children and
correlates with the severity of ASD. Bacterial and fungal diversity differ between ASD
and NT children, indicating a difference in taxonomic abundance profiles, which have
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been reported at all bacterial phylogenetic levels. However, studies analyzing gut
microbiota have a heterogeneous methodology and several limitations that could account
for the variety of findings for each taxon. Also, covariate analysis reveals influence of
demographics, diet, disease severity, GI comorbidity and allergies. Integration of these
findings with changes in metabolome and genetic risk factors allowed for a better
understanding of microbiota involvement in ASD pathogenesis for future research.
Keywords: Autism Spectrum Disorder, Microbiota, Gut-brain Axis, Functional
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Gastrointestinal Disorders, Dysbiosis
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1. INTRODUCTION
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Autism spectrum disorders (ASD) is an umbrella term that incorporates a set of
neurodevelopmental disorders previously referred to as autistic disorder (AD), Asperger´s

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syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), and
childhood disintegrative disorders (Gyawali and Patra 2019). The global prevalence of
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ASD was estimated at 1 in 160 in 2000 (Elsabbagh et al. 2012) and rates have shown an
increasing trend over time. In 2016, ASD was found to affect 1 in every 37 US children
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(Xu et al. 2018), with a significantly higher prevalence in boys (Christensen et al. 2018).
Although this upward trend seems to have leveled off (Tsai 2014), this group of disorders
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has become a major concern in the scientific community.
Along with the core diagnostic traits, a wide range of comorbid conditions varying in
severity and combination can be observed in people with ASD. Among the more
commonly reported ones are sleep problems (Elrod and Hood 2015), atypical feeding
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patterns (Sharp et al. 2013), psychiatric disorders such as obsessive compulsive disorder,
bipolar disorder, psychotic spectrum disorders, anxiety and depression (Nahar et al.
2019), epilepsy and gastrointestinal (GI) disorders (McElhanon et al. 2014). Several
emerging studies have identified an imbalanced composition of the intestinal microbiota
in ASD individuals, often reported under the controversial term “dysbiosis”, providing a
plausible explanation for the more frequent occurrence of functional GI disorders
(FGIDs) in ASD (Coury et al. 2012; Ding, Taur, and Walkup 2016; Roman, Rueda-
ruzafa, and Cardona 2018). Three large millionaire research projects on the link between
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gut microbiota to neurodevelopmental and psychiatric disorders have been launched since
2013. Recent studies support that changes in gut microbiota could affect the brain
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functions and development through the gut-brain axis (H. X. Wang and Wang 2016),
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which refers to the bidirectional interaction pathways between the central nervous system
(CNS) and the trillions of microorganisms that inhabit the gut. However, characterization
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of a distinctive ASD microbial pattern and its possible role on ASD remains unclear
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(Hsiao 2014; Luna, Savidge, and Williams 2016; Yang, Tian, and Yang 2018).
In view of this emerging evidence in recent years, this narrative review seeks to synthesize
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what is currently known about: 1) FGIDs in ASD, 2) evidence of a distinctive gut
microbial signature in ASD patients, and 3) the possible role of gut microbiota dysbiosis
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and gut-brain axis dysfunction in ASD pathogenesis.

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1.1 Functional Gastrointestinal Disorders in Autism Spectrum Disorder
FGIDs comprise a heterogeneous group of chronic recurrent gastrointestinal symptoms
which could not be explained by any underlying anatomical or biochemical abnormalities
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(Gorrindo et al. 2012). Several studies found that the prevalence of any GI disorder in
patients with ASD ranged from 23% to 70% (Chaidez, Hansen, and Hertz-picciotto 2014;
Coury et al. 2012). The wide variation for each symptom across studies has been
attributed to methodological limitations, such as retrospective study design and
inappropriate control groups, enrollment of clinically heterogeneous ASD children, bias
incase selection and reliance on parental reports of symptoms (Holingue et al. 2018). This
breadth of estimates was also apparent in a meta-analysis, where it was reported that ASD
children had a four-fold higher risk for general GI concerns, a three-fold risk for
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constipation and diarrhea, and a two-fold risk for abdominal pain (McElhanon et al.
2014); in most cases, these complaints received a diagnosis of a FGID.
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In the absence of any identified objective biomarker, the diagnosis of GI disorders is
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usually foreshadowed by the emergence or exacerbation of certain behavioral problems
which could indicate a child’s attempt to communicate the discomfort (Maenner et al.
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2012). In fact, it has been found that ASD patients who showed signs of GI comorbidity
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displayed increased sleep difficulties, abnormal mood and argumentative, oppositional,
defiant or destructive behavior, anxiety, sensory responsivity, rigid compulsive

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behaviors, self-injury, aggression, lack of expressive language and social impairments
when compared to ASD patients without GI comorbidities (Nikolov et al. 2009).

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Moreover, in some studies, the presence and intensity of abdominal pain was directly
associated with the severity of ASD core symptoms (Ding, Taur, and Walkup 2016;
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Vuong and Hsiao 2016), and the occurrence of constipation correlated with rigid-
compulsive behavior (Srikantha and Hasan Mohajeri 2019). Despite the fact that
behavioral disturbances are not efficient predictors of GI problems, as they are already
frequent in ASD children without GI complaints (Maenner et al. 2012), they could
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indicate a more than subtle association between GI dysfunction in ASD and behavioral
output through the gut-brain axis (Hsiao 2014; Vuong and Hsiao 2016).
2. EVIDENCE OF AN ALTERED GUT MICROBIOTA IN AUTISM SPECTRUM
DISORDERS
2.1 Description of included studies
A summary of the characteristics of 33 original studies examining gut bacterial and fungal
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profile in children and adolescents with ASD is shown in Table 1.
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In most cases (69.7%), unrelated NT children were recruited for the control group (Adams
et al. 2011; Carissimi et al. 2019; Coretti et al. 2018; Finegold et al. 2002; Inoue et al.
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2016; Iovene et al. 2017; D.-W. Kang et al. 2013; D. W. Kang et al. 2018; Kantarcioglu,
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Kiraz, and Aydin 2016; Kong et al. 2019; Kushak et al. 2017; Lee et al. 2017; Liu et al.
2019; Luna et al. 2017; Ma et al. 2019; Pärtty et al. 2015; Plaza-Díaz et al. 2019; Sandler
et al. 2000; Song et al. 2003; Song, Liu, and Finegold 2004; Strati et al. 2017; Williams
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et al. 2011; Williams, Hornig, and Parekh 2012; Zhang et al. 2018). However, some
studies contrast their results with a group of NT siblings (9%) (De Angelis et al. 2013a;
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Gondalia et al. 2012; Son et al. 2015), NT family members (6%) (Kong et al. 2019;
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Pulikkan et al. 2018), or both related and unrelated controls (15.2%) (Finegold et al. 2010;
Helena M R T Parracho et al. 2005; Tomova et al. 2015; Lv Wang et al. 2011, 2013). In
some cases, ASD sample was stratified by severity of ASD (Finegold et al. 2010;
Gondalia et al. 2012; D.-W. Kang et al. 2013; Ma et al. 2019; Strati et al. 2017; Tomova
et al. 2015). Likewise, two studies were confined to specific clinical subtypes, such as
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mild (Lee et al. 2017), severe (Pulikkan et al. 2018) and late-onset (Finegold et al. 2002;
Sandler et al. 2000; Song et al. 2003a; Song, Liu, and Finegold 2004) ASD forms, subjects
with Aspergers and attention deficit hyperactivity disorder (ADHD) (Pärtty et al. 2015)
or ASD children with developmental delay (Carissimi et al. 2019). Similarly, two studies
stratified their sample by PDD-NOS and AD (De Angelis et al. 2013), and ASD with and
without mental regression (Plaza-Díaz et al. 2019) respectively.
Most studies suffered from methodological issues, since 81.8% had small sample size
(n<50 subjects on the ASD group), 15.2% had a modest amount of subjects enrolled
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(Adams et al. 2011; Gondalia et al. 2012; H M R T Parracho et al. 2010; Son et al. 2015;
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Strati et al. 2017), and the one study with a larger sample size included subjects with
suspected ASD diagnosis (Kantarcioglu, Kiraz, and Aydin 2016). Moreover, all but the
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latter suffered from sex bias, as female representation in sample was either low (Adams
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et al. 2011; Coretti et al. 2018; Finegold et al. 2010b; Gondalia et al. 2012; Iovene et al.
2017b; D.-W. Kang et al. 2013; D. W. Kang et al. 2018; Kong et al. 2019; Kushak et al.
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2017; Lee et al. 2017; Liu et al. 2019; Ma et al. 2019; H M R T Parracho et al. 2010;
Pulikkan et al. 2018; Son et al. 2015; Strati et al. 2017; Tomova et al. 2015; Lv Wang et
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al. 2011, 2013; Zhang et al. 2018), none (Carissimi et al. 2019; Luna et al. 2017; Pärtty
et al. 2015b; Williams et al. 2011; Williams, Hornig, and Parekh 2012) or unspecified
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(De Angelis et al. 2013; Finegold et al. 2002; Inoue et al. 2016; Plaza-Díaz et al. 2019;
Sandler et al. 2000; Song et al. 2003; Song, Liu, and Finegold 2004). Regarding study
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design, only 15.2% of studies were age and gender-matched (Liu et al. 2019; Ma et al.
2019; Plaza-Díaz et al. 2019; Song et al. 2003a; Song, Liu, and Finegold 2004), 12.1%
were age-matched only (Kushak et al. 2017; Lee et al. 2017; Pärtty et al. 2015; Pulikkan
et al. 2018) and 6% were family-matched (Pulikkan et al. 2018; Son et al. 2015).
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However, it should be noted that subjects enrolled in ASD and control groups were often
of similar ages.
Verification of ASD diagnosis was often carried out in the selection process by clinical
observation or using several validated tests. One study included suspected ASD patients
(Kantarcioglu, Kiraz, and Aydin 2016), whereas other three performed no validating
assessment of a previous ASD diagnosis (Adams et al. 2011; Gondalia et al. 2012; Son et
al. 2015), directly studying severity of disease. Four earlier studies claimed to include
children diagnosed with ASD but did not report any diagnostic method (Finegold et al.
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2002; Helena M R T Parracho et al. 2005; Song et al. 2003; Song, Liu, and Finegold
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2004).
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Microbiota changes and overall bacterial diversity were assessed by quantification of
targeted bacterial and/or fungal species from a single or several stool samples (84.8%),
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urine (3%) and/or biopsies (15.2%), which were taken from the stomach and small bowel
(Finegold et al. 2002) cecum and ileum (Williams et al. 2011; Williams, Hornig, and
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Parekh 2012), duodenum (Kushak et al. 2017) and rectum (Luna et al. 2017). One study
analyzed stool fungal profile exclusively (Kantarcioglu, Kiraz, and Aydin 2016), four
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examined both stool bacteria and fungi (Adams et al. 2011; Finegold et al. 2002; Iovene
et al. 2017; Strati et al. 2017) and the remaining 28 studied bacterial population only. The
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most common methods for bacterial identification were sequencing or pyrosequencing of
the 16s rRNA gene, a very sensitive technique to obtain reliable results (Hang et al. 2014;
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Rosselli et al. 2016). By contrast, all but one study (Strati et al. 2017) investigating fungi
relied on cultural approaches for yeast detection, given that molecular biology methods
were not available until very recently (Iovene et al. 2017). It should also be noted that,
although statistical analyses were performed on nearly all studies, covariates adjustment
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was only carried out in some of the latest ones, reaching more conservative significance
levels. Moreover, in some cases, some microbiota-modulating factors were not taken into
consideration in the exclusion criteria, such as previous and/or concomitant intake of
antibiotics (Iovene et al. 2017; Kushak et al. 2017; Lee et al. 2017; Pärtty et al. 2015; Lv
Wang et al. 2011, 2013), probiotics and prebiotics, also known as functional foods
(Finegold et al. 2002, 2010; Iovene et al. 2017; D.-W. Kang et al. 2013; Kantarcioglu,
Kiraz, and Aydin 2016; Kushak et al. 2017; Luna et al. 2017; Pulikkan et al. 2018; Sandler
et al. 2000; Tomova et al. 2015; Lv Wang et al. 2011; Williams et al. 2011; Williams,
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Hornig, and Parekh 2012); presence of concomitant pathologies (Gondalia et al. 2012;
Kantarcioglu, Kiraz, and Aydin 2016; Kushak et al. 2017; Pärtty et al. 2015; Sandler et
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al. 2000; Lv Wang et al. 2011, 2013; Williams et al. 2011; Williams, Hornig, and Parekh
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2012) and dietary status (Adams et al. 2011; De Angelis et al. 2013; Finegold et al. 2002;
Gondalia et al. 2012; Inoue et al. 2016; Iovene et al. 2017; D.-W. Kang et al. 2013; D. W.
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Kang et al. 2018; Kantarcioglu, Kiraz, and Aydin 2016; Kushak et al. 2017; Lee et al.
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2017; Luna et al. 2017; H M R T Parracho et al. 2010; Pärtty et al. 2015; Sandler et al.
2000; Son et al. 2015; Tomova et al. 2015; Lv Wang et al. 2011, 2013; Williams et al.
2011; Williams, Hornig, and Parekh 2012). Nevertheless, some of these studies analyzed
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their association or effect on bacterial outcomes, as will be discussed later. In addition,
studies that did consider prior antibiotic intake displayed variable discontinuation periods,
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ranging from 2 weeks to 3 months, with the average amount of days being 32 days and 1
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month being the most frequent duration (52%), followed by 3 months (30%). In some
studies, other medications were assessed, and subjects taking antipsychotics (Lee et al.
2017), dietary supplements (Helena M R T Parracho et al. 2005), steroids (Luna et al.
2017), anti-inflammatories and antioxidants (Pulikkan et al. 2018), sedatives and muscle
relaxants (Plaza-Díaz et al. 2019) were ruled out. However, antifungal-taking subjects
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were only excluded in two studies (Finegold et al. 2010b; D.-W. Kang et al. 2013; D. W.
Kang et al. 2018; Liu et al. 2019), none of which was among those analyzing fungal
populations (Adams et al. 2011; Iovene et al. 2017b; Kantarcioglu, Kiraz, and Aydin
2016; Strati et al. 2017). Furthermore, some studies did not provide information on some
of these inclusion or exclusion criteria (Finegold et al. 2002, 2010; Inoue et al. 2016; D.
W. Kang et al. 2018; Helena M R T Parracho et al. 2005; Song et al. 2003; Song, Liu,
and Finegold 2004; Tomova et al. 2015).
Following microbial sequencing, assessment of bacterial diversity was performed in over
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60% of the cases. Species richness and diversity, which make up alpha diversity, were
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estimated in 58% and 33% of studies respectively. For the former, bacterial count was
performed by assemblage into operational taxonomic unit (OTU) clusters, abundance-

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based coverage (ACE) estimator metric or Chao1 index, whereas, for the latter, microbial
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evenness was measured by Shannon, phylogenic diversity (PD) or Fisher indexes. On the
other hand, beta diversity, measured by unweighted and weighted UniFrac distances,
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Jaccard distance and Bray–Curtis dissimilarity was carried out in 45% of studies and
allowed for identification of differences among microbial communities within groups,

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which enabled analysis of the effect of covariates in microbial composition. Only two
studies analyzed fungal diversity (Kantarcioglu, Kiraz, and Aydin 2016; Strati et al.
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2017).
Lastly, additional assessment of GI function was carried out in 26 studies using parental
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symptom reports and standardized questionnaires in all cases but five (Inoue et al. 2016;
Lee et al. 2017; Plaza-Díaz et al. 2019; Pulikkan et al. 2018; Zhang et al. 2018), whose
measuring methods were not provided, and one ground-breaking earlier study, which only
considered loose stool history (Sandler et al. 2000). Strict inclusion or exclusion of
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children with GI disturbances or stratification by this variable prompted classification of
the studies in Table 1 for further analysis of the implication of these GI conditions in
ASD microbial profile.
2.2 Major findings of included studies
The results of studies focusing on bacterial diversity in ASD patients are inconsistent.
20% of them reported a significant reduction of species richness (Carissimi et al. 2019;
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D.-W. Kang et al. 2013; D. W. Kang et al. 2018; Ma et al. 2019), one of which attributed
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this finding to a generally lower number of taxa in ASD, rather than the depth of
sequencing, since this reduction was not supported by the reads count within groups
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(Carissimi et al. 2019). Opposite changes were found in 10% of studies (De Angelis et al.
2013; Finegold et al. 2010). This, however, is not supported by the remaining 70% of
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studies reporting no significant differences in species richness, a trend that seemed to be
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consistent regardless of the kinship shared between ASD and controls or GI function,
since 29% of them included an unrelated group of NT children without GI dysfunction

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(Coretti et al. 2018; Liu et al. 2019; Plaza-Díaz et al. 2019; Zhang et al. 2018) 21%
established comparisons with unrelated NT-GI children (Kushak et al. 2017; Strati et al.
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2017; Williams et al. 2011), 29% of them had a control group of related individuals
(Gondalia et al. 2012; Kong et al. 2019; Pulikkan et al. 2018; Son et al. 2015) and 21%
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analyzed both unrelated and sibling controls (Helena M R T Parracho et al. 2005; Tomova
et al. 2015; Lv Wang et al. 2013). Similar tendencies were found for species diversity,
with a decreased reported by 33% of studies (Carissimi et al. 2019; D.-W. Kang et al.
2013; D. W. Kang et al. 2018; Liu et al. 2019; Ma et al. 2019), an increase in only one
study (7%) (Coretti et al. 2018) and no overall differences in the remaining 60%
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(Gondalia et al. 2012; Kong et al. 2019; Kushak et al. 2017; Plaza-Díaz et al. 2019;
Pulikkan et al. 2018; Son et al. 2015; Strati et al. 2017; Williams et al. 2011; Zhang et al.
2018). It should be noted that only one study with a control group of relatives found
significant changes in species richness (De Angelis et al. 2013), and none regarding
species diversity or beta diversity (Gondalia et al. 2012; Kong et al. 2019; Pulikkan et al.
2018; Son et al. 2015). This underpins the hypothesis that changes in siblings to ASD
children could be intermediate between NT and ASD ones (Finegold et al. 2010). As
microbiota is influenced by genetic and environmental factors, certain similarities in
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microbiome between ASD and their NT siblings could be expected. By contrast, analysis
of the beta diversity between ASD and unrelated NT children unanimously revealed a
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separate clustering for healthy and autistic samples (Coretti et al. 2018; D. W. Kang et al.
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2018; Liu et al. 2019; Luna et al. 2017; Ma et al. 2019; Strati et al. 2017; Zhang et al.
2018). In one case, however, this distinction was only significant in the unweighed
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UniFrac analysis (Ma et al. 2019), which exclusively considers the presence or absence
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of bacterial reads and does not account for their abundance.

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Overall, all but one study (Kushak et al. 2017) agreed that bacterial diversity did not show
any significant differences regarding age (D.-W. Kang et al. 2013; Luna et al. 2017;
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Helena M R T Parracho et al. 2005; Pulikkan et al. 2018) and sex (D.-W. Kang et al.
2013; Luna et al. 2017; Helena M R T Parracho et al. 2005). This could be ascribed to the
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fact that samples in the discrepant study are collected from adolescents aged 12.7 to 17.3
years, who could be in pubertal age, and sexual maturation and hormones are considered
as major determinants for gender differences. Likewise, no effect was caused by antibiotic
history, intake of probiotics and other supplements (Helena M R T Parracho et al. 2005),
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body mass index (Pulikkan et al. 2018), diet pattern (D.-W. Kang et al. 2013; Helena M
R T Parracho et al. 2005) or the severity of the autistic phenotype (Gondalia et al. 2012;
Strati et al. 2017). Moreover, half of the studies analyzing the effect of GI dysfunction
concluded that bacterial diversity variations could not be attributed to the latter. However,
in some cases, richness was found to negatively correlate with GI severity (D.-W. Kang
et al. 2013) and stratification by constipation was found to increase alpha diversity within
ASD subjects (Kong et al. 2019), as well as to separate clustering of samples within NT
subjects (Strati et al. 2017) and between the ASD-C, ASD-NC and NT-NC groups (Liu
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et al. 2019) in regard to beta diversity.
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Changes at the phylum level are shown in Figure 1 and Supplementary Table S1. The
gut microbiota is mainly defined by two bacterial phylotypes: Bacteroidetes and

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Firmicutes, whose ratio was only reported to vary significantly in five studies (Coretti et
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al. 2018; Kong et al. 2019; Strati et al. 2017; Tomova et al. 2015; Williams et al. 2011),
which found opposite tendencies. When both phyla are examined separately,
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contradictory findings replicate in all cases but when comparing ASD individuals to their
relatives. Indeed, the only study including family members to ASD in the cohort which
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found a significant phylum change relative to ASD, specifically an increase in Firmicutes,
claimed to have a heterogeneous control group regarding kinship (Pulikkan et al. 2018).
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Likewise, replication of findings was rare within the class, order, family, genera and
species levels, which are shown in Figure 2, Figure 3 and Table S1.
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Covariate analysis for correlation between demographics and microbial profile
predominantly concluded that there were no associations between age (Coretti et al. 2018;
Finegold et al. 2002; D.-W. Kang et al. 2013; Helena M R T Parracho et al. 2005), gender
(Adams et al. 2011; Coretti et al. 2018; Finegold et al. 2002; D.-W. Kang et al. 2013;
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Luna et al. 2017; Helena M R T Parracho et al. 2005) or ethnicity (D.-W. Kang et al.
2013) and any specific taxa. However, a prospective follow-up study of newborns for 13
years revealed that, while no single constant microbiota composition component or
change was detected, some significant differences were found within the ASD and NT
groups between the third and eighteenth month of life, namely, decreased levels of
Bifidobacterium and decreased Bacteroides and cumulative levels of Enterococcus and
Lactobacillus (Pärtty et al. 2015) at 6 and 18 months respectively. Since microbial
colonization in offspring achieves its stability between the 6th and 36th month of life
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(Mangiola et al. 2016), these changes could partake in a chain of pathological events
leading to ASD development, which will be further discussed in the following section.
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Although these differences found by Pärtty et al 2015 were not upheld at older ages
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(Pärtty et al. 2015), changes in those genera have been reported by several studies in
young children and adolescents (see Figure 2). Similarly, after stratification by age, Luna
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et al 2017 found decreased Parabacteroides distasonis and increased Alistipes putrednis
and Clostridium perfringens in the 13-18th year old group in contrast with higher
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Ruminococcus spp. counts in the <6 years old cohort (Luna et al. 2017).
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The effect of diet on ASD has been gaining broad attention, since it is one of the key
modulators of gut microbiota and has been linked to the development of ASD in humans
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and mice (Buffington et al. 2017; Coury et al. 2012). In fact, elimination diets and intake
of dietary supplements such as probiotics, prebiotics and symbiotics has gained popularity
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among ASD children. Early studies included in our analysis conclude that there is no
relationship between the absolute levels of any bacterial populations and diet type
(Finegold et al. 2002; Helena M R T Parracho et al. 2005). This finding has been
contradicted by a recent and thorough study using the mathematical model principal
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component analysis (PCA), which found that dissimilarity between ASD and NT children
was driven by distinct dietary components and bacterial variables within each cohort.
Furthermore, relationships have been established between Bifidobacterium and a high
consumption of dairy products, Prevotella and a plant-rich diet and, specifically in ASD
children, fish consumption and Lactobacillus (Adams et al. 2011), as well as pastry,
processed cold meat and fish, and low vegetable consumption with Hespellia (Plaza-Díaz
et al. 2019). This essentially implies that diet variability could be a confounding factor
for microbiota assessment and might contribute to the heterogeneity of results across the
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studies analyzed, since they were conducted in different geographical areas. For instance,
the genus Sutterella, which has been put forward as relevant in ASD dysbiosis (D.-W.
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Kang et al. 2013; Lv Wang et al. 2013; Williams et al. 2011), was absent from all subjects
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consuming a Mediterranean diet (Plaza-Díaz et al. 2019).
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Half of the studies assessing the correlation between ASD severity and bacterial taxa did
not observe any association (Carissimi et al. 2019; D.-W. Kang et al. 2013; Strati et al.
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2017). On the contrary, Iovene et al 2017 found a significant positive correlation between
autism severity measured by Childhood Autism Rating Scale (CARS) with Clostridium
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spp. and calprotectin value, which is a marker of gut inflammation (Iovene et al. 2017).
Likewise, Autism Diagnostic Observation Schedules (ADOS) scores positively

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correlated with Faecalibacterium prasusnitzii and Bacteroides uniformis in another study
(Coretti et al. 2018). Involvement of these two species in autism is, however, unclear,
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since evidence on the direction of change in ASD relative to NT is inconsistent (De
Angelis et al. 2013; D. W. Kang et al. 2018). Interestingly, Tomova et al 2015 observed
fluctuating results depending on the tool used to measure the severity of symptoms. That
is, children with CARS>50 had a nearly significant higher Clostridia and Desulfovibrio
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amount and lower Bacteroidetes/Firmicutes ratio, while Autism Diagnostic Interview
(ADI) scores tended to a positive correlation with the relative amount of Desulfovibrio,
emanating from the strong association between this genus and the ADI
restricted/repetitive behavior subscale score (Tomova et al. 2015). Conflictingly, other
studies revealed that levels of metabolically active Desulfovibrio genus and
Desulfovibrionaceae family were 11 and 9.7 times greater in mildly autistic children (Lee
et al. 2017), compared to an increase of 2.7 of the relative family levels in severely
affected ones (Pulikkan et al. 2018).
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The onset of behavioral signs of autism is usually conceptualized as occurring in either
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an early onset pattern, in which children show abnormalities in social and communicative
development in the first years of life, or a regressive pattern (previously referred to as

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“late onset”), in which children develop typically for a certain period of time and then
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lose previously acquired skills. Information about microbial changes between these two
phenotypes is scarce. In this review, we found three early studies to exclusively include
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a group of late-onset ASD (Finegold et al. 2002; Song et al. 2003a; Song, Liu, and
Finegold 2004), and a recent one which stratified its ASD sample into subgroups by
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mental regression (AMR) and no regression (ANMR) phenotype (Plaza-Díaz et al. 2019).
Although no obvious similarities among them were found for the differentially present
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taxa in ASD relative to NT, the latter study showed a higher relative abundance of
Proteobacteria in AMR and augmented levels in Actinobacteria phylum and class in

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ANMR. Likewise, isolated results suggested differences in microbiota between different
clinical subtypes included under the term ASD; more specifically, it was found that
changes in microbiota and bacterial diversity in PDD-NOS were intermediate between
those found in AD and those found in NT siblings (De Angelis et al. 2013).
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For the purposes of this paper, we reviewed studies that stratified their ASD sample by
comorbidity with FGIDs. In the ASD-FGIDs group, an increase was observed in the
absolute numbers or Ruminococcus torques and the relative abundances of Bacteroides
fragilis (Lv Wang et al. 2011). Interestingly, the increase of R. torques did not reach
significance before stratification by FGID presence (Lv Wang et al. 2011) or in studies
with a mixed ASD cohort in terms of GI function (D. W. Kang et al. 2018; Lv Wang et
al. 2011). The rise of B. fragilis, however, was also significant in another study where
neither ASD nor NT children reported FGIDs (De Angelis et al. 2013). Another study
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found the mean relative abundance of the Chloroplast genus pertaining the
Cyanobacteria phylum to be increased in ASD-FGIDs and to dominate first-order
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interactions between ASD and FGIDs, a finding that could have been confounded by chia
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seed consumption (Son et al. 2015). Additionally, the severity of GI symptoms inversely
correlated with Clostridia and Desulfovibrio amounts and the Bacteroidetes/Firmicutes

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ratio in one study (Tomova et al. 2015), although this finding did not reach statistical
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significance and was not supported by a posterior study (Carissimi et al. 2019).
Furthermore, cecal and ileal relative levels of Clostridiales and cumulative levels of
Lachnospiraceae and Ruminococcaceae in the FGIDs subset were significantly higher in

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children whose first episode of GI symptoms occurred before or at the same time (within
the same month) as the onset of autism rather than after, and this difference remained
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significant after adjustment by age of onset of GI symptoms (Williams et al. 2011).

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When FGID symptoms are assessed individually, significant associations are found
between irritable bowel syndrome and aerophagia and increased Clostridium aldenense;
aerophagia and decreased Blautia luti, Bifidobacterium adolescentis, Eubacterium
ventriosum, Anoxystipes fissicatena, Coprococcus comes, Eubacterium ramulus, and
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Phascolarctobacterium faecium; as well as abdominal migraine and decreases in
Akkermansia muciniphila, Coprococcus catus, Odoribacter splanchnicus, Clostridium
lactatifermentans and Ruminococcus lactaris. Notably, none of these organisms
contributed to the separation of the ASD and NT groups (Luna et al. 2017). A greater
amount of evidence has focused on functional constipation in ASD children, which has
been associated with higher relative abundances of Escherichia/Shigella and Clostridium
cluster XVIII (Strati et al. 2017), the genera Fusobacterium, Barnesiella, Coprobacter,
Olsenella and Allisonella, the family Actinomycetaceae and the order Fusobacteriales
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(Liu et al. 2019), as well as decreases in Oscillospira plautii, Bacteroides eggerthii,
Bacteroides uniformis, Faecalibacterium prausnitzii and Clostridium clariflavum (Luna
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et al. 2017). Remarkably, non-constipated NT children harbored differentiating bacteria
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from ASD and NT constipated individuals, with higher abundances of Gemmiger (Strati
et al. 2017) and a preponderance of Eubacterium rectale group, Streptococcus,

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Butyricicoccus, Eubacterium ventriosum group, Propionibacterium and
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Lachnospiraceae NC2004 group (Liu et al. 2019). Moreover, children suffering from
abdominal pain had higher levels of Turicibacter sanguinis, Clostridium lituseburense,
Clostridium disporicum, C. aldenense and O. plautii. The latter two, whose link to other
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GI symptoms has already been reflected in this review, together with Tyzzerella sp. and
Parasutterella excrementihominis were further enriched in the ASD-FGID pain group

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compared to the ASD-FGID no pain group (Luna et al. 2017). On the contrary, ASD and
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NT subjects without this symptom had higher levels of Roseburia and Bacteroides, the
latter emanating from an increase in ASD patients without abdominal pain, but not in NT
(Kong et al. 2019).
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Interestingly, some of the positively correlated bacteria with GI symptoms have been
found at very high levels in some ASD children (i.e. Turicibacter sanguinis) (De Angelis
et al. 2013; D.-W. Kang et al. 2013), whereas they displayed lower levels of the negatively
correlated ones such as Bifidobacterium adolescentis (De Angelis et al. 2013),
Phascolarctobacterium faecium (Ma et al. 2019), Blautia luti (Luna et al. 2017),
Ruminococcus lactaris (non-significant) (Finegold et al. 2002) and Roseburia (D.-W.
Kang et al. 2013; D. W. Kang et al. 2018). For the latter, at a species level, an increase of
R. inulinovorans (De Angelis et al. 2013) and the decrease of R. faecis, R. hominis and R.
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intestinalis (De Angelis et al. 2013; Luna et al. 2017) has been observed. In regard to the
pathogenesis of GI disorders, some studies have hypothesized a role of disaccharidase
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activity as a connection between microbial abundance, dysbiosis and malabsorption
(Kushak et al. 2017; Williams et al. 2011). -p

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Finally, given the higher prevalence of allergies in the ASD population, especially food
relates ones, two studies evaluated the effect of this comorbidity on cecal and ileal
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(Williams et al. 2011) and stool (Kong et al. 2019) microbial profile. ASD subjects with
allergies showed increased relative abundance of stool Proteobacteria, a phylum

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previously associated with autoimmune conditions (Kong et al. 2019). Also, food
allergies seemed to increase cecal Betaproteobacteria, as well as ileal and cecal

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Firmicutes and the Firmicutes/Bacteroidetes ratio (Williams et al. 2011). This effect was
stronger after stratification by milk related allergies, whereas wheat-related ones only
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exerted a significant effect on ileal proportions. Further evidence in ASD children was
provided by the negative correlation of the Firmicutes/Bacteroidetes ratio with
allergy/immune function in stool (Kong et al. 2019) and with overall atopic disease
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manifestations (asthma, allergic rhinitis and atopic dermatitis) in the cecum (Williams et
al. 2011).
Fungal diversity was only specifically studied by Strati et al 2017, who, unlike previous
culture-based studies by Kantarcioglu et al 2016 or Iovene et al 2017, did not observe
significant differences in alpha diversity between ASD and NT children. Nonetheless, he
found significant dissimilarity when beta diversity was analyzed (Strati et al. 2017), a
distinction that seemed to be mainly driven by a relative two times increase of the genus
Candida in ASD (uncorrected p value = 0.006, FDR-corrected p value = 0.09), which is
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consistent with the two previously mentioned studies, where a higher number of isolates
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was recovered for every species studied and Candida was isolated from 79.8% of ASD
versus 19.6% of NT children, and 57.5% versus 0% respectively (Iovene et al. 2017;
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Kantarcioglu, Kiraz, and Aydin 2016). Candida albicans was largely the most
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represented species in these two studies, whereas Strati et al 2017 did not provide a
species-level classification of Candida. Correlation analyses among the most abundant

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fungi and bacteria showed no significant association within ASD individuals, but a
significant positive correlation between the genera Aspergillus and Bifidobacterium was
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found within NT subjects (Strati et al. 2017). Candida levels seemed to be increased in
ASD independent of GI symptoms (Iovene et al. 2017) and, specifically, constipation

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status (Strati et al. 2017), since no difference was found when ASD and NT groups were
stratified by this symptom, and difference remained significant when ASD-not

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constipated (NC) and NT-NC and AD-constipated (C) vs NT-C populations were
compared (p=0.007 and p=0.05 respectively) (Strati et al. 2017). Furthermore, correlation
analysis with the most abundant and widely distributed fungi genera revealed that,
whereas Aspergillus and Malassezia did not differ between NT and ASD children,
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regardless of constipation status, Penicillum levels showed a strong correlation with this
symptom in NT children (p<0.01), and a trend to correlate in ASD ones (p=0.07). Further
underpinning this association, higher levels of this genus in NT versus ASD children
(p=0.08) lost significance when NT-C and ASD-C children were compared (p=0.88) but
remained in the absence of constipation (p=0.07) (Strati et al. 2017).
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3. GUT MICROBIOTA DYSBIOSIS AND GUT-BRAIN-AXIS DYSFUNCTION IN
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AUTISM SPECTRUM DISORDER
Currently, there seems to be a consensus about the multifactorial etiology of ASD, and
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that heritability and environmental contributors seem to equally influence its occurrence
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and interact through epigenetics (Kim and Leventhal 2015; Sandin et al. 2014).
Microbiota lies at the intersection between genes and environment, as its role and
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composition are dependent on genetic background and crucially shaped by environmental
factors (Vuong and Hsiao 2016).

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Regarding genetic factors, there are some gene polymorphisms that potentially increase
the risk of ASD and are distinctively associated with ASD individuals with co-occurring
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GI dysfunction. For instance, a variant of the Chromodomain helicase DNA binding

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protein 8 gene (CHD8) has been associated with GI complaints, especially constipation,
among ASD children (Bernier et al. 2014). CHD8 is a chromatin regulator enzyme which
is essential during human fetal development. As evidenced on zebrafish and mice,
disruptive mutations of this gene result in a reduced colonization of the GI tract by enteric
neurons, and hence, slower intestinal transit and reduced intestine length along with
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altered social interaction, increased anxiety and higher brain weight (Bernier et al. 2014;
Nithianantharajah et al. 2017), a feature that occurs in 15-35% of ASD children (Bernier
et al. 2014). The variant of c-MET is another example of gene that has been related to
ASD and comorbid FGID. It encodes the MET tyrosine kinase, leading to MET
hypofunction in the GI tract, whose endogenous ligand is called hepatocyte growth factor
(HGF), and decreases in MET expression in temporal cortex (Hsiao 2014). Similarly, the
autism-associated polymorphisms in SLC6A4, encoding the serotonin transporter
(SERT), can lead to SERT hyperfunction not only in the brain but also in the
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gastrointestinal tract, resulting in abnormalities in behavior and gastrointestinal function,
as well as autism-related increase in blood serotonin levels (Hsiao 2014). As shown in
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animal models, transgenic mice expressing a human ASD-associated SERT variant
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exhibit the hyperserotonemia phenotype, along with core ASD-related behavioral
abnormalities and altered serotonergic signaling, causing slower motility and GI transit,
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reduced intestinal permeability and a decrease in the number of neurons in both the
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myenteric and submucosal plexuses of the enteric nervous system (Adamsen et al. 2014;
Hsiao 2014).
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On the other hand, several environmental risk factors for the development or aggravation
of ASD have been associated with a dysbiotic microbial community. It has been
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hypothesized that environmental factors that favor Candida colonization, such as a
prolonged antibiotic usage or reduced early life encounters with foodborne and
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environmental bacteria and fungi in urban areas, could be a risk factor for ASD
development (Adams et al. 2011; Allen et al. 2017; Ding, Taur, and Walkup 2016; Strati
et al. 2017). According to these hypotheses, the increased incidence of ASD cases in the
last decades may be partially attributable to “Western” habits (i.e., high-fat diet,
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gestational maternal obesity and diabetes, and excessive overall hygiene). Changes in gut
microbiota in babies born by Cesarean section delivery, from mothers taking drugs such
as Valproate (VPA) and ethanol during pregnancy, and in formula-fed infants have also
been proposed as possible factors related to the increased incidence of ASD (Coury et al.
2012; Li et al. 2017; Macfabe 2012; Nithianantharajah et al. 2017; Strati et al. 2017).
However, changes in reporting practices are likely to account for most of the increasing
ASD rates over the years (Hansen, Schendel, and Parner 2015; Roman, Rueda-ruzafa,
and Cardona 2018; Sadelhoff et al. 2019).
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Maternal infections during pregnancy might be a factor linked to ASD (Patterson 2012).
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Recent findings showed that offspring have an altered fecal microbiota similar to ASD
individuals in a MIA model (mimic viral infection) (Hsiao et al. 2013). In addition,
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different microbial patterns were found in male and female ASD-like mice, but both
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presented increased gut permeability, evident inflammatory cells infiltration and ASD-
like behavioral abnormalities (Coretti et al. 2017). These disturbances were reversed by
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treatment with Bacteroides fragilis (Hsiao et al. 2013). The relevance of the findings in
the MIA model is due to the clinically-common etiology and sex-specificity incidence of
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ASD after maternal infections, which is 4 times higher in males (Ruskin et al. 2017).
Surprisingly, early in life, male infants are more likely to suffer infections than females
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(Klein and Flanagan 2016). In fact, it has been postulated that this sex-specific immune
sensitivity could be related to higher male prevalence in early-onset neurodevelopmental

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disorders (Schwarz and Bilbo 2013). There is strong evidence that early life infections in
males can alter microglia, the resident immune cells of the brain, resulting in an
exaggerated pro-inflammatory response from microglia and impairment of learning and
memory (Bilbo et al. 2005; Williamson et al. 2011). Interestingly, microglial
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development and myelination in the prefrontal cortex seems to be regulated by microbiota
(Roman, Rueda-ruzafa, and Cardona 2018). Adult mixed-sex groups of mice raised in a
germ-free background or depleted gut microbes have perpetually “immature” microglia
(Erny et al. 2015). This immature profile, characterized by increased proliferation and
decreased immune reactivity, can be reversed by re-colonization with a diverse bacterial
population or by treatment with metabolites derived from commensal bacteria (Erny et
al. 2015).
The gut microbiota interacts with the brain through the named gut-brain axis, that refers
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to the bidirectional interaction pathways comprising the autonomic nervous,
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neuroendocrine, immunological and metabolic (via microbial toxin production) systems.
Herein, we describe the different hypothesized pathways and, when available, specific
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finding for ASD patients. Figure 4 shows the integration of the impaired gut-brain-axis
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pathways in ASD individuals.
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3.1 Increased permeability of barrier paths

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In general terms, homeostasis of gut microbiota is achieved when 70% of microorganisms
are Gram-negative, and the remaining 30% are Gram-positive (Mirielys and Gutiérrez
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2018). The microbial signature of ASD individuals, with a higher proportion of Gram-
negative bacteria (Coretti et al. 2018), increases production of lipopolysaccharides (LPS)

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and pro-inflammatory cytokines (see section 3.4), which have been suggested as
contributing factors for the impairment of both the intestinal and blood-brain barrier
(BBB) found in ASD patients (Fiorentino et al. 2016). In fact, by measuring serum levels
of adhesion proteins, it has been shown that increased abnormal intestinal permeability
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or “leaky gut” is present in 37% of ASD patients (Hsiao et al. 2013). This could be related
to lower number of Lactobacilli in patients with ASD (De Angelis et al. 2013; Iovene et
al. 2017; Ma et al. 2019; Pärtty et al. 2015), since they contribute to the maintenance of
tight junction in the intestinal epithelial barrier (Srikantha and Hasan Mohajeri 2019) and
its depletion has been directly related to chronic constipation in NT children (Kushak et
al. 2017). As a result of the impairment of the intestinal barrier, entrance of toxins and
bacterial products into the bloodstream is allowed (see below, metabolic pathway) and
bacterial translocation into the mesenteric lymphoid tissue is favored, where they activate
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the immune system.
It should be noted that, though mucosal barrier impairment is the most studied mechanism
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connecting GI comorbidity and intestinal dysbiosis in ASD, this relationship appears to
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be much more complex. In light of this, recent research suggests a relationship between
ASD microbial profile and an altered metabolism and absorption of disaccharides in their
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gut epithelium (Kushak et al. 2017; Srikantha and Hasan Mohajeri 2019).
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3.2 Anatomical pathway: The gut-brain’s neural network

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Two neuroanatomical routes are known to deliver the signals from the intestine to the
brain. The first is the autonomic nervous system and the vagus nerve, and the second is
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the enteric nervous system, including the enteroglial cells and the autonomic nervous
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system and vagal nerve in the spinal cord. The gut luminal contents and events and the
mucosal constituents create the signals to be transmitted by hierarchic integrative levels
cephalically (Lerner, Neidhöfer, and Matthias 2017). Regarding this pathway, certain
anatomical changes have been found in the brains of autistic individuals, such as impaired
GABAergic functioning (Horder et al. 2018) and increased activation of microglial cells
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and a lower count of Purkinje cells in the cerebellum, which could be related to the first
(Srikantha and Hasan Mohajeri 2019).
3.3 Neuroendocrine pathway:
3.3.1 Hypothalamic-pituitary-adrenal (HPA) axis
The primary function of the HPA axis is to regulate the response to environmental factors,
such as stress. Under stress conditions, the hypothalamus releases Corticotropin-
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releasing-hormone (CRH) and vasopressin that signal the release of Adrenocorticotropin
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(ACTH) from the pituitary gland, which in turn influences the secretion of hormones from
the adrenal glands, such as cortisol, a glucocorticoid that affects many human organs,
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including the brain, where it can regulate the activity of the intestinal functional effector
cells, modulating GI motility, permeability, immunity and mucus. These same cells are
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under the influence of the gut microbiota (Carabotti et al. 2015) (Figure 5). Germ free
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(GF) mice show an increased stress response with augmented levels of ACTH and
cortisol, which can be reversed after germ colonization in young mice(Lerner, Neidhöfer,
and Matthias 2017), by fecal microbial transplant or by Bifidobacterium infantis

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(Evrensel and Ceylan 2016). GF mice also display reduced brain-derived neurotrophic
factor (BDNF) and N-methyl-D-aspartate (NMDA) receptor expressions in cortex and

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hippocampus, which affect the release and expression of the CRH in the hypothalamus
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and thus change the function of the HPA axis (Sudo et al. 2004). Some studies,
specifically in ASD patients, have found altered mRNA levels of the glucocorticoid
receptor and CRH receptor 1 (Patel et al. 2016), which essentially implies an alteration
of this pathway.
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3.3.2 Neurotransmitters and Neural regulators
Gut bacteria seem to regulate several key neurotransmitters such as gamma amino butyric
acid (GABA), glutamate, serotonin, dopamine (Yano et al. 2015; Yunes et al. 2016),
which have shown altered levels in ASD patients (Mohamadkhani 2018; Srikantha and
Hasan Mohajeri 2019). In fact, an imbalance in the CNS between excitation (glutamate)
and inhibition (GABA) has been postulated to contribute to ASD (El-Ansary et al. 2018;
Horder et al. 2018; Srikantha and Hasan Mohajeri 2019). In addition, bacteria can produce
a wide range of neuroendocrine hormones which can intervene in intestinal homeostasis
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and modulate mood and behavior (Lyte 2014; Oleskin, Shenderov, and Rogovsky 2017).
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More specifically, it has been theorized that both the SLC6A4 gene polymorphism
discussed above and the increase of serotonin-producing microbes, such as Candida,

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Streptococcus, Escherichia, Enterococcus and Clostridiales in ASD may rise intestinal
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production of serotonin at the expense of a lower synthesis in the brain (due to
consumption of its precursor tryptophan), leading to hyperserotoninemia, intestinal

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dysmotility and a still inconsistently reported behavioral outcome (Fattorusso et al. 2019;
Luna et al. 2017; Srikantha and Hasan Mohajeri 2019).

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3.4 Immunological pathway: cytokine release

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The development of the immune system heavily relies on gut microbiota; hence, GF mice
have limited immune activity (Lerner, Neidhöfer, and Matthias 2017). Most bacteria and
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host communication is carried out by TLRs (Toll-Like Receptors), which are found in a
variety of cells from the innate immune system, to intestinal epithelial cells or neurons
(both from the ENS and CNS). TLRs can recognize the microbiota triggering appropriate
intracellular signaling pathways and immune responses. Among other bacterial products,
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increased LPS in ASD subjects could activates TLR4 in the ENS, where stimulates pro-
inflammatory cytokine production, and in the CNS, where it can lead to inflammation
through activating microglia once crossed the impaired mucosal and blood-brain-barriers
(Li et al. 2017; Srikantha and Hasan Mohajeri 2019), as it has been shown in postmortem
ASD brain biopsies (Morgan et al. 2010). Likewise, cytokines can bind TLRs in neurons,
inducing changes in the electric layout of neuronal membranes and altering the regulation
of emotion and behavior (Evrensel and Ceylan 2016).
The microbial imbalance in ASD patients could induce an inadequate immune activation
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leading to cytokine dysregulation, thereby activating the HPA axis and altering the
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permeability of the gut and blood-brain barrier through MLCK and MAPCK-mediated
regulation of tight junction (Ahmad et al. 2017; Barrier et al. 2014; Carabotti et al. 2015;
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Ulluwishewa et al. 2018) (Figure 6). Further evidence is provided by studies carried out
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in both ASD models and ASD individuals who displayed increased LCR, plasma or serum
pro-inflammatory and Th2 cell-derived cytokines, such as IL-1, IL-4, IL-5, IL-6, IL- 8,
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TNF-α, IFN- and IL-17 , as well as low IL-10, TGF- and Treg cells levels, which are
involved in synapse, survival and differentiation of neurons, and correlate with the
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severity of ASD symptoms (Eftekharian, Ghafouri-fard, and Noroozi 2018; Sadelhoff et
al. 2019). Further correlation between faecal TNF-α and the severity of GI symptoms was
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reported in ASD children (Srikantha and Hasan Mohajeri 2019). Moreover,
Faecalibacterium abundance in ASD children was strongly correlated with a greater

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number of differentially expressed genes involved in both the interferon (IFN)-γ and type-
I IFN signaling pathways, which were enriched in this cohort (Inoue et al. 2016).
T-helper 1 and T-helper 17 cells affect the reactivity of peripheral immune cells in the
CNS and the integrity of blood-brain barrier, induce apoptosis in oligodendrocytes and
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increase glutamate excitotoxicity (Eftekharian, Ghafouri-fard, and Noroozi 2018;
Kamada et al. 2013; Stolp et al. 2005); their hyper-activation has been linked to the
increase of Clostridiales (Luna et al. 2017) and disruptions in the mammalian target of
rapamycin (mTOR) pathway in some ASD phenotypes. Enhanced mTOR activity is also
responsible for increased autophagy in the intestine and disbalance in allergy-associated
Th2 and Treg cells (Sadelhoff et al. 2019). This evidence, together with a ten-fold
incidence of mastocytosis found in ASD patients, the similarities in gut biopsies of
children with ASD and individuals with immunodeficiency and food allergies, and the
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previously mentioned increased prevalence of food allergies in this population, points to
a relationship between intestinal allergic responses and brain circuits involved in social
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functioning, which can be targeted by rapamycin and amino-acid administration
(Fattorusso et al. 2019; Sadelhoff et al. 2019). -p

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3.5 Metabolic pathway: microbial metabolome
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The gut microbiota produces constantly changing metabolites that impact host physiology
and susceptibility to disease (Lerner, Neidhöfer, and Matthias 2017). ASD patients show
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altered levels of potentially toxic phenol compounds (Alabdali, Al-Ayadhi, and El-
Ansary 2014) produced, among others, by Bifidobacterium, C. difficile, C. histolyticum

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clusters II and I and some Lactobacillus (Ding, Taur, and Walkup 2016; Fattorusso et al.
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2019; Mohamadkhani 2018). Among them, the increased abundance of urinary and fecal
para-cresol (p-cresol) and its conjugated derivative p-cresylsulfate, which might impair
oligodendrocyte differentiation in vitro and can inhibit the enzyme dopamine-beta-
hydroxylase, are considered putative metabolic markers for ASD, especially useful in
females and severely autistic males (Persico and Napolioni 2013; Roman, Rueda-ruzafa,
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and Cardona 2018). Along with it, increased Clostridia derived metabolite 3-(3-
hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) could lead to a depletion of
catecholamines in the CNS and its reduction has been shown to decrease ASD severity
(Roman, Rueda-ruzafa, and Cardona 2018; Xiong et al. 2016). Another significant
metabolite increased in stool of children with ASD is isopropanol, which has been related
to GI disturbances (Mohamadkhani 2018), whereas relevant findings in tue urinary profile
point to the alteration of nicotinamide, tryptophan, vitamine B6 and purine metabolism
(Srikantha and Hasan Mohajeri 2019).
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Other metabolites plausibly involved in ASD pathogenesis could be short-chain fatty
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acids (SCFAs), which are fermentation products of dietary carbohydrates produced by
intestinal bacteria such as Clostridia, Bifidobacteria, Bacteroidetes, the families

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Ruminococcaceae and Lachnospiraceae, and the genus Desulfovibrio (Fattorusso et al.
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2019; Roman, Rueda-ruzafa, and Cardona 2018; Williams et al. 2011). Interestingly, the
overall structure of ASD children was found to be dominated by carbohydrate

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metabolism, among other functions (Ma et al. 2019), and the abundance of SCFA-
producing bacteria has been reported to be altered in ASD individuals (Zhang et al. 2018).
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Despite data being somewhat discordant, findings suggest decreased butyrate, and
increased acetate and propionate (PPA) levels in ASD patients (De Angelis et al. 2013b;
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Grimaldi et al. 2016; L Wang 2010). Although PPA is beneficial at appropriate levels
(den Besten et al. 2013), excessive amounts are associated with the severity of ASD (Lv
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Wang et al. 2012). In fact, its acidic properties may make them liposoluble enough to
cross the gut and blood-brain barriers and gain access to the brain, where it could induce
several ASD-linked behavioral and neurochemical changes, such as neurotransmitter
alteration (dopamine, serotonin and glutamate), increased oxidative stress,
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neuroinflammation, and intracellular acidification, which can inhibit mitochondrial
function and may contribute to causing gut dysmotility and altering development and
behavior (Foley et al. 2014; Macfabe 2012; Roman, Rueda-ruzafa, and Cardona 2018;
Srikantha and Hasan Mohajeri 2019).
4. CONCLUSIONS
The lack of consistent knowledge inherent to the emerging nature of the field and
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incomplete understanding of the gut-brain biological signaling pathways, together with
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the disparity of sample sizes, demographics and methodologies used, translates into
discordant differences reported for bacterial gut microbiota in ASD individuals. In fact,
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measuring tools varied widely, not only for autism diagnosis, microbial and GI
characterization, but also for the statistical analysis of findings, leading to heterogeneous
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p-values in terms of conservatism. Besides, the effect of change for each taxon targeted
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could not always be given, since not all studies provided calculations or enough data to
calculate them. In one case, this data, while existing, could not be accessed (Carissimi et
al. 2019). Finally, it should be kept in mind that bacterial abundances should be
na
segregated by absolute and relative amounts in order to accurately establish comparisons.
All in all, inferences regarding changes of individual bacteria at each level should be
ur
cautiously made.
Jo
Despite more research being needed to shed light on the many questions that remain
unanswered, the present review concludes that there is general agreement on the existence
of a distinctive microbial pattern in ASD individuals that can be affected by
demographics, autistic phenotypical profile and symptomatology, diet pattern as well as
GI and autoimmune comorbidity. However, for a deeper clarification regarding the

31
32
relationship between GI disorders and ASD, studies investigating the correlation between
GI symptoms and gut microbiota in NT groups should have been included.
Overall, there is a need for further research on the interactions between gut bacterial and
fungal microbiota, the role of the bacterial and fungal components in the neuroplastic
changes and gastrointestinal physiology in ASDs, and the integration of such data with
genetics, immunology, and metabolomics. Not only can this provide novel insights into
the intricate gut microbiota-brain axis, whose relevance to ASD is herein evaluated, but
it can also help us thoroughly identify the distinctive ASD microbiome and ASD specific
of
biomarkers, useful in the determination of at-risk population, early ASD detection and
treatment development.
ro
Declarations of interest: none
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re
Acknowledgements
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The authors are grateful to Dr Manuel Maynar for his decisive role in the genesis and
development of this research through his critical reading of the first drafts. Also, we thank
na
Dr Jeffery for her critical reading of the manuscript. This research did not receive any
specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
ur
Jo
32
33
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Figure 1: overview of phyla whose change in ASD was reported as significant in at least
one study. The rooted tree shows the phylogenetic relationships of the different phyla. In
the heatmap, colors blue and red denote direction of change (decreased and increased
respectively), whereas color intensity refers to the strength of the evidence, light meaning
p<0.05 and dark meaning p<0.01. Squares colored in grey correspond to studies finding
no significant differences between ASD and controls. Folds change relative to controls
are given when data was significant and available and control values differed from 0.
Letters R and A in the first row indicate if studies provide relative or absolute bacterial
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levels. Abbreviations: mild=mild ASD, sib=sibling, unr=unrelated, sev=severe ASD,
ANMR=ASD with no mental regression, AMR=ASD with mental regression. Asterisks:
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*= Gene Ontology classification used to assign each gene to a GO term, depending on its
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functional characteristics, rather than calculating a relative abundance for each gene;
*1=cumulative level of Firmicutes and Proteobacteria.

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Figure 2: overview of genera whose change in ASD was reported as significant in at least
one study. The rooted tree shows the phylogenetic relationships of the different genera.
Color ranks indicate the pertaining phylum: dark blue = Verrucomicrobia, purple =
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Bacteroidetes, green = Proteobacteria; brown = [Thermi]; red = Armatimonadetes, light
blue = Actinobacteria, orange = Firmicutes. In the heatmap, colors blue and red denote
direction of change (decreased and increased respectively), whereas color intensity refers
to the strength of the evidence, light meaning p<0.05 and dark meaning p<0.01. Squares
colored in grey correspond to studies finding no significant differences between ASD and
controls. Folds change relative to controls are given when data was significant and
available and control values differed from 0. Letters R and A in the first row indicate if
studies provide relative or absolute bacterial levels. Abbreviations: mild=mild ASD,
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sib=sibling, unr=unrelated, sev=severe ASD, PDD=pervasive developmental disorder,
AD=autism disorder, ANMR=ASD with no mental regression, AMR=ASD with mental
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regression. Asterisks: *1=cumulative level of Clostridium and Ruminococcus;
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*2=cumulative level of Bacteroides, Porphyromonas and Prevotella; *3=cumulative level
of Pseudomonas and Aeromonas; *4=cumulative level of Enterococcus and

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Lactobacillus; *5=cumulative level of Pseudomonas and Aeromonas; *6= retrieved at 18
months old; *7= retrieved at 6 months old; *8= cumulative levels of Enterococcus and
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Lactobacillus retrieved at 18 months old; *9= quotient calculated after removal of outlier
(previous value of 83).

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Figure 3: overview of species whose change in ASD was reported as significant in at
least one study. The rooted tree shows the phylogenetic relationships of the different
species. Color ranks indicate the pertaining phylum: dark blue = Verrucomicrobia, yellow
= Fusobacteria, green = Proteobacteria, purple = Bacteroidetes, light blue =
Actinobacteria, orange = Firmicutes. In the heatmap, colors blue and red denote direction
of change (decreased and increased respectively), whereas color intensity refers to the
strength of the evidence, light meaning p<0.05 and dark meaning p<0.01. Squares colored
in grey correspond to studies finding no significant differences between ASD and
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controls. Folds change relative to controls are given when data was significant and
available and control values differed from 0. Letters R and A in the first row indicate if
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studies provide relative or absolute bacterial levels. Abbreviations: mild=mild ASD,
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sib=sibling, unr=unrelated, sev=severe ASD, PDD=pervasive developmental disorder,
AD=autism disorder, FGIDs= functional gastrointestinal disorders.

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Figure 4: Integration of the impaired gut-brain-axis pathways in ASD individuals and
mouse models.
Abbreviations:
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48
BBB: blood-brain barrier; CHD8: chromodomane helicase DNA binding protein 8
gene; CNS: central nervous system; CRH: corticotropin-releasing hormone; G-
bacteria: Gram negative bacteria; G x E: interactions between genes and environmental
factors; GABA: gamma amino butyric acid; GI: gastrointestinal; HGF: hepatocyte
growth factor, ligand of the MET receptor; LPS: lipopolysaccharides; MET
rs1858830: variant of the MET gene which encodes the c-MET tyrosine-protein
kinase, also called hepatocyte growth factor receptor; poly I:C: polyinosinic-
polycytidylic acid, used to mimic maternal viral infection in mice; PPA: propionic
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acid; SERT: serotonin transporter; SLC6A4: variant of the gene which encodes the
serotonin transporter; 5-HT: serotonin; Th1, 2, 17: T-helper cell 1, 2 and 17; VN: vagus
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nerve; VPA: Valproate.
Color code:
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Navy: increased permeability of barrier pathways: intestinal mucosal barrier and BBB.
Orange: anatomical pathways: the gut-brain’s neural network.

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Green: neuroendocrine pathway: HPA axis (colored in grey) and neurotransmitters
and neural regulators (colored in light green).

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Light blue: immunological pathway: cytokine release.
Red: metabolic pathway: microbial metabolome.

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Yellow: microbial and clinical outcomes in ASD.

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Figure 5: Hypothalamic-pituitary-adrenal (HPA) axis. Under stress conditions, the
hypothalamus releases vasopressin and corticotropin-releasing hormone (CRH) that

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signal the release of adrenocorticotropic hormone (ACTH) from the pituitary gland,
which in turn influences the secretion of hormones from the adrenal glands, such as
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cortisol. Cortisol is a glucocorticoid that affects many human organs, including the brain,
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where it can regulate the activity of the intestinal cells, such as the immune cells, the
epithelial cells, the enterochromaffin cells, the interstitial cells of Cajal, the enteric
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neurons and the smooth muscle cells. These same cells are under the influence of the gut
microbiota, which, when dysbiotic, can induce an early immune activation leading to
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increased pro-inflammatory cytokines. As will be discussed later, cytokines gain access
to the hypothalamus throughout the impaired blood-brain barrier, acting on the HPA axis
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similarly to stress.
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Figure 6: Interaction pathway between bacterial and viral components and the nervous
and immune systems: subepithelial dendritic cells, one of the basic cells of the gut
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immune system, extend their dendrites through epithelial cells into the gut lumen and
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collect bacteria and their metabolites, which are then processed into proteins, nucleic
acids, sugars and lipids and carried in exosomes. This content is transferred from dendritic
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cells to T cells in the lymph nodes, allowing exosomes to enter the systemic circulation
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via the lymphatic system. Once they have reached the brain by passing through the blood-
brain barrier, they bind toll-like receptors (TLRs) in neurons and induce changes in the
electrical layout of neuronal membranes which are responsible for the regulation of
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emotion and behavior. On the upper side of the diagram, interactions between bacterial
and viral components and the immune system are represented, as they are the first step to
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produce cytokine response. When cytokines are spread in the bloodstream and have
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gained access to the brain, they can activate the HPA axis.
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Author/ yeara Sample size and ASD diagnostic and Measuring tools and sample for Measuring tools for Limitations 52
demographics categorizing tools microbial analysis GI function
GI function not studied

Song et al 2003 Late-onset AD (n=13); Not reported. Bacterial culture, genotypic (HPLC, NA 1. Small sample size.
f
NT unrelated (n=8) PCR) and phenotypic 2. Unknown sample age and sex
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characterization of 15 isolates on 3. Unknown exclusion criteria
stool, blood and an intra- 4. No stratification by ASD severity
abdominal abscess. 5. Number of samples per subject not specified (presumably
one).
Song et al 2004 Late-onset AD (n=13); Not reported. Clostridium quantification by qPCR NA 1. Small sample size.
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NT unrelated (n=8). on stool 2. Unknown sample age and sex
3. Unknown exclusion criteria
4. No stratification by ASD severity
5. Number of samples per subject not specified.
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Tomova et al ASD (m:f = 9:1, aged 2-9y); ICD-10, CARS, ADI qPCR on stool bacterial population NA 1. Small sample size.
2015 NT siblings (m:f = 7:2, aged 5- 2. Sex bias.
17y); 3. Not considered as exclusion criteria: concomitant intake of
NT unrelated (m:f = 10:0; 2- other medication besides antibiotics
11y). 4. Number of samples per subject not specified.
Pr
Pärtty et al 2015 Aspergers and ADHD (m:f = ICD-10 FISH and qPCR on stool bacterial NA 1. Small sample size.
6:0); population 2. Sex bias.
NT unrelated (m:f = 34:35). 3. Not considered as exclusion criteria: concomitant antibiotic
and intake of other medications, such as probiotics
(supplemented in 40 NT subjects), dietary status, concomitant
pathologies
l 4. No stratification by ASD severity

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5. Single sample per subject.
Kantarcioglu et al ASD suspected/ diagnosed No verification of ASD Yeast culture from stool samples NA 1. Unverified diagnosis of ASD subjects
2015 (m:f = 879:676, aged 9mo- diagnosis and identification by 2. Not considered as exclusion criteria: functional foods besides
17y); morphological and biochemical kefir, dietary status, concomitant pathologies
NT unrelated (m:f = 234:169; tests. 3. No stratification by ASD severity
2-18y). 4. Number of samples per subject not specified.
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Ma et al 2019 ASD (m:f = 39:6, aged 6.2- DSM-V, CARS 16S rRNA gene pyrosequencing by NA 1. Small sample size.
8.34y); PCR on stool bacterial population 2. Sex bias
NT unrelated-wGI (m:f = 39:6; 3. Not considered as exclusion criteria: dietary status, but p-value
aged 5.85-8.23y) adjusted accordingly
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Mixed sample (not stratified by GI function)
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Finegold et al ASD (m:f = 24:9 – 3 excluded); Clinical observation (no Bacterial tag-encoded FLX Presence of 1. Small sample size.
2010 NT siblings (m:f= 5:9); specific test used) amplicon pyrosequencing on stool symptoms. 2. Sex bias.
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NT unrelated (m:f=5:3). samples 3. Not considered as exclusion criteria: functional food intake,
Age range: 2-13y, *some with dietary status, unclear if concomitant pathologies
GI 4. Number of samples per subject not specified.
Gondalia et al AD or Asperger (m:f = 42:9, No validating assessment Bacterial tag-encoded FLX Presence of 1. Modest sample size.
2012 *28 ASD-GI); (usage of CARS scores amplicon pyrosequencing on stool symptoms. 2. Sex bias.
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NT siblings (m:f = 19:34, * dated up to 12 months samples 3. Not considered as exclusion criteria: concomitant medication
NTsib-GI). prior) intake (antibiotic intake only of one child on the control
Age range: 2-12y group), dietary status, food allergies
5. No unrelated NT control group
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Iovene et al 2016 ASD (m:f=40:7; aged 6±2.8y, DSM-V-TR, ADI-R, CARS, Bacterial and yeast culture from QPGS-RIII and 1. Small sample size.
*33 ASD-GI) ADOS stool samples with identification LA/MA for intestinal 2. Sex bias.
NT unrelated-wGI (m:f=23:9; of colonies by VITEK2 system. permeability 3. Not considered as exclusion criteria: concomitant medication
aged 7.3±3.1y) intake (including antibiotics), dietary status
Pr
Lee et al 2017b ASD (m:f = 18:2, aged 17.5- Diagnosis: DSM-V Isolation and 16S rRNA Not reported 1. Small sample size.
27.3y); Characterization: K-CARS pyrosequencing of bacteria- 2. Sex bias
NT unrelated-wGI (m:f = 24:4; derived extracellular membrane 3. Not considered as exclusion criteria: concomitant antibiotic in
aged 11.6-30.6y vesicles from urine samples ASD group, other medication besides antipsychotics, dietary
status
l 4. Number of samples per subject not specified.
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Kang et al 2018 ASD (m:f = 22:1, aged 6-14.2y, ATEC, PDD-BI 16S rRNA gene pyrosequencing by 6-GSI 1. Small sample size.
*21 ASD-GI); Genome Sequencer FLX-Titanium 2. Sex bias
NT unrelated (m:f = 15:6; System on stool bacterial 3. Not considered as exclusion criteria: functional foods, dietary
aged 5-11.8y, *10 NT-GI) population status, unclear if concomitant pathologies
Pulikkan et al Severe ASD (m:f = 28:2, aged CARS, INDT-ASD, ISAA 16S rRNA gene sequencing (NGS) Not reported 1. Small sample size.
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2018b 3-16y); on stool bacterial population 2. Sex bias.

NT mostly related - wGI (m:f = 3. Not considered as exclusion criteria: functional foods and
15:9; 3.5-16y) antifungal intake
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5. Single sample per subjects

Zhang et al 2018 ASD (m:f = 29:6, aged 3.4- DSM-V 16S rRNA gene sequencing (NGS) Not reported 1. Small sample size.
6.4y, *21 ASD-GI); on stool bacterial population 2. Sex bias
NT unrelated-wGI (m:f = 5:1; 3. Not considered as exclusion criteria: antifungal intake
aged 3.5-5.7y) 4. No stratification by ASD severity
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Coretti et al 2018 ASD (m:f = 9:2, aged 2.4-3.4y, Diagnosis: DSM-V, ADOS-2 16S rRNA and rDNA gene QPGS-RIII 1. Small sample size.
*2 ASD-GI); Characterization: ADI-R, sequencing by droplet digital PCR 2. Sex bias
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NT unrelated-wGI (m:f = 8:6; GMDS, VABS, CARS on stool bacterial population 3. Not considered as exclusion criteria: antifungal intake
aged 2.2-3.6y)
Plaza-Díaz 2019 ASD-MR (n=18); DSM-V, ADI-R, ICD-10, 16S rRNA gene pyrosequencing by Not reported 1. Small sample size.
ASD-wMR (n=30); ADOS, PDDBI, CARS, PCR on stool bacterial population 2. Unknown sample sex
NT unrelated (n=48; ged 3.5- Battelle developmental test 3. Not considered as exclusion criteria: concomitant medication
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3.9y; *2 ASD-abdominal pain) (developmental delay), 5- intake (including antibiotics), dietary status (but analysis of its
item questionnaire effect on microbial pattern).
(developmental regression) 4. Number of samples per subject not specified.
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AD-GI versus NT-GI
Williams et al AD-GI (m:f= 15:0, aged 3.5- DSM-IV-TR, ADI-R, 16S rRNA gene pyrosequencing by Standardized 1. Small sample size.
2011 5.9y); Shortened CPEA qPCR on bacterial population from questionnarie 2. Sex bias.
NT-GI (m:f= 7:0, aged 3.9- Regression Interview ileal and cecal biopsies. 3. Not considered as exclusion criteria: concomitant medication
Pr
5.5y); (regression status) intake (antibiotic intake only of one child on the control
group), dietary status, concomitant pathologies (but analysis
of their effect on microbial pattern)
4. No stratification by ASD severity.
Williams et al AD-GI (m:f= 15:0, aged 3.5- DSM-IV-TR, ADI-R, 16S rRNA gene pyrosequencing by Standardized 1. Small sample size.
2012 5.9y + 8m aged 6-10 for Shortened CPEA qPCR on bacterial population from questionnarie 2. Sex bias.
Suterella assessment); Regression Interview ileal and cecal biopsies. 3. Not considered as exclusion criteria: concomitant medication
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NT-GI (m:f= 7:0, aged 3.9-5.5y (regression status) intake (antibiotic intake only of one child on the control
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+ 2m aged 6-10 for Suterella group), dietary status, concomitant pathologies
assessment). 4. No stratification by ASD severity.
Kushak et al 2017 ASD-GI (m:f = 19:2, aged DSM-IV 16S rRNA gene pyrosequencing on Presence of 1. Small sample size.
12.7—16.1y); duodenal bacterial population symptoms. 2. Sex bias.
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NT-GI (m:f = 10:9, aged 14.8- 3. Not considered as exclusion criteria: concomitant medication
17.3y). intake (including antibiotics), dietary status, concomitant
pathologies
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AD-wGI versus NT-wGI

De Angelis et al PDD-NOS-wGI (n=10); DSM-IV-TR, ADI-R, ADOS, Bacterial tag-encoded FLX Presence of 1. Small sample size.
2013 AD-wGI (n=10); CARS amplicon (16S rDNA and 16S symptoms. 2. No information about age and sex distribution across groups
NT siblings-wGI (n=10); rRNA) pyrosequencing on stool 3. Not considered as exclusion criteria: antifungal intake, dietary
Age range: 4-10y, m:f = 14:16 samples status
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Inoue et al 2016 ASD-wGI (n=6); DSM-V, PARS, M-CHAT 16S rRNA gene pyrosequencing by Not reported 1. Small sample size.
NT unrelated - wGI (n=6) PCR on stool bacterial population 2. Unknown sample sex
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Age range: 3-5y 3. No information about age and sex distribution across groups
4. Not considered as exclusion criteria: dietary status, unclear if
concomitant pathologies
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AD-GI versus NT-wGI
Sandler et al Late-onset AD + antecedents DSM-IV, behavioral Quantification of aerobic and Loose stool history 1. Small sample size.
2000 of antibiotic exposure questionnaire anaerobic species on stool 2. Unknown age from control group
followed by chronic diarrhea (unreported exact method). 3. Unknown sample sex
(m:f = 10:1 – 7 excluded, aged 4. Not considered as exclusion criteria: dietary status,
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2-8y); concomitant pathologies
NT adults n=104. 5. No stratification by ASD severity
6. Number of samples per subject not specified (presumably
one).
Pr
7. No statistical analysis between groups
Finegold et al Late-onset AD-GI n = 13 (stool Not reported (possibly not Bacterial culture, analysis of Presence of 1. Small sample size.
2002 data) and n = 7 (gastric, small validated) bacterial metabolites and cellular symptoms. 2. Unknown sample age and sex
bowel data); fatty acids, PCR of the 16S and 16S 3. Not considered as exclusion criteria: concomitant intake of
NT unrelated n = 8 and n= 4 rRNA gene sequencing on stool, other medication besides antibiotics, dietary status, unclear If
respectively. gastric and small-bowel samples. concomitant pathologies
l 5. Number of samples per subject not specified.
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Adams et al 2011 AD (55), Aspergers (3)-GI (m:f No validating assessment Bacterial culture, quantification 6-GSI 1. Modest sample size.
= 50:8, aged 3.5-10.3) for diagnosis (previous and identification of bacteria and 2. Sex bias.
NT unrelated-wGI (m:f = diagnosis by physician), yeast from stool samples. 3. Not considered as exclusion criteria: antifungal intake, dietary
18:21, aged 3.3-12.1) ATEC (severity) status
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Carissimi et al ASD-severe develop-mental Clinical observation 16S rRNA gene sequencing (NGS) GIH 1. Small sample size.
2019 delay (m:f = 16:0, aged 2-6y, GMDS, ADOS-2 on stool bacterial population 2. Sex bias
*12 ASD-GI, *3mild, 9 3. Not considered as exclusion criteria: antifungal intake, dietary
moderate, 4 severe); status, unclear if concomitant pathologies
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NT unrelated-wGI (m:f = 2:5; 4. Number of samples per subject not specified.

aged 5-16y)
AD-GI versus AD-wGI
Wang et al. 2011 AD (17), Aspergers (6) (m:f = DSM-V, CARS qPCR on stool bacterial population FGID questionnaire 1. Small sample size.
21:2, aged 3-17y, *9 ASD-GI); 2. Sex bias.
NT siblings (m:f = 11:11, aged 3. Not considered as exclusion criteria: concomitant medication
4.5-18.5y, *6 NTsib-GI); intake (including antibiotics), dietary status, concomitant
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NT unrelated (m:f = 4:5, aged pathologies (epilepsy and intellectual disability included in ASD
3.5-15y, *1 NT-GI). group)
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Wang et al. 2013 AD (17), Asperger (6) (m:f = DSM-V, CARS qPCR on stool bacterial population FGID questionnaire 2. Small sample size.
21:2, aged 3-17y, *9 ASD-GI); 3. Sex bias.
NT siblings (m:f = 11:11, aged 4. Not considered as exclusion criteria: concomitant medication
4.5-18.5y, *6 NTsib-GI); intake (including antibiotics), dietary status, concomitant
pr
NT unrelated (m:f = 4:5, aged pathologies (epilepsy and intellectual disability included in ASD
3.5-15y, *1 NT-GI). group Epilepsy and intellectual disability included in ASD
group)
e-
AD-GI versus NT-GI versus NT-wGI
Parracho et al. ASD (m:f = 48:10, aged 3-16y, Not reported FISH analysis and microscopic Questionnaire 1. Modest sample size.
2005 *53 ASD-GI); examination of stool bacterial 2. Sex bias.
NT siblings (m:f = 7:5, aged 2- population 3. Not considered as exclusion criteria: concomitant medication
Pr
10y, *3 NTsib-GI); intake (including antibiotics), dietary status, but analysis of
NT unrelated - wGI (m:f = 6:4; their association with bacterial profiles. Unclear if concomitant
aged 3-12y) pathologies were considered.
Kang et al. 2013 AD-GI (m:f = 18:12, aged 3- ADI-R, ADOS, PDD-BI qPCR analysis of 16S rDNA stool Modified version of 1. Small sample size.
11y); bacterial population. GSI 2. Sex bias.
NT unrelated (m:f = 17:3,
l 3. Not considered as exclusion criteria: functional foods, dietary
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aged 3-16y, *7 NT-GI). status and dietary supplements
Liu et al 2019 ASD (m:f = 25:5, aged 3-5.9y, DSM-V, ICD-10 16S rRNA gene pyrosequencing by Modified versión of 1. Small sample size.
*15 ASG-GI); PCR on stool bacterial population 6-GSI 2. Sex bias
NT unrelated-wGI (m:f = 16:4; 3. No stratification by ASD severity
aged 3.3-5.3y) 4. Number of samples per subject not specified.
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Kong et al 2019 ASD (m:f = 15:5, aged 13- DSM-V 16S rRNA gene pyrosequencing by GSI 1. Small sample size.
18y); PCR on stool bacterial population 2. Sex bias
NT related (m:f = 8:11, aged 3. Not considered as exclusion criteria: antifungal intake, dietary
11-50y) status and allergies, but analysis of the effect of the last two
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on bacterial profiles
AD-wGI versus NT-GI versus NT-wGI
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Luna et al 2017c ASD-GI (m:f = 14:0, aged 4- ADOS; SRS for NT children 16S rDNA characterization from QPGS-RIII 1. Small sample size.
13y); rectal bacterial population 2. Sex bias.
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NT unrelated-GI (m:f = 12:3, 3. Not considered as exclusion criteria: concomitant intake of
aged 3-18y); other medication besides antibiotics and steroids, dietary
NT unrelated-wGI (m:f = 6:0; status
3-14y) 4. No stratification by ASD severity
AD-GI versus AD-wGI versus NT-GI versus NT-wGI
pr
Son et al 2015 ASD (m:f = 52:7, *25 ASD-GI); Unclear verification of qPCR and 16S rRNA gene QPGS-RIII 1. Modest sample size.
NT siblings (m:f = 21:23, *13 diagnosis, behavior pyrosequencing of stool bacterial 2. Sex bias.
NT-GI). problems assessed by population. 3. Not considered as exclusion criteria: antifungal intake, dietary
Age range: 7-14y, only 37 CBCL status, concomitant pathologies
family-matched pairs. 4. No stratification by ASD severity
e-
Strati et al 2017 AD (m:f = 31:9, aged 5-17y, *5 DSM-V, ADOS, ABC, CARS 16S rRNA gene pyrosequencing on Constipation 1. Modest sample size.
constipated, *29 non-cons., bacterial and fungal stool sample defined according 2. Sex bias.
Pr
*36 severe AD); populations. to Rome III 3. Not considered as exclusion criteria: antifungal intake, dietary
NT unrelated (m:f = 28:12, status, celiac disease (present in 2 ASD subjects
aged 3.6-12y, *11 cons.). 4. Number of samples per subject not specified.
6-GSI: 6-item gastrointestinal severity index; ABC: Autism Behavior Checklist; AD: autism disorder; ADI/ ADI-R: Autism Diagnostic Interview/ Revised Version; ADOS-2: Autism Diagnostic Observation Schedules – 2;
ASD: autism spectrum disorder; ATEC: Autism Treatment Evaluation Checklist; CARS: Childhood Autism Rating Scale; CBCL: Child Behavior Checklist; DSM-IV/V/TR: Diagnostic and Statistical Manual of Mental
Disorders 4th Edition/ 5th Edition/ Text revision; FGID: functional gastrointestinal disorders; FGIDs: functional gastrointestinal disorders; FISH: fluorescence in situ hybridization; GI: gastrointestinal disorders; GIH:
CHARGE Gastrointestinal History Questionnaire; GMDS: Griffiths Mental Development Scales; HPLC: high performance liquid chromatography; ICD-10: International Statistics Classification of Diseases and Related
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Health Problems, 10th Revision; INDT-ASD: INCLEN Diagnostic Tool for Autism Spectrum Disorder (DSM-V approved); ISAA: Indian Scale for Assessment of Autism; KCARS: Korean Childhood Autism Rating Scale;
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LA/MA: lactulose/mannitol test; M-CHAT: Modified Check-list for Autism in Toddlers; m:f: male/female ratio; MR: mental regression; NA: not applicable; NGS: next-generation sequencing; NT: neurotypical; PARS:
Pervasive Developmental Disorders Autism Society Japan Rating Scale; PCoA: principal coordinate analysis; PCR: Polymerase Chain Reaction; PDD-NOS: pervasive developmental disorder not otherwise specified;
PDDBI: Pervasive Developmental Disorders Behavior Inventory; qPCR quantitative polymerase chain reaction, also known as real-time PCR (RT-PCR), QPGS-RIII: Questionnaire on Pediatric Gastrointestinal
Symptoms-Rome III version; SRS: Social Responsiveness Scale; VABS: Vineland Adaptive Behavior Scales; wGI: without gastrointestinal disorders; wMR: without mental regression
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Table 1: gut bacterial and fungal profile in children and adolescents with ASD. a Studies classified by assessment of GI function on microbial analysis and
displayed in ascending order of publication date; b Unclear whether ASD children suffer from GI conditions, c For specific analysis of correlation between
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microbiota and abdominal pain, stratification on four groups was conducted: ASD-GI with no pain reported (n=5), ASD-GI with abdominal pain (n=9), NT with
no pain reported (n=10), and NT with abdominal pain (n=11).
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Microbiota and gut-brain axis dysfunction in Autism Spectrum Disorder:

I. Lasheras, P. Seral, E. Latorre, E. Barroso, P. Gracia-Garcı́a, J.

To appear in: Asian Journal of Psychiatry

Received Date: 12 August 2019

© 2019 Published by Elsevier.

Microbiota and gut-brain axis dysfunction in Autism Spectrum

Disorder: Evidence for Functional Gastrointestinal disorders

Lasheras I a, Seral P a, Latorre E b,c,d*

*Corresponding and reprint author:

Department of Biochemistry and Molecular and Cell Biology

Pedro Cerbuna 12, 50009, Zaragoza, Spain

distinctive ASD microbial pattern is still unclear.

METHODOLOGY: First, we searched the PubMed database in peer-reviewed journals

included. Lastly, research on the role of microbial dysbiosis as an interface between

genetic and environmental risk factors in the pathogenesis of neuropsychiatric disorders,

and specifically ASD, was examined.

and NT children, indicating a difference in taxonomic abundance profiles, which have

demographics, diet, disease severity, GI comorbidity and allergies. Integration of these

understanding of microbiota involvement in ASD pathogenesis for future research.

Keywords: Autism Spectrum Disorder, Microbiota, Gut-brain Axis, Functional

neurodevelopmental disorders previously referred to as autistic disorder (AD), Asperger´s

syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), and

has become a major concern in the scientific community.

emerging studies have identified an imbalanced composition of the intestinal microbiota

plausible explanation for the more frequent occurrence of functional GI disorders

what is currently known about: 1) FGIDs in ASD, 2) evidence of a distinctive gut

and gut-brain axis dysfunction in ASD pathogenesis.

1.1 Functional Gastrointestinal Disorders in Autism Spectrum Disorder

FGIDs comprise a heterogeneous group of chronic recurrent gastrointestinal symptoms

which could not be explained by any underlying anatomical or biochemical abnormalities

attributed to methodological limitations, such as retrospective study design and

inappropriate control groups, enrollment of clinically heterogeneous ASD children, bias

2014); in most cases, these complaints received a diagnosis of a FGID.

displayed increased sleep difficulties, abnormal mood and argumentative, oppositional,

defiant or destructive behavior, anxiety, sensory responsivity, rigid compulsive

behaviors, self-injury, aggression, lack of expressive language and social impairments

when compared to ASD patients without GI comorbidities (Nikolov et al. 2009).

2. EVIDENCE OF AN ALTERED GUT MICROBIOTA IN AUTISM SPECTRUM

2.1 Description of included studies

without mental regression (Plaza-Díaz et al. 2019) respectively.

most common methods for bacterial identification were sequencing or pyrosequencing of

consideration in the exclusion criteria, such as previous and/or concomitant intake of

their association or effect on bacterial outcomes, as will be discussed later. In addition,

and Finegold 2004; Tomova et al. 2015).

Following microbial sequencing, assessment of bacterial diversity was performed in over

performed by assemblage into operational taxonomic unit (OTU) clusters, abundance-

allowed for identification of differences among microbial communities within groups,

children with GI disturbances or stratification by this variable prompted classification of

ASD microbial profile.

2.2 Major findings of included studies

since 29% of them included an unrelated group of NT children without GI dysfunction

microbiota is influenced by genetic and environmental factors, certain similarities in

of bacterial reads and does not account for their abundance.

gut microbiota is mainly defined by two bacterial phylotypes: Bacteroidetes and

found a significant phylum change relative to ASD, specifically an increase in Firmicutes,

Covariate analysis for correlation between demographics and microbial profile

years revealed that, while no single constant microbiota composition component or

Bifidobacterium and decreased Bacteroides and cumulative levels of Enterococcus and

Lactobacillus (Pärtty et al. 2015) at 6 and 18 months respectively. Since microbial

Furthermore, relationships have been established between Bifidobacterium and a high

Likewise, Autism Diagnostic Observation Schedules (ADOS) scores positively

correlated with Faecalibacterium prasusnitzii and Bacteroides uniformis in another study

since evidence on the direction of change in ASD relative to NT is inconsistent (De