The Blastocystis PubMLST database now has a new look!

Thanks to Keith Jolley and colleauges, the PubMLST site now has a new look! Why not try and visit the Blastocystis section? It's available here. 

The Blastocystis database has mostly been used to identify whether subtypes that are shared between humans are animals reflect the same strains or not based on intra-subtype genetic variation (allele analysis), for which is works quite well. For this purpose, you should use DNA sequences reflecting the barcode region.

 You can query a single sequence or a whole batch of sequences. On the landing page, simply press 'Typing' and then opt for 'Single sequence' or 'Batch seqeunces', depending on what you want to analyse.

It's our ambition to expand the database with more subtype alleles, since this will allow all Blastocystis researchers interested in Blastocystis epidemiology to query their barcode sequences against a curated, standardised database. However, the genetic universe of Blastocystis is rapidly expanding, so it can be difficult to keep up! 

This site works best with Sanger sequences, or, possibly, consensus sequences generated based on next-generation sequencing of the 5'-end of the SSU rRNA gene of Blastocystis; we still haven't very much experience with the latter.

 

Pre-empting Pandora's Box - Update on Blastocystis Subtypes and Reference Data

here Back in 2006, when we came up with the subtype terminology for Blastocystis, the spectrum of and boundaries between Blastocystis subtypes were quite clear and distinct. Since then, the genetic make-up of Blastocystis has appeared to be an even bigger universe than we (or at least I) expected, and we may be far from having explored the entire 'galaxy' yet.

New technologies make it easier to sequence DNA, and sequences attributed to Blastocystis are accumulating in the publicly available databases with great speed. While this situation is one of the things that stimulate research (genetic diversity, co-evolution, host specificity, parasite-host-microbiome interaction, etc.), issues have emerged when it comes quality-controlling DNA sequences and putting taxonomic identifiers on these sequences.

For Blastocystis, the main taxonomic identifier is the 'subtype'. In 2013, 17 subtypes of Blastocystis had been acknowledged based on SSU rDNA analysis, and since then, quite a few more have been suggested by independent researchers all around the world. While it's great to see the field advance and more and more researchers 'checking in' on Blastocystis, care should be taken to ensure that Blastocystis terminology remains a useful one. And this... is not an easy task!

Some things are relatively straightforward though. For instance, sequence quality control. A simple BLAST query in GenBank (NCBI Database) should tell you whether your sequence is Blastocystis or something else. Like banana. Or asparagus. DNA sequence chimeras are sequences where one piece of DNA is combined with a piece of DNA from another strain/species/genus/etc., which can happen during PCR-based amplification of DNA. Suppose you have a sequence that is 75% Blastocystis and 25% banana. If you BLAST such a sequence, you might get Blastocystis as the top hit, but with a modest amount of sequence identity - maybe 85%. If you're not cautious, you might jump to the conclusion that this might be a new subtype, since 85% similarity is a lot less than the 95-97% similarity that is used pragmatically to delimit the boundary between subtypes. But if you look carefully at the alignment of the query sequence and the reference sequence, you'll probably note that a large part of the sequence aligns very well to the most similar reference sequence, while a minor part of it has great dissimilarity. This should be a warning sign, and you should try and BLAST only the bit of the sequence not aligning up well... and when you do this, you might end up with... banana! In which case you would have to discard this part of the sequence. Please also see one of my recent posts for more on this. If you do not check for chimeras, you might end up including chimeric DNA sequences in your phylogenetic analyses that will distort and confuse the interpretation and - in the worst case - lead to erroneous calling of new subtypes.

What is less easy is to set a 'one-fits-all' threshold for sequence similarity... how similar can Blastocystis DNA sequences be to be considered the same subtype? When do you have evidence of a 'new' subtype? It's difficult to know, as long as the data available in public databases is so limited as it is. Moreover, researchers do not always use the same genetic markers. It's still common practice to amplify and sequence only about 1/3 of the SSU rRNA gene and use that as a taxonomic identifier. But if it's not the same 1/3 then it gets tricky to compare data. Moreover, we actually need near-complete SSU rDNA sequences (at least 1600 bp or so) to be able to infer robust phylogenetic relationships between reference sequences and sequences potentially reflecting new subtypes. Obviously, this is because variation can exist across the entire SSU rRNA gene.

One subtype that has proven particularly challenging is ST14, a subtype which is common in larger herbivourous mammals, is very difficult to delimit. It may easily be confused with other subtypes, if sufficiently long sequences are not used for investigation. To this end, we try to keep a pragmatic approach to Blastocystis subtype terminology, and it may turn out that it would be more practical and relevant to refer to ST24 and ST25 as ST14 (see figure below). For now, we suggest keeping them as separate subtypes. Near-complete Blastocystis SSU rDNA sequences from a lot of larger herbivorous mammals will help us resolve the taxonomy in the top part of the tree shown in the figure above.

In terms of acquiring near-complete SSU rDNA sequences, I would personally recommend MinION sequencing of PCR products obtained by the universal eukaryotic primers RD5 + RD3. And if DNA from cultures isused (yes, it IS possible to culture Blastocystis not only from human hosts, but also from a variety of animals), then then MinION sequencing and analysis of the data output should be a straight-forward and relative cost-effective task.

Figure. As of January 2020, 'real' Blastocystis subtypes are most likely subtypes 1–17, 21, 23–26. This simplified phylogeny gives and indication of the relatedness of the subtypes and the relative host specificity. Humans can host subtypes 1–9 and also 12; when subtypes other than 1–4 are encountered in human samples, this may reflect cases of zoonotic transmission.

Graham Clark and I just published an article in Trends in Parasitology on this, and we concluded that some of the newly proposed subtypes are in fact invalid. Invalid subtypes (subtypes 18, 19, 20, 22) typically reflected DNA sequence chimeras.

In the figure above, you can see the subtypes identified to date that we consider valid.

We also provided updated guidelines on Blastocystis subtyping. One very important thing to include here is reference sequence data. It would be very useful if our wonderful Blasto colleagues could all try and use the same reference sequences when they develop multiple sequence alignments for phylogenetic analyses. We have already done all the work for you, so all there is to it, is to download the sequences from London School of Hygiene and Tropical Medicine's server available here and align them with your own DNA sequences. It would make life easier for all of us!

🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞🌞

Corrected proofs of the article can be downloaded here.

Thanks for reading!

Website for 2nd International Blastocystis Conference is now live!

I'm happy to be able to point your attention towards the website for the 2nd International Blastocystis Conference in Bogotá in October 2018. You can access the website here.

On this site, you'll find information about the venue, the preliminary programme, speakers, registration, and sponsors. You will also find some information about Bogotá.

Please visit the website on a regular basis for potential updates/changes.

See you there!

Trends in Blastocystis Research

I thought I'd post two current conference abstracts to exemplify some of the trends in  Blastocystis research.

The first is from Dr Pauline D Scanlan, who will be speaking at 15th International Congress of Protistology currently taking place in Prague. Go here for more info about the meeting.

In the symposium 'The eukaryome, bringing protists into the spotlight of microbiome research' taking place today, Pauline will be giving a talk with the title:

Inter-Kingdom Interactions in the human gut microbiome-the prevalence of the intestinal protist Blastocystis is linked to host age, antibiotic use and gut bacterial diversity and composition

and the abstract reads like this:

The human gut is host to a complex microbial ecosystem that plays a central role in host health. In addition to bacteria, viruses and archaea, the gut microbiota includes a diversity of fungal and protist species that are collectively referred to as the gut ‘eukaryome’. Although research into the gut eukaryome is in its infancy, emerging data indicates that the intestinal protist Blastocystis is perhaps the most common member of the human gut eukaryome worldwide. Despite its association with intestinal disease, asymptomatic carriage is common with Blastocystis frequently observed in surveys of the healthy adult gut microbiome. Furthermore, Blastocystis is less prevalent in chronic diseases such as Irritable Bowel Syndrome compared to healthy controls. Antibiotic administration significantly reduces Blastocystis prevalence rates between case and controls groups with the reduction in Blastocystis prevalence in the antibiotic treated group possibly due to direct effects on Blastocystis and/or secondary loss due to loss of bacteria that Blastocystis interacts with. In support of this latter hypothesis, data showing correlations between the presence of Blastocystis and specific features of the bacterial component of the gut microbiome (high diversity and a specific bacterial composition) are suggestive of inter-kingdom interactions between bacteria and Blastocystis in the gut microbiome. Blastocystis is less prevalent in infant populations relative to contemporaneous adult populations indicating that Blastocystis is not adapted to the infant gut. Given the difference in microbiome composition and diversity in infants compared to adults perhaps Blastocystis requires a more adult-like gut microbiome for successful colonisation. Collectively, emerging data suggests that successful colonisation of the gut by Blastocystis is linked to the composition and diversity of the bacterial fraction of human gut microbiome. Consequently, interactions between Blastocystis and bacteria in the gut microbiome may account for some of the variation in prevalence rates observed across age, health and geography.

 
Along similar lines, I will be giving a talk at the EMBO conference 'Anaerobic protists: Integrating Parasitology with mucosal microbiota and immunology' running from 31 AUG to 03 SEP in Newcastle, UK. You can read about the conference here.

The title and abstract of my talk are as follows:

The diversity of the most common intestinal protists, Blastocystis and Dientamoeba, and their interactions with the microbiota: what role in health and disease?

The integration of DNA-methods in Clinical Microbiology has enabled a more detailed and accurate snapshot of the protists colonising and infecting our guts. Parasites like Blastocystis and Dientamoeba are much more common than previously known, when detection relied mainly on microscopy of faecal concentrates and smears only.

While Dientamoeba isolated from humans exhibits very little genetic variation across the small subunit ribosomal gene, Blastocystis displays a perplexing amount of genetic heterogeneity, and nine subtypes, which are arguably separate species, have been found in humans. Subtypes 1 to 4 account for about 95% of human Blastocystis carriage.

It is expected that over 1 billion people are colonised by Blastocystis, and based on DNA-based detection, prevalence figures of up to 100% have been reported in developing countries. Conversely, the prevalence of Blastocystis appears relatively low in e.g. the US, and it has been suggested that the low prevalence is indicative of the defaunation of indviduals adapting a Westernized life style.

In a developed country like Denmark, the prevalence of Blastocystis and Dientamoeba is highest in individuals without gastrointestinal, while the prevalence of these parasites is lower in patients with functional and organic bowel disease, suggesting that these parasites are in fact markers of gastrointestinal health.

This is also in part exemplified by recent independent data linking high gut microbiota diversity to the presence of these parasites. Certain bacterial populations appear to be linked to parasite carriage, and studies are emerging that try to look into the association between these parasites and the remaining gut microbiota.

Moreover, higher age appears to be linked to Blastocystis colonisation. Blastocystis is more common in older children and adults, while in younger children, Dientamoeba is much more common.

Whether these parasites are able to modulate gut microbiota structure and function remains unknown, and it also remains to be demonstrated whether certain microbiota communities and/or metabolites are required for successful establishment of these parasites. More research data on these topics will inform future advances in probiotics in particular and gut microbiota manipulation in general.

 Thanks for your time.