That 'gut bacteria transplant provokes autistic signs in mice' paper is not perfect but...

The paper by Gil Sharon and colleagues [1] has certainly created headlines and discussion in equal measure (see here and see here and see here and see here). Concluding that: "Mice harboring human ASD [autism spectrum disorder], but not TD [typically developing], microbiomes exhibit ASD-like behaviors", the idea of a gut-brain connection in relation to autism (see here) potentially gains some research traction.

The Sharon study involved transplanting gut bacteria - the gut microbiome - from a small number of participants - "from 5 control volunteers and 11 patients diagnosed with autism spectrum disorder" - into mice lacking a microbiome and breeding said mice. They then analysed the behaviour and other biological parameters of those offspring mice according to whether their mother mice had received a transplant from controls or participants with various 'degrees' of autism. They also looked at 'metabolite profiles' based on "analyses of colon contents from oTD [offspring typically developing] and oASD [offspring autism spectrum disorder] mice."

Results: "colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors." By 'hallmark autistic behaviors' researchers observed that said mice showed "increased repetitive behavior, decreased locomotion, and decreased communication... compared to mice colonized with samples from TD controls (oTD), as tested by marble burying (MB), open-field testing (OFT), and ultrasonic vocalization (USV), respectively." Researchers also observed specific differences across the mouse group gut microbiomes, some of which were consistent with that noted in other independent studies.

Also: "Twenty-seven out of 313 detected metabolites were significantly different in the colon contents of oASD mice, compared to oTD mice." They specifically focused in on two metabolites - taurine and 5-aminovaleric acid (5-AV) - both of which were reported in lower levels in the oASD mice, and how these compounds show a *connection* to GABA, a compound potentially important to autism (see here). Further they showed that supplementation of 5-AV and taurine to another strain of mouse that serves as a 'mouse model of autism' (BTBR T+ tf/J (BTBR) mouse model) resulted in "improved repetitive and social behaviors." I should add the word 'mouse' into the sentence "improved repetitive and social behaviors."

Insofar as the limitations of the Sharon studies and paper, various people have been keen to point out that the results should be viewed cautiously and as preliminary. This on the basis of the number of animals included for study, the reliance on mouse models of autism (and the logical fallacies that can sometimes follow) and some of the generalisations made in the study write-up by the authors. I wouldn't disagree with such cautions, bearing in mind that some mouse models of autism - the valproic acid autism mouse model for example - actually seem to be pretty good at mimicking some facets of (induced) autism. I'd also point out that the metabolomics work undertaken by Sharon and colleagues looks to be pretty wide-ranging (GC-MS and NMR are discussed) and findings related to taurine have also been noted in other independent study (see here). I also observed that there was a research tie-up with Arizona State University in the Sharon study, as the name Dae-Wook Kang is mentioned and 'poo transplants for [some] autism' makes yet another appearance (see here and see here).

"While ours is a limited study, with 16 donor samples from a pediatric cohort, the results support a hypothesis that the human gut microbiota contributes to ASD phenotypes." I'd agree that the Sharon results add a further layer to the idea that the new triad - intestinal permeability, mucosal immunology and intestinal microbiota - could be important to at least some autism. The results offer a road map for further investigation in this area and perhaps eventually, yet another avenue for screening and intervention to complement other recent initiatives (see here); all set with the view of the (plural) 'autisms'.

Finally, I note that another study [2] mentioning the words 'mouse' and 'autism' has been published recently. With some media attention mentioning how: "Exercise reversed autistic behaviors in an animal model of the condition" there didn't seem to be the same 'keenness' to point out the flaws of the Andoh study, despite once again a reliance on 'mouse autism' and all which that entails. It makes me wonder whether the focus on the second brain (gut) and autism detailed in the Sharon study might still have the ability to raise hackles in some quarters?

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[1] Sharon G. et al. Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice. Cell. 2019 May 30;177(6):1600-1618.e17.

[2] Andoh M. et al. Exercise Reverses Behavioral and Synaptic Abnormalities after Maternal Inflammation. Cell Reports. 2019; 27: 10. June 4.

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"a probiotic (Bifidobacterium infantis) in combination with a bovine colostrum product (BCP)" for autism?

The study findings reported by Megan Sanctuary and colleagues [1] caught my eye recently, and their aim to "assess tolerability of a probiotic (Bifidobacterium infantis) in combination with a bovine colostrum product (BCP) as a source of prebiotic oligosaccharides and to evaluate GI [gastrointestinal], microbiome and immune factors in children with ASD [autism spectrum disorder] and GI co-morbidities."

I appreciate that such a study is probably not going to be everyone's cup of tea given, for example, the rather *interesting* history of colostrum and autism (including the words 'transfer factor' [2]). The Sanctuary study however, should be taken on its own merit regarding "concurrent supplementation with both the probiotic B. infantis and bovine colostrum product (BCP) as a source of immune factors and prebiotic glycans could alter the microbiota to a more beneficial composition in order to improve gut health in children with ASD and GI symptoms." The rationale behind such work is that (a) what goes on the in the deepest, darkest recesses of the GI tract in a microbial sense could impact on the functional gut symptoms, and (b) said functional gut symptoms seem to be 'over-represented' in relation to autism (see here) and *could* in some cases, be linked to behavioural presentation (see here). Ergo, try and impact on functional gut symptoms and one *might* be able to impact on behaviour...

Sanctuary et al report preliminary findings designed to "assess tolerability" and "to evaluate GI, microbiome and immune factors in children with ASD and GI co-morbidities." This work represented a first step towards a bigger research trial to ascertain whether such a supplemental combination *might* be useful for some people on the autism spectrum in a 'clinically relevant' sense. Despite being a pilot study, researchers did conduct what is considered a gold-standard study insofar as it being a "double-blind, crossover, randomized clinical trial (RCT)." The study protocol was also research registered (see here), so quite a few methodological boxes were ticked.

Given that this study was carried out at the MIND Institute, an institution that has quite a lot of experience in all-manner of different autism research areas (see here and see here), researchers were pretty precise when it came to diagnosing autism/ASD and ascertaining the presence or not of GI symptoms in their small cohort (N=11). The authors also provide quite a bit of information about the supplements used including "the bovine colostrum product (Imucon)" and details of its safety: "The product was tested and found to be negative for Escherichia coli, Salmonella, Listeria, coagulase positive Staphylococcus and antibiotic residue." Insofar as dosages, we are told that: "The colostrum powder dose administered in this study was 0.15 g/lb body weight per day" and "The probiotic dose administered in this study was 20 billion CFU [colony forming units] per day." A couple of supplemental combinations were examined during the study including BCP on its own and BCP+probiotic.

Results: there were quite a few different types of results reported on for 8 of the original 11 participants. Importantly: "Bovine colostrum product appears to be well-tolerated in these children [diagnosed with autism] as its own treatment as well as when combined with the probiotic B. infantis." 'Well-tolerated' means that there were "no participants needing to withdraw due to adverse events" despite a small number of reports of things like gassiness. A couple of kids were also reported to find the taste of the products not too great.

Also: "Some participants on both treatments saw a reduction in the frequency of certain GI symptoms, as well as reduced occurrence of particular aberrant behaviors." Being really careful here because of the small participant number included for study for example, researchers reported that "87.5% (7/8) of participants exhibited some improvement in GI symptoms while on the BCP only arm and 100% (8/8) of participants exhibited some improvement in GI symptoms while on the combination treatment arm." The sorts of GI effects mentioned included reductions in "pain with stooling, frequency of diarrhea, and consistency." Researchers also reported that appetite seemed to be improved for some kids too, particularly the consumption of fruit and meat.

A few other changes were noted in the study, but on the basis of the small participant size and the aims of the study I'm gonna leave them for now. As the authors mention: "the lack of a clear control group receiving a placebo" means that this was a "cross-over study where each participant was his own control" and therefore one needs to be cautious for now. What is needed next is a larger trial and more focus on the behavioural presentation side of autism...

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[1] Sanctuary MR. et al. (2019) Pilot study of probiotic/colostrum supplementation on gut function in children with autism and gastrointestinal symptoms. PLoS ONE 14(1): e0210064.

[2] Fudenberg HH. Dialysable lymphocyte extract (DLyE) in infantile onset autism: a pilot study. Biotherapy. 1996;9(1-3):143-7.

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Lactobacillus (L.) reuteri and mouse-modelled autism: spotlight on the vagus nerve?

I am a little late getting to the findings reported by Martina Sgritta and colleagues [1] but I eventually arrived at them. As per the title of this post - "Lactobacillus (L.) reuteri and mouse model autism: spotlight on the vagus nerve?" - there were a number of important elements to this research covering mouse-modelled autism, the "gut-microbiota-brain axis", and something called the vagus nerve. Pretty good fodder for this blog by all accounts, given some past discussions (see here) including mention of previous research from some of the Sgritta paper co-authors (see here).

So, continuing a theme from this research group suggesting that, in mice at least, some pregnancy 'risk factors' for autism might have a microbial connection [2], authors set out to examine whether the inclusion of a specific bacterial species called Lactobacillus reuteri or L. reuteri for short might have some important effect on the social behaviour of a mouse model of autism. L. reuteri has been the source of quite a bit of study down the years [3] as a function of it's antimicrobial activity and connection to inflammatory conditions. Outside of the Baylor College group research, this bacterial species has also been talked about with autism in mind [4] in other studies, as per conclusions like: "This study identifies bacterial species that are sensitive to an autism-related mutation." I'll say no more on that study for now aside from offering a viewpoint from elsewhere on the web.

Alongside their observation that "treatment with L. reuteri selectively rescues social deficits in genetic, environmental, and idiopathic ASD [autism spectrum disorder] models" Sgritta et al also put a little scientific flesh on what underlying mechanism(s) might be pertinent to such 'rescuing' of social issues. You might think it was something to do with the contribution of L. reuteri to the gut microbiome of the [mouse] host? Well, yes and no, as authors highlighted how "L. reuteri acts in a vagus nerve-dependent manner" so bringing in the notion that bacteria in the gut *talk to* the brain (or the wider central nervous system) via the vagus nerve. The paper by Bruno Bonaz and colleagues [5] provides as good an explanation as any of how bacteria talk to the brain and what this means for the "gut-microbiota-brain axis." Interestingly too, authors mention how when the vagus nerve was 'disrupted' in said mouse-modelled autism, so the the gut-microbiota-brain link was also disrupted, and onward that L. reuteri didn't seemingly work as well at rescuing those social issues. They also talk about a role for oxytocin receptors in their findings similar to their last research occasion, but I'm going to leave that for now. All I will say is that oxytocin has also been mentioned in other autism research circles (see here).

Obviously, there are caveats to the Sgritta findings; not least that mouse-modelled autism is just mouse-modelled autism and probably not totally representative of real-life human autism (see here). Generalisations of the social behaviour of mice raised and living under laboratory conditions to people (children and adults) traversing the big, wide real world always require a degree of caution.

But I am interested in this area and would like to see more study done on the "gut-microbiota-brain axis" with autism in mind. Given that L. reuteri is freely available to anyone and everyone and seems to have quite a good safety record, I'd perhaps suggest that a clinical trial could be something that a young, upcoming researcher or research group might possibly want to consider exploring...

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[1] Sgritta M. et al. Mechanisms Underlying Microbial-Mediated Changes in Social Behavior in Mouse Models of Autism Spectrum Disorder. Neuron. 2018 Dec 3. pii: S0896-6273(18)31009-2.

[2] Buffington SA. et al. Microbial Reconstitution Reverses Maternal Diet-Induced Social and Synaptic Deficits in Offspring. Cell. 2016 Jun 16;165(7):1762-1775.

[3] Mu Q. et al. Role of Lactobacillus reuteri in Human Health and Diseases. Front Microbiol. 2018;9:757. 

[4] Tabouy L. et al. Dysbiosis of microbiome and probiotic treatment in a genetic model of autism spectrum disorders. Brain Behav Immun. 2018 Oct;73:310-319.

[5] Bonaz B. et al. The Vagus Nerve at the Interface of the Microbiota-Gut-Brain Axis. Front Neurosci. 2018;12:49. 

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Exclusion diet plus prebiotics for [some] autism?

"To our knowledge, this is the first study where the effect of exclusion diets and prebiotics has been evaluated in autism, showing potential beneficial effects."

So said the results reported by Roberta Grimaldi and colleagues [1] and the findings of their study attempting to "understand the impact of diet on GM [gut microbiota] composition and metabolism in ASD [autism spectrum disorder] children and to investigate the modulating potential of B-GOS® intervention on these parameters."

The study by Grimaldi et al was research registered (see here) and included the 'gold-standard' research design: "A randomised, double-blind, placebo-controlled" study. Researchers divided 30 children diagnosed with an ASD into two groups (A and B), which were subsequently further divided into two groups depending on whether they followed an exclusion diet - "mainly gluten and casein free" - or an unrestricted diet based on the analysis of 4-day food diaries. Within those subdivided A and B groups, half received a placebo supplement of maltodextrin whilst the other half received a "prebiotic B-GOS® mixture" over a 6-week period. As well as including 'run-in', 'beginning of treatment' and 'end of treatment' periods, researchers also included a follow-up period of 2 weeks at the end of the study where various behavioural and physiological measures complemented their use during the experimental period.

Results: there were quite a few results reported as a function of baseline variables such as whether or not participants were following an exclusion diet or not, and as a function of the intervention(s). Behaviourally (probably most importantly), researchers reported that: "Results showed consistent reduction over time in anti-sociability score in children on the combination of the exclusion diet and B-GOS intervention, with the most apparent difference occurring at follow-up." This was measured using the autism 'rising' instrument called the A-TEC (see here) and also complemented other results based on the use of the autism spectrum quotient (AQ). As far as I can make out, all other behavioural and psychometric measures used - the "empathy and systemising quotient (EQ-SQ)... and the Spence’s Children Anxiety Scale-Parent version (SCAS-P)" - did not show any significant changes over the study duration.

Gastrointestinal (GI) symptoms were also examined during the Grimaldi study as per the use of "daily questionnaires for GI function and symptoms" and the utilisation of that fabulous graphical resource, the Bristol stool chart. Authors reported no significant changes/differences as a consequence of intervention, although: "Significantly lower scores of abdominal pain (P < 0.05) and bowel movement (P < 0.001) were reported in children following exclusion diets" at baseline. Interesting, in light of other independent results (see here).

Grimaldi and colleagues also provide quite a lot of data following their examination of fecal and urine samples taken over the course of their investigation. This is perhaps not unexpected given their previous research interests in this area and the proud reputation earned at one of the affiliated institutions. The results? Lots of them. Perhaps the most important intervention-wise was the finding of a "significant increase of Lachnospiraceae family" following B-GOS® intervention. The authors talk about this in terms of the production of butyrate (as per their previous research) and the (positive) reputation this stuff is starting to garner.

Other details? Well, going back to the baseline assessment of samples as a function of the use of an exclusion diet or not, there are some interesting findings. So: "Before prebiotic B-GOS® intervention, we evaluated the nutritional impact of exclusion diets (GFCF) and our results showed deficiency in vitamin D intake, which was significant in children on unrestricted diets." This kinda ties into other research which observed that the 'horror' that is a gluten-free, casein-free (GFCF) diet in relation to autism might actually not be that horrible in nutritional terms (see here). I'm also minded to bring in other recent research which suggested that a GF diet might be a bit of a 'fixer' when it comes to vitamin D deficiency issues under certain clinical circumstances [2]. I say this minus any sweeping generalisations or universal application to the label of autism.

Overall the Grimaldi findings are interesting and suggest further investigations are required on the use of prebiotics and diet in autism; perhaps complementing the still-growing interest in the gut microbiota and autism (see here for one example). Mindful also that diet can itself be seemingly affect gut bacterial composition too [3]. As they stand however, the current results aren't yet a glowing endorsement of B-GOS® intervention 'for' autism, mindful of the small participant group eventually included for study and the relatively short-term nature of the Grimaldi trial. We'll see where this goes... although next time, I'd also like to see a more prominent statement about any harms or side-effects encountered or not during the study period. I assume 'not' in the current study but...

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[1] Grimaldi R. et al. A prebiotic intervention study in children with autism spectrum disorders (ASDs). Microbiome. 2018 Aug 2;6(1):133.

[2] Zingone F. & Ciacci C. The value and significance of 25(OH) and 1,25(OH) vitamin D serum levels in adult coeliac patients: A review of the literature. Dig Liver Dis. 2018 Aug;50(8):757-760.

[3] Berding K. & Donovan SM. Diet Can Impact Microbiota Composition in Children With Autism Spectrum Disorder. Front Neurosci. 2018 Jul 31;12:515.

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'Comprehensive metabolomics' and ME/CFS: lipid and energy production turn up again

The findings reported by Dorottya Nagy-Szakal and colleagues [1] describing the results of "biomarker discovery and topological analysis of plasma metabolomic, fecal bacterial metagenomic, and clinical data from 50 ME/CFS [myalgic encephalomyelitis/chronic fatigue syndrome] patients and 50 healthy controls" provide the rather long blogging fodder today.

Just in case that opening quote sounds like gibberish, this was a study that in effect examined two quite prominent biological 'systems' alongside looking at symptom profiles of participants diagnosed with ME/CFS compared with controls. Metabolomics is a discipline that is no stranger to this blog, and is focused on the analysis of small molecule metabolities in a range of biological fluids (see here). The interface between the technology used to separate out and analyse said metabolites and the statistical analysis of the huge amounts of data generated as a result, are what make metabolomics the science that it is. 'Fecal bacterial metagenomics' also known as microbiomics (see here) refers to the science of cataloguing what bacterial species are present in poo(p) samples. Yes, bacteria have their own genomes too, and stool samples can therefore be a rather informative medium.

It's important to realise that this isn't the first time that metabolomics has been spoken in the same breath as CFS/ME (see here and see here for examples); something alluded to in the Nagy-Szakal paper. Indeed, this most recent paper adds to the authors other work in this area [2] (see here for my take) where the focus was on immune-related parameters and their *association* with CFS/ME in the context of the gut and its bacterial inhabitants. And once again, there are some eminent research names included on the authorship list as last time...

So, fifty participants diagnosed with CFS/ME were compared with 50 asymptomatic (I hate the words 'healthy control') participants, and their blood (plasma) and stool were analysed. Mass spectrometry played an important role in their metabolomic work, as over 550 compounds were initially separated out from the samples provided and identified.

Results: "Among the top plasma biomarkers differentiating ME/CFS patients from controls were decreased levels of betaine, complex lipids (lysophosphatidylcholine [LPC], phosphatidylcholine [PC]) and sphingomyelin (SM), and increased levels of triglycerides (TG), α-N-phenylacetyl-glutamine, ε-caprolactam and urobilin." I'm not going to go through all of those compounds individually as to their possible relevance but there are some important classes of compound being mentioned (i.e. lipids and triglycerides).

Authors also mention another group of compounds as also potentially being important: ceramides. You may have heard the word 'ceramide' before if you are/were a user of certain brands of shampoo in recent times (see here). Outside of any hair care role, ceramide "is a waxy lipid implicated in suppression of electron transport, insulin and leptin resistance and apoptosis." Among the many roles they play 'in' the body, there is some research literature to suggest that ceramides "may play a role in gut barrier dysfunction and increased gut permeability." Interesting (see here). And going back to the Nagy-Szakal results we are told that "patients with ME/CFS and IBS [irritable bowel syndrome] have increased plasma levels of ceramide." Even more interesting.

Having mentioned the gut and gut issues in the form of IBS, it's also important to note that the authors made allowances for the presence of such gut dysfunction in their participant groups. And yes, one needs to remember that it was "based on self-reported diagnosis of IBS on the medical history form". As probably expected, the introduction of IBS (self-reported) did seem to affect the metagenomic (microbiomic) data obtained (something authors talked about in their last paper). More than that: "Chemical enrichment analysis of plasma metabolites revealed that metabolomic profiles of ME/CFS patients with IBS were distinguished from controls by levels of TG, ceramides, phosphatidylethanolmines (PE) and metabolites in the carnitine-choline pathway." Indeed also, take away the IBS bit from the ME/CFS findings and: "ME/CFS patients without IBS co-morbidity showed disturbances in PCs and carnitine-choline pathways, similar to the disturbances found in the overall ME/CFS cohort." Again, interesting.

Authors conclude that their results draw attention to a few areas already pertinent to CFS/ME, in particular, "lipid and energy metabolism." The word 'mitochondria' figures a few times in their results write-up and specifically how: "compounds in the choline-carnitine pathway were decreased in ME/CFS patients regardless of their IBS status." I've written about quite a bit of research on mitochondria and CFS/ME (see here and see here for examples) and how even if there aren't genetic reasons for mitochondrial issues (see here), this does not mean that there may not be more other issues with this system (see here).

We really need much more research in the area of metabolomics and ME/CFS. And patients really need it now, not some time later in the future...

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[1] Nagy-Szakal D. et al. Insights into myalgic encephalomyelitis/chronic fatigue syndrome phenotypes through comprehensive metabolomics. Sci Rep. 2018 Jul 3;8(1):10056.

[2] Nagy-Szakal D. et al. Fecal metagenomic profiles in subgroups of patients with myalgic encephalomyelitis/chronic fatigue syndrome. Microbiome. 2017; 5: 44.

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Omega-3 fatty acids, 'antibiotic exposure-induced gut microbiota dysbiosis' and obesity

The findings reported by Kaliannan and colleagues [1] provide food for thought today and the suggestion that in mice at least: "elevated tissue levels of omega-3 fatty acids significantly reduce body weight gain and the severity of insulin resistance, fatty liver, and dyslipidemia resulting from early-life exposure to azithromycin (AZT)."

Azithromycin is an antibiotic quite commonly used for treating a number of bacterial infections. The idea that antibiotic use, and specifically the effects on the trillions of wee beasties that call our gut home (the gut microbiome), might impact on weight is not a new one. The precise mechanism(s) are still a point of some speculation. Evidence from various sources however (see here for example) seems to indicate that there may be something about the gut microbiome that seems to have an important bearing on weight gain and obesity.

Kaliannan et al started with the premise that "increased tissue levels of omega-3 fatty acids may prevent antibiotic-induced alteration of gut microbiota and obesity later in life." I know some people might be a little puzzled as to why supplementing a fat might actually stop someone (or at least something) from 'becoming fat', but these are times where the sweeping notion that 'fat makes you fat' is being re-analysed (see here). Using a specific type of mouse - "the fat-1 transgenic mouse model" [2] - that is "capable of producing n-3 fatty acids from the n-6 type, leading to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in their organs and tissues, without the need of a dietary n-3 supply", researchers exposed mice to AZT during their earliest days. They found that this particular type of mouse that is able to synthesize its own omega-3 fatty acids, potentially showed some important effects as a result of its naturally high levels of omega-3 fatty acids in tissues. Further: "These effects were associated with a reversal of antibiotic-induced dysbiosis of gut microbiota in fat-1 mice."

Reiterating that this was a mouse study focused on a very specific type of mouse, these are interesting findings. It's not necessarily new news that "dietary lipids affect specific populations of gut microbes and their metabolic end products" [3] or that specific types of bacteria might also affect fat composition of host tissues [4] but more scientific flesh needs to be put on the bones of the relationship. Insofar as how these results might transfer outside of the fat-1 transgenic mouse, well, the authors speculate about "the potential utility of omega-3 supplementation as a safe and effective means for the prevention of obesity in children who are exposed to antibiotics." I would however be minded to suggest that quite a bit more investigation is needed before fish oils are routinely prescribed alongside/following antibiotics in early childhood to 'prevent' microbiota-associated obesity. That also agricultural methods used might be a relevant factor is food for thought [5] as is the focus on fish oils and Akkermansia muciniphila (see here) among other things.

But speaking of our microbial masters, I might also draw your attention to the paper by Laura Blanton and colleagues [6] talking about the other extreme of weight and gut bacteria. Feed the world eh?

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[1] Kaliannan K. et al. Omega-3 fatty acids prevent early-life antibiotic exposure-induced gut microbiota dysbiosis and later-life obesity. Int J Obes (Lond). 2016 Feb 15.

[2] Kang JX. Fat-1 transgenic mice: a new model for omega-3 research. Prostaglandins Leukot Essent Fatty Acids. 2007 Nov-Dec;77(5-6):263-7.

[3] Shen W. et al. Influence of dietary fat on intestinal microbes, inflammation, barrier function and metabolic outcomes. J Nutr Biochem. 2014 Mar;25(3):270-80.

[4] Wall R. et al. Metabolic activity of the enteric microbiota influences the fatty acid composition of murine and porcine liver and adipose tissues. Am J Clin Nutr. 2009 May;89(5):1393-401.

[5] Średnicka-Tober D. et al. Composition differences between organic and conventional meat: a systematic literature review and meta-analysis. Br J Nutr. 2016 Feb 16:1-18.

[6] Blanton LV. et al. Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children. Science. 2016. Feb 19.

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Kaliannan K, Wang B, Li XY, Bhan AK, & Kang JX (2016). Omega-3 fatty acids prevent early-life antibiotic exposure-induced gut microbiota dysbiosis and later-life obesity. International journal of obesity (2005) PMID: 26876435

What exercise might do to the gut microbiome and gut permeability in CFS/ME

"These findings suggest a role for an altered gut microbiome and increased bacterial translocation following exercise in ME/CFS [myalgic encephalomyelitis/chronic fatigue syndrome] patients that may account for the profound post-exertional malaise experienced by ME/CFS patients."

That was the conclusion reached by Sanjay Shukla and colleagues [1] (open-access available here) who analysed the "microbiomes of blood and stool samples" for a small group of "clinically characterized ME/CFS patients" (n=10) and ten matched asymptomatic controls undertaking an exercise test ("a maximal exercise test on an electronically braked cycle ergometer"). Clinically characterised by the way, refers to a diagnosis of CFS using the 1994 CDC case definition.

Blood and stool samples were provided by participants at various time points including baseline, 15 minutes and some days following the exercise test. Indeed we are told that: "Stool samples were collected at baseline (pre-exercise) and 48 and 72 hours post-exercise" which is certainly quite a feat getting 20 people to er, poo(p) on demand on such a timescale.

Analyses of samples and importantly, the state of participants following the exercise test, yielded some potentially interesting results. So: "Following maximal exercise challenge, there was an increase in relative abundance of 6 of the 9 major bacterial phyla/genera in ME/CFS patients from baseline to 72 hours post-exercise compared to only 2 of the 9 phyla/genera in controls." The authors interpret this as meaning that bacterial load in those with ME/CFS may be "preferentially enhanced during post-exertional malaise." This was to some degree complemented by blood results "with high levels of bacterial sequences maintained at 72 hours post-exercise in ME/CFS patients versus clearance in the controls." Again the authors tentatively interpret this as meaning that "bacteria may have translocated into the blood stream from the gut after the maximal exercise challenge" and hence there may be a preferential role for intestinal dysbiosis and altered intestinal permeability in relation to at least some cases of CFS/ME.

These are interesting if preliminary findings. I've previously touched upon the idea that the gut microbiome might be a research target for CFS/ME before on this blog (see here). Allied to the idea that altered intestinal permeability (a.k.a the leaky gut - yes it is a real thing) might also be a feature of 'some' CFS/ME (see here) and one has some intriguing potential investigations to be done alongside the notion that new therapeutic targets could be on the horizon. I say this acknowledging again the small participant group included for study in the Shukla study.

I must also pay further attention to the exercise element of the Shukla paper. Exercise and post-extertional fatigue/malaise is a core feature of CFS/ME (even SEID) and has been the topic of quite a lot of discussion and even arguments when it comes to certain 'therapeutic' options put forward in recent years. Other studies looking more generally at the effects of vigorous exercise on measures of intestinal permeability have suggested that there may be some important physiological changes associated with undertaking a strenuous workout [2]. A specific measure of intestinal permeability is not reported by Shukla and colleagues on this occasion but one wonders whether there may be some important further research to do in this area particularly when one notices the lack of statistical difference in various physiological measures between the ME/CFS participants and controls on this [preliminary] research occasion (see here)?

To close, remembering Alan Rickman...

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[1] Shukla SK. et al. Changes in Gut and Plasma Microbiome following Exercise Challenge in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). PLoS One. 2015 Dec 18;10(12):e0145453.

[2] Pals KL. et al. Effect of running intensity on intestinal permeability. J Appl Physiol (1985). 1997 Feb;82(2):571-6.

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Shukla SK, Cook D, Meyer J, Vernon SD, Le T, Clevidence D, Robertson CE, Schrodi SJ, Yale S, & Frank DN (2015). Changes in Gut and Plasma Microbiome following Exercise Challenge in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). PloS one, 10 (12) PMID: 26683192

Recurrent antibiotic exposure and risk of depression and/or anxiety?

Accepting the notions that (i) correlation is not necessarily the same as causation and that (ii) case-control observational studies in particular are not the best way to ascertain whether A causes B, I was rather interested in the findings reported by Ido Lurie and colleagues [1] and that: "Recurrent antibiotic exposure is associated with increased risk for depression and anxiety but not for psychosis."

Starting from the idea that: "Changes in the microbiota (dysbiosis) were suggested to increase the risk of several psychiatric conditions through neurologic, metabolic, and immunologic pathways", y'know, the old microbiota-gut-brain axis that everyone seems to be talking about these days (see here), researchers set about looking at how one particular set of agents - antibiotics - known to affect the balance of the trillions of wee beasties that call out gut home, might play a role in said relationship. Analysing "3 nested case-control studies during the years 1995–2013 using a large population-based medical record database from the United Kingdom" Lurie et al pulled in several thousand participants ("202,974 patients with depression, 14,570 with anxiety, and 2,690 with psychosis and 803,961, 57,862, and 10,644 matched controls"). Receipt of antibiotic therapy - 1 of 7 antibiotic classes - more than 1 year before an index date was the "primary exposure of interest" taking into account various other potential confounding variables.

The results: "Treatment with a single antibiotic course was associated with higher risk for depression with all antibiotic groups." The risk, or rather odds ratios, wasn't massively increased but given the participant numbers was deemed significant enough to report. Further, the risk stats increased when multiple antibiotic exposures figured, potentially indicating something of a dose-dependent relationship. Authors also noted that further attention to the use of anti-fungal medicines might also figure in any further research in this area.

Going back and reiterating the caveats with which I started this post, these are interesting results. The idea that the various bacteria, fungi and viruses that inhabit our gut might be doing much more than we ever truly realised is gaining some significant research traction these days (see here for example). Yes, we have to beware of the hype and inevitable sweeping generalisations that accompany the whole 'gut bugs doing more than digesting food' mantra but data are data and this recent data strengthens the calls for more research inspection in this area including on the longer-term effects of 'swallowing a grenade'. I might add that facets of the gut bacteria and depression have been talked about before in the research literature (see here).

Although perhaps not looking at precisely the same aspect of the gut bacteria - behaviour link, it is timely that the Lurie paper coincides at the time of writing with some new reports coming out of this years Society for Neuroscience (SfN) conference. The headline ran with: "Probiotic bacteria may aid against anxiety and memory problems" highlighting some interesting results on the use of a daily capsule containing Bifidobacterium longum 1714 on aspects of mild anxiety and other parameters. What this and other research suggests is that when it comes to mental health and wellbeing, it may be advisable to pay due consideration to the idea of a 'gut feeling' alongside the idea that supplementing with probiotics or even eating certain foods (see here) might actually be heading to a psychiatrist near you...

Music: Saint Etienne - Nothing Can Stop Us.

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[1] Lurie I. et al. Antibiotic Exposure and the Risk for Depression, Anxiety, or Psychosis: A Nested Case-Control Study. J Clin Psychiatry. 2015. October 15.

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Lurie, I., Yang, Y., Haynes, K., Mamtani, R., & Boursi, B. (2015). Antibiotic Exposure and the Risk for Depression, Anxiety, or Psychosis The Journal of Clinical Psychiatry DOI: 10.4088/JCP.15m09961

Microbes passing traits to host babies?

The paper by Clara Moon and colleagues [1] has garnered a lot of headlines with their findings suggesting that [mouse] mothers pass on bacteria to their [mouse] offspring. Their results reported in Nature, focused on a bacterium not unfamiliar to this blog, Sutterella (see here) and how, through the transmission of Sutterella via their poo(p), a specific trait might also be passed on. In this case, low levels of immunoglobulin A (IgA) (something else that has cropped up on this blog) were noted in offspring. The idea being that Sutterella might have an effect on IgA production and low IgA is a factor in quite a few conditions not least the inflammatory bowel diseases [2] and other autoimmune related conditions.

I have to say that I did a bit of double-take when reading this study and some of the accompanying media given the potential implications. "When we study mice, we have to account for the possibility that inherited bacteria and their genes could be influencing the trait we're trying to learn about," is a quote from one of the researchers involved in this work. Certainly this is the most pressing issue to come from the Moon study but the results are potentially much more profound than just where you house mice in a research setting. First though, I'd like to see some independent confirmation of these findings please.

Although there is quite a bit of hype around the microbiome these days, and particularly those trillions of beasties which reside in our gastrointestinal (GI) tract, there is something of a greater appreciation that their role(s) appears to extend well beyond just helping us digest food and producing the odd nutrient or two. My coverage of the 'poo transplant [potentially] made me obese' paper (see here) kinda hints at one possible alternative action for our gut bacteria or some other, related component of the gut. I'd also draw your attention back to the paper by Desbonnet and colleagues [3] talked about in this post, which brought the concept of 'psychobacteriomics' (my definition!) to the world and how gut bacteria might also impact on [mouse] social behaviour too.

Going back to the the Moon paper, I'm also drawn to the possible implications for a wide range of issues and diagnoses based on their findings. Genetics, more traditional structural genetics, based on investigations of how the structural differences of the genome might confer elevated/reduced risk of good or adverse health issues, is undergoing a bit of a revolution at the moment. Not only are we talking more about the epigenome (see the Nature special on this and a very informative video here) but as per some other comments from the paper authors, science is potentially thinking of an: "expanded model of heredity to produce a more complicated but also much more insightful picture of how human, bacterial and viral genes influence human health." This is all the more important when you consider how many bacterial genes there are vs. 'human' genes alongside the idea that just as viruses have a history of passing genes to humans (think HERVs) so bacteria may also be able to 'transfer' genes to us too. I wonder if the Moon findings might also account for those peculiar 'elevated levels of autoimmune conditions in the spouses of those with coeliac disease' findings [4] too?

Given that Sutterella is the starting point for this possible association, and with something of a potential role for this bacteria in some cases of autism [5] replicated even [6], one might entertain the idea that autism should perhaps figure in follow-up work in this area. I'm not suggesting that autism as a label (or labels) is somehow the product of Sutterella but as per the discussions linking Sutterella to potential bowel pathology in some cases of autism, whether the mechanism talked about by Moon and colleagues might also apply to humans too.

Then there is the tantalising idea of 'altering' the microbiome to alter specific traits...

So then, it missed out on an Oscar but the song is still awesome...

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[1] Moon C. et al. Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation. Nature. 2015 Feb 16.

[2] Ludvigsson JF. et al. Association between IgA deficiency & other autoimmune conditions: a population-based matched cohort study. J Clin Immunol. 2014 May;34(4):444-51.

[3] Desbonnet L. et al. Microbiota is essential for social development in the mouse. Mol Psychiatry. 2014 Feb;19(2):146-8.

[4] Emilsson L. et al. Autoimmune Disease in First-degree Relatives and Spouses of Individuals with Celiac Disease. Clin Gastroenterol Hepatol. 2015 Jan 30. pii: S1542-3565(15)00112-3.

[5] Williams BL. et al. Application of novel PCR-based methods for detection, quantitation, and phylogenetic characterization of Sutterella species in intestinal biopsy samples from children with autism and gastrointestinal disturbances. MBio. 2012 Jan 10;3(1).

[6] Wang L. et al. Increased abundance of Sutterella spp. and Ruminococcus torques in feces of children with autism spectrum disorder. Mol Autism. 2013 Nov 4;4(1):42.

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Moon C, Baldridge MT, Wallace MA, Burnham CA, Virgin HW, & Stappenbeck TS (2015). Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation. Nature PMID: 25686606

Metabolomics and autism: the continuing search for biomarkers

I'm always a happy bunny when some of my own research findings receive something like independent replication. So it was when I read the monster paper from Paul West and colleagues [1] (open-access) reporting results based on not one, not two, not three, not even four, but five mass spectrometric methods looking for potential biomarkers for autism. Metabolomics in action (see here for an introduction to this topic).

Rosina @ Wikipedia 

The particular reason for my excitement was the quote: "Creatinine was decreased in children with ASD [autism spectrum disorder] and is consistent with the findings of Whitely et al, who observed similar changes in urinary creatinine in children diagnosed with PDD [pervasive developmental disorder]" based on the results of our paper a few years back [2] (see here for further details of how I spend my spare time talking about urine). The caveat being that we (the Royal We) looked in urine and West et al looked at blood (plasma). I can also forgive the authors for spelling my name wrong too... WHITELEY.

The West paper is open-access but I'm gonna give you a few pointers nonetheless. Stick with me on this one because although quite a long post, this is important work...

• So: "The aim of the study was to perform a broad evaluation of small molecules in blood plasma to discover metabolites that may lead to biomarkers associated with ASD." 
• The value-added bit was that this was study from the MIND Institute which meant that participant groups were very well-defined in terms of diagnosis and presenting symptoms. Indeed, as per other studies of biomarkers (see here), the talk was all about study groups (ASD vs. asymptomatic controls denoted as 'typically developing' TD) and also the use of training and test sets, where: " 82 patient samples (52 ASD and 30 TD samples) were split into two sets, (1) a training set of 61 samples (39 ASD and 22 TD) for identification of statistically significant features and classification modeling and (2) a 21-sample independent validation set (13 ASD and 8 TD) used to evaluate performance of the classification models."
• So, then to the interesting bit... the mass spec methods used and data handling. A combination of liquid chromatography-high resolution mass spectrometry (LC-HRMS) and gas chromatography-mass spectrometry (GC-MS) were used. Actually the LC-HRMS was based on separation using C8 and HILIC column chromatography (the LC part) on both occasions coupled to "electrospray ionization" (the MS part) in positive and negative ion mode so giving "4 separate data acquisitions per sample." That and the GC-MS data makes 5 methods. 
• Various methods/software were used to identify potential metabolites of interest including a couple of programs we use in our lab such as "Agilent Technologies MassHunter Qualitative Analysis software" and the METLIN database.
• Results: as one might imagine, quite a few compounds/metabolites/signals were picked up across the 5 methods used. Table 2 of the paper gives you some idea of the sorts of numbers talked about. That being said, assigning a molecular formula to all those metabolites is rather another matter as per the authors note: "... 179 features comprised 3% of the LC-HRMS and 8% of the GC-MS preprocessed set of features". 'Features' by the way referred to "a moiety detected by the mass spectrometer that is defined by 2 properties 1) the detected mass-to-charge ratio (m/z) and 2) the chromatographic retention time".
• Those 179 'features' formed the basis of the statistical analyses used to try and differentiate autism from control samples. These were subsequently whittled down to: "an 80 feature set [that] exhibited the best combined classification performance metrics... with an average accuracy of 90%, an average sensitivity of 92%, an average specificity of 87%, and an average AUC [area under the curve] of 0.95."
• When moving from training to validation sets, the previous 80 feature model did not work as well. Indeed, some further statistical modelling was used and: "The results suggest that at least 40 features are needed to reach an accuracy of 70% and that a range of 80 to 160 features had the best performance with this independent validation sample set as well as the training set of samples."
• To get to the juicy details of which compounds might be the ones to watch with autism biomarkers in mind, well: "a variety of molecular classes including amino acids, organic acids, sterols, and fatty acids" came up. I've already mentioned creatinine but other prominent mentions were given to "aspartate, glutamate, DHEAS, citric acid, succinic acid, methylhexa-, tetra- and hepta-decanoic acids, isoleucine, glutaric acid, 3-aminoisobutyric acid" and homocitrulline. The authors provide a handy overview of where their results might fit with other autism research areas (e.g. mitochondrial dysfunction, the gut microbiome) which I would encourage interested readers to further peruse. I'm gonna highlight isoleucine as one example where a form of autism has already been talked about with the words 'branched chain amino acids' in mind (see here).
• The authors conclude with a need for quite a bit more study in this area: "This initial study provides proof of concept to further pursue development of metabolic biomarkers of ASD." Personally, I'd like to think that proof-of-concept is perhaps too preliminary a way of introducing metabolomics to autism research given previous research forays (see here and see here and see here) and their potentially important findings. Certainly, things need to be scaled up in terms of participant numbers [3] and also delving into those all-important subgroups of 'the autisms'. Challenges however do remain in assigning molecular formulae to all those compounds detected.

What's more to say? Well, as has been mentioned in a previous post (see here) one always needs to be a little careful when talking about biomarkers for autism as if we're talking about a homogeneous diagnosis and the search for compound X supposedly covering all that heterogeneity (and comorbidity). If we've learned anything from the genetic research on autism for example, it is that simple, universal objective markers are probably not going to be present. Given that the metabolome is to quite a large extent determined by the proteome potentially also intersecting with the microbiome, complexity is probably going to be the keyword.

That being said, I do see merit in the continued use of metabolomics as part of all that system biology kerfuffle (see here) when applied to autism research. I'd personally suggest a few tweaks to how this kind of research is carried out on the basis for example, of not necessarily using the diagnostic label of 'autism' or 'autism spectrum disorder' as a primary starting point. I've talked before on this blog about the notion of best responders and non-responders to intervention for example (see here) and how if one chose to use this as an important variable differentiating those on the spectrum, one might just see a few differences across groups. Such research might also help further inform researchers / clinicians / parents / people on the spectrum who might be best suited for certain types of intervention. Interestingly with dietary intervention in mind, the authors reported that: "Ten of the 52 ASD subjects were on a gluten and/or casein-free (GFCF) diet". Mmm...

Then to some music... Go Your Own Way by Fleetwood Mac. Or if you prefer, the Seaweed version...

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[1] West PR. et al. Metabolomics as a Tool for Discovery of Biomarkers of Autism Spectrum Disorder in the Blood Plasma of Children. PLoS One. 2014 Nov 7;9(11):e112445.

[2] Whiteley P. et al. Spot urinary creatinine excretion in pervasive developmental disorders. Pediatr Int. 2006 Jun;48(3):292-7.

[3] Roessner V. Large sample size in child and adolescent psychiatric research: the way of salvation? European Child & Adolescent Psychiatry. 2014. November 6.

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West, P., Amaral, D., Bais, P., Smith, A., Egnash, L., Ross, M., Palmer, J., Fontaine, B., Conard, K., Corbett, B., Cezar, G., Donley, E., & Burrier, R. (2014). Metabolomics as a Tool for Discovery of Biomarkers of Autism Spectrum Disorder in the Blood Plasma of Children PLoS ONE, 9 (11) DOI: 10.1371/journal.pone.0112445

The gut microbiome and autism... so far

A micropost if you will, to provide readers with a link to the paper by Xinyi Cao and colleagues* (open-access) reviewing where autism research is up to when it comes to those trillions of beasties - the various gut bacteria - which call our deepest, darkest recesses home.

Regular readers probably already know about my borderline obsession with the gastrointestinal tract (gut) and its inner workings when it comes to at least some cases of the autisms and how the gut microbiome represents a potentially valuable new research frontier for lots of different states and conditions.

The paper by Cao et al lists just about everything, research-wise, related to the analysis of gut bacteria pertinent to autism up to October 2013, with all the big players so far included - from Williams (see here) to Wang (see here) and lots in-between. The final conclusions are a familiar one: more research needed and a requirement to ensure that "confounding variables" are controlled for as best they can be. Of course, as is common in research, one might argue that the Cao paper is already out of date given for example, the replication of that Sutterella finding by Wang and colleagues (see here). And slightly outside of studies of looking at gut bacteria in real people, there is the emerging evidence from mouse models too (see here) which sparked talk about probiotics and the feasibility of trying to alter gut bacterial arrangements**. I'm not yet entirely convinced about this probiotic method in the longer-term but alongside other suggestions (see here) will be keeping my research eyes open for any trial results.

A final quote to leave you with: "There does, however, appear to be a ‘signal’ suggesting significant differences in the GI microbiome between ASD [autism spectrum disorder] children and children without ASD, so there would be value in continuing this line of research". I can't argue with that.

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* Cao X. et al. Characteristics of the gastrointestinal microbiome in children with autism spectrum disorder: a systematic review. Shanghai Archives of Psychiatry. 2013; 25: 342-353.

** Critchfield JW. et al. The potential role of probiotics in the management of childhood autism spectrum disorders. Gastroenterol Res Pract. 2011;2011:161358.

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Xinyi CAO, Ping LIN, Ping JIANG, & Chunbo LI (2013). Characteristics of the gastrointestinal microbiome in children with autism spectrum disorder: a systematic review Shanghai Archives of Psychiatry Other: http://www.saponline.org/upload/2013/1231/342.pdf

Gut bacterial diversity and autism

Let's face it, those who regularly read this blog probably knew that I was always going to be interested in the paper published by Dae-Wook Kang and colleagues* (open-access) discussing the gut microbiome and autism.

Holding it in @ Wikipedia  

For quite a while now I've been going on (and on and on) about how those trillions of wee beasties which call us home might be doing so much more than just helping to digest our food and producing the odd vitamin or two.

Indeed it's come to the point that questions have started to be asked about whether gut bacteria might not just shape or influence behaviour (see here) but indeed whether our very psychological development might be linked to what goes on in the deepest, darkest recesses of our bowels: psychobacteriomics anyone?

Appreciating that such work still needs to go some before we proclaim that social development for example, is linked to gut bacteria (it was a study of mice** after all) and put quite a few psychologist-theorists out of business, there is nevertheless a growing tide of research highlighting how important the symbiotic relationship between bacteria and human might be. Just call me Dax.

The Kang paper has, as one might expect, already received some media attention (see here). Indeed, the authorship list also includes a favourite autism researcher of mine - Prof. Jim Adams - who aside from publishing that pretty good gold-standard RCT of vitamin supplementation for autism a while back (see here), has himself already dabbled in the science of gut bacteria and autism as per other articles*** (open-access).

• In the latest study, the name of the game was stool analysis; said stools provided by 20 children diagnosed with an autism spectrum disorder (ASD) and 20 age- and sex-matched aysmptomatic control kids. DNA was extracted from the stools (what a lovely job that must have been!) and analysed to discern what bacteria and families of bacteria were present across the two groups. If you really want more information about 16S rDNA sequencing method used in the Kang study, I've got another post scheduled soon on more gut microbiomics in chronic fatigue syndrome (CFS) which gives a little more information on the science.
• The results: well, after some fancy analysis based on the various groupings of bacteria and the "microbial richness and diversity" present between the groups, one of the main conclusions was that the control asymptomatic group "harbored more diverse gut microbiota than the autistic group did". 
• When they looked at the presence of gastrointestinal (GI) issues related to cases of autism there was some hint of an effect too on gut bacterial diversity but it appeared that the severity of autism was a more important factor to potentially account for the microbial differences detected.
• The authors note findings of: "significantly lower abundances of the genera Prevotella, Coprococcus, and unclassified Veillonellaceae in autistic samples". Not being an expert on the various types of bacteria which colonise our gut, I can't necessarily suggest anything more than what the authors noted about the link between some of these bacterial families and things like the digestion of carbohydrate-rich foods. Interesting though that the name Brent Williams appears in the paper text and his 'carbs and dysbiosis' work in autism (see here). 
• The authors conclude that the reduced microbial diversity and specific differences across the groups should be further investigated taking into account issues like dietary effects (see here) and the 'cross-talk' between bacteria and other biological functions.

OK you can perhaps appreciate that this was a relatively small study based on the participant numbers included. That and the fact that unlike the hunt for autism-related genes and genetic mutations (yep, lots of them potentially) when we talk about the gut microbiome, we are 'generally' (see here) talking about a dynamic system influenced by all manner of environmental effects not just the food we eat (see here). Kinda more like the methylome me thinks. That gut bacteria might also only be one part of the 'triad' of issues (pathogenic gut bacteria, gut hyperpermeability / leaky gut, immune response) which seem to be discussed with autism and the gut in mind is also worthwhile remembering.

But on the positive side of things, the Kang paper represents a lot of hard work and is yet another brick in the autism research wall suggesting that we should be looking more 'whole-body' when it comes to cases and phenotypes. Of particular note is the suggestion that behavioural symptom severity might trump GI symptom presentation as being 'correlated' to gut microbiome diversity. The authors do qualify this assertion by suggesting that "autism-related GI disorders may be linked to a unique shift in microbial balance", part of which they detected in their study. Whether or not this statement ties into those Stephen Walker 'distinctive features' bowel findings reported recently (see here) is perhaps further source for speculation and investigation.

To close Franz Ferdinand and Take Me Out.

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* Kang D-W. et al. Reduced Incidence of Prevotella and Other Fermenters in Intestinal Microflora of Autistic Children. PLoS ONE 8(7): 2013; e68322. doi:10.1371/journal.pone.0068322

** Desbonnet L. et al. Microbiota is essential for social development in the mouse. Molecular Psychiatry. 2013. doi: 10.1038/mp.2013.65

*** Adams JB. et al. Gastrointestinal flora and gastrointestinal status in children with autism -- comparisons to typical children and correlation with autism severity. BMC Gastroenterology 2011, 11:22 doi:10.1186/1471-230X-11-22

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Kang D-W (2013). Reduced Incidence of Prevotella and Other Fermenters in Intestinal Microflora of Autistic Children PLoS ONE DOI: 10.1371/journal.pone.0068322