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!

Is this a new Blastocystis subtype? Maybe not! Here's Why!

The genetic diversity of Blastocystis is becoming comparable to the universe! Seventeen subtypes (which are likely separate species or even genera) have been acknowledged so far, but quite a few more have been mentioned.

However, before assigning new Blastocystis subtype numbers to your SSU rDNA sequences, you'd need to do some QC work on your data. Sometimes we notice sequences deposited in the NCBI Database or included in articles that may look like new Blastocystis subtypes.... but they're most likely not!

I asked Prof Graham Clark from London School of Hygiene and Tropical Medicine, who has more than 20 years' experience in the Blasto business, to give a couple of examples, explaining where issues may arise. He says:

'One of the tasks I do when I have a few minutes to spare is to look at new Blastocystis sequences that have been deposited into GenBank. I am always hoping to stumble across some exciting new subtypes or new hosts that will expand our understanding of diversity in Blastocystis. Only rarely does this happen, however. I do, occasionally, come across sequences that are problematic and it is these that I want to focus on.

Chimaeras: This problem occurs during PCR amplification when one primer binds to a Blastocystis subtype DNA and the other primer binds to a different source of DNA. In the first case I came across the other source was a different Blastocystis subtype, meaning that the sequence at one end of the PCR product matched one subtype and the sequence at the other end matched a different subtype. This observation is mentioned in the paper describing barcoding of Blastocystis (Scicluna et al, 2006). Since then I have seen other chimaeric sequences: one recently was a mixture of Blastocystis plus a plant while another was Blastocystis plus a free-living protist.

Chimaeras are produced when there is incomplete replication of a DNA strand during a cycle. After denaturation in the next cycle, the single stranded partial product can bind to another single stranded product from a different source and synthesis results in a product combining sequences from two sources. The conservation of ribosomal RNA genes means there can be sufficient similarity to allow binding between sequences from distantly related organisms.

Chimaeras are generally only found when the sequences are from cloned ribosomal RNA gene sequences obtained by PCR, although they also occur in some forms of Next Generation  Sequencing. When mixed PCR products are sequenced directly the sequence obtained is the average of all the products in that reaction, and so chimaera sequences will usually be ‘diluted out’ by the major product of the reaction. Only when a single sequence from that mixture is isolated and studied will chimaeras be detected.

If the ‘alien’ region makes up a significant percentage of the sequence then the result of BLAST analysis will show a percentage divergence from known subtypes that indicates it may represent a new subtype. A quick way to evaluate this is to compare the BLAST results using the first and last thirds of the sequence. If it is a new subtype the results should be similar. In a recently detected chimaera, the first third was a 100% match to a known Blastocystis subtype while the last third was a 95% match to asparagus. This approach is an easy way to check whether there is something to get excited about.

A chimaera sequence can sometimes be detected because of its impact on phylogenetic trees. The sequence will be on its own branch, often at the base of a clade containing the subtype found at the Blastocystis-matching end.

Non-Blastocystis Blastocystis sequences: Like chimaeras these are often PCR artefacts, most commonly encountered when amplifying from stool DNA, especially if the stool is non-human. There is an expectation that Blastocystis-specific primers will only amplify Blastocystis DNA but, sadly, that is not always the case. I have personally seen this many times - if Blastocystis DNA is a minority of the eukaryotic DNA in the sample then the likelihood of artefacts increases greatly. These are generally identified easily if the sequence is compared using BLAST against the full nr/nt nucleotide collection in GenBank. However, there is a temptation to limit the search to the genus Blastocystis to speed up the identification process, because that is what you expect it to be. Again because of the conservation of ribosomal RNA genes, if ribosomal RNA genes are amplified there will be a match to Blastocystis, and the divergence will likely suggest, again, a new subtype.  Comparing against the full nucleotide collection will always show whether the sequence is of Blastocystis origin.

Both chimaeras and non-Blastocystis products are easily identified if the correct steps are taken. In conclusion, be suspicious of anything that is significantly divergent to known Blastocystis – it could be an indication of an artefact.'

Fig. 1. A 'Blastaragus' (a chimaera of a Blastocystis and an asparagus)

Fig. 2. An example of a chimaeric DNA sequence (the 'Blastaragus' from Fig. 1). Notice how the consensus sequence starts out as Blastocystis ST14, shifts to asparagus, and then shifts back again to Blastocystis ST14.

I thank Graham, and I really hope that this information will be picked up by many of our colleageus. And please share! Research into Blastocystis is rapdily expanding, and we should all take on the responsibility of QCing our data.

Thanks for listening!

By the way... if you're interested in tutorials on Blastocystis subtyping from our recent workshop in Colombia, please look up Workshop Session 4 in the manual available at this link. 

Hope to be back before Christmas!

2nd International Blastocystis Conference Wrap-Up - Part II

So, a lot of people would like to know about the take-home messages from the recent 2nd International Blastocystis Conference in Bogotá. There were many, and I might develop one more post to make room for more.

The first - and most important - thing I'd like to emphasise is that the community interested in Blastocystis is growing. And we're seeing a clearly multidisciplinary approach to studying the parasite. I think that this is what we need. The initial ideas about having Blastocystis-specific conference were developed by Funda Dogruman-Al and myself, and we both have a background in clinical microbiology. We have realised that in order to make sense of Blastocystis in a clinical microbiology (and infectious disease) context, we need research input from bordering fields, such as biology (genomics, cell biology, etc.), veterinary medicine (host specificity and impact of Blastocystis on animal health), gastroenterology (connection to microbiota and the extent of Blastocystis being involved in functional and inflammatory bowel diseases), bioinformatics (processing NGS data such as those pertaining to the profiling of gut microbiota communities), and ecology (people who are used to study interactions between organisms). At the conference, I believe that all (or at least most) of these fields were represented.

I was also thrilled to realise that many researchers have now adapted to the subtype terminology, - and even the allele terminology appears to be useful and pragmatic.

Status on the Blastocystis genome project. Slide by Andrew Roger.

Andrew Roger highlighted that the genomes of Blastocystis are more different than the genomes of human and mouse! Well-annotated genomes are available for ST1, ST4, and ST7, while draft genomes are available for subtypes 2, 3, 6, 8 and 9. 

 

What use are genomes? Summary provided by Andrew Roger.

Animal experimental modelling is possible. We know that rats can be colonised/infected by Blastocystis ST1 strain from a human and shed cysts in stool for more than one year.

Blastocystis is one of the few parasites that are really easy to culture and easy to get by. If we can learn to induce cysts in culture, these can be separated by sucrose gradient centrifugation or other methods and used for inoculation into volunteers, pigs, or rats, for instance. This can be used to study the impact of Blastocystis on the host, including immune system and gut microbiota. Baseline microbiota profiling is necessary prior to inoculation to know about the background variation in study individuals.

In terms of Blastocystis and gut microbiota: Since we published our conspicuous observations in 2015, many researchers have now corroborated our findings: Blastocystis is typically linked to increased microbiota richness and diversity; - something, which is generally considered a benefit and which is linked not only to gut health, but also to leanness. Especially the negative association between Blastocystis and Bacteroides has been highlighted by many now. It will be very interesting to learn why this is so. It also seems that Blastocystis are more common in individuals with a gut microbiota dominated by strictly anaerobes rather than facultative aerobes.

Faecal microbiota transplantaion (FMT): The recommendation of excluding FMT donors based on the finding of Blastocystis came up many times and was discussed in the context of the microbiota studies. It appear relevant to investigate further whether FMT donors should really be dismissed if they are Blastocystis-positive.

Some of the take home messages from Raul Tito Tadeo's talk.

In many animal groups, Blastocystis is a very common finding. These include mostly omnivores or herbivores. On the contrary, Blastocystis is very rare in strict carnivores, with no consistency in subtype distribution, indicating that these animals are not natural hosts of Blastocystis.The Blastocystis incidentially found in these hosts might stem from the prey that they have eaten.

Finally, I wish to highlight that there are excellent resources available from the pre-conference workshop, including an R script for microbiota analysis, and some tools for Blastocystis genome annotation. Please visit my previous blog post for links to these.

We cannot totally dismiss pathogenicity of Blastocystis; if existing, it may involve both strain- and host-specific factors.

And.... it's out: The time and venue for the 3rd International Blastocystis Conference will be Crete in 2021 (possibly June), with Eleni Gentekaki and Anastasios Tsaousis being involved in both the scientific and local organising committees... ! Please mark you calendars!

Andrew Roger, Raul Tito Tadeo, Kevin Tan and myself (taking the picture) enjoying some Club Colombia.

This Month in Blastocystis Research (NOV 2016)

Oftentimes, I receive emails from colleagues wanting to know how you subtype Blastocystis, how to grow them in culture, and how to freeze down cultures.

I'm very pleased to announce that Dr Graham Clark and I have developed protocols for exactly these activities and published them in Wiley's 'Current Protocols in Microbiology'; please go here for the subtyping protocol and here for the culture and cryopreservation protocols.
These should not only be seen as SOPs but also as a resource that enables standardization within the field.

Unfortunately, we have not yet come up with a protocol on how to axenise Blastocystis cultures, i.e., get rid of metabolically active organisms other than Blastocystis in cultures while keeping Blastocystis alive and multiplying.

We are well aware that many might not have access to these protocols because they haven't subscribed to Wiley Online Library; good news is that reprints will be available on request!


References:

Stensvold CR, & Clark CG (2016). Molecular Identification and Subtype Analysis of Blastocystis. Current Protocols in Microbiology, 43 PMID: 27858971  

Clark CG, & Stensvold CR (2016). Blastocystis: Isolation, Xenic Cultivation, and Cryopreservation. Current Protocols in Microbiology, 43 PMID: 27858970

This Month in Blatstocystis Research (OCT 2016)

A few things to highlight:

I'm very pleased to announce the Special Issue on Blastocystis recently appearing in Parasitology International - go here for the list of contents. The papers included in this issue represent the breadth of the contributions made to the 1st International Blastocystis Symposium, which took place last year in Ankara. A couple of review and opinion articles written by members of the Scientific Committee are accompanied by several articles outlining original research findings that were presented at the symposium. This special issue is particularly useful for younger researchers who wish to familiarise themselves with some of the methods that are currently in use in surveys of Blastocystis.
Readers should not expect to find articles on Blastocystis in a microbiota context; nor should they expect to see data from seminal studies that challenge the view that Blastocystis is a possible pathogen. Nevertheless, there is an interesting opinion paper with the title "Eradication of Blastocystis in humans--really necessary for all?"

Led by Dr Alison Jacob and Dr Graham Clark, London School of Hygiene and Tropical Medicine, our group just published an article on a comparative study of Blastocystis mitochondrial genomes. In general, mitochondrial genomes differ vastly in length, structure, and gene content across organisms, and by studying these genomes it has been possible to develop hypotheses on how these organisms have evolved including the adaptive/non-adaptive processes involved in shaping organismal and genomic complexity. Unlike most anaerobic eukaryotes, Blastocystis does not have true mitochondria but has mitochondrion-related organelles (MROs; also referred to as mitochondrion-like organelles [MLO]) that contain a genome. In the study in question, we sequenced and compared mitochondrial genomes from subtypes 1, 2, 3, 4, 6, 7, 8, and 9. All of them have the same genes in the same order, but two curiosities were noted. One gene, called orf160, as stop codons near the beginning of the coding region in most subtypes. A second gene, coding for ribosomal protein S4, lacks a start codon in some subtypes.
In both cases, these characteristics would normally prevent a gene from being expressed, but because these genes are otherwise conserved and most of the gene is 'intact', it seems likely that the genes are functional. Ribosomal protein S4 is considered an essential component of the ribosome needed for protein synthesis in the organelle. How the genes are expressed to produce functional proteins remains a mystery, - just one more peculiarity of Blastocystis!

In the growing pool of articles exploring relationships between intestinal parasites and gut microbiota, I was pleased to discover an article by Iebba et al. (2016) on "Gut microbiota related to Giardia duodeanlis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d'Ivoire". In this observational study, the authors used qPCR to detect groups of bacteria that are indicative of dysbiosis vs eubiosis, dysbiosis being a perturbed, imbalanced microbiota and eubiosis being a healthy, balanced gut microbiota. The authors found that individuals with Blastocystis and Entamoeba were characterised by eubiosis, while individuals with Giardia were characterised by dysbiosis. It says that samples (n = 20) were randomly chosen, but even so, the number of samples tested was low, and care should be taken when interpreting the results. The overall approach, however, is interesting, and somewhat resembles the work that we have been doing in our lab (ref). I also recently blogged about another study with a similar aim (go here to view the post).

I would also like to bring your attention to the EMBO Conference "Anaerobic protists: Integrating parasitology with mucosal microbiota and immunology", which will take place in Newcastle upon Tyne, UK in Aug/Sep 2017 (image). I will be there doing my best to deliver a stimulating talk on current knowledge and advances in Blastocystis and Dientamoeba research. You can visit the conference website by folloing this link. 

References:

Dogruman-Al F, Stensvold CR, & Yoshikawa H (2016). Editorial - PAR INT - special issue on Blastocystis. Parasitology international, 65 (6 Pt B) PMID: 27742000

Iebba V, Santangelo F, Totino V, Pantanella F, Monsia A, Di Cristanziano V, Di Cave D, Schippa S, Berrilli F, & D'Alfonso R (2016). Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d'Ivoire. Journal of infection in developing countries, 10 (9), 1035-1041 PMID: 27694739

Jacob AS, Andersen LO, Pavinski Bitar P, Richards VP, Shah S, Stanhope MJ, Stensvold CR, & Clark CG (2016). Blastocystis mitochondrial genomes appear to show multiple independent gains and losses of start and stop codons. Genome biology and evolution PMID: 27811175

Smith DR (2016). The past, present and future of mitochondrial genomics: have we sequenced enough mtDNAs? Briefings in functional genomics, 15 (1), 47-54 PMID: 26117139