Showing posts with label metagenomics. Show all posts
Showing posts with label metagenomics. Show all posts

Wednesday, July 1, 2015

This Month in Blastocystis Research (JUN 2015)

I started developing this blog more than three years ago. After a bit more than a year, I collected a bunch of the posts, edited them and published them as a book on Amazon. Recently, I logged into my Amazon profile to see how the book was doing, and I was very pleased to notice that there were no less than four reviews of the book, and very positive ones too! Thank you to everyone who read/browsed it.

Blastocystis research is currently a quickly moving field, and I'm please to be able to inform you that one of the most interesting contributions to Blastocystis research coming out from our intstitute has just been published in Fems Microbiology Ecology. The article appearing in this journal was first-authored by PhD student Lee O'Brien Andersen (Statens Serum Institut) and post doc Ida Bonde (Danish Technical University) and describes how Lee and Ida took a retrospective approach to analysing metagenomics data originally generated by the MetaHIT Consortium and published in the often cited paper by Arumugam et al. (2012).

The abstract reads as follows:
Blastocystis is a common single-celled intestinal parasitic genus, comprising several subtypes. Here, we screened data obtained by metagenomic analysis of faecal DNA for Blastocystis by searching for subtype-specific genes in co-abundance gene groups, which are groups of genes that co-vary across a selection of 316 human faecal samples, hence representing genes originating from a single subtype. The 316 faecal samples were from 236 healthy individuals, 13 patients with Crohn's disease (CD), and 67 patients with ulcerative colitis (UC). The prevalence of Blastocystis was 20.3% in the healthy individuals and 14.9% in patients with UC. Meanwhile, Blastocystis was absent in patients with CD. Individuals with intestinal microbiota dominated by Bacteroides were much less prone to having Blastocystis-positive stool (Matthew's correlation coefficient = -0.25, P < 0.0001) than individuals with Ruminococcus- and Prevotella-driven enterotypes. This is the first study to investigate the relationship between Blastocystis and communities of gut bacteria using a metagenomics approach. The study serves as an example of how it is possible to retrospectively investigate microbial eukaryotic communities in the gut using metagenomic datasets targeting the bacterial component of the intestinal microbiome and the interplay between these microbial communities.

As far as we know this is the first study to sift out data on Blastocystis from data originally intended for analysis of bacterial communities only, and in the paper we describe how this was done. We believe that this approach has imminent potential for quickly advancing our knowledge on Blastocystis in a gut ecology context, including knowledge on the role of Blastocystis in terms of impacting/manipulating one or more types of intestinal bacteria.

I have a feeling that this is the first study in a string of similar studies that will soon hit PubMed, and within a year or two, we should be able to with confidence to hypothesise on the relationship between the structure and function on of the gut microbiota and Blastocystis, and–hopefully–other intestinal micro-eukaryotes.

Lastly, it was very interesting to note the article by Paramsothy et al. on donor recruitment for faecal microbiota transplantation (FMT; never heard of this? Watch the video below to learn more), recently appearing in the journal Inflammatory Bowel Disease. The study is interesting because it shows that most FMT donors are seemingly ineligible due to a variety of reasons, including colonisation by intestinal parasites such as Blastocystis... Given emerging data suggesting that Blastocystis is more common in healthy invididuals than in patients with gastrointestinal disease, the question remains whether Blastocystis-positivity should be a limiting factor for stool donation?



References:

Andersen LO, Bonde I, Nielsen HB, Stensvold CR. A retrospective metagenomics approach to studying Blastocystis. Published online 30 June 2015. DOI: http://dx.doi.org/10.1093/femsec/fiv072

Paramsothy S, Borody TJ, Lin E, Finlayson S, Walsh AJ, Samuel D, van den Bogaerde J, Leong RW, Connor S, Ng W, Mitchell HM, Kaakoush N, & Kamm MA (2015). Donor Recruitment for Fecal Microbiota Transplantation. Inflammatory bowel diseases, 21 (7), 1600-6 PMID: 26070003

Wednesday, January 8, 2014

2014 Prospects

Happy New Year!

So, what's in store for us in 2014?

Difficult to say, but as least I can try and say a little about what is going on in our lab. Firstly, we are trying to publish what we are think are very interesting data on how gut bacteria may select for Blastocystis colonisation, a hypothesis we have developed based on studies of metagenomic data.

We are also working with the assembly and annotation of mitochondrial and nuclear genomes in collaboration with our international colleagues; something that will definitely take a while, since we have so few people in our lab to do it (literally one-two persons) but oceans of data (!!) - it's a pity that we cannot speed this up, since genomes are expected to hold keys to some of the great gates of Blastocystis enlightenment. Of course, a constant aim is to attract funding that can help us employ one or more PhD students/post docs interested in genomics and parasites. As always, I encourage my readers to come up with suggestions for funding.

Funding-wise we are also going to try and establish a Marie Curie ITN-network on the roles of intestinal microbial eukaryotes in health and disease and we are also awaiting decisions on other applications; hopefully, we will get some money for gut microbiome and immunological host profiling in experimental animals challenged with Blastocytis cysts. There may also be some work in our lab dealing with the impact of Blastocystis on bacterial communities in in-vitro studies.

Epidemiological data are produced as we speak; luckily, quite a few colleagues in different parts of the world are taking an interest in characterisation of Blastocystis in various cohorts so that we will know more about its epidemiology.

Those are the seminal things. Of course, there will be some exciting conferences, which I've mentioned before, and I'm also looking forward to putting together a Blastocystis review.

Sunday, September 8, 2013

Fellowships in Blastocystis Research

We are continually looking into the opportunity for funding for research in Blastocystis and we are on the lookout for young researchers with a MSc or PhD degree who want to spend at least a couple of years in Blastocystis research. Right now, taking an omics approach to studying the clinical significance of Blastocystis is extremely relevant of course, given the amount of genetic diversity of the parasite, its apparent association to groups of bacteria/bacterial richness, its varied distribution across different cohorts, and the general availability of ngs technology and data pipelines.

I'm going to focus my next funding application on the integration of metagenomics, metabolomics, comparative genomics, and transcriptomics, and I'm hoping to find one or two persons with track records documenting extensive experience with one or more of these disciplines and who take an interest in parasites/parasitic protists in general and/or in Blastocystis in particular.

Please note that this is NOT a job offer, but merely an invitation to get into some sort of a dialogue. What we can offer is access to samples, strains, technology, and a Blastocystis-centred network.

Please do not answer in the comments section, but contact me directly (mail/phone) for further info + expression of interest. You'll find a link to my contact details in the previous blog post. Thanks.

Wednesday, May 15, 2013

Wrap-Up of Cell Symposium on Microbiome and Host Health

For a parasitologist with a major interest in novel technology like me the Cell Symposium on Microbiome and Host Health (#CMHH) was a challenging, yet stimulating tour de force in bacteriology and immunology, and I realise that gut fungi and protists still fly below the radar of intestinal microbiome research.

The announced line-up of speakers was impressive, and although we missed e.g. Drs Peter Turnbaugh and Fergus Shanahan, we were still spoiled with brilliant talks.

Most of the projects and results presented on the meeting were based on studies on bacterial diversity and structure by either targeted 16S 454 sequencing or metagenomics, while studies of gene function and the 'super-organism' that is the complete microbiome (including the  fungome and protistome I should say, since these genomes are much larger than bacterial ones) were still scarce if represented at all.

Since my focus is on intestinal parasites, my main interest in the vast universe of the human microbiome naturally orbits around the intestinal microbiome. Although there is still a long way to go - due to e.g. significant differences in methodologies and lack of consensus on the analytical basis for 'enterotypes'  - we are slowly but steadily building up a picture of the effect that the human microbiome has on health and disease. Hundreds of species live and have important functions in our gut, to cite Dr Peer Bork, but these species have also been associated with more than 30 human diseases, even neurological ones. Shifts in the composition of the microbiome are associated with an expanding list of chronic diseases that includes obesity, inflammatory bowel disease, and diabetes (Dr Ruth Ley).

Many things may influence our susceptibility to intestinal pathogens, including competition between species (colonisation resistance), the ability of some bacteria to synthesise antimicrobial compounds or stimulate innate immune defenses. Differences in susceptibility to infection may boil down to differences in antimicrobial compounds secreted by our individual microbiota (Dr Michael Fischbach). Bacteroides fragilis is a commensal immunoregulatory microbe mediating major effects through a single molecule, polysaccharide A (Dr Dennis Kasper); polysaccharide A mediates immunoregulation via innate and cognate immune system collaboration.

The list of buzz words was endless, and patterns of cause and effect in this fascinating hubbub of cutting edge science difficult to keep apart - but then again, - many pathways and interactions leading to alterations in gut flora and thereby alteration in host clinical phenotype may result from the complex interplay of any type of intervention (diet, antibiotics, surgery (gastric bypass), microbe exposure, etc.) and host genetics. Dr Wendy Garrett used some of her time to address the fact that antibiotic treatment may lead to more significant perturbation of the intestinal microbiota than e.g. diets and immunoregulation, and she also encouraged thoughts on how to approach causality in studies of microbial communities.

Other things that are interesting include how bacteria "talk" together by quorum sensing to control gene expression and crosstalk between beneficial bacteria (e.g. probiotics) and the intestinal ecosystem, and how these systems can be influenced altogether.

Computer technology - the Creed of today: The Barcelona Supercomputing Centre (with 'Mare Nostrum') located in a former chapel. Source.


So, while focus is still on the trillions of bacteria we have in our gut, we hope that it won't be long before common eukaryotic components of the intestinal microbiome will be studied and analysed alongside with bacterial communities. It says on Wikipedia that targeted studies of eukaryotic and viral communities are limited and subject to the challenge of excluding host DNA from amplification and the reduced eukaryotic and viral biomass in the human microbiome. Excluding host DNA is challenging, but not impossible, and who has actually documented that eukaryotic biomass in the human microbiome is 'reduced'?

The meeting was very well organised and took place at the Sheraton Hotel in Lisbon. I've storified a list of the #CMHH tweets here in case you are interested in more 'headlines'. I apologise for any misquotes.

Further reading:

Koren O, Knights D, Gonzalez A, Waldron L, Segata N, Knight R, Huttenhower C, & Ley RE (2013). A guide to enterotypes across the human body: meta-analysis of microbial community structures in human microbiome datasets. PLoS Computational Biology, 9 (1) PMID: 23326225

Andersen LO, Vedel Nielsen H, & Stensvold CR (2013). Waiting for the human intestinal Eukaryotome. The ISME Journal PMID: 23407309

Ivanov II, & Honda K (2012). Intestinal commensal microbes as immune modulators. Cell Host & Microbe, 12 (4), 496-508 PMID: 23084918

Brown J, de Vos WM, Distefano PS, Doré J, Huttenhower C, Knight R, Lawley TD, Raes J, & Turnbaugh P (2013). Translating the human microbiome. Nature Biotechnology, 31 (4), 304-8 PMID: 23563424

Blaser M, Bork P, Fraser C, Knight R, & Wang J (2013). The microbiome explored: recent insights and future challenges. Nature Reviews. Microbiology, 11 (3), 213-7 PMID: 23377500

Saturday, February 16, 2013

Waiting For The Human Intestinal Eukaryotome

We were lucky enough to have a paper accepted for publication in the ISME Journal (Nature Publishing Group) in which we call for data on the "human intestinal eukaryotome".

In the paper, we start out:

"Recent developments in Next Generation Sequencing (NGS) technologies have allowed culture-independent and deep molecular analysis of the microbial diversity in faecal samples, and have provided new insights into the bacterial composition of the distal gut microbiota. Studies of the microbiome in different patient groups using metagenomics or 16S rRNA gene sequencing are increasing our knowledge of how the microbiota influences health and disease. The majority of recent advances in our understanding of human microbiota structure and dynamic changes in disease were made through phylogenetic interrogation of small subunit (SSU) rRNA (Paliy and Agans 2012). However, until recently such studies have generally failed to include data on common eukaryotic, endobiotic organisms such as single-celled parasites and yeasts ('micro-eukaryotes'). This deficiency may strongly bias the interpretation of results and ignoring an entire kingdom of organisms is a major limitation of human microbiome studies."

Sunday, September 2, 2012

Bugs Galore!

After spending more than 8 years in clinical microbiology with special reference to parasitology, I’ve come to realise that it truly is a bug’s life! Use of nucleic acid-based methods such as PCR in routine clinical microbiology diagnostic labs have revealed that single-celled parasites are colonising the intestine of up to 50% of the Danish population! And so what? Well, this finding has several implications.

A couple of months ago I revisited Why it is a bugs life by Jörg Blech (The Guardian (2002)). Speaking of numbers, - I wonder which one is the most successful eukaryote in terms of numbers? Blastocystis? Dientamoeba? Or any other “Parasite sp.”? After realising that microscopy methods allow us to see only the very tip of the iceberg and after adding PCR to our routine diagnostics, we have found a few examples of “novel” parasitic species and many more may be in store for us. Morphologically identical organisms, such as those belonging to Iodamoeba bütschlii, may be found in both human and non-human hosts and may differ genetically across the nucelar small subunit rRNA gene by up to more than 30%! This is quite astonishing given the fact that the difference between human and murine small subunit rDNA is about 1%! Since these data have been established only recently, obviously no one knows the respective clinical significance of these morphologically similar but genetically very different lineages, and further studies may reveal differences in pathogenicity as seen in other amoebic genera. Blastocystis and Entamoeba coli are somewhat similar examples.

Our results reveal that faecal-oral transmission is much more common in Denmark - a highly industrialised country where drinking water comes from waterworks (i.e. no surface water supplies), where outbreaks even due to bacteria are scarce, and where authorities spend 1.2 billion DKK on food safety and control. Today, 90% of dwellings in Denmark (5.6m citizens) are connected to efficient sewage systems, and Denmark has more than 1,400 treatment plants to purify wastewater from households, businesses and institutions. But somewhere the chain pops off… Even in Denmark it is “bugs galore”, which means that faecal exposure is much more common that we would probably like to think. Intestinal protists (primarily Blastocystis and Dientamoeba) are telltales of exposure to faecal contamination and faecal-oral transmission.

In Denmark, 90% of dwellings are connected to efficient sewage systems, and the country has more than 1,400 treatment plants.

However, we might also learn to see these parasites as other types of indicators. In our experience Danish patients with inflammatory bowel disease (IBD) represent a cohort of people whose gut flora is remarkably different from that of other cohorts (patients with irritable bowel syndrome (IBS) and patients with non-IBD/non-IBS diarrhoea): Apparently IBD patients don’t harbour parasites. This can in part be explained by the fact that some IBD patients have had bowel resection, but even IBD patients with in intact bowel system are generally negative for parasites.

We know that in highly developed countries the prevalence of helminth infections has gone down over the past few decades due to improved hygiene measures, but maybe also due to other reasons, which have not been clarified, but as we have seen, many of us are still positive for one or more intestinal parasites. However, most IBD patients do not have any parasites at all. This correlates well with the hygiene hypothesis, and it may be so that not only helminths, but also amoebae, which are able to colonise our guts for months and even years, may be co-responsible for 1) preventing us from developing inflammatory bowel disease and other autoimmune diseases by immunomodulatory mechanisms, and 2) maintaining a sound intestinal flora and ecology. Or is it so that these protists are dependent on a certain gut ecology or gut flora in order to colonise our intestines for a longer period, and in this way, they can be seen as indicators of a certain gut microbiota? Do they have any modulatory functions or do they happen to "lead their own life"?

As a parasitologist and worshipper of most things eukaryotic, I was both pleased and disconcerted after leaving the MetaHIT conference in Paris in March. Pleased, since the stratification of people into enterotypes and correlation of enterotypes to disease phenotypes suited my naïve, B/W perception of the world, but disconcerted since all presentations and posters addressed only bacteria (and virus to a minor extent, - maybe one on archaea even?). But, how about intestinal yeasts and parasites? Where in the gene catalogues and pools of metagenomic data could I find information on eukaryotes? Nowhere. Which hopefully boils down to methodological limitations rather than absence of interest.

The concept of paving an avenue of new knowledge with metagenomics data is holistic in its approach, but it currently fails to encompass a common part of the intestinal microbiota, possibly due to methodological limitations. However, we are probably facing the imminent inclusion of eukaryotic data in metagenomic studies, and this will enable us to investigate the potential role of intestinal protists and maybe yeasts as biomarkers of certain enterotypes and maybe even disease or health phenotypes.

Further reading:

Stensvold CR, Lebbad M, & Clark CG (2012). Last of the human protists: the phylogeny and genetic diversity of Iodamoeba. Molecular biology and evolution, 29 (1), 39-42 PMID: 21940643

Stensvold CR (2012). Thinking Blastocystis out of the box. Trends in parasitology, 28 (8) PMID: 22704911

Sunday, August 19, 2012

The Potential Role of Our Microbiome Ecosystems

For those who like these pop-sci articles on the still somewhat conjecture-like but very inspiring theories about the role of our intestinal microbiome in health and disease, here's a link to an article from The Economist (18 AUG 2012):

The Human Microbiome: Me, myself, us

And let me reiterate: We still don't know much about mikro-eukaryotes in all this... do they play a role as well? And how do they cope with different types of microbiomes?

Anyways, enjoy!

Friday, June 22, 2012

More Bits And Pieces On The Microbiome... Or Maybe Mycobiome...

I promised to include some more stuff from some of the many recent publications in Science and Science Translational Medicine on the intestinal microbiome and its potential role in health and disease, and I've chosen two papers that could have broad public interest; for those who need an introduction to the microbiome, please go here (Wikipedia entry).

Because the microbiome has been more or less exclusively synonymous with the "bacteriome" it's very refreshing to discover a paper on fungal diversity in the gut. Like Blastocystis, and other single-celled parasites, intestinal fungi are also micro-eukaryotes, and we are continuously searching for the role of micro-eukaryotes in health and disease.

In general, very little is known about fungi in the intestine, and most clinicians, even mycologists, hardly bother about the fungi that may be present in our intestine, - I think I can say that without offending anyone! Maybe one of the most interesting things in a clinical respect is the fact that antibodies against the yeast Saccharomyces cerevisiae (see below) is a common finding in patients with Crohn's Disease, but relatively uncommon in patients with ulcerative colitis and healthy individuals.

Now, Iliev et al. (2012) start out by confirming the fact that fungi are indeed common commensals and thus a part of our normal intestinal flora. They then showed that colitis chemically induced in mice led to circulating antibodies against S. cerevisiae, which suggested that fungal antigens commonly found in the gut might be responsible for the induction of these antifungal antibodies during colitis.
The innate immune receptor Dectin-1 appears to have a key role in fungal recognition and combating. Therefore the authors wanted to further explore the role of this receptor by studying mice with and without Dectin-1. They found that Dectin-1 deficiency led to increased susceptibility to chemically induced colitis, including weight loss, tissue destruction and cell infiltration by inflammatory cells, etc. Moreoever, evidence was found of fungal invasion of inflamed tissue in the Dectin-1 knockout mice and taken together, their data suggest that Dectin-1 deficiency leads to altered immunity to commensal gut fungi.
To cut a long story short, results from these experiments in mice led the investigators to search for mutations in CLEC1A (the human Dectin-1 gene) in patients with ulcerative colitis, and they found that mutations were significantly more common in patients with severe ulcerative colitis (patients requiring colectomy) than in those with a less aggressive disease progression. This suggests that not only bacteria but also intestinal fungi interact with the intestinal immune system and may thereby influence health and disease. If this can be confirmed by others, this is an example of how biomarkers can predict the disposition towards/progression of disease and the results may have profound consequences for diagnostic strategies (e.g. screening for mutations in the Dectin-1 gene) and therapeutic management of patients with severe ulcerative colitis. Maybe it would have been interesting to know about such mutations in patients with Crohn's Disease as well...

Next, the investigators took to identifying what types of fungi were actually present in the colon of these mice. What may be a little bit controversial is the fact that the authors - by amplification and deep sequencing of  ITS1-2 (genetic marker commonly used to identify and taxonomically group fungi) - appear to have found not only species representing a staggering 50 well-annotated fungal genera in the mouse microbiome, but an additional 100 "novel and/or un-annotated fungi" as well - this does sound like a lot, but somehow the reader is calmed down a bit, when the authors later tell us that 97.3% of all fungi detected in the mouse faeces belonged to only 10 species, with 65.2% of the fungal sequences belonging to Candida tropicalis. So, whether the 100 novel fungi are indeed fungi colonising the intestinal tract is unknown, but they may very well represent fungi "on transit", so to say, acquired from food, drink or environment maybe... we know that fungi are ubiquitous - we inhale fungi every day for instance, and when deep sequencing is applied, it may be possible to trace even fungi only present in very small quantities; also ITS-2 analysis does not tell us whether the sequences are from "intact/live" (i.e. colonising) fungi or from degraded fungi (i.e. ingested); a classic example is Saccharomyces cerevisiae (Brewer's or Baker's yeast), which we may often acquire from food and drink, but which may also colonise (settle and proliferate) our intestines. Contamination of the faecal samples from fungi present in the environment and during processing is also a possibility (one of the reasons why PCR-based diagnostics for fungal infections is a tricky task...). Well so, all of these new species/genera may not necessarily represent the "mouse mycobiome". However, the authors found only few of the fungi in the food that was fed to the mice, so this still may remain a bit of a mystery... it would have been interesting to know whether the fungi detected were yeasts or molds, for instance, and very little information can be extracted from the supplementary material (phylogenetic analysis) accompanying the paper. Anyway, it's all very stimulating and further studies will assist in exploring fungal diversity and, hopefully, the diversity of micro-eukaryotes in general.

Saccharomyces cerevisiae is used in food and drink, but may also colonise our guts.

The next paper is one of many recent papers heralding the implementation of microbiome-based therapies in future personalised and precision medicine, possibly relevant to diseases such as inflammatory bowel disease, obesity and diabetes. Microbiome manipulation, so to say, is key to this concept and includes controlled diet, pre- and probiotic interventions, bariatric surgery (e.g. gastric bypass), faecal transplants (see my recent blog post on feacal bacteriotherapy), helminth therapy (yes!) or ecological engineering. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, and these may be known to many as lactobacilli or bifidobacteria (or simply "yoghurt"!) that protect us against harmful bacteria by inhibiting their growth and by helping reduce cholesterol levels, synthesise vitamins and sustain immune responses. Prebiotics are non-digestible dietary sugar molecules (oligosaccharides) that can enhance the activity of for instance lactobacilli and bifidobacteria. While the potential benefits of pre- and probitics have been known for many years, it is only with current available technology that we are starting to get a mechanistic understanding of their impact on our bodies.

The article picks up on host-gut microbiota metabolic interactions and the so-called "host-microbe metabolic axes", which include pathways and interactions responsible for gut permeability, formation of blood vessels (angiogenesis) in the gut mucosa, ion transports, sulfation ability of xenobiotics, and many other things; sulfation ability is a key component in metabolising of drugs, for instance. Differences in our individual abilities to sulfate certain compounds give us at least one explanation as to why different people may respond differently two drugs treatment (see previous posts), and our ability to metabolise a common drug such as acetaminophen (paracetamol) can apparently be predicted form our preinterventional excretion of the microbial co-metabolite 4-cresyl sulfate; other gut microbial contributions that can alter the absorption, metabolism, and safety of drugs have been demonstrated recently.

Gastric bypass (Roux-en-Y) is a surgical procedure carried out to delay and reduce the absorption of calories and includes bypassing a large part of the stomach and a part of the small intestine by a procedure known as "stapling". Roux-en-Y appears to be associated with major and stable changes in the microbiota and in many microbially generated compounds, all of which are key components in the host-microbe metabolic axes. "This suggests that the microbiota is an essential part of the "gearbox" that connects the physical effects of bariatric surgery to the resulting beneficial effects."

Gut ecology changes with age, and current investigations seek to define the rationale of and potential for manipulating the microbiome of older people, for instance with pre- and probiotics, to secure higher microbiome diversity (high microbiome diversity appears to be beneficial) and resilience to antibiotics-induced changes in gut flora.

For those of you who nearly choked on "helminth therapy" - I may put up a post in the future on how helminths (and maybe other intestinal eukaryotes such as amoebae?) apparently play a role in the presentation and regulation of diseases such as asthma and inflammatory bowel diseases...

The cells of our intestinal microbiome outnumber our own body cells by 10 to 1. Within the next decade or so we will be able to extract a lot of information about how the bacteria and other "bugs" in our guts influence and contribute to health and disease. Importantly, we may have to realise now more than ever that "germs and bugs" and their actions and interactions can hold the key to a healthy life in ways that we wouldn't think were possible only a few years ago. This means that we should acknowledge that some bacteria and parasites may be a sign of a healthy intestinal environment / a healthy gut function, and that consumption of drugs such as antibiotics may produce shifts in our microbiota that may not necessarily be beneficial.

References:

Iliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, Brown J, Becker CA, Fleshner PR, Dubinsky M, Rotter JI, Wang HL, McGovern DP, Brown GD, & Underhill DM (2012). Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science (New York, N.Y.), 336 (6086), 1314-7 PMID: 22674328
 
Holmes E, Kinross J, Gibson GR, Burcelin R, Jia W, Pettersson S, & Nicholson JK (2012). Therapeutic modulation of microbiota-host metabolic interactions. Science translational medicine, 4 (137) PMID: 22674556

Saturday, June 9, 2012

On Faecal Bacteriotherapy

For those of you who read my most recent blog post and who went on to read Carl Zimmer's article in The New York Times about gut flora transplantation on a woman suffering from chronic Clostridium difficile diarrhoea: The concept of faecal bacteriotherapy is maybe not that new. Allegedly, it dates back to Pliny the Elder and others, who prescribed orally ingested faeces to cure maladies! Stools were, however, incinerated first, and only the ashes ingested.

Pliny the Elder and others with him allegedly recommended  ingesting the ashes of faeces to cure disease.

In less ancient times - in 1989 to be more precise - Tvede and Rask-Madsen from Copenhagen, Denmark (Statens Serum Institut and The Danish State Hospital) reported on bacteriotherapy for chronic relapsing C. difficile diarrhoea in six patients. They hypothesised that absence of Bacteroides results in chronic relapsing C. difficile diarrhoea, and that its presence may prevent colonisation by C. difficile. In the current issue of Microbe Magazine, Young and Aronoff describe some of the mechanisms that may be involved in our indigenous gut flora's ability to prevent the colonisation of potentially pathogenic bacteria such as C. difficile. These include: (1) occupying space (physically preventing contact by newly arrived microbes with the host), (2) directly impairing the growth or germination of C. diffıcile, (3) withholding nutrients or germinants from C. diffıcile, and (4) shaping the host adaptive and innate immune responses.

Hence, the concept of dysbiosis and the ideas of manipulating the gut flora in order to "restore order" have been going on for a long time. Metagenomics, however, will assist us in exploring exactly what is happening in much more detail and in a much broader and standardised context than previously possible. We will be able to predict shifts in the structure, function and interaction of microbial communities - hopefully including micro-eukaryotes such as fungi (the "mycobiome") and common protists such as Blastocystis and Dientamoeba (maybe we can call it the "protistome"?), - and any influence of diet, pro- and antibiotics.

And fortunately, the focus on metagenomics continues: While CMI just launched a themed issue on metagenomics advances (see previous blog post), even Science and Science Translational Medicine now dedicated an entire joint issue to "The Gut Microbiota", and I hope to be able to address one or two of these papers soon. Until then, here's a bit of suggested reading:

'Bugs as Drugs'

Tvede, M., & Rask-Madsen, J. (1989). Bacteriotherapy for chronic relapsing Clostridium difficile diarrhoea in six patients The Lancet, 333 (8648), 1156-1160 DOI: 10.1016/S0140-6736(89)92749-9

Young and Aronoff (2012). Clostridium difficile linked to disrupted gut microbiota. Microbe Magazine (ASM): http://goo.gl/FIZmC 

Mueller, K., Ash, C., Pennisi, E., & Smith, O. (2012). The Gut Microbiota Science, 336 (6086), 1245-1245 DOI: 10.1126/science.336.6086.1245

Saturday, June 2, 2012

Blastocystis and Microbiomology

Speaking of metagenomics: The July 2012 issue of one of the most prestigious journals in the field of clinical microbiology, Clinical Microbiology and Infection (CMI – published by European Society of Clinical Microbiology and Infectious Diseases), focuses entirely on recent advances in metagenomics, including its implications on clinical microbiology. Several of the keynote speakers from the MetaHIT conference in Paris (March, 2012) have contributed with papers, including Rob Knight, Willem M. de Vos and Paul W. O’Toole. In his editorial, Didier Raoult, puts emphasis on mainly two things: 1) that we need to be patient with data obtained from studies using metagenomics, since currently some conclusions are pointing in different directions and data are still scarce, and 2) that metagenomic studies should be independent of financial support from commercial sources, such as the industry of antibiotics and probiotics.

Although it may be too early to make b/w inferences from data already published, I think that the pioneers in metagenomics teach us to re-think or at least modify several hypotheses about the role of intestinal microbes in gastrointestinal health and disease and pursue new and exciting trajectories. In this blog post I would like to highlight a few things that may be interesting to people who are not familiar with metagenomics, but who are interested in our gut flora and how it may impact our lives.

So, what is metagenomics? Well, only a few years ago, microbiologists were used to looking at one single organism at a time, when exploring the potential role of an organism in health and disease. They were dependent on isolating the organism, for instance by culture, in order to have sufficient material for molecular studies, and in order to avoid mix-up of data from contaminating organisms. However, the human intestinal microbiome (gut flora) is made up by a plethora of organisms, mainly prokaryotes (bacteria), but also to some extent eukaryotes (parasites and fungi), archaea and viruses. Metagenomics, facilitated by massive high-throughput parallel sequencing of nucleic acids extracted from human faecal samples, allows us to get a holistic picture of the entire gut flora of a person. I.e.: We move from examining one single species or organism at a time, to be analysing entire eco-systems. We get to know not only the composition of microbic species inhabiting our gut, but also how they impact our body physiology: Interestingly, Gosalbes et al. (2012) describe how the composition of the intestinal flora may differ significantly from person to person, but later shows that the active intestinal flora is fairly similar among healthy individuals. So, what’s the active flora? Briefly: while metagenomics analyses the DNA (16s rDNA) from the microbiome and hence provides us with data on the mere composition of microbes, including a quantification, metatranscriptomics looks at RNA communities by looking at 16S rRNA and mRNA transcripts. In this way, we get to know the function of the intestinal microbiota and can temporarily ignore the part of the microbial community that is in “stand-by” mode only. The collective genome of the intestinal microbes vastly surpasses the coding capacity of the human genome with more than 3 million genes - in comparison the human genome comprises 20,000-25,000 protein-coding genes.

So far, metagenomic studies have focused mainly on bacteria, and hence we know very little about how intestinal parasites directly or indirectly impact the remaining gut flora and the host, and, importantly, how the bacterial flora influences the presence and activity of parasites. This is due in part to methodological limitations, but mainly to the fact that the bacterial microbiome can be viewed as an organ of the human body (Baquero et al., 2012) taking care of vital and irreplaceable functions that the host is not otherwise capable of, ranging from energy and vitamin metabolism to epithelial barrier integrity and immune modulation (Salonen et al., 2012). Like any other organ, the microbiome has physiology and pathology, and the individual (and collective?) health might be damaged when its collective population structure is altered (Baquero et al, 2012). This is one of the reasons why studies of host-gut flora interactions have focused on bacteria.

One of the striking findings in metagenomic studies is that humans can be more or less successfully stratified into three enterotypes based on their intestinal flora (Arumugam et al., 2011):


We see that the three enterotypes are dominated by mainly three different types of bacteria (Bacteroides, Prevotelia and Ruminocoocus, respectively). However, as mentioned earlier, functional analysis (and probably a lot more sampling) is required to understand microbial communities. One of the interesting topics in this respect is how enterotypes correlate to different health/disease phenotypes; i.e. whether people with a certain gut flora are more prone to (a) certain type(s) of disease(s).There is preliminary evidence that variations in the microbiota are linked to diseases including bowel dysfunction and obesity.

In terms of parasites, I believe that in the near future we will see data revealing to which extent - if any - common intestinal micro-eukaryotes such as Blastocystis and Dientamoeba correlate with these enterotypes or other subsets of bacteria which will enable us to generate hypotheses on the interaction of micro-eukaryotes and the bacterial flora, which in turn may impact host physiology. I will expand a little more on this in an upcoming letter in Trends in Parasitology (article in press).

Interested in more: Why not have a look at Carl Zimmer's article in The New York Times about gut flora transplantation, or read about modulating the intestinal microbiota of older people to promote enhanced nutrition utilisation and to improve general health (O'Toole et al., 2012)... Also, have a look at my most recent blog post.

Literature:

O’Toole, P. (2012). Changes in the intestinal microbiota from adulthood through to old age Clinical Microbiology and Infection, 18, 44-46 DOI: 10.1111/j.1469-0691.2012.03867.x  

Gosalbes, M., Abellan, J., Durbán, A., Pérez-Cobas, A., Latorre, A., & Moya, A. (2012). Metagenomics of human microbiome: beyond 16s rDNA Clinical Microbiology and Infection, 18, 47-49 DOI: 10.1111/j.1469-0691.2012.03865.x  

Baquero, F., & Nombela, C. (2012). The microbiome as a human organ Clinical Microbiology and Infection, 18, 2-4 DOI: 10.1111/j.1469-0691.2012.03916.x  

Salonen, A., Salojärvi, J., Lahti, L., & de Vos, W. (2012). The adult intestinal core microbiota is determined by analysis depth and health status Clinical Microbiology and Infection, 18, 16-20 DOI: 10.1111/j.1469-0691.2012.03855.x

Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D., Yamada, T., Mende, D., Fernandes, G., Tap, J., Bruls, T., Batto, J., Bertalan, M., Borruel, N., Casellas, F., Fernandez, L., Gautier, L., Hansen, T., Hattori, M., Hayashi, T., Kleerebezem, M., Kurokawa, K., Leclerc, M., Levenez, F., Manichanh, C., Nielsen, H., Nielsen, T., Pons, N., Poulain, J., Qin, J., Sicheritz-Ponten, T., Tims, S., Torrents, D., Ugarte, E., Zoetendal, E., JunWang, ., Guarner, F., Pedersen, O., de Vos, W., Brunak, S., Doré, J., Consortium, M., Weissenbach, J., Ehrlich, S., & Bork, P. (2011). Enterotypes of the human gut microbiome Nature, 474 (7353), 666-666 DOI: 10.1038/nature10187

Sunday, May 20, 2012

Brave New World

Using Blastocystis as an example, we have only recently realised the fact that conventional diagnostic methods in many cases fail to detect Blastocystis in faecal samples, which is why we have started using molecular diagnostics for Blastocystis. I was also surprised to realise that apparently no single drug can be used to treat Blastocystis, and that in fact we do not know which combo of drugs will actually consistently eradicate Blastocystis (Stensvold et al., 2010).

There will come a time - and it will be soon - where it will be common to use data from genome sequencing of pathogenic micro-organisms to identify unique signatures suitable for molecular diagnostic assays and to predict suitable targets (proteins) for chemotherapeutic intervention; in fact this is already happening (Hung et al., in press). However, despite already harvesting the fruits of recent technological advances, we will have to bear in mind that the genetic diversity seen within groups of micro-organisms infecting humans may be quite extensive. This of course will hugely impact our ablility to detect these organisms by nucleic acid-based techniques. For many of the micro-eukaryotic organisms which are common parasites of our guts, we still have only very little data available. For Blastocystis, data is building up in GenBank and at the Blastocystis Sequence Typing Databases, but for other parasites such as e.g. some Entamoeba species, Endolimax and Iodamoeba, we have very little data available. We only recently managed to sequence the small subunit ribosomal RNA gene of Iodamoeba, and we demonstrated tremendous genetic variation within the genus; it is now clear that Iodamoeba in humans comprises a species complex rather than "just" Iodamoeba bütschlii (Stensvold et al, 2012).

Cysts of Iodamoeba
Ribosomal RNA is present in all living cells and is the RNA component of the ribosome. We often use this gene for infering phylogenetic relationships, i.e. explaining how closely or distantly related one organism is to another. This again assists us in hypothesising on transmission patterns, pathogenicity, evolution, drug susceptibility and other things. Since ribosomal RNA gene data are available for most known parasites, we often base our molecular diagnostics on such data. However, the specificity and sensitivity of our molecular diagnostic assays such as real-time PCRs are of course always limited by the data available at a given point in time (Stensvold et al., 2011). Therefore substantial sampling from many parts of the world is warranted in order to increase the amount of data available for analysis. In terms of intestinal micro-eukaryotes, we have only seen the beginning. It's great to know data are currently builiding up for Blastocystis from many parts of the world, - recently also from South America (Malheiros et al., 2012) - but the genetic diversity and host specificity of many micro-eukaryotes are still to be explored. It may be somewhat tricky to obtain information, since conventional PCR and sequencing offer significant challenges in terms of obtaining sequence data; such challenges can potentially be solved by metagnomic approaches - today's high throughput take on cloning; however, although the current next generation sequencing technology hype makes us feel that we are almost there, it seems we still have a long way to go - extensive sampling is key!

Cited literature:

Hung, G., Nagamine, K., Li, B., & Lo, S. (2012). Identification of DNA Signatures Suitable for Developing into Real-Time PCR assays by Whole Genome Sequence Approaches: Using Streptococcus pyogenes as a pilot study Journal of Clinical Microbiology DOI: 10.1128/JCM.01155-12

Malheiros AF, Stensvold CR, Clark CG, Braga GB, & Shaw JJ (2011). Short report: Molecular characterization of Blastocystis obtained from members of the indigenous Tapirapé ethnic group from the Brazilian Amazon region, Brazil. The American journal of tropical medicine and hygiene, 85 (6), 1050-3 PMID: 22144442

Stensvold, C., Lebbad, M., & Clark, C. (2011). Last of the Human Protists: The Phylogeny and Genetic Diversity of Iodamoeba Molecular Biology and Evolution, 29 (1), 39-42 DOI: 10.1093/molbev/msr238  

Stensvold, C., Lebbad, M., & Verweij, J. (2011). The impact of genetic diversity in protozoa on molecular diagnostics Trends in Parasitology, 27 (2), 53-58 DOI: 10.1016/j.pt.2010.11.005

Stensvold, C., Smith, H., Nagel, R., Olsen, K., & Traub, R. (2010). Eradication of Blastocystis Carriage With Antimicrobials: Reality or Delusion? Journal of Clinical Gastroenterology, 44 (2), 85-90 DOI: 10.1097/MCG.0b013e3181bb86ba