Elsevier

Physiology & Behavior

Volume 138, January 2015, Pages 179-187
Physiology & Behavior

Gastrointestinal microbiota in children with autism in Slovakia

https://doi.org/10.1016/j.physbeh.2014.10.033Get rights and content

Highlights

  • Fecal microbiota in autistic children in Slovakia differs from controls and siblings.

  • There is a correlation of the autism severity with the severity of GI dysfunction.

  • In our study Desulfovibrio spp. abundance is associated with the severity of autism.

  • Probiotic supplementation normalizes bacterial balance in fecal microbiota.

  • No joined influence of oxytocin, testosterone, DHEAS and fecal microbiota on autism.

Abstract

Development of Autism Spectrum Disorders (ASD), including autism, is based on a combination of genetic predisposition and environmental factors. Recent data propose the etiopathogenetic role of intestinal microflora in autism. The aim of this study was to elucidate changes in fecal microbiota in children with autism and determine its role in the development of often present gastrointestinal (GI) disorders and possibly other manifestations of autism in Slovakia. The fecal microflora of 10 children with autism, 9 siblings and 10 healthy children was investigated by real-time PCR. The fecal microbiota of autistic children showed a significant decrease of the Bacteroidetes/Firmicutes ratio and elevation of the amount of Lactobacillus spp. Our results also showed a trend in the incidence of elevated Desulfovibrio spp. in children with autism reaffirmed by a very strong association of the amount of Desulfovibrio spp. with the severity of autism in the Autism Diagnostic Interview (ADI) restricted/repetitive behavior subscale score. The participants in our study demonstrated strong positive correlation of autism severity with the severity of GI dysfunction. Probiotic diet supplementation normalized the Bacteroidetes/Firmicutes ratio, Desulfovibrio spp. and the amount of Bifidobacterium spp. in feces of autistic children. We did not find any correlation between plasma levels of oxytocin, testosterone, DHEA-S and fecal microbiota, which would suggest their combined influence on autism development. This pilot study suggests the role of gut microbiota in autism as a part of the “gut-brain” axis and it is a basis for further investigation of the combined effect of microbial, genetic, and hormonal changes for development and clinical manifestation of autism.

Introduction

Autism spectrum disorders (ASD) are pervasive developmental disorders, characterized by social abnormalities, communication impairments, and stereotyped and repetitive behaviors. The incidence of ASD in 2010 was 1.47% in the USA [1] and it has been increasing worldwide for last decade. The interest in autism is based firstly, on the escalating incidences of ASD and secondarily, on the common gastrointestinal (GI) manifestations in these people. Up to 90% of children with ASD suffer from GI disorders such as gastroesophageal reflux, constipation, diarrhea, abdominal pain, vomiting and nutrition issues [2], [3], [4]. A direct correlation between the severity of autism and gastrointestinal symptoms has been shown [4], [5]. Understanding the pathophysiology of the GI morbidity in ASD might be important for the early identification of ASD-related pathology and for guiding the therapy of GI symptoms and perhaps ASD.

There is considerable evidence that GI disorders are linked to intestinal dysbiosis. Gut microbiota plays a significant role in modulating human metabolism and in the development of the immune system. The cellular and biochemical pathways of gut-brain interaction provide a basis for the influences of normal gut microbiota on development, neurochemistry, gene expression, and functioning of the brain [6], [7], [8], [9], [10]. The variations in the composition of gut microbes are associated with changes in the normal functioning of the nervous system and behavior [11], [12]. For the last several years an escalating number of studies show the changes of gut microbiota in patients with ASD.

Possible involvement of a microbial element in pathogenesis of autism was shown for the first time in 1998, when the hypothesis was introduced that Clostridium tetani neurotoxin ascends along the vagus nerve route from the intestinal tract to the CNS causing the symptoms of autism [13]. Later the positive effect of the antibiotic vancomycin on the GI symptoms in autistic patients confirmed the involvement of gut bacteria [14]. Unfortunately, the effect was temporal and could be explained by bacteria spore formation [15]. Establishment of dysbiosis in autistic children [16], and health improvement after using probiotics [17] only rapidly increased the evidence, that the composition of gut microbiota is associated with changes in the normal functioning of the nervous system and behavioral changes in ASD. A recent pyrosequencing study serves as convincing evidence of dysbiosis in autistic subjects, including not only a change of ratio of normally present microorganisms, but a significant increase of bacterial diversity [18]. The investigation of the dominant intestinal bacterial phyla in metagenomic analyses disclosed a significant change in the Bacteroidetes/Firmicutes ratio in the feces of autistic children comparing to healthy or neurotypical children with GI symptoms [18], [19]. Various bacterial species have been shown to be involved in dysbiosis in children with autism, particularly they had a higher bacterial incidence of Desulfovibrio spp. [20] and some Clostridium clusters [13], [16], [21] than the control children population. The diversity of bacterial species was also higher in autistic compared to neurotypical children. For example, they had more Clostridia species [18], [22]. Conversely, other species were significantly reduced in gut microbiota of autistic children, like Akkermansia muciniphila and Bifidobacterium spp. [23]. And finally some bacterial species have been shown to be present almost exclusively in autistic gut microbiota, such as Alkaliflexus [18] and Sutterella [24] or some other bacteria were present but only in healthy subjects, as Weissella [18].

The hypothesis of the pathogenesis of ASD includes multiple mechanisms, a combination of genetic predisposition and environmental factors. Although there have been advances in identifying a genetic cause in ASD, recent studies of concordant twins suggest there is a stronger environmental component than previously believed [25], [26]. Our study is aimed to make a step forward to elucidating the importance of environmental factors and possibly in combination with other factors leading to developing the disease.

Recent data suggest the connection of intestinal microflora content on health status of autistic individuals, and its possible etiological role in people with autistic predisposition. One of the latest theories of autism pathogenesis includes the etiopathogenetic role of specific bacteria (Clostridia, Desulfovibrio and Bifidobacterium) [27]. This is the reason why in our pilot study we investigated the intestinal microflora in children with autism in Slovakia and the specificity of their dysbacteriosis, compared to their siblings and to control group of neurotypical children. Moreover, we have also investigated the changes of microflora in a group of autistic children after probiotic therapy. Although over 50 bacterial phyla have been described in the human gut microbiota, we focused our study on the two most dominant, the Bacteroidetes and the Firmicutes. Special attention was paid to the bacteria described as being involved in manifestations of ASD (Lactobacillus, Bifidobacterium, Clostridia, and Desulfovibrio). For more complex investigation in the development of autism, we also looked at these children's oxytocin plasma levels and at such neurosteroids as testosterone and DHEAS, that were implicated to play an important role in autism development [28], [29], [30] and their possible coaction with intestinal microflora.

Section snippets

Subjects

We enrolled in our study 10 autistic children, their 9 non-autistic siblings, and 10 non-autistic children as a control. 9 of 10 autistic children underwent probiotic intervention. The age of autistic children was from 2 to 9 years, siblings — from 5 to 17 years and control children from 2 to 11 years old. In the group of autistic subjects were included 9 boys and one girl, in the group of siblings — 7 boys and two girls, the control group consisted of 10 boys. Children with autism were recruited

Results

In our study 9 out of 10 children with autism, 7 out of 9 in the siblings group and 6 out of 10 in the control group, had some kind of GI symptoms. The scoring, including the number of GI symptoms and their severity showed a significantly higher level of GI dysfunction in autistic children, as well as in their siblings, compared to the controls (Fig. 1). The significance of GI disorders was shown by a strong positive correlation between the intensity of GI symptoms and the severity of autism,

Discussion

Although the major deficits in ASD are social and cognitive, individuals with autism often suffer from GI disorders [2], [3], [4], and it remains to be determined whether they are directly related to ASD pathophysiology, or are strictly comorbid condition. GI complains are accompanied by intestinal microflora changes and, therefore, it is important to identify microbiome and specific microorganisms' changes that can be targeted for diagnosis as well as for treatment of autism-related GI

Conclusion

This is the first investigation of gastrointestinal microbiota in children with autism in Slovakia. We found that the gut microflora assessed from stool samples in children with autism differs from the one in children without autism: the Bacteroidetes/Firmicutes ratio was decreased and the Lactobacillus abundance was elevated. We also observed trends for elevated Clostridia cluster l and Desulfovibrio incidences in children with autism. Interestingly, gut microbiota of siblings had significant

Acknowledgements

A particular thanks to Dr. Perďochová, HPL spol. s.r.o., Slovakia and Dr. Gordon Webster, Cardiff University, UK for providing the reference bacterial strains. We thank Dr. P. Celec, Institute of Molecular Biomedicine FM CU, Slovakia, for his suggestions in modeling the experiment, Dr. B. Mravec, Institute of Physiology, FM CU, Slovakia, and Dr. A. Tillinger, SAS, Slovakia, for their manuscript suggestions, and Dr L. Levy, College of Osteopathic Medicine of Nova Southeastern University, USA,

References (62)

  • C.P. Braegger et al.

    Tumour necrosis factor alpha in stool as a marker of intestinal inflammation

    Lancet

    (1992)
  • K.Z. Long et al.

    Vitamin A supplementation modifies the association between mucosal innate and adaptive immune responses and resolution of enteric pathogen infections

    Am J Clin Nutr

    (2011)
  • E.Y. Hsiao et al.

    Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders

    Cell

    (2013)
  • D.F. MacFabe et al.

    Effects of the enteric bacterial metabolic product propionic acid on object-directed behavior, social behavior, cognition, and neuroinflammation in adolescent rats: Relevance to autism spectrum disorder

    Behav Brain Res

    (2011)
  • ADDMNS

    Prevalence of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2010

    MMWR Surveill Summ

    (2014)
  • D.L. Coury et al.

    Gastrointestinal conditions in children with autism spectrum disorder: developing a research agenda

    Pediatrics

    (2012)
  • K. Babinská et al.

    The effects of probiotic administration on gastrointestinal functions in children with autism

    Act Nerv Super Rediviva

    (2012)
  • J.B. Adams et al.

    Gastrointestinal flora and gastrointestinal status in children with autism–comparisons to typical children and correlation with autism severity

    BMC Gastroenterol

    (2011)
  • T. Buie et al.

    Evaluation, diagnosis, and treatment of gastrointestinal disorders in individuals with ASDs: a consensus report

    Pediatrics

    (2010)
  • J.G. Mulle et al.

    The gut microbiome: a new frontier in autism research

    Curr Psychiatry Rep

    (2013)
  • J.F. Cryan et al.

    The microbiome-gut-brain axis: from bowel to behavior

    Neurogastroenterol Motil

    (2011)
  • R. Diaz Heijtz et al.

    Normal gut microbiota modulates brain development and behavior

    Proc Natl Acad Sci U S A

    (2011)
  • S.H. Rhee et al.

    Principles and clinical implications of the brain-gut-enteric microbiota axis

    Nat Rev Gastroenterol Hepatol

    (2009)
  • E.A. Mayer

    Gut feelings: the emerging biology of gut-brain communication

    Nat Rev Neurosci

    (2011)
  • A.V. Rao et al.

    A randomized, double-blind, placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue syndrome

    Gut Pathog

    (2009)
  • R.H. Sandler et al.

    Short-term benefit from oral vancomycin treatment of regressive-onset autism

    J Child Neurol

    (2000)
  • H.M. Parracho et al.

    Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children

    J Med Microbiol

    (2005)
  • J.W. Critchfield et al.

    The potential role of probiotics in the management of childhood autism spectrum disorders

    Gastroenterol Res Pract

    (2011)
  • B.L. Williams et al.

    Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances

    PLoS One

    (2011)
  • Y. Song et al.

    Real-time PCR quantitation of clostridia in feces of autistic children

    Appl Environ Microbiol

    (2004)
  • S.M. Finegold et al.

    Gastrointestinal microflora studies in late-onset autism

    Clin Infect Dis

    (2002)
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