Posts

What is a strain in microbiology and why does it matter?

By Prof. Colin Hill, Microbiology Department and APC Microbiome Ireland, University College Cork, Ireland

At the recent ISAPP meeting in Sitges we had an excellent debate on the topic of ‘All probiotic effects must be considered strain-specific’. Notwithstanding which side of the debate prevailed, it does raise the question: what exactly is a strain? As a card-carrying microbiologist I should probably be able to simply define the term and give you a convincing answer, but I find that it is a surprisingly difficult concept to capture. It is unfortunately a little technical as a topic for a light-hearted blog, but here goes. Let me start by saying that the term ‘strain’ is important largely because we like to name things and then use those names when we share information, but that the concept of ‘strain’ may have no logical basis in nature where mutations and changes to a bacterial genome are constantly occurring events.

Let’s suppose I have a culture of Lactobacillus acidophilus growing in a test-tube, grown from a single colony. This clonal population is obviously a single strain that I will name strain Lb. acidophilus ISAPP2022. That was easy! I am aware of course that within this population there will almost certainly be a small number of individual cells with mutations (single nucleotide polymorphisms, or SNPs), cells that may have lost a plasmid, or cells that have undergone small genomic rearrangements. Nonetheless, because this genetic heterogeneity is unavoidable, I still consider this to be a pure strain. If I isolate an antibiotic resistant version of this strain by plating the strain on agar containing streptomycin and selecting a resistant colony I will now have an alternative clonal population all sharing a SNP (almost certainly in a gene encoding a ribosomal subunit). Even though there is a potentially very important genotypic and phenotypic difference I would not consider this to be a new strain, but rather it is a variant of Lb. acidophilus ISAPP2022. To help people in the lab or collaborators I might call this variant ISAPP2022SmR, or ISAPP2022-1. In my view, I could continue to make changes to ISAPP2022 and all of those individual clonal populations will still be variants of the original strain. So, the variant concept is that any change in the genome, no matter how small, creates a new variant. When I grow ISAPP2022 in my lab for many years, or share it with others around the word, it is my view that we are all working with the same strain, despite the fact that different variants will inevitably emerge over time and in different labs.

Where the strain concept becomes more difficult is when I isolate a bacterium from a novel source and I want to determine if it is the same strain as ISAPP2022. If the whole genome sequence (WGS) is a perfect match (100% average nucleotide identity or ANI) then both isolates are the same strain and both can be called ISAPP2022. If they have only a few SNPs then they are variants of the same strain. If the two isolates only share 95% ANI then they are obviously not the same strain and cannot even be considered as members of the same species (I am using a species ANI cut-off of 96% that I adopted from a recent paper in IJSEM.

Where it gets really tricky is when the ANI lies between 96% (so that we know that the isolates are both members of the same species) and 100% (where they are unequivocally the same strain). Where should we place the cut-off to define a strain? At what point is a threshold crossed and an isolate goes from being a variant to becoming a new strain? Should this be a mathematical decision based solely on ANI, or do we have to consider the functionality of the changes? If it is mathematical then we could simply choose a specific value, say 99.95% or 99.99% ANI, and declare anything below that value is a new strain. Remember that the 2Mb genome size of Lb. acidophilus would mean that two isolates sharing 99.99% ANI could differ by up to 200 SNPs. This could lead to a situation where an isolate with 199 SNPs compared to ISAPP2022 is considered a variant, but an isolate with 201 SNPs is a new strain (even though it only differs from the variant with 199 SNPs by two additional SNPs). This feels very unsatisfactory. But what about an isolate with only 50 SNPs, but one that has a very different phenotype to ISAPP2022 because the SNPs are located in important genes? Or what about an isolate with an additional plasmid, or missing a plasmid, or with a chromosomal deletion or insertion? I would argue we should not have a hard and fast cut-off based on SNPs alone, but we should continue to call all of these variants, and not define them as new strains.

So, by how much do two isolates have to differ before we no longer consider them as variants of one another, but as new strains? I will leave that question to taxonomists and philosophers since for me it falls into the territory of ‘how many angels can dance on the head of pin?’

All this may seem somewhat esoteric, but there are practical implications. Can we translate the findings from a clinical trial done with a specific variant of a strain to all other variants of the same strain? If Lactobacillus acidophilus ISAPP2022 has been shown to deliver a health benefit (and is therefore a probiotic), can we assume that Lb acidophilus ISAP2022-1 or any other variant will have the same effect? What if a variant has only one mutation, but that mutation eliminates an important phenotype required for the functionality of the original strain? I am afraid that at the end of all this verbiage I have simply rephrased the original debate topic from ‘All probiotic effects must be considered strain-specific’ to ‘All probiotic effects must be considered variant-specific’. Looks like we might be heading back to the debate stage in 2023!

Research on the microbiome and health benefits of fermented foods – a 40 year perspective

By Prof. Bob Hutkins, PhD, University of Nebraska Lincoln, USA

Many ISAPPers remember when fermented foods attracted hardly any serious attention from scientists outside the field. Certainly, most clinicians and health professionals gave little notice to fermented foods. In the decades before there were artisan bakeries and microbreweries proliferating on Main Street USA, even consumers did not seem very interested in fermented foods.

When I began my graduate program at the University of Minnesota in 1980, I was very interested in microbiology, but I did not know a lot about fermented foods. Accordingly, I was offered two possible research projects. One involved growing flasks of Staphylococcus aureus, concentrating the enterotoxins, feeding that material to lab animals, and then waiting for the emetic response.

My other option was to study how the yogurt bacterium, Streptococcus thermophilus, metabolized lactose in milk. This was the easiest career choice ever, and the rest, as they say, is history.

Indeed, that lab at Minnesota was one of only a handful in North America that conducted research on the physiology, ecology, and genetics of microbes important in fermented foods. Of the few labs in North America delving into fermented foods, most emphasized dairy fermentations, although some studied vegetable, meat, beer, wine, and bread fermentations. Globally, labs in Europe, Japan, Korea, Australia, and New Zealand were more engaged in fermented foods research than we were in North America, but overall, the field did not draw high numbers of interested researchers or students.

That’s not to say there weren’t exciting and important research discoveries occurring. Most research at that time was focused on the relevant functional properties of the microbes. This included carbohydrate and protein metabolism, flavor and texture development, tolerance to acid and salt, bacteriocin production, and bacteriophage resistance. Despite their importance, even fewer labs studied yeasts and molds, and the focus was on lactic acid bacteria.

Other researchers were more interested in the health benefits of fermented foods. Again, yogurt and other cultured dairy foods attracted the most interest. According to PubMed, there were about 70 randomized clinical trials (RCTs) with yogurt as the intervention between 1981 and 2001. Over the next 20 years, there were more than 400 yogurt RCTs.

Fast forward a generation or two to 2021, and now fermented foods and beverages are all the rage. Certainly, having the molecular tools to sequence genomes and interrogate entire microbiomes of these foods has contributed to this new-found interest. Scanning the recent literature, there are dozens of published papers on microbiomes (and metabolomes) of dozens of fermented foods, including kombucha (and their associated symbiotic cultures of bacteria and yeast, known as SCOBYs), kefir, kimchi, beer (and barrels), cheese (and cheese rinds), wine, vinegar, miso and soy sauce, and dry fermented sausage.

It’s not just fermentation researchers who are interested in fermented foods. For ecologists and systems biologists, fermented foods serve as model systems to understand succession and community dynamics and how different groups of bacteria, yeast, and mold compete for resources.

Moreover, consumers can benefit when companies that manufacture fermented foods take advantage of these tools. The data obtained from fermented food microbiota analyses can help to correlate microbiome composition to quality attributes or identify potential sources of contamination.

Importantly, it is also now possible to screen microbiomes of fermented foods for gene clusters that encode potential health traits. Indeed, in addition to microbiome analyses of fermented foods, assessing their health benefits is now driving much of the research wave.

As mentioned above, more than 400 yogurt RCTs were published in the past two decades, but alas, there were far fewer RCTs reported for other fermented foods. This situation, however, is already changing. The widely reported fiber and fermented foods clinical trial led by Stanford researchers was published in Cell earlier this year and showed both microbiome and immune effects. Other RCTs are now in various stages, according to clinicaltrials.gov.

Twenty years ago, when ISAPP was formed, I suspect few of us would have imagined that the science of fermented foods would be an ISAPP priority. If you need proof that it is, look no further than the 2021 consensus paper on fermented foods. It remains one of the most highly viewed papers published by Nature Reviews Gastroenterology and Hepatology.

Further evidence of the broad interest in fermented foods was the recently held inaugural meeting of The Fermentation Association. Participants included members of the fermented foods industry, culture suppliers, nutritionists, chefs, food writers, journalists, retailers, scientists and researchers.

Several ISAPP board members also presented seminars, including this one who remains very happy to have made a career of studying fermented foods rather than the emetic response of microbial toxins.

The future is microbial: A post-pandemic focus on identifying microbes and metabolites that support health

By Prof. Maria Marco, Department of Food Science and Technology, University of California Davis, USA

The COVID-19 pandemic has been a sobering reminder of the significance that microorganisms have on human life. Despite the tremendous scientific and medical advances of the twentieth century, our best precautions against the virus have been to practice the oldest and most simplistic of all public health measures such as washing hands and maintaining physical distance from others. At the same time, the effectiveness of the new SARS-CoV-2 vaccines and the speed in which they were developed show how sophisticated and advanced our understanding of viruses has become. Taken together, the limitations and successes of responses to the pandemic underscore the power of investment in microbiology research. This research, which was first catalyzed by the pioneering work of Louis Pasteur, Robert Koch, and contemporaries in the late 1800s, was the basis for the overall reduction in infectious diseases during the twentieth century. Continued investment in these efforts will prepare us for the next pandemic threat.

Beyond pathogens to health-promoting microbes

As our attention turns to the promise of the New Year, we may also take this moment to appreciate the fact that microorganisms can also do good. Our “microbial friends” were first promoted by the lauded biologists Élie Metchnikoff, Henry Tissier, and Issac Kendall at the turn of the twentieth century. Since then, nearly another century passed before the power of microorganisms to benefit human health reached wider acceptance.

Marked by the emergence of laboratory culture-independent, nucleic-acid based methods to study microbial communities, there is now excitement in the identification of microorganisms that are important for health promotion. This interest is catalyzed by the urgency to find ways to prevent and treat cardiovascular diseases, cancers, and other non-communicable, chronic conditions that are now the leading causes of death worldwide. Much like the pressure to address infectious diseases as the primary cause of mortality prior to the twentieth century, so too is the need today for sustained research investments in studying how certain microorganisms contribute to, or may be essential for, preventing and treating the greatest threats to public health in the modern era.

Exemplified by the growing number of human microbiome studies, it is now broadly understood that the human microbiome contributes positively to digestive, immune, and endocrine systems function. Systematic reviews and meta-analyses of clinical trials support the use of probiotics for a variety of conditions and there are positive associations between the consumption of fermented dairy foods and good metabolic health. To understand how microbes can be beneficial, numerous mechanisms have been proposed (for example, modulation of the immune system and production of neurochemicals that can impact the gut-brain axis), and these mechanisms apply to both autochthonous microbiota and probiotics alike. However, our understanding of exactly how this occurs lags far behind what is currently known about microorganisms that cause harm.

Identifying microbes & metabolites that maintain health

The future of beneficial microbes is in identifying the specific, health-promoting metabolites, proteins, and other compounds that they make. Presently only a handful of such examples are known. Perhaps most recognized are the short chain fatty acids, butyrate, propionate, and acetate, which are known to bind specific human cell receptors to modulate numerous cell pathways including those that affect metabolism. Other microbial compounds generated as intermediate or end products of microbial metabolism (such as metabolites of amino acids), secondary metabolites (such as bacteriocins), and bacterial cell surface constituents (such as certain membrane proteins) were shown to benefit health, although a more complete description of mechanistic details for their effects remains to be discovered. Precise mechanistic descriptions of “beneficial factors”, or the microbial enzymatic pathways and molecules that induce desired cellular and systemic responses in the human body, will be pivotal for elucidation of the precise ways microorganisms sustain health and well-being (for more detail on this topic see here).

Based on what we know about the complexity of the human microbiome and the now many decades of probiotics research, this effort will require innovation and multi-disciplinary coordination. Just as early microbiologists raced to address the high rates of mortality due to microbial pathogens, we are in a new age where again microorganisms are regarded as emerging public health threats. However, we now have to our advantage the knowledge that not all microorganisms cause harm but instead the majority may offer solutions to the greatest health challenges of the twenty-first century.

 

 

What makes a synbiotic? ISAPP provides a sneak peek at the forthcoming international scientific consensus definition

By Kristina Campbell, science and medical writer

The word ‘synbiotic’ is found on the labels of many different products, from supplements to chocolate bars, and it has generally been understood to be a combination of a probiotic and a prebiotic. But what happens when scientists want to test whether these combination products really deliver any health benefits? Can these products be tailored to have specific effects on the body or on the human gut microbiota? Agreeing on a clear definition of synbiotics is needed to provide focus for scientific research in this area, to facilitate the design of studies, and to allow for progress wherein their health effects are uncovered.

The scientific definition of synbiotic was the central topic of the international scientific panel brought together by ISAPP in May 2019 in Antwerp, Belgium. Members of the panel, eleven of the top academic experts in the field of probiotics and prebiotics, gathered to clarify a scientifically valid approach for use of the word ‘synbiotic’, and to communicate this by position paper. The outcome of this consensus panel is currently in press at Nature Reviews Gastroenterology & Hepatology.

Kelly Swanson, Professor in the Department of Animal Sciences and Division of Nutritional Sciences at University of Illinois at Urbana-Champaign, chaired the panel and led the paper’s publication. Swanson has been studying gastrointestinal health in both humans, companion animals (dogs and cats) and rodent models for the past 20 years—and having followed the rapid advances in the field of probiotics and prebiotics during those two decades, he knew the task of creating a synbiotic definition would not be easy.

He says, “The field is highly complicated, so an interdisciplinary panel was essential. The main areas of expertise included microbiology and microbial ecology; gastrointestinal physiology; immunology; food science; nutritional biochemistry and host metabolism.”

A timely discussion

According to Swanson, an increase in research interest, built on a foundation of recent scientific and technical gains, made this the right time to come to consensus on a synbiotic definition. He says, “Over the past decade, technological advances have allowed scientists to study the gut microbiome at a molecular level. In addition to characterizing the composition of the gut microbes, researchers are learning more about their biological activity and how they may impact host health.”

Furthermore, clarity about the definition was urgently needed because of the rapidly growing synbiotics market. Consumers seem to be more aware of synbiotics than ever, but they face a bewildering array of product offerings labeled as ‘synbiotic’ without a clear understanding of what that term entails and with no framework for establishing scientific efficacy. Swanson says, “As the field has moved forward and the sales of probiotics and prebiotics have increased, there has been more interest in combining substances to enhance efficacy. Some of these combinations may function as synbiotics, but it is not guaranteed. Rather than randomly combining substances together, there should be scientific rationale supporting their use.”

Clarifying the concept

One of the first questions the panel members had to tackle was whether to stick to the idea of a synbiotic as ‘probiotic plus prebiotic’, thus leaning heavily on the ISAPP-led international consensus definitions of probiotics and prebiotics published in 2014 and 2017, respectively. But the panel members decided this narrow scope would ultimately limit innovation in the synbiotic category.

Swanson explains, “While many synbiotics may be composed of an established prebiotic and established probiotic, the panel did not want to restrict scientific advances in the synbiotic category by requiring use of components already established on their own.”

As a result, he says, previously untested live microbes and potential prebiotic substances could be considered a synbiotic if the combination showed efficacy, and if the health benefit came from administering both the live microbe and the substrate it utilized—that is, the microbe together with its ‘food’.

Another conclusion from the panel is that probiotics (with known health benefits) and prebiotics (with known health benefits) cannot be called synbiotics unless they have been tested together. “There should be a rationale supporting the combination used, and then testing of the combination to confirm its efficacy,” says Swanson.

The panel suggests a synbiotic may be composed of either of the following, as long as efficacy is demonstrated for the combination:

  • Established probiotic + established prebiotic (each component meeting the efficacy and mechanistic criteria for each)
  • Previously untested live microbe + a substrate that is selectively utilized by the co-administered live microbe

Further details, including two different ‘categories’ of synbiotics, will be provided in the published paper.

In addition to the definition, the publication will cover the history of synbiotic-type products, how these products can be characterized, levels of evidence that currently exist versus levels of evidence desired, points about safety documentation and reporting, and relevant characteristics of the target hosts.

A remaining challenge—not just for the expert group, but also across the field—is the difficulty of establishing causal links between substances’ effects on the gut microbiota (e.g. ‘selective utilization’ of a substrate) and health outcomes.

While the publication of the synbiotic definition will be an important milestone, Swanson anticipates further discussion in the years ahead. “As more is learned, I expect the criteria for assessing synbiotic efficacy will continue to change,” he says.

An update on the scientific consensus definition of synbiotic was presented to ISAPP members at the 2020 virtual meeting in June.

 

The Art of Interpretation

By Prof. Gregor Reid, BSc Hons PhD MBA ARM CCM Dr HS, Lawson Research Institute, University of Western Ontario, Canada

It takes a certain degree of intelligence to become a scientist, and certainly hard work to be able to fund a lab and students. Yet, is it not bemusing when scientists cannot interpret simple things like definitions and the results of human studies?

I’ve written repeatedly, as have others, about the definition of probiotics (in case you forgot – “Live microorganisms that, (or which) when administered in adequate amounts, confer a health benefit on the host”),1,2 and yet people look at it and must think that ‘dead’ fits, as does ‘consume’, as does ‘colonize’. It beggar’s belief how such a simple definition can be so badly interpreted by intelligent people.

Time after time papers I review mis-write and/or misinterpret the definition. Conference after conference, I hear dieticians, pharmacists, physicians, scientists not only get the definition wrong, but say things like ‘the probiotics in kombucha’ when there are none, ‘we have lots of probiotics in our gut’ when you don’t unless you consumed them, ‘the lactobacilli need to colonize’ when this was never a prerequisite nor does it happen except in rare instances.

The interpretation gets more difficult when people use terms that are completely undefined like ‘psycho-biotics’ and ‘post-biotics’. Even ‘dead probiotics’ have been used in clinical trials – God help us when the authors can’t even define it. Why stop at killing probiotic strains? Why not just kill any bacterial strain? Even the gut-brain axis which is now mentioned everywhere in the literature is undefined and unproven. The vagus nerve links to many body sites as does the nervous system, making it exceedingly difficult to prove that brain responses are only due to the gut microbes.

Everyone can site a manuscript that has been badly analyzed, interpreted or peer-reviewed, or whose findings are overblown. But let’s not excuse this as ‘it’s just science’ or ‘it’s just the way it is.’ No, it is not. When a paper uses a product that is stated to be ‘probiotic’, there is an onus on the authors to make sure the product meets the appropriate criteria. These have been stated over and over again and reiterated this March, 2019.3

If scientists and science writers are really that smart, then how do they keep getting this wrong? How do we let a poorly analyzed paper get published and allow authors to say that Bacteroides fragilis is a probiotic that can treat autism?4,5 And when this leads to companies claiming probiotics can treat autism, why do other scientists convey cynicism for the field instead of against their colleagues and specific companies making the false claims?

Where does opinion cross the line with ignorance or stupidity? Martin Luther King Jr. must have predicted life today when he said, “Nothing in all the world is more dangerous than sincere ignorance and conscientious stupidity.”

Is it envy or anger that drives the anti-probiotic sentiments? It seems to go far beyond a difference of opinion. When the BBC and JAMA fail to comment on two much better and larger studies on the effects of probiotics published6,7 at the same time as the ones in Cell8,9 that were promoted by press releases, what is driving opinion? The science or the press releases? Are the journalists and communications’ people interpreting study results vigorously? One cannot believe they are.

In an era where anyone can write anything at any time and pass it along to the world, what are we recipients to do? Just go with our instincts? Soon, we will not know the difference between fact and fake news. The avatars will be so real, we will act on falsehoods without knowing. When all news is fake, where does that leave us as people, never mind scientists?

Manuscripts are sent for peer-review but how many reviewers are experts in bioinformatics, molecular genetics, clinical medicine, biostatistics and what happens on the front line of products to consumers or patients? Like it or not, poor studies will get out there and it will be the media who will tell the story and interpret the findings or press releases.

One must hope that confirmatory science will continue and if it fails, the writers and readers will stop citing the original incorrect report. But how often does that happen? And what are we left with?

It takes effort to object or fight back, but if we don’t then the fake news will become the norm.

Try interpreting that if you will.

 

Literature Cited

  1.  FAO/WHO. 2001. Probiotics in food.  http://www.fao.org/food/food-safety-quality/a-z-index/probiotics/en/
  2. Hill C. et al. 2014. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotics. Nat. Reviews Gastroenterol. Hepatol. 11(8):506-14.
  3. Reid G. et al. 2019. Probiotics: reiterating what they are and what they are not. Front. Microbiol. 10: article 424.
  4. Hsiao et al. 2013. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell. 155(7):1451-63.
  5. Sharon G, et al. 2016. The central nervous system and the gut microbiome. Cell. 167(4):915-932.
  6. Korpela K. et al. 2018. Probiotic supplementation restores normal microbiota composition and function in antibiotic-treated and in caesarean-born infants. Microbiome. 6(1):182.
  7. De Wolfe, T.J. et al. 2018. Oral probiotic combination of Lactobacillus and Bifidobacterium alters the gastrointestinal microbiota during antibiotic treatment for Clostridium difficile infection. PLoS One. 13(9):e0204253.
  8. Suez J. et al. (2018). Post-antibiotic gut mucosal microbiome reconstitution is impaired by probiotics and improved by autologous FMT. Cell. 2018 Sep 6;174(6):1406-1423.e16.
  9. Zmora N. et al. 2018. Personalized gut mucosal colonization resistance to empiric probiotics is associated with unique host and microbiome features. Cell. Sep 6;174(6):1388-1405.e21.