Elsevier

Anaerobe

Volume 16, Issue 4, August 2010, Pages 444-453
Anaerobe

Molecular biology, genetics and biotechnology
Pyrosequencing study of fecal microflora of autistic and control children

https://doi.org/10.1016/j.anaerobe.2010.06.008Get rights and content

Abstract

There is evidence of genetic predisposition to autism, but the percent of autistic subjects with this background is unknown. It is clear that other factors, such as environmental influences, may play a role in this disease. In the present study, we have examined the fecal microbial flora of 33 subjects with various severities of autism with gastrointestinal symptoms, 7 siblings not showing autistic symptoms (sibling controls) and eight non-sibling control subjects, using the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) procedure. The results provide us with information on the microflora of stools of young children and a compelling picture of unique fecal microflora of children with autism with gastrointestinal symptomatology. Differences based upon maximum observed and maximum predicted operational taxonomic units were statistically significant when comparing autistic and control subjects with p-values ranging from <0.001 to 0.009 using both parametric and non-parametric estimators. At the phylum level, Bacteroidetes and Firmicutes showed the most difference between groups of varying severities of autism. Bacteroidetes was found at high levels in the severely autistic group, while Firmicutes were more predominant in the control group. Smaller, but significant, differences also occurred in the Actinobacterium and Proteobacterium phyla. Desulfovibrio species and Bacteroides vulgatus are present in significantly higher numbers in stools of severely autistic children than in controls. If the unique microbial flora is found to be a causative or consequent factor in this type of autism, it may have implications with regard to a specific diagnostic test, its epidemiology, and for treatment and prevention.

Introduction

Autism is a complex disorder and probably embraces a number of differing entities. There are no specific diagnostic tests so the disease is defined by its characteristics—cognitive defects, including impairment of spoken and/or receptive language; social, communication and behavioral problems; repetitive behaviors; unusual sensitivity to stimuli such as noises; and restricted interests [1]. Autism research to date has mainly focused on finding a genetic association but it has been recognized that, in addition to heritable predisposition to the disease [2], environmental factors are likely important [3]. There has been a striking increase in incidence of autism worldwide; CDC’s Autism and Developmental Disabilities Monitoring (ADDM) Network released data that noted that approximately 1% (1 in 110) of children in multiple areas of the U.S. in 2006 had an autistic spectrum disorder, up 57% from 2002 [4]. This increase has not been explained in a satisfactory way. We are inclined to feel that changes in diagnostic criteria and increased attention to autism in the media fail to satisfactorily explain the marked increased in incidence. It should be noted that autistic children are susceptible to various infections, particularly ear infections and that there is considerable use of antibiotics in these children. Indeed, anecdotally, parents have noted the onset of regressive disease in their children after the use of antibiotics. This could account for the changes in bowel flora to be noted in this manuscript. As with other infections involving the gastrointestinal tract, there may be transmission of infectious agents to other children who come in contact with the index case. The financial impact of autism on U.S. society is dramatic; it costs about $3.2 million to take care of an autistic person over his or her lifetime. Caring for all people with autism costs an estimated $35 billion per year according to a Harvard School of Public Health Press Release [5]. A recent consensus report from a multidisciplinary panel concluded that evidence-based recommendations for evaluation, diagnosis, and treatment of gastrointestinal disorders in subjects with autism are not yet available and that these individuals deserve the same thoroughness and standard of care for diagnostic workup and treatment as patients without autism spectrum disorders [6]. The Interagency Autism Coordinating Committee (IACC) points out the need for additional studies on the role of the environment, of epidemiology, and of specific treatments for autism [7]. Published data lend credence to the notion that an alteration in bowel microflora is associated with autistic symptoms [8], [9], [10], [11], [12]. The studies presented herein are concerned with detailed analysis of the fecal microflora of children with autism with gastrointestinal abnormalities.

Section snippets

Subjects

33 Autistic subjects, 7 non-autistic siblings and 8 control subjects considered here were enrolled in the study at the Evergreen Center in Oregon City, OR and the Center for Autism and Related Disorders (CARD) in Tarzana, CA with signed, informed consent of their parents or guardians and approval of VA Greater Los Angeles Healthcare System Institutional Review Board IRB B. All autistic children participating in this study had an educational or developmental pediatrician evaluation and were

Principal component analysis

To assess the separability of the samples, Principal Component Analysis was implemented. Principal Component Analysis (PCA) [27] is widely used for dimensionality reduction to help with visualization of high dimensional data. PCA is defined as the orthogonal projection of the data onto two or three dimensional space such that the variance of the projected data is maximized. Custom Python scripts tailored for next generation data (distance matrices and taxonomic abundance) were implemented to

Results and discussion

The results indicate there is a significantly higher diversity of bacteria found in the feces of autistic subjects compared to controls (Table 1). Even when relatively large genetic distances (5% divergence) are considered, these estimates predict that even at the genus level there is significantly less diversity (at a 5% significance level) and richness of microbial communities in control subjects than in the autistic group. These diversity results were similar when using three separate

Conclusions

An issue that needs to be decided is does the autism lead to the altered flora and/or does the altered flora play a role in the disease or its syndromes. In other situations with major changes in bowel microflora associated with disease (e.g., Clostridium difficile-asssociated pseudomembranous colitis [41] and trinitrobenzenesulphonic acid-induced colitis in mice) [42], fecal enemas or implantation of individual members of the fecal flora leads to improvement in the disease process [42], [43].

Potential problems or pitfalls

A factor of major potential, not evaluated in the current study, may be the immune status of the host; there may be predisposition to suffer ill effects from an abnormal intestinal microbial flora, related to hereditary and/or environmental factors; we presume these to be present. Other factors not evaluated in this study, but potentially important are mucosa-associated microflora and the possibility of biofilm formation (we studied only luminal flora). It should also be noted that

Acknowledgments

SF would like to express appreciation to Mrs. Ellen R. Bolte and Richard Sandler, M.D. who first got him interested in studying autism. The authors express their appreciation to Jeffrey Gornbein, Dr.P.H., UCLA SBCC/Dep’t of Biomathematics, David Geffen School of Medicine, Los Angeles, CA for his review of the manuscript and statistical assistance.

References (43)

  • D.B. Campbell et al.

    A genetic variant that disrupts MET transcription is associated with autism

    Proc Natl Acad Sci U S A

    (2006 Nov 7)
  • ADDM. Autism and Developmental Disabilities Monitoring (ADDM) Network. ADDM 8 A.D. October 20. Available from: URL:...
  • HSPH

    Autism has high costs to U.S. society

    (2006)
  • M. Hornig et al.

    Lack of association between measles virus vaccine and autism with enteropathy: a case-control study

    PLoS ONE

    (2008)
  • IACC

    Strategic plan for autism spectrum disorder research

    (2008)
  • R.H. Sandler et al.

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

    J Child Neurol

    (2000 Jul)
  • S.M. Finegold et al.

    Gastrointestinal microflora studies in late-onset autism

    Clin Infect Dis

    (2002)
  • Y. Song et al.

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

    Appl Environ Microbiol

    (2004)
  • 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 Oct)
  • R.D. Wolcott et al.

    Bacterial diversity in surgical site infections: not just aerobic cocci any more

    J Wound Care

    (2009 Aug)
  • R.D. Wolcott et al.

    Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and titanium amplicon pyrosequencing and metagenomic approaches

    BMC Microbiol

    (2009)
  • Cited by (743)

    View all citing articles on Scopus
    View full text