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Episode 31: Microbial species and strains: What’s in a name?

/in Podcast, Season Two /by Laura

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The Science, Microbes & Health Podcast 

This podcast covers emerging topics and challenges in the science of probiotics, prebiotics, synbiotics, postbiotics and fermented foods. This is the podcast of The International Scientific Association for Probiotics and Prebiotics (ISAPP), a nonprofit scientific organization dedicated to advancing the science of these fields.

Microbial species and strains: What’s in a name? with Dr. Jordan Bisanz PhD

Episode summary:

In this episode, the ISAPP podcast hosts speak with Dr. Jordan Bisanz PhD, Assistant Professor of Biochemistry and Molecular Biology at Penn State University in State College, USA. They discuss how to define a bacterial strain, the diversity of strains within a species, and how genetic differences correspond with functional differences. They also talk about manipulating microbial communities for insights about health and disease.

Key topics from this episode:

  • Dr. Bisanz says just because strains within a species are genetically related doesn’t mean they do the same things. Bacteria gain and lose genes rapidly, but we don’t yet know what a lot of those genes do.
  • Natural variation in strains can be used as a tool to find out the functions of genes. 
  • Metagenomics illuminates strain-level differences, but that assumes we know what makes a strain. There’s no single accepted definition of a strain.
  • Knowing the mechanisms behind the effects of a strain on a host is important for predicting if closely related strains will have the same effect.
  • Moving forward, it could be useful to have functional information to go along with strains and their taxonomic descriptors.
  • Dr. Bisanz’s lab tests experimentally how microbial genes are gained and lost in vivo, both through wetlab experiments and computational approaches.
  • Experiments on strains are essential – for example, two strains with differences in 1000 SNPs might be functionally the same, while differences in 2-3 key SNPs might make a big difference.
  • When testing probiotic effects, you may be testing something derived from the original microbial genome but not identical. How can this be managed in industry? Understanding the mechanisms is important, strains that function similarly can qualify as the same strain.
  • A microbiome involves multiple microbes working together, acting differently from all the strains in isolation.
  • Dr. Bisanz studies tractable microbial communities: find the microorganisms that are different in a disease state compared to a healthy state, and create a synthetic community of the microbes that are absent. What are the functions of this community?
  • The challenge is that microbiologists need to be able to manipulate the microbes but cannot do this in a whole human fecal sample.
  • Is gut microbiome sequencing useful? At the level of individual, it may not provide value. But putting the data all together, in the future it may provide interesting information. The challenge with interpretation is that the microbiome is driving, but also responding to, dietary inputs.
  • In the microbiome field, gnotobiotic models (using humanized mice) need to be taken a step further than they currently go – specifying not only which microbes established in the host, but also how they could plausibly affect the mechanism.

Episode abbreviations and links:

Additional resources:

About Dr. Jordan Bisanz PhD:

Jordan Bisanz is an assistant professor of Biochemistry and Molecular Biology at the Pennsylvania State University and the One Health Microbiome Center. The Bisanz lab combines computational analyses and wet lab experimentation to understand how gut microbes interact with each other and their host. The lab specializes in coupling human intervention studies with multi ‘omics approaches and gnotobiotic models to understand how host-microbe interactions shape health generating both mechanistic insights and translational targets.

Five things scientists should know about the future of probiotics and prebiotics

/in ISAPP Science Blog /by KC

By Marla Cunningham​, Metagenics Global R&D Innovation Manager and 2021 ISAPP Industry Advisory Committee representative

As anyone connected with probiotics and prebiotics knows – there’s a lot happening in this space.

After a well-attended discussion group at the 2019 ISAPP Annual Meeting in Antwerp, a collaboration of 16 industry and academic scientists came together to produce a broad overview of current and emerging trends that were covered in this discussion. Just released online by Trends in Microbiology, the open access paper identifies some top trends across multiple spheres of influence on the future of probiotics and prebiotics.

  1. Discovery: Prebiotics and probiotics are emerging from unexpected sources – naturally occurring as well as synthesised or engineered. Food, human and animal microbiome-derived probiotics feature heavily in probiotic development through top-down microbiome data-driven approaches as well as physiological target-driven screening approaches. Prebiotic sources have expanded beyond traditional plant sources to include food waste streams, animal gut-derived glycans and mammalian milk as well as increasingly sophisticated synthesis techniques, involving sonication, high pressure, acid, enzyme and oxidation treatments. A growing understanding of the implications of carbohydrate structure on microbial metabolism is driving the emergence of designer prebiotics, as specific substrates for microbes of interest or the production of target metabolites, such as polyphenol-derived bioactives.
  2. Evaluation: Calls for integrated systems biology -omic approaches to the evaluation of probiotic and prebiotics effects continue to increase, utilising whole genome and metabolite approaches, with a focus on better understanding of mode of action as well as differential host and microbial responses that serve to improve host health.
  3. Product development: Quality assurance techniques continue to undergo evolution as the challenges of divergent product formats and increasingly complex formulations necessitate innovation in the field. There is a focus on techniques beyond cell culture enumeration for probiotic product verification as well as on the identification of functional markers of probiotic and prebiotic activity, which can be applied in complex food matrices.
  4. Regulation: Recent regulatory challenges with claim approval are understood to have driven corresponding evolution in clinical science and an increased focus on mechanistic elucidation. However, the converse is also occurring, with the development of novel probiotic species, therapeutics for disease treatment and increasingly microbiome-driven modes of action having implications for regulatory frameworks. This ‘give and take’ between science and regulatory requirements will likely accelerate into the future as the field continues to evolve.
  5. Implementation: Interest continues to grow in precision and personalised approaches to nutrition and healthcare, especially in the field of microbiome-related interventions where there is significant appreciation of host-to-host variability. The identification of putative microbial signatures of health and disease continues to fuel the development of health-associated microbes as candidate probiotics and as targets for novel prebiotic substrates. Further, a focus beyond microbial composition and into microbial function is driving interest in interventions which can correct metabolomic profiles, such as probiotics with specific enzyme activity to boost synthesis or catabolism of key microbial metabolites in vivo, including purine and monoamine compounds.

These and other trends create a rich and evolving landscape for scientists within the field and provide the promise of a bright future for prebiotics and probiotics.

Read the full paper here

Reference:

Cunningham, M., Azcarate-Peril, M. A., Barnard, A., Benoit, V., Grimaldi, R., Guyonnet, D., Holscher, H. D., Hunter, K., Manurung, S., Obis, D., Petrova, M. I., Steinert, R. E., Swanson, K. S., van Sinderen, D., Vulevic, J., & Gibson, G. R. (2021). Shaping the Future of Probiotics and Prebiotics. Trends in microbiology, S0966-842X(21)00005-6. Advance online publication. https://doi.org/10.1016/j.tim.2021.01.003

 

 

 

Limitations of microbiome measurement: Prof. Gloor shares insights with ISAPP

/in featured, ISAPP Science Blog, News /by Mary Ellen Sanders

February 20, 2019

The number of papers published on the human microbiome is growing exponentially – but not all of the studies are equally well designed or reported. Evaluating the latest research requires a basic understanding of the latest approaches to microbiome methods and data analysis.

To help equip scientists not conducting microbiome research with the tools to understand microbiome-focused publications, ISAPP hosted a webinar titled Understanding microbiome experiments: a critical assessment of methods and data analysis. The webinar featured Gregory Gloor, PhD., Professor, Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, Canada.

Prof. Gloor’s slides are available here.

Prof. Gloor opened his talk with a sobering perspective: the current body of microbiome publications is fraught with problems. There is a fundamental lack of reproducibility in the microbiome field (Sinha et al. 2017). This is largely due to the large number of tools available and a lack of an a priori established research plan for microbiome analysis, which should be consistently followed throughout a project. At every step of the way, many decisions must be made regarding wet lab methods, bioinformatics toolsets and statistics to use. Different choices lead to different results. Once the biological specimens are assayed, choices for bioinformatics and statistical analyses can greatly influence the conclusions. In short, it’s possible to view the data through so many different lenses that eventually a researcher can find a story worth telling. How close that story comes to the truth is a principle that sometimes is sacrificed for the sake of an interesting story.

Another important challenge to the field is representative sampling. Too few samples are typically taken, often because of cost limitations, so that the samples do not reasonably approximate the truth about the environment being sampled. Conclusions from such studies result in both many false positives and many false negatives.

Prof. Gloor also warned about outsourcing microbiome analysis. Commercial entities often use every metric, hoping the customer will get some outcome they hoped for. Further, their tools are often outdated or proprietary. So caution must be used – there is no substitute for expertise.

Some suggestions for improving outcomes were offered:

  • Each project should stipulate a research approach and outcome a priori, which is consistently followed throughout the project.
  • Methodological consistency is important within a lab, but analytical methods do not necessarily need to be standardized across all labs. If all labs use the same methods, consistent, but incorrect, outcomes may result. So use of different metrics is good, but methods should be consistent within a project. The value of different research groups using different methods to ask particular research questions is that if the same result emerges from different approaches, it increases confidence that the results are true.
  • Gloor cautioned that microbiome datasets are compositional, and compositional data approaches must be used (Gloor et al 2017).
  • Functional readouts have less methodological variation than taxonomic readouts. Therefore, functional analysis of shotgun metagenomics or shotgun metatranscriptomics is typically a more reproducible, and also more informative, readout.
  • Recent advances have significantly decreased the cost of performing shotgun metagenomics for both taxonomic and functional readouts (Hillmann et al 2018).
  • There are now near-complete microbial genomic datasets available for European, North American and Asian populations (Almeida et al 2019) that will make it easier to functionally map datasets.

Prof. Gloor mentioned an interesting aside: prior clinical trial registration, ~60% of large clinical trials showed benefit of the intervention being tested. After the registration process required declaration of primary research outcomes, that number dropped to closer to 10% (Kaplan and Irvin 2015). This suggests that primary outcomes and analysis methods need to be in place to restrict researcher bias. Right now such mechanisms are insufficient in the microbiome field.

Prof. Gloor’s paper, Microbiome Datasets Are Compositional: And This Is Not Optional, provides great background reading for this webinar.

This webinar was developed by ISAPP Industry Advisory Committee representatives as an extension of the annual IAC Learning Forum.

Dr. Gloor is a professor of biochemistry with broad experience in molecular biology, genetics and genomics. His research is focused on the development of tools to examine 16S rRNA gene composition, gene expression of mixed population samples and metabolomic analysis of clinical samples. He is currently working on developing and adapting principled methods to characterize correlation and differential abundance in sparse, high throughput sequencing data as generated in 16S rRNA gene sequencing surveys, meta-genomics and meta-transcriptomics. One of his primary contributions has been the ALDEx2 tool in Bioconductor for the analysis of high-throughput experiments that generate counts per sequence tag: 16S rRNA gene sequencing, metagenomics, transcriptomics and selex-type experiments.

Humpty Dumpty and the Microbiome

/in featured, ISAPP Science Blog /by Mary Ellen Sanders

Prof. Colin Hill, Microbiology Department and Alimentary Pharmabiotic Centre, University College Cork, Ireland (@colinhillucc)

“When I use a word,” Humpty Dumpty said, in rather a scornful tone, “it means just what I choose it to mean—neither more nor less.”

Microbiome science is an evolving discipline, and new terminology is an important part of any developing field.  But precise language is important, especially in a multidisciplinary field with researchers from many diverse scientific backgrounds.  Language provides us a means of communicating with brevity and accuracy, but this is effective only if the reader is deriving the correct (intended) information from the author.

For example, is there a difference between ‘microbiome’, ‘microbiota’ and ‘microflora’?  Are the terms interchangeable, or would it be useful to have them mean related but distinctly different concepts?  I have heard people state that ‘microbiota’ refers to the microbial content of an environment, whereas ‘microbiome’ refers to the microbes AND their environment (the biome).  I have heard others suggest that ‘microbiome’ actually refers to the genetic content of a particular microbiota, in the same way that the genome is the genetic content of an organism.  Some definitions assert that the microbiome/microbiota/microflora only describes the microbial cells (bacteria, archaea and fungi) in a particular niche, while others include non-cellular microbes such as viruses and bacteriophage in their definition.  It has also been pointed out that ‘microflora’ is a misnomer, since technically the term ‘flora’ is reserved for the kingdom Plantae.

A few other examples.  Do we all know what is meant when someone uses the term ‘metagenomics’?  Also, people often refer to analysing the microbiome by 16S – but they are really only analysing the bacterial fraction of the microbiome, the ‘bacteriome’.  Of course ‘16S’ itself is not a valid term – it is 16S rRNA genes that are being analysed.  Would a clear distinction between microbiome, bacteriome, phageome, mycome, virome, archaeome and all the other ‘omes’ help or hinder our understanding of the subject under discussion?  Should most studies actually use the term ‘faecal bacteriome’ rather than ‘gut microbiome’, since it is almost always faeces that is under investigation, and usually only the bacterial component?

I am not going to call out any individuals for abuse of language, since I am pretty sure I could look at my own output and find lots of examples of poorly expressed concepts.  But does any of this matter or am I simply being pedantic? I think it does matter, since if terms are poorly defined it may lead to confusion on the part of the reader (or listener), whereas the authors (or speakers) may know exactly what they mean – neither more or less, as suggested by Humpty Dumpty.

ISAPP has convened consensus panels on the meaning of some very commonly used terms such as probiotic1 and prebiotic2, but there is a limit to this activity, and consensus panels cannot be convened for every new term.  Even with these consensus papers, we still have a plethora of additional terms surrounding beneficial microbes, including paraprobiotics (killed microbes), psychobiotics (originally defined as probiotics with a mental health benefit, but the definition has recently been expanded to any exogenous influence whose effect on the brain is bacterially-mediated3), synbiotics (probiotics and prebiotics administered simultaneously – a term for which ISAPP is convening another Consensus Panel in 2019), live biotherapeutics, etc, etc.  One site I saw referred to bacteriophage as a prebiotic, using the argument that they can influence a microbiome in a selective manner to achieve a beneficial outcome.  This is surely a good example of where the ISAPP definition could provide clarity since prebiotics have to be utilised in order to qualify for the term. Other terms we often use without an agreed consensus as to their meaning are ‘dysbiotic’ (when we could use disturbed, or different, or disrupted), ‘unculturable’ (when we usually mean ‘not yet cultured as far as I know but I haven’t really tried’), ‘hypothetical genes’ (when we actually mean ‘function unknown’), ‘stability’, ‘resilience’, etc.  It may be useful to have some kind of standardised microbiome dictionary, or an accepted glossary of terms.  This is not a new idea (so few of mine ever are), and Julian Marchesi and Jacques Ravel published a lovely short paper to this effect in 20154.  The World Microbiome Day website also has a very short Glossary5.

Obviously, words must be the servants of the author and should not restrict expression or limit our ideas, and in many instances context can make it abundantly clear what meaning is intended by the author.  But in general, a strict definition is not the enemy of understanding, but makes it easier for author and reader to share common ground.

Who should create and curate such a Microbiome Glossary?  Ideally it would be interactive, perhaps along the line of a wiki page, where people could provide their newly coined terms along with a strict definition and arrive at a consensus for commonly used terms.  Reviewers of journal papers and reviews could help, by challenging authors on what terms they use, and whether or not they are the appropriate ones.

Meanwhile, I have to go back to the lab to do some comprehensive metagenomics on the gut microbiome – by which I mean that a competent scientist who works with me is going to go into the lab and conduct a particular form of 16s rRNA gene analysis to profile the more abundant members of the bacteriome of a portion of a faecal sample which has been collected, stored and extracted according to our in-house protocols.  Obviously!

 

  1. Hill et al., 2014. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the scope and appropriate use of the term probiotic.  Nat. Rev. Gastroenterol. Hepatol. 11, 506.
  2. Gibson et al., 2017. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics.  Nat. Rev. Gastroenterol. Hepatol. 14, 491.
  3. Sarkar et al., 2016. Psychobiotics and the Manipulation of Bacteria–Gut–Brain Signals.  Trends in Neurosciences 39, 763
  4. Marchesi JR and J. Ravel. 2015. The vocabulary of microbiome research: a proposal.  Microbiome 3, 31
  5. http://worldmicrobiomeday.com/glossary-of-microbiome-terms/