Gut Flora and Autism
Autism Spectrum Disorder
Autism spectrum disorder (ASD) is the category of which autism falls under. ASD describes a range of neural development disorders which normally manifests in children before 18 months of age but late onset can occur after 18 months (1,2). There are different degrees of ASD with varying symptoms among individuals. It is usually characterized by difficulties in social interaction, repetitive behaviour and delays in understanding language (2). The etiology of autism is unknown. However, autistic children have been reported to suffer from severe and frequent gastrointestinal (GI) problems which suggests that there may be a link between autism and the gut (3,4).
The Human Gut
It is estimated that there are about 300-500 different species of bacteria that form a mutualistic interaction with the human digestive tract to provide metabolic, immune and protective functions (5). Gut flora aid in the fermentation of non-digestible dietary residue and carbohydrates. They also help produce short chain fatty acids. The gut microbiome consists mainly of bacteria from two phylum Firmicutes and Bacteroidetes (5).
GI problems include diarrhea, unformed stools, constipation, bloating and/or gastroesophageal reflux. Among the autistic population, GI problems are a common occurrence, as shown in a 2011 study where 63% of the autistic children had GI problems compared to the 2% of the control group that had GI problems(3). The control group refers to the group of children who do not have autism.
In 2002, stool, gastric juice and duodenal fluid samples from collected from various children hospitals from autistic patients with a history of GI problems and were examined (6). In total, 25 different species of Clostridium were encountered, 9 of which were found exclusively in the gut of autistic children while 3 species were only found in the control (6). Clostridium difficile, a species that was found only in autistic stools, is known to be a major cause of severe GI diseases through its production of enterotoxin A, which is responsible for various GI symptoms through an unknown mechanism (7). Another study published in April of 2012 found that those with late onset autism have been shown to have higher counts of Desulfovibrio in their fecal matter via pyrosequencing but Clostridium was not isolated (1).
Clostridium is a genus of Gram-positive, obligate anaerobic, spore-forming bacteria while Desulfovibrio is a genus of Gram-negative and aerotolerant bacteria (8). Their metabolism produces short-chain fatty acids (SCFA) from carbohydrates and amino acids. Increased levels of SCFA present in circulating blood can cross the blood brain barrier where they affect brain development and function (9). Propanoic acid (PPA) is a type of SCFA known to influence cell signaling, neurotransmitter synthesis/ release, mitochondrial function and gene expression (9). It interferes with cellular metabolism by overwhelming the metabolic functions of the mitochondria to cause oxidative dysfunction and stress (8). Increased PPA over the developmental time period may play a role in the development of autism and other neurodevelopment disorders (7).
An experiment performed in 2008 tested the hypothesis that increased levels of PPA resulted in autistic-like characteristics such as impaired social behaviour (10). 114 adult male rats were randomly selected into the following groups: PPA, propanol control, sodium acetate (SA) control and phosphate buffered saline control (PBS) (10). Sodium acetate is produced in the gut from carbohydrate fermentation and propanol is a non-acidic analog of PPA.
Behaviour was analyzed with two rats of the same experimental conditions and data was collected on the following variables via a video tracking software: mean distance between rats, percent of time rats spent within 5cm proximity of each other and total distance travelled by an individual rat (10). Additionally, the rats were videotaped and evaluated for playfulness based on frequency of play initiations, number and type of defenses evoked (10).
The results showed that the rats with injected PPA had consistently impaired social behaviour. PPA injected rats exhibited a greater distance apart, spent less time within a 5cm proximity to each other, had less play interactions, and were more likely to show evasive defenses (10). These findings support that increased PPA levels somehow impair social behaviour. SA rats initially showed these behaviours as well, but they decreased as time progressed (10). Propanol and PBS injected rats did not show any of the above behaviours (10). The rats were not tested for the presence of Clostridium or Desulfovibrio.
There are no studies published on the use of antimicrobial agents against Desulfovibrio but there was a trial study based on the use of oral vancomycin on late onset autistic children with a history of chronic diarrhea. Vancomycin is an antibiotic used to treat infections caused by Gram-positive bacteria, making it very effective against Clostridium but ineffective against Desulfovibrio (11). The hypothesis was that the protective function of gut flora was compromised due to the use of antimicrobial agents, which allowed the colonization of Clostridium (11).
Post and pre-treatment behaviours of the patients were videotaped and evaluated by a clinical psychologist. It was found that 8 out of 10 of the autistic patients had improved behaviour (11). They were less aggressive, had increased eye contact and improved in their receptive language and speech. Most of their GI problems also went away. Unfortunately, follow up indicated that the effects of vancomycin were short term and the children returned to their previous behaviour (11). It is speculated that the spores of Clostridium were unaffected by the treatment which may have led their recolonization (11).
Ignicoccus species are chemolithoautotrophs that use molecular hydrogen as the inorganic electron donor and elemental sulphur as the inorganic terminal electron acceptor . The reduction of the elemental sulphur results in the production of hydrogen sulphide gas.
Ignicoccus are autotrophs in that they fix their own carbon dioxide into organic molecules. The carbon dioxide fixation process they use is a novel process called a dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle that involves 14 different enzymes .
Members of the Ignicoccus genus are able to use ammonium as a nitrogen source.
Because members of the Ignicoccus genus are hyperthermophiles and obligate anaerobes, it is not surprising that their growth conditions are very complex. They are grown in a liquid medium known as ½ SME Ignicoccus which is a solution of synthetic sea water which is then made anaerobic.
Grown in this media at their optimal growth temperature of 90C, the members of the Ignicoccus genus typically reach a cell density of ~4x107cells/mL .
Ignicoccus hospitalis is the only member of the genus Ignicoccus that has been shown to have an extensive symbiotic relationship with another organism.
Ignicoccus hospitalis has been shown to engage in symbiosis with Nanoarchaeum equitans . Nanoarchaeum equitans is a very small coccoid species with a cell diameter of 0.4 µm . Genome analysis has provided much of the known information about this species.
To further complicate the symbiotic relationship between both species, it’s been observed that the presence of Nanoarchaeum equitans on the surface of Ignicoccus hospitalis somehow inhibits the cell replication of Ignicoccus hospitalis . How or why this occurs has not yet been elucidated .
Nanoarchaeum equitans has the smallest non-viral genome ever sequenced at 491kb . Analysis of the genome sequence indicates that 95% of the predicted proteins and stable RNA molecules are somehow involved in repair and replication of the cell and its genome .
Analysis of the genome also showed that Nanoarchaeum equitans lacks nearly all genes known to be required in amino acid, nucleotide, cofactor and lipid metabolism. This is partially supported by the evidence that Nanoarchaeum equitans has been shown to derive its cell membrane from its host Ignicoccus hospitalis cell membrane. The direct contact observed between Nanoarchaeum equitans and Ignicoccus hospitalis is hypothesized to form a pore between the two organisms in order to exchange metabolites or substrates (likely from Ignicoccus hospitalis towards Nanoarchaeum equitans due to the parasitic relationship). The exchange of periplasmic vesicles is not thought to be involved in metabolite or substrate exchange despite the presence of vesicles in the periplasm of Ignicoccus hospitalis .
These analyses of the Nanoarchaeum equitans genome support the fact of the extensive symbiotic relationship between Nanoarchaeum equitans and Ignicoccus hospitalis. However, it has not yet been proven that it is a strictly parasitic relationship and further research may prove that there is a commensal relationship between the two species.
(1) Burggraf S., Huber H., Mayer T., Rachel R., Stetter K.O. and Wyschkony I. ” Ignicoccus gen. nov., a novel genus of hyperthermophilic, chemolithoautotrophic Archaea, represented by two new species, Ignicoccus islandicus sp. nov. and Ignicoccus pacificus sp. nov.” International Journal of Systematic and Evolutionary Microbiology, 2000, Volume 50.
(2) Naether D.J. and Rachel R. “The outer membrane of the hyperthermophilic archaeon Ignicoccus: dynamics, ultrastructure and composition.” Biochemical Society Transactions, 2004, Volume 32, part 2.
(3) Giannone R.J., Heimerl T., Hettich R.L., Huber H., Karpinets T., Keller M., Kueper U., Podar M. and Rachel R. “Proteomic Characterization of Cellular and Molecular Processes that Enable the Nanoarchaeum equitans- Ignicoccus hospitalis Relationship.” PLoS ONE, 2011, Volume 6, Issue 8.
(4) Eisenreich W., Gallenberger M., Huber H., Jahn U., Junglas B., Paper W., Rachel R. and Stetter K.O. “Nanoarchaeum equitans and Ignicoccus hospitalis: New Insights into a Unique, Intimate Association of Two Archaea.” Journal of Bacteriology, 2008, DOI: 10.1128/JB.01731-07.
(5) Grosjean E., Huber H., Jahn U., Sturt H, and Summons R. “Composition of the lipids of Nanoarchaeum equitans and their origin from its host Ignicoccus sp. strain KIN4/I.” Arch Microbiol, 2004, DOI: 10.1007/s00203-004-0725-x.
(6) Briegel A., Burghardt T., Huber H., Junglas B., Rachel R., Walther P. and Wirth R. “Ignicoccus hospitalis and Nanoarchaeum equitans: ultrastructure, cell–cell interaction, and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography.” Arch Microbiol, 2008, DOI 10.1007/s00203-008-0402-6.
(7) Burghardt T., Huber H., Junglas B., Naether D.J. and Rachel R. “The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex.” Molecular Microbiology, 2007, Volume 63.
(8) Berg I.A., Eisenreich W., Eylert E., Fuchs G., Gallenberger M., Huber H.,Jahn U. and Kockelkorn D. “A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis.” PNAS, 2008, Volume 105, issue 22.
(9) Brochier C., Gribaldo S., Zivanovic Y., Confalonieri F. and Forterre P. “Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?” Genome Biology 2005, DOI:10.1186/gb-2005-6-5-r42.
(10) Huber H., Rachel R., Riehl S. and Wyschkony I. “The ultrastructure of Ignicoccus: Evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon.” Archaea, 2002, Volume 1.