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

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.

A recording of the webinar is available here, and 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 Further, their tools are often outdated or proprietary.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.

The Children of Masiphumelele Township

Gregor Reid PhD MBA FCAHS FRSC, Professor, Western University and Scientist, Lawson Health Research Institute, London, Canada

Just off the main road from Cape Town, South Africa to Simon’s Town, sits Masiphumelele township where challenges of poverty, malnutrition, HIV and the risk of violence face people every day.

It is also the location for the Desmond Tutu HIV Foundation Youth Centre, a safe haven that provides adolescent-friendly sexual and reproductive health services alongside educational and recreational activities for youth living in Masiphumelele and surrounding areas.

To understand some of the dangers that children face, in 2017, about 270,000 people in South Africa were newly infected with HIV, adding to one of the highest HIV prevalence rates in the world. The Tutu Youth Centre aims at helping educate youth to reduce their risk of becoming another HIV statistic.

I was invited there by University of Cape Town Professor Jo-Ann Passmore, a woman not only recognized for her research but whose passion for helping others is reflected in her warm smile (4th from left in group photo). She asked if I would be interested in holding a workshop to illustrate to the youth how using sachets of probiotic bacteria could empower them. I jumped at the chance. On an afternoon break from the Keystone Symposium, thirty researchers joined me along with Jo-Ann and my wife Debbie, a teacher of children with learning disabilities.

After a tour of the areas where children learn on computers, play games in safety, or have personal discussions about sexual health, everyone filled the room with a stunning backdrop of the Nobel Laureate’s image. Having been privileged to meet the Archbishop when he was hosted by St. Joseph’s Healthcare Foundation in 2008, it was a nerve-tingling experience for me.

Giving a lecture on beneficial microbes is hard enough to peers sitting in the back of the room, but to do so with young South Africans was more somewhat daunting. However, it proved to be a lot of fun especially when we had to identify kids who were good leaders (the boys all pointed to a girl), who liked to make stuff and sell it to others (two boys stood out). By the end, we had picked the ‘staff’ of a new company.

The next step was for four groups to decide on the company’s name, what products they’d make from the probiotic sachets (the options were many including yoghurt, cereals, fruit juices, maize), what marketing tools they would use and who they would target to obtain a respectable income.

Interestingly, several of the conference participants seemed less engaged, as if they had never considered how microbiology research could affect real lives. In front of them were children facing huge challenges on a day-to-day basis. In one group, the kids were quiet until my wife brought out pens and paper, then they went to town designing products, names and labels. A lesson for me on how different people need different stimuli to become engaged. The faculty left early to beat the traffic back to Cape Town, so unfortunately, they did not hear the outcome of the children’s work.

When we re-assembled to present the results, I was impressed with what could be created in such a short time. My favourite was the Amazing Maize, a bottle shaped like a corn cob with the idea it would contain fermented maize. It emphasized the importance of marketing and for products to taste and look good to be purchased.

It has been over ten years since Archbishop Tutu applauded us for the Western Heads East project and thanked us for empowering women and youth and contributing to nutrition in Africa. Since then, thanks to the huge efforts of Western staff and students, and more recently IDRC funding and partnerships especially with Yoba-for-life, Heifer International and Jomo Kenyatta University of Agriculture and Technology, over 260,000 people in east Africa are now consuming probiotic yoghurt every week. The children of the South African townships were maybe too young to join in this new wave of microenterprises, but at least now they have heard about it and the importance of fermented food and beneficial bacteria.

In the background of the workshop several wonderful women committed to start up a new production unit using the Yoba/Fiti sachets developed by Yoba-for-life. I left them some sachets for them to try out the process.

But it was me who left with the biggest lesson on how precious each life is, and how those of us with the knowledge, need to provide the means for others to use their own talents to fulfill the purposes of their lives.

No better way than to start with the children.

Prof. Maria Marco joins the ISAPP board of directors

ISAPP happily announces that Prof. Maria Marco Ph.D. from the Department of Food Science and Technology at UC Davis has joined the ISAPP board of directors.

Prof. Marco has broad expertise in probiotics, prebiotics, and fermented foods. She has a special interest in lactic acid bacteria (from plant and animal sources) and the mechanisms of their interaction with their hosts.  She is one of the few researchers globally to tackle the important issue of the role of delivery matrix in probiotic functionality. She has more than 70 publications and book chapters.

Prof. Marco serves as the Chair of the Food Science Graduate Group and has mentored over 50 undergraduates, MS students, and visiting scholars, 11 PhD students, and 13 post-docs. She is active in education and public outreach on fermented foods and is a founder of a start-up company on microbial detection.

Prof. Marco serves as an Editor of mSphere and has served as the Guest Editor – Special issue on Food Biotechnology for Current Opinion in Biotechnology (2018). As guest editor of COB, she mediated publication of outcomes from several ISAPP discussion groups.

See her profile here. For more details on what her lab is up to, see here.

See here for the list of ISAPP’s all-academic board of directors.

ISAPP’s prebiotics & probiotics infographic now available in Russian

‘International’ is the first word in ISAPP’s title—and the organization takes seriously its commitment to advancing education about probiotics and prebiotics in countries around the world. ISAPP members are happy to announce that the infographic “Effects of Prebiotics and Probiotics on our Microbiota” is now available in Russian. See here.

In an effort to reach broader global populations with its science-based communications on probiotics, prebiotics and fermented foods, ISAPP is undertaking steps to translate its infographics into multiple languages. Expected in the next month are translations of ISAPP’s popular “Probiotics” and “Prebiotics” infographics, which will be available in Bulgarian, Chinese, Dutch, French, Indonesian, Italian, Polish, Portuguese, Russian, and Spanish. (See here for all available translations of ISAPP infographics.)

The translation efforts, led by Dr. Roberta Grimaldi from University of Reading (UK), are made possible by many translators who are contributing generously of their time and skills.

Humpty Dumpty and the Microbiome

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/
hospital_room

Late initiation of probiotic therapy for acute pediatric gastroenteritis may account for null results

Francisco Guarner, MD, PhD, University Hospital Vall d’Hebron, Barcelona, Spain; Michael Cabana, MD, MPH, University of California, San Francisco, CA, USA; and Mary Ellen Sanders, PhD, International Scientific Association for Probiotics and Prebiotics, Centennial, CO, USA. 

Schnadower et al (1) and Freedman et al (2) conclude that probiotics given to children who presented to emergency departments with gastroenteritis are not effective; however, these new well-conducted trials used probiotics in children who were symptomatic much longer than when current recommendations suggest initiating therapy.  Both studies recruited children that were symptomatic for up to 72 hours or more at time of randomization. Half the cohort of Freedman (2) had diarrhea for 43 hours at randomization. In the study by Schnadower, (1) children were symptomatic at randomization for a median of 53 hours. It is not surprising that probiotic intervention at this late stage was not successful, since most children were close to spontaneous remission. Acute gastroenteritis in high-income countries is usually benign and after 48 hours typically remits spontaneously. These new studies should not change current recommendations (3,4,5) to use probiotics early after onset of pediatric gastroenteritis in conjunction with oral rehydration, consistent with previous beneficial trials.

 

  1. Schnadower D, Tarr PI, Casper TC, et al. Lactobacillus rhamnosus GG versus placebo for acute gastroenteritis in children. N Engl J Med. 2018; 379(21):2002-2014.
  2. Freedman SB, Williamson-Urquhart S, Farion KJ, et al. Multicenter trial of a combination probiotic for children with gastroenteritis. N Engl J Med. 2018;379(21):2015-2026.
  3. Allen SJ, Martinez EG, Gregorio GV, Dans LF. Probiotics for treating acute infectious diarrhoea. Cochrane Database Syst Rev. 2010 Nov 10;(11):CD003048.
  4. Szajewska H, Guarino A, Hojsak I, et al. Use of probiotics for management of acute gastroenteritis: a position paper by the ESPGHAN Working Group for Probiotics and Prebiotics. J Pediatr Gastroenterol Nutr. 2014;58(4):531-9.
  5. Lo Vecchio A, Dias JA, Berkley JA, et al. Comparison of recommendations in clinical practice guidelines for acute gastroenteritis in children. J Pediatr Gastroenterol Nutr. 2016;63(2):226-35.

 

Importance of understanding probiotic mechanisms of action

By Prof. Sarah Lebeer, Universiteit Antwerpen, Belgium

At present, we do not fully understand the mechanistic basis of many well established probiotic health benefits. This limits our ability to predict which probiotics are likely to be effective.

For instance, prevention of antibiotic-associated diarrhea and necrotizing enterocolitis are health benefits that are well substantiated by meta-analyses, which combine results on many probiotic strains. But what the effective strains have in common from a mechanistic perspective is not known. We cannot yet pinpoint one or a few molecules produced by these strains that might drive the clinical effects. This is likely due to interplay between both host and probiotic factors. These health conditions are complex pathologies and the probiotic strains are living micro-organisms likely working through multiple mechanisms and molecules.

This is in contrast to some more clearly defined situations. Lactose maldigestion results from a deficiency in the enzyme lactase, which is required for converting lactose to glucose and galactose in the small intestine. If lactose is not broken down, it reaches the colon and is fermented by the gut microbiota, leading to symptoms. Some probiotic bacteria (including those present in yoghurt) contain lactase, which can reduce the typical symptoms of lactose digestion.

Several colleagues and I published a recent paper (Kleerebezem et al. 2019) discussing the importance of understanding mechanisms of action. We argue that such knowledge will enable: “(i) selection of more effective probiotic strains; (ii) optimization of probiotic product manufacturing and quality assurance, (iii) improved design of probiotic formulation, and (iv) support of the design of effective clinical trials with the best chance of realizing benefits to human health.”

While knowledge of the mechanism of action is not necessary for translation to effective products, it provides important insights that can improve actions throughout the translational pipeline.

The strain-specificity of different mechanisms of action is another point that will be clarified by future mechanism-focused research. Different probiotic strains clearly express different mechanisms, but some mechanisms are also shared (Sanders et al. 2018). How different host- and probiotic-specific factors interact to achieve a clinically successful intervention remains to be unraveled.

ISAPP recognizes Prof. Michael Cabana’s contributions during his board of directors tenure

Prof. Michael Cabana’s service on ISAPP’s board of directors has come to an end in 2018—and the remaining board members wish to affirm his rich legacy of contributions, which furthered ISAPP’s mission of advancing the science of probiotics and prebiotics.

Dr. Cabana, Professor of Pediatrics, Epidemiology and Biostatistics and the Director of the Division of General Pediatrics at the University of California, San Francisco (UCSF), joined the ISAPP board in 2008. He served as secretary for five years and treasurer for one year, and was local host for the 2011 ISAPP annual meeting in Berkeley, USA. He chaired discussion groups at eight different annual meetings:

  • 2009: Designing human clinical trials for probiotics
  • 2010: Prebiotics and probiotics in perinatal nutrition
  • 2012: From clinical trials to clinical guidelines:  Reconciling the evidence
  • 2013: Use of probiotics and/or prebiotics to program fetal and newborn health / first 1000 days of life
  • 2014: Infant colic:  Is there enough clinical evidence to support probiotic interventions?
  • 2015: Technology transfer and academic-industry partnerships
  • 2016: Colic update:  IPDMA and mechanisms
  • 2019: Prebiotic applications in children

 

Dr. Cabana was proactive in developing ISAPP responses to media misrepresentations of research by co-authoring letters to the editor in the New England Journal of Medicine (under review) and JAMA Internal Medicine (in press):

  • Guarner F, Cabana MD, Sanders ME. Late initiation of probiotic therapy for acute pediatric gastroenteritis may account for null results. New England J Med. Submitted.
  • Cabana MD, Salminen S, Sanders, ME. Probiotic safety – reasonable certainty of no harm. JAMA Internal Med. In Press.

As outcomes of ISAPP discussion groups or as part of other ISAPP initiatives, Dr. Cabana coauthored several papers, including:

 

Always a congenial and collaborative colleague, Dr. Cabana will be missed by the ISAPP board as he now turns his focus to other professional activities. Dr. Cabana’s UCSF lab has several ongoing trials related to the microbiome and probiotics in pediatric populations.

Prof. Hania Szajewska joins the ISAPP board of directors

ISAPP is pleased to announce that Prof. Hania Szajewska MD from the Department of Paediatrics of The Medical University of Warsaw has joined the ISAPP board of directors.

Prof. Szajewska’s depth and breadth of experience in probiotics, prebiotics, and the effects of early dietary interventions on long term health will greatly facilitate ISAPP’s ability to fulfill its mission to advance the science of probiotics and prebiotics.

In addition to conducting clinical trials on probiotics in pediatric populations, Prof. Szajewska has led numerous efforts to systematically review the totality of evidence on probiotics and prebiotics in order to develop evidence-based recommendations where warranted. She has more than 285 publications and 25 book chapters.

Prof. Szajewska serves as the Editor-in-Chief (Europe) of the Journal of Pediatric Gastroenterology and Nutrition and is involved in European Society for Paediatric Gastroenterology, Hepatology and Nutrition in numerous capacities.

Prof. Szajewska replaces Prof. Michael Cabana MD MPH, who is stepping down from the board to embrace new professional opportunities.

See here for the list of ISAPP’s all-academic board of directors.

kelly_swanson

ISAPP plans consensus panel on synbiotics

The term ‘synbiotic’ – which refers to a substance that combines both a probiotic and prebiotic – lacks a concise, modern definition. Stakeholders, including researchers, regulatory experts, consumers, marketers, industry scientists and healthcare providers, would benefit from a clear definition of synbiotics, a concise review of the state of the science of synbiotics, and a clarification of what kinds of products fall under the synbiotic scope.

ISAPP will convene a panel of top scientific experts on May 13th in Antwerp to develop a consensus around this topic. This panel will be chaired by Prof. Kelly Swanson, The Kraft Heinz Company Endowed Professor in Human Nutrition, Professor in the Department of Animal Sciences and Division of Nutritional Sciences, and Adjunct Professor in the Department of Veterinary Clinical Medicine at the University of Illinois at Urbana-Champaign. Prof. Swanson is known for his research on the mechanisms by which nutritional interventions affect health outcomes in both animals and humans. He is a co-author of the 2017 ISAPP consensus statement on the definition and scope of prebiotics.

As with the ISAPP consensus statements on probiotics (Hill et al. 2014) and prebiotics (Gibson et al. 2017), ISAPP is working with Nature Reviews Gastroenterology and Hepatology to publish the outcome of the synbiotics panel.

ISAPP’s focus on the science of probiotics and prebiotics makes it uniquely positioned to champion a panel of experts to discuss the definition and scientific justification for synbiotics.

The consensus panel members are:

  • Kelly Swanson, University of Illinois at Urbana-Champaign, USA (chair)
  • Glenn Gibson, University of Reading, UK
  • Gregor Reid, University of Western Ontario, Canada
  • Kristin Verbeke, University of Leuven (KU Leuven), Belgium
  • Nathalie Delzenne, Université Catholique de Louvain, Belgium
  • Robert Hutkins, University of Nebraska-Lincoln, USA
  • Karen Scott, University of Aberdeen, UK
  • Raylene Reimer, University of Calgary, Canada
  • Hannah Holscher, University of Illinois at Urbana-Champaign, USA
  • Meghan Azad, University of Manitoba, Canada
  • Mary Ellen Sanders, ISAPP

ISAPP’s 2019 Annual Meeting Program Released

ISAPP is pleased to announce the release of the official program for its 2019 Annual Meeting, scheduled for May 14-16, 2019, in Antwerp. Unlike the 2018 ISAPP meeting in Singapore, which was an open registration meeting, the 2019 event will comprise only invited academic experts and industry scientists from member companies. For program details, see the meeting website.

The 2019 program offers a strong lineup of probiotic, prebiotic and microbiome presentations. Featured topics include human milk oligosaccharides, learnings from the Flemish Gut Flora project, and leveraging political infrastructure to advance important science and public health messaging. Half-day breakout discussion groups are scheduled for May 15th, covering timely topics relevant to both industry and clinical practice, such as recommended dietary allowance (RDA) for live cultures, and the use of probiotics and prebiotics as adjuncts to drugs. Prof. Glenn Gibson will host the “fishbowl”, a session designed to integrate audience and experts’ perspectives in an interactive format; this year’s topic is: What can scientists and industry do to spring probiotics and prebiotics into mainstream health management?

For companies interested in participating in this meeting, now’s the time to join ISAPP and become part of its active industry advisory committee. Details on industry membership can be found here. ISAPP’s industry members help ISAPP achieve its mission of advancing the science of probiotics and prebiotics—see  here for a summary of our latest accomplishments.

Students and fellows will constitute an important presence at the annual meeting. Members of the ISAPP students and fellows association (SFA) will be keen participants, having organized a poster session as well as two SFA oral presentations. The group will also run a half-day parallel student-focused program.

The local host for ISAPP’s 2019 Annual Meeting, Prof. Sarah Lebeer, University of Antwerp, is excited to welcome her ISAPP colleagues to Antwerp. The history of Antwerp goes back to the 4th century and today the city remains an important European cultural and trade center. ISAPP Annual Meeting participants are invited to join a riverboat trip and dinner to get to know this historic city.

 

 

Do you know the difference between fiber and prebiotics? A new ISAPP infographic explains

Many people think prebiotics and fiber are the same thing. But according to leading scientists, they’re not. Fiber and prebiotics are both dietary tools to promote health, but you need to know some key differences between these two types of nutrients in order to make the best decisions for your health.

This new infographic summarizes what fiber and prebiotics have in common, and how they are different (including their distinct effects on the gut microbiome). And most importantly of all: you’ll learn how to get them in your daily diet so you can take advantage of their proven health benefits.

The infographic was written by ISAPP board of directors with input from several outside experts and coordinated by the ISAPP science translation committee.

ISAPP Releases a Mission-Based Summary of 2018 Activities

The mission of ISAPP is to advance scientific excellence in probiotics and prebiotics. ISAPP is an independent, science-based voice for the probiotic and prebiotic fields. The newly released short summary details ISAPP’s accomplishments in 2018 based around the core value of Stewardship, Advancing the Science, and Education. See here for the summary, also featuring ISAPP’s recent publications.

Thank you to the ISAPP Board of Directors for their leadership, dedication and scientific expertise, making these accomplishments possible.

Thank you to the Industry Advisory Committee for their ongoing support of ISAPP, providing the resources needed for ISAPP to accomplish its mission to advance the science of probiotics and prebiotics.

Click here to see the 2018 Summary.

See all Annual Reports and Short Summaries here.

YOGURITO –the Argentinian social program with a special yogurt

Dra. María Pía Taranto, CERELA-CONICET, Argentina and Prof. Seppo Salminen PhD, University of Turku, Finland

It is widely accepted that technologies play a central role in the processes of social change. The Argentinian experience has documented that yogurt can be a promising tool for promoting social development.  The program is called “Scholar Yogurito, the social probiotic” and the probiotic product is called “Yogurito”. This social program began with the development of a probiotic food, in the form of yogurt. This yogurt contains the probiotic strain Lactobacillus rhamnosus CRL1505, whose functional and technological characteristics are widely documented by CERELA-CONICET researchers. These researchers conducted clinical studies that demonstrated that the consumption of this probiotic product improves natural defenses and prevents respiratory and intestinal infections, the infectious events of greatest relevance in childhood. The “Yogurito Social Program” benefits some 300,000 schoolchildren in the province of Tucumán and some 50,000 in other provinces and municipalities of Argentina. This social transfer project, implemented in 2008 in the province of Tucumán, is a paradigm of interaction between the scientific sector, the manufacturing sector and the state, to improve the quality of life of highly vulnerable populations.

The social and economic implications for such translational research are significant and especially pertinent for people living in poverty, with malnutrition and exposure to environmental toxins and infectious diseases including HIV and malaria. This example of probiotic applications illustrates the power of microbes to positively impact the lives of women, men, and children, right across the food value chain. The researchers are looking for grants that would enable them to compare outcomes of schools given Yogurito to schools with no participation in the program.

 

Additional reading:

Julio Villena, Susana Salva, Martha Núñez, Josefina Corzo, René Tolaba, Julio Faedda, Graciela Font and Susana Alvarez. Probiotics for Everyone! The Novel Immunobiotic Lactobacillus rhamnosus CRL1505 and the Beginning of Social Probiotic Programs in Argentina. International Journal of Biotechnology for Wellness Industries, 2012, 1, 189-198.

Reid G, Kort R, Alvarez S, Bourdet-Sicard R, Benoit V, Cunningham M, Saulnier DM, van Hylckama Vlieg JET, Verstraelen H, Sybesma W. Expanding the reach of probiotics through social enterprises. Benef Microbes. 2018 Sep 18;9(5):707-715. doi: 10.3920/BM2018.0015.

 Senior Researcher Maria Pia Taranto and the Yogurito product

 

Maria Luz  Ovejero, a teacher at Primary School 252 Manuel Arroyo y Pinedo, explains probiotics to 4-6 year old children in Tucuman province in Argentina

Where does our food come from – why should we care?

Dr. Karen Scott, The Rowett Institute, University of Aberdeen,  Scotland

The food we eat feeds our microbes, gives us energy and nutrition, and keeps us healthy. The choices we make about our food clearly affects our health, but also has a huge effect on the world around us. We need to make more effort to choose correctly.

Sometimes it seems that everywhere we look, someone has an opinion on what we should be eating. Television is full of programmes telling us how and what to cook – suitable for a range of abilities. In supermarkets we are continually targeted with special offers and promotions, encouraging us to buy things we do not need, that are not on our shopping list. In magazines there are page long adverts, letting us know many reasons why our lives will be enriched if we purchase product Y, and perhaps even how we will be missing out if we do not. Even newspapers print articles telling us which foods are “super” this week, and will endow us with youthful skin, long life, and/or a svelte figure. Next week there will be another article with a new superfood, and one demoting last week’s superfood to the “standard” food, or even demonising it completely.

Yet even with all this focus on what we should be eating, do we really care about where our food comes from? Shouldn’t we really be more concerned with the provenance and sustainability of our food, rather than whether it is “super”?

Quinoa is a grain with a high nutrient content, high protein content (including all nine essential amino acids) and is also a source of some essential micronutrients and vitamins. By popular measures, a “superfood”. Quinoa is primarily grown in South America (Peru, Chile and Bolivia) where it is an important dietary staple. The increased demand and resultant export of quinoa has contributed considerably to the Peruvian economy. On the other hand, the cost increases associated with the increased worldwide demand means that the local Andean population now struggle to afford to include this healthy food in their own diets. Additionally the enlarged land area now used for quinoa production has reduced the amount of land available to grow alternative crops, and this reduced diversity has a negative impact on soil quality and on wildlife. Not so “super”.

Another healthy food-fad with a negative environmental impact is avocado. The current demand for avocados as part of the ‘green smoothie’ revolution has resulted in considerable deforestation in Mexico to make way for avocado plantations. Avocado trees also need a lot of water, which, given that they are frequently grown in climates with problems of drought, is clearly not sustainable.

The other factor is price – we are constantly persuaded that we should be looking for the best deal, getting those “2-for-1 offers”, or buying our food in the specific supermarket “saving you the most on your weekly shop”. The reality is that we spend a smaller % of our income on food today than we ever have – and this is not because we eat less, far from it. But if we think about it, it is not the large supermarket that loses money when it introduces offers. Buy one get one free offers on, for example fruit, usually mean that the farmer is only getting paid for one of every two oranges sold. Is this fair? If you ask a people doing their food shopping if they think that milk should cost more than water – most people would say “yes of course”. Yet at the milk counter in the supermarket they automatically reach for the “special offer”, cheapest product. Sometimes the farmer gets paid less for the milk he sells the supermarket than it costs to produce. Again if you asked people in the shop if they thought this was fair, they would no doubt say no, but they still reach for the “special offer”, cheapest product. This is already driving smaller dairy farmers out of business. Is this what we want? We as consumers, as well as the supermarkets, have to take responsibility.

Similarly with meat products and eggs. Most people, when asked about the best and most humane ways to look after animals on farms, prefer the low density, outside methods often depicted in children’s story books. Yet when we reach the meat counter in the supermarket we are more likely to reach for the cheaper product than the one from the farm which assures humane conditions, but which may cost twice as much. Such farming methods are more expensive to run, so the products have to cost more. We have to make more effort to include our instinctive morality when we are actually making purchases of food.

We have also become accustomed to being able to buy anything, at any time of year. If we want to buy fruit that is out-of-season in our own country, it will be in-season somewhere else and can be flown across the world for display in our local supermarket. When we ask people if they care about global warming – most will agree that it is a big problem, threatening the world. Yet they will buy specific fruits or vegetables that have been flown 1000s of miles, in aeroplanes contributing CO2 emissions, without a thought. Locally produced food, eaten in season, completely avoids this non-essential contribution to global warming.

Feeding our microbes is easy – they just eat our leftovers. But perhaps we also need to think about them. Food produced in intensively farmed conditions contains more pesticide and antibiotic residues than foods produced less intensively. Depending where we live, imported foods may have fewer controls on additives and production methods than those produced locally. Although specific studies have not been carried out to gauge the effect of such residues on our microbes, it is likely that there will be an effect. The healthy compounds in fruits develop best when they are allowed to ripen on the bush/tree and are not harvested unripe and then transported across the world. Our ancestors ate fresh foods in season and produced locally. People living in remote areas of the modern world without access to the diverse range of foods in a supermarket have a more diverse, healthy microbiota than those of us consuming “western diets”. Our microbes do not need, and potentially do not want, intensively produced foods.

Many of us are in the fortunate position of being able to afford to pay a bit more for our food, and thus to support it being produced in the way we would prefer if we stopped to think about it. This is why we DO have to stop to think and not automatically reach for the cheapest product on the shelf.  If we do not support farmers who are producing food in the most humane way, they will go out of business and we will be left with no choice but to buy mass-produced, often imported, food. Is this really what we want?

We have become so accustomed to paying less for our food, and looking for bargains, that we seem to care less about the quality and provenance than the price. Unless we change our outlook we will affect whole populations and environments forever. We need to stop the disconnect between our thoughts about what our foods should be, and what we actually buy, and we need to do it before it is too late.

Conference Focusing on the Microbiome in Women

By Prof. Gregor Reid, University of Western Ontario

It started with an idea for a mini symposium as an add-on to the PhD defence of Jessica Younes in 2015. It would be an event that focused on the impact of microbes on women’s health.

It had never been done before. Held in Artis, the Amsterdam Zoo and Microbiology museum, the 2015 conference attracted close to 100 people.

Following two more successful meeting in The Netherlands, “Women and their Microbes” is now coming to North America.

On March 6th and 7th next year an exciting program awaits at McMaster University’s campus in Hamilton, Ontario, a 90 minute drive from downtown Toronto.

See the program here.

Last year, I was happy to pass membership on the organizing committee to young clinicians and scientists such as Dr. Ruben Hummelen, who along with Jessica, have prepared an outstanding and practical program for 2019. Winclove B.V. remains the key sponsor, and ISAPP continues to add its voice.

As you will see from the program, there are a number of internationally recognized speakers, but also some outstanding Canadians you may not have had the pleasure to yet hear. The first day has split sessions with an emphasis on clinical practice. The second day features aspects of pregnancy influenced by microbes, including the exciting gut-brain axis research.

It is a great opportunity for scientists who have enjoyed ISAPP meetings and for members of our Students and Fellows Association to participate. At only $50 for students and $120 for faculty, you’ll be hard pressed to find a meeting with such value for money.

 

International Dairy Summit 2018 in Daejeon in South Korea

By Prof. Seppo Salminen PhD, University of Turku, Finland

The International Dairy Federation (IDF) convenes annual meetings that bring together scientists and industry professionals to discuss issues foremost to the production of safe and nutritious dairy products globally. Since probiotics find a home in so many dairy foods worldwide, ISAPP and IDF have some overlapping interests.

ISAPP president, Prof. Seppo Salminen of University of Turku, spoke at IDF’s International Dairy Summit 2018 on the potential for fermented foods to fight diseases and improve nutrition. He emphasized that many fermented foods contain a diverse collection of live microorganisms, which likely support our gut microbiota, perhaps even promoting gut microbiota resilience. Further, he stated, “Fermented dairy products, especially yoghurt, which combines milk, microbial starter cultures and pre-digested nutrients for human use, can be considered for future food-based dietary guidelines or recommendations focusing on beneficial microbe intake for gastrointestinal and other health effects.”

Another speaker, Prof Bruno Pot, discussed the global situation with regard to health claims for fermented dairy products. He focused on the situation in the European Union, where the only allowed health claim for probiotics is the benefit from live bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) in yoghurt reducing symptoms of lactose maldigestion. He reported that yoghurt is becoming a mainstream food in Asia. Key growth drivers in Asia are the perceptions that yoghurt is a healthy product with its beneficial impact on the digestive and immune systems, and they offer a good source of protein and calcium. The symposium also explored ways to enrich food through product development and innovation, particularly to provide nourishment for vulnerable populations. The potential for new ingredients such as milk protein hydrolysate-calcium complexes as calcium sources in yoghurt production was recognized.

David Everett, Chair of IDF’s Standing Committee on Dairy Science and Technology, reported: “Holding the 6th edition of the Symposium on Fermented Milks in Asia is of tremendous value as the scientific research on fermented dairy and the interest in these products is growing in the region.”

Forthcoming changes in Lactobacillus taxonomy

Mary Ellen Sanders PhD, Executive Science Officer, ISAPP

I was privileged to be included in a small meeting of scientists, both academic and industry, who met last week in Verona to discuss changes in Lactobacillus taxonomy. The first objectives of the meeting were to clarify with industry the need for the proposed changes and to clarify the methodology that will be used. The second objectives were to discuss at large potential consequences and approaches to address them.

Changes to the Lactobacillus genus

Experts from the Taxonomic Subcommittee for Lactobacilli, Bifidobacteria and Related Organisms agreed that the genus Lactobacillus is too heterogeneous and dividing this genus into several genera is inevitable. The need for this taxonomic ‘correction’ has been known for a long time, but until recently, the methodologies needed to reliably group the current Lactobacillus species into new genera were not available. But earlier this year, a paper by Salvetti et al (2018) analyzed 269 Lactobacillus and related (e.g., Pediococcus, Leuconostoc, Fructobacillus, Oenococcus) species and showed that the Lactobacillus genus comprises 10 phylogroups (see box). Each of these phylogroups represents at least one new genus. These same 10 phylogroups were observed using three separate approaches [phylogenetic analysis of 16S ribosomal DNA sequences, whole genome sequence analysis, leading to the comparison of 72 shared housekeeping genes (the core genome), and the comparison of average amino acid identity and percentage of conserved proteins], providing strong evidence that these groupings are robust. Commercially important Lactobacillus probiotic strains span at least 7 of those newly defined phylogroups; food fermentation lactobacilli cover even more.

lactobacillus_info

Although these 10 phylogroups were identified by this study, the current genus Lactobacillus could ultimately be resolved into 10 or up to 23 genera, depending on the cut-off values used for the different approaches. If researchers choose to split the genus into fewer new genera, it increases the chance that taxonomic changes will be needed in the nearer future. If they split the genus into more genera, it increases the chance that nomenclature will remain stable.

The names of the new genera are not decided. New names must be published (or validated) in the International Journal of Systematic and Evolutionary Microbiology. The authors of the publication will propose the new genus names. All species will be retained and their species names will not change. To minimize disruption, researchers will try to propose new genera names that begin with the letter “L”. Because “Lactobacillus” is a masculine Latin noun, the new genus names must be masculine for the species names to be retained.

A silver lining

Critics of these changes may suppose that adhering to taxonomic convention is their only purpose. But a classification system that better reflects genetic relatedness of the species may reap other benefits. As evidence for clinical benefits accumulates (summarized in open access review “Probiotics for Human Use”, 2018) and investigations provide insight into probiotic mechanisms of action, a clearer image of mechanisms and functions associated with particular taxonomic groups may emerge. The concept of core, shared benefits that were not strain-specific but linked to higher taxonomic groupings was explored in two ISAPP publications [Hill et al. (2014) and more in depth in Sanders et al. (2018)]. Reconsideration of clinical evidence and its relationship to new genera might prove enlightening.

What can be done to minimize confusion?

The meeting attendees brainstormed potential complications that might result from changing genus names. Company representatives in general considered that internal changes could be managed, although resources would be required to update names on all different paperwork and labels associated with commercial products (for example, marketing materials, product information, certificates of analysis, labeling, import/export certificates). The 2002 WHO/FAO probiotic guidelines, as well as the 2017 CRN/IPA guidelines, indicate that the genus, species and strain designation should be included on product labels. Further, the name used should reflect current nomenclature. This requirement is reflected in some national regulations. Therefore, genus name changes will necessitate label changes.

Further, it was emphasized that a clear document should be prepared and endorsed by reputable organizations (EFSA, NIH, FDA, medical organizations, and others). The document should: (a) indicate the name changes, (b) provide a clear, concise statement of why the changes were needed, and (c) emphasize that only the names, not the strains, would be different. This could be leveraged by companies to communicate with all stakeholders. End-users of probiotic products would likely not be a significant communication challenge. Authorities involved with probiotic safety (FDA with GRAS and EFSA with QPS) likely will manage these changes, as they are science-based. More of a concern was communication with other regulators, both at the level of national agencies responsible for probiotic-specific regulations (including countries with positive lists of species that are acceptable as probiotics) as well as authorities involved in import/export of product. Some potential issue with intellectual property may be envisaged, especially in a transition period during which the new names are not routinely used yet.

The bottom line: Name will change but the strains will stay the same 

The current Lactobacillus genus will be split into at least 10, and perhaps as many as 23, genera. No species names will change, but many species – including commercially important ones – will have a different genus names, hopefully beginning with the letter “L”.  Because of the tremendous heterogeneity of the current Lactobacillus genus, Prof. Paul O’Toole concluded his presentation saying “the status quo is not an option.” Some disruptions can be expected from this massive change, but the probiotic field would benefit from embracing these changes and developing strategies to minimize any difficulties resulting from them.

 

Additional information:

The International Committee on Systematics of Prokaryotes (ICSP) and the International Code of Nomenclature of Bacteria are responsible for the naming of bacteria. The subcommittee of the ICSP responsible for naming lactobacilli is the Taxonomic Subcommittee for Lactobacilli, Bifidobacteria and Related Organisms.

The meeting was convened by the Lactic Acid Bacteria Industrial Platform and chaired by Esben Laulund of Chr Hansens, who also chairs IPA Europe. A full report of meeting conclusions is expected to be published in a scientific journal by the end of 2018. Abstracts and program will to be posted on the LABIP website in due time.

The taxonomic hierarchy for Lactobacillus currently is: Domain: Bacteria; Division/Phylum: Firmicutes; Class: Bacilli; Order Lactobacillales; Family: Lactobacillaceae; Genus: Lactobacillus. The lowest order of taxonomy is the subspecies; the strain designation has no official standing in nomenclature. There are currently over 230 recognized species of Lactobacillus, and approximately 10 new species are added each year.

ISAPP Releases New Infographic – Probiotic Checklist: Making a Smart Selection

Not all products labelled “probiotic” are true probiotics. ISAPP just released a new infographic focused on helping consumers make smart selections when examining probiotic products. The infographic addresses identifying products backed by science, effective dosing, and more.

See and download the full infographic here.

See all ISAPP infographics here.

 

happy_baby

Probiotics and D-lactic acid acidosis in children

Prof. Hania Szajewska PhD, The Medical University of Warsaw, Department of Paediatrics, Poland and Prof. Seppo Salminen PhD, Faculty of Medicine, Functional Foods Forum, University of Turku, Finland

See related post ‘Brain Fogginess’ and D-Lactic Acidosis: Probiotics Are Not the Cause

In their recent study, Rao and colleagues1 incriminated probiotics in the induction of D-lactic acidosis (1). Many who benefit from probiotics could be frightened—on the basis of this report—into stopping them, with potentially negative impacts on their health (2). Some probiotic bacteria, including some specific components of the intestinal microbiota, may produce D-lactic acid. Indeed, if plasma D-lactic acid rises sufficiently, it is clinically relevant, causing D-lactic acidosis. D-lactic acidosis has mainly been observed in subjects with short bowel syndrome. However, some authorities have regulated the use of D-lactic acid producing bacteria in infant and weaning foods, but the reasoning for normal infant population has been debated. Even in adults, the safety of D-lactic acid producing bacteria has been challenged, but apart from short bowel patients no evidence on clinical problems has been reported (3).

For this reason, we conducted a review and examined whether D-lactic acid-producing bacteria, acidified infant formulas and fermented infant formulas were potential causes of paediatric D-lactic acidosis (4).

We identified five randomised controlled trials conducted between 2005-2017 with 544 healthy infants. Additionally, some case reports and experimental studies were considered. No clinically relevant adverse effects of D-lactic acid-producing probiotics or fermented infant formulas in healthy children were identified. The only known cases of paediatric D-lactic acidosis were observed in patients with short bowel syndrome (4). It is of importance that human milk also contains lactic acid bacteria and bifidobacteria, some of which may produce D-lactic acid. Some stress situations, such as exercise, may elevate human milk lactate concentrations.  Thus, breast milk D-lactate content needs to be analysed more carefully to compare with fermented infant formulas.

Taken together, our results suggest that neither the probiotics that were evaluated in the studies we reviewed nor fermented infant formulas cause D-lactic acidosis in healthy children.

 

  1. Rao, S. S. C., Rehman, A., Yu, S. & Andino, N. M. Brain fogginess, gas and bloating: a link between SIBO, probiotics and metabolic acidosis.  Transl. Gastroenterol.9, 162 (2018). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006167/
  2. Sanders, M. E., Merenstein, D. & Merrifield, C. A. Probiotics for human use.  Bull.43, 212–225 (2018). https://onlinelibrary.wiley.com/doi/10.1111/nbu.12334
  3. Quigley E.M.M, Pot B., Sanders M.E. ‘Brain fogginess’ and D-lactic acidosis: probiotics are not the cause. Transl. Gastroenterol.9, 187 (2018). https://www.nature.com/articles/s41424-018-0057-9
  4. Łukasik, J., Salminen S., Szajewska H. Rapid review shows that probioticsand fermented infant formulas do not cause D-lactic acidosis in healthy children. Acta Pediatrica 107, 1322-1326 (2018). https://www.ncbi.nlm.nih.gov/pubmed/29603358

FDA/NIH Public Workshop on Science and Regulation of Live Microbiome-based Products: No Headway on Regulatory Issues

September 20, 2018

By Mary Ellen Sanders, PhD, Executive Science Officer, ISAPP

On September 16, 2018, the US Food and Drug Administration’s Center for Biologics Evaluation and Research (CBER) and National Institute of Allergy and Infectious Diseases (NIAID) collaborated on the organization of a public workshop on “Science and Regulation of Live Microbiome-based Products Used to Prevent, Treat, or Cure Diseases in Humans”.  I was present at this meeting along with ISAPP vice-president, Prof. Daniel Merenstein MD, who lectured on the topic of probiotics and antibiotic-associated diarrhea.

Prof. Dan Merenstein speaking at CBER/NIAID conference

While regulatory issues are often discussed at other microbiome conferences, the fact that this meeting was organized by the FDA suggested it was a unique opportunity for some robust discussions and possible progress on regulatory issues involved with researching and translating microbiome-targeted products. The regulatory pathways to drug development seem clear enough, but regulatory issues for development of functional foods or supplements are less clear. Jeff Gordon and colleagues have previously pointed out regulatory hurdles to innovation of microbiota-directed foods for improving health and preventing disease (Greene et al. 2017), and at the 2015 ISAPP meeting, similar problems were discussed (Sanders et al. 2016).

The meeting turned out to be mostly about science. Some excellent lectures were given by top scientists in the field (see agenda below), but discussion about regulatory concerns was a minimal component of the day. Questions seeding the panel discussions focused on research gaps, not regulatory concerns: an unfortunate missed opportunity.

Bob Durkin, deputy director of the Office of Dietary Supplements (CFSAN), left after his session ended, suggesting he did not see his role as an important one in this discussion. One earlier question about regulatory perspectives on prebiotics led him to comment that the terms ‘probiotic’ and ‘prebiotic’ are not defined. From U.S. legal perspective he is correct, as there are no laws or FDA regulations that define these terms. But from a scientific perspective, such a statement is disappointing, as it shows the lack of recognition by U.S. regulators of the widely cited definitions developed by top researchers in these fields and published in 2014 and 2017, respectively.

Two issues not addressed at this meeting will require clarification from the FDA:

The first is how to oversee human research on foods or dietary supplements. CBER’s oversight of this research has meant most studies are required to be conducted under an Investigational New Drug (IND) application. From CBER’s perspective, these studies are drug studies. However, when there is no intent for research to lead to a commercial drug, the IND process is not relevant. Even if endpoints in the study are viewed as drug endpoints by CBER, there should be some mechanism for CFSAN to make a determination if a study fits legal functions of foods, including impacting the structure/function of the human body, reducing the risk of disease, or providing dietary support for management of a disease. When asked about this, Durkin’s reply was that CFSAN has no mechanism to oversee INDs. But the point was that without compromising study quality or study subject safety, it seems that FDA should be able to oversee legitimate food research without forcing it into the drug rubric. CBER acknowledged that research on structure/function endpoints is exempt from an IND according to 2013 guidance. But FDA’s interpretation of what constitutes a drug is so far-reaching that it is difficult to design a meaningful study that does not trigger drug status to them. For example, CBER views substances that are given to manage side effects of a drug, or symptoms of an illness, as a drug. Even if the goal of the research is to evaluate a probiotic’s impact on the structure of an antibiotic-perturbed microbiota, and even if the subjects are healthy, they consider this a drug study. With this logic, a saltine cracker eaten to alleviate nausea after taking a medication is a drug. Chicken soup consumed to help with nasal congestion is a drug. In practice, many Americans would benefit from a safe and effective dietary supplement which they can use to help manage gut disruptions. But in the current regulatory climate, such research cannot be conducted on a food or dietary supplement in the United States. There are clearly avenues of probiotic research that should be conducted under the drug research oversight process. But for other human research on probiotics, the IND process imposes research delays, added cost, and unneeded phase 1 studies, which are not needed to assure subject safety or research quality. Further, funders may choose to conduct research outside the United States to avoid this situation, which might explain the low rate of probiotic clinical trials in the United States (see figure).

The second issue focuses on actions by CBER that have stalled evidence-based use of available probiotic products. This issue was discussed by Prof. Merenstein in his talk. He pointed out that after the tragic incident that led to an infant’s death from a contaminated probiotic product (see here; and for a blog post on the topic, see here), CBER issued a warning (here) that stated that any probiotic use by healthcare providers should entail an IND. This effectively halted availability of probiotics in some hospital systems. For example, at Johns Hopkins Health-system Hospitals, the use of probiotics is now prohibited (see below). Patients are not allowed to bring their own probiotics into the hospital out of concern for the danger this poses to other patients and staff. This means that a child taking probiotics to maintain remission of ulcerative colitis cannot continue in the hospital; an infant with colic won’t be administered a probiotic; or a patient susceptible to Clostridium difficile infection cannot be given a probiotic. Available evidence on specific probiotic preparations indicates benefit can be achieved with probiotic use in all of these cases, and denying probiotics can be expected to cause more harm than benefit.

It might be an unfortunate accident of history that probiotics have been delivered in foods and supplements more than drugs. The concept initially evolved in food in the early 1900’s, with Metchnikoff’s observation that the consumption of live bacilli in fermented milk had value for health. Probiotics have persisted as foods through to the modern day, likely because of their safety. The hundreds of studies conducted globally, including in the U.S. until 10-15 years ago, were not conducted as drug studies, even though most would be perceived today as drug studies by CBER. This has not led to an epidemic of adverse effects among study subjects. True, serious adverse events have been reported, but the overall number needed to harm due to a properly administered probiotic is negligible.

According to its mission, the FDA is “…responsible for advancing the public health by helping to speed innovations that make medical products more effective, safer, and more affordable and by helping the public get the accurate, science-based information they need to use medical products and foods to maintain and improve their health.” Forcing human research on products such as yogurts containing probiotics to be conducted as drug research, when there is no intent to market a drug and when the substances are widely distributed commercially as GRAS substances, does not advance this mission. Further, CBER actions that discourage evidence-based use of available probiotics keeps effective and safe products out of the hands of those who can benefit.

A robust discussion on these issues was not part of the meeting earlier this week.  Researchers in the United States interested in developing probiotic drugs will find CBER’s approaches quite helpful. Yet researchers interested in the physiological effects of, or clinical use of, probiotic foods and supplements will continue to be caught in the drug mindset of CBER. CFSAN does not seem interested. But without CFSAN, human research on, and evidence-based usage of, probiotic foods and supplements will continue to decline (see figure), to the detriment of Americans.

Human clinical trials on “probiotic”
1992-September 20, 2018

 

 

 

ISAPP-initiated systematic review and meta-analysis shows the association of probiotic consumption with reduced antibiotic prescriptions

At the ISAPP meeting in Turku, Finland in 2016, scientists convened a working group led by Dan Merenstein of Georgetown University (USA) along with Irene Lenoir-Wijnkoop of University of Utrecht (the Netherlands) and Danone Research. In their discussions, the group identified a gap in the literature: a systematic review of randomized, controlled trials to determine how antibiotic prescriptions are associated with probiotic consumption for the prevention of common acute infections. The protocol was registered with PROSPERO (registration number CRD42016052694).

The analysis, authored by ten scientists, was recently published—and results showed that infants and children who received probiotics were at least 29% less likely to be prescribed antibiotics. Find the paper here in the European Journal of Public Health.

ISAPP scientists say probiotics deserve consideration as a public health intervention that may reduce the widespread over-prescription of antibiotics.

See the ISAPP press release here, and the Georgetown University press release here.

See here for media coverage of this paper:

http://www.microbiometimes.com/scientific-analysis-shows-probiotic-use-is-associated-with-fewer-antibiotic-/

https://www.pharmacytimes.com/resource-centers/vitamins-supplements/daily-probiotics-may-reduce-kids-need-for-antibiotics

https://www.news-medical.net/news/20180914/Probiotics-could-reduce-the-need-for-antibiotics.aspx

Clinical evidence and not microbiota outcomes drive value of probiotics

By ISAPP Board of Directors, plus Prof. Francisco Guarner and Dr. Bruno Pot

September 10, 2018

Two recent papers have generated much adverse publicity for the probiotic field. Headlines driven by sensationalism, not data, claim “Probiotics labelled ‘quite useless’” (BBC) and “Probiotics ‘not as beneficial for gut health as previously thought’” (The Guardian). The quotes are from author Eran Elinav, who generalizes the study findings to all ‘probiotics’ as a class – a generalization that ignores that specific probiotic are meant for specific purposes. This research was published this month in Cell (here and here).

The scope of these papers is limited to microbiome data; no clinical endpoints are assessed. Without clinical evidence, it is not possible to conclude about the tested probiotic’s usefulness, and it is certainly not possible to conclude about probiotic usefulness in general. Stating that probiotics are ‘quite useless’ or ‘not as beneficial’ is, quite simply, wild and factually inaccurate. The authors discount the existing body of evidence for probiotic health benefits, including Level 1 placebo-controlled, randomized trials. Cochrane reviews (the gold standard used by physicians and public health policy makers) of the totality of evidence show that specific probiotics can prevent antibiotic associated diarrhea (AAD) and C. difficile diarrhea. This evidence has been translated into evidence-based recommendations for probiotics issued by medical groups. Regardless of an effect on the microbiota, these are established, evidence-based benefits of probiotics.

No clinical endpoints tracked in either study

What these papers provide is extensive data about the impact of one product containing 11 common probiotic species on different microbiome measures. To the authors’ credit, they analyzed mucosal and luminal samples from humans, in addition to samples from stool.  Nonetheless, the probiotic definition [live microorganisms that, when administered in adequate amounts, confers a health benefit on the host (Hill et al 2014)] does not require that probiotics function via interaction with the microbiota, nor is there much evidence that they alter the microbiota composition in an appreciable manner. Absence of impact on microbiome measures is not evidence that probiotics lack clinical or physiological effects. Probiotics function via many mechanisms that might not be revealed by the measures made in these papers.

Methodological concerns

A careful reading of this paper reveals many methodological concerns.

The extensive data in the paper is an assortment of different types of analyses. For example, for a beta-diversity metric, they sometimes use weighted Unifrac, sometimes unweighted Unifrac, and sometimes Bray-Curtis, without an explanation for their choice. These approaches to presenting the data can give very different results. With the transcriptomics data, sometimes the authors choose samples from the duodenum and sometimes the jejunum. For example, in figure 6, panels C-E compare the difference in gene expression between the naïve group and the treatment in the duodenum, whereas in panels F-H they compare the antibiotic state with the treatment in the jejunum. Such an approach leads the reader to speculate that the authors picked the metrics and data that best fitted the story they wanted to tell. In a well-conducted clinical trial, the statistical plan is registered before the study starts, to assure readers that the scientific process of advancing a hypothesis and designing a study to test the hypothesis is respected.

The probiotic was not administered to human subjects until 7 days after the treatment with antibiotics commenced, after the damage by the antibiotics has been done. Dozens of human studies with specific probiotics have documented that probiotics prevent AAD or C. difficile infection. In most clinical trials, the probiotic is administered together with the antibiotics. A recent meta-analysis concluded that “administration of probiotics closer to the first dose of antibiotic reduces the risk of (Clostridium difficile infection) by >50% in hospitalized adults.” (Emphasis added) The approach in the Suez et al paper is not consistent with the aforementioned clinical studies, with how probiotics are used in clinical practice or with the knowledge of how probiotics most likely prevent AAD. When provided on the same days as antibiotics, probiotics have the opportunity to prevent overgrowth of opportunistic, antibiotic-resistant microbes by competitive exclusion in the ecosystem. Therefore, the microbiome findings of Suez et al likely cannot be applied to clinical trials with such different time course of antibiotic/probiotic administration.

Several conclusions about the effect of probiotics on the microbiota were based on relative abundance measures, which do not relate to actual bacterial numbers or metabolic activity of all relevant species in the gut.

The antibiotic treatment used was potent for a study population that would otherwise not need antibiotics. Volunteers were administered oral ciprofloxacin 500 mg bi-daily and oral metronidazole 500 mg tri-daily for a period of 7 days. They are both very strong and indiscriminate antibiotics, having a severe impact on the gut microbiota.  One could question if this drug therapy might have a different impact on the microbiome of a healthy person compared to a patient likely to receive this treatment, i.e., one whose microbiota ecosystem is disrupted by disease or fever.

The probiotic product

A serious issue is that the authors chose a product for this study that has no demonstrated clinical benefits. At a minimum, the product used for this study should have evidence for impact on antibiotic associated conditions, including symptoms or emergence of opportunistic pathogens. The 3 (possibly 2, as the latter 2 appear to be the publication of the same data) human studies conducted on this product (here, here and here), showed no clinical benefit. Thus, the investigators tested the potential benefits of a product for which no benefits had been previously shown. Further, the papers do not adequately describe the product; only a total count (25 billion) is given; counts of each strain – through the end of the administration period – should have been provided. Furthermore, the authors state about the product that “B. longum was probably represented by two strains.” This constitutes imprecise characterization unacceptable in a well-defined probiotic product.

Appearance of author bias

The conclusions reached in the papers promote a personalized approach to probiotic use. In an article on the BBC, the lead author stated, “In the future probiotics will need to be tailored to the needs of individual patients. And in that sense just buying probiotics at the supermarket without any tailoring, without any adjustment to the host, at least in part of the population, is quite useless.” The authors did not disclose they are involved with a company promoting this personalized approach.

Probiotic colonization

The authors suggest that their finding that probiotics do not colonize long term is noteworthy. In fact, researchers in this field have known this for 30 years: most probiotics do not colonize or become established as part of the resident microbiota. A 2016 paper by Madonado-Gomez et al was notable precisely because a Bifidobacterium longum strain was found that did persist. In most cases, probiotic effects are likely mediated by transient effects.

Responders and non-responders

A well-established concept in medicine is that some people respond clinically and physiologically to interventions and others don’t. This is the case with much of probiotic as well as pharmaceutical literature. (See review on responders and non-responders to probiotics by Reid et al.) An individual’s response is likely impacted by diet, resident microbes, host genes and host physiology/health. The validity of a personalized approach to probiotic administration remains to be determined, as evidence for a clinical benefit to the approach is needed. Microbiome data alone are not sufficient.

Need for future research

In the Cell publications, the authors acknowledge their study was limited due to lack of clinical endpoints and the testing of only a single product. It is unfortunate that the press marched ahead with inflammatory stories about the negative effects of probiotics based on such paltry evidence. The scientific community understands that this is one study, on a small number of human subjects, by one research group. Sweeping conclusions cannot be made. There are many hypotheses that can be generated from this study that can lead to follow up studies, which we hope will ensue.

Conclusions

Hundreds of human trials have demonstrated clinical benefits of probiotics and several evidence-based recommendations have been issued by medical organizations. Of course, not all studies are positive. Not all probiotics work for all conditions. But the safety record of probiotics administered to healthy as well as many patient populations is well-established. Numerous media outlets have reported on these two studies as if they are proof that probiotics are useless at best and harmful at worst. This irresponsible reporting may lead people who are benefitting from probiotics to stop using them, potentially causing real harm.

The erroneous interpretation of the current study and previous research by the primary author is disingenuous, as he states,  “Contrary to the current dogma that probiotics are harmless and benefit everyone, these results reveal a new potential adverse side effect of probiotic use with antibiotics that might even bring long-term consequences.” This comment and the papers’ conclusions are not corroborated by the totality of safety and efficacy clinical evidence on probiotics, which includes thousands of probiotic-treated subjects. In comparison, the data in Suez et al come from microbiome assessments from only eight probiotic-treated subjects.

Furthermore, this paper evaluated just one product of limited provenance and containing a combination of multiple, incompletely characterized strains. This is in sharp contrast to numerous studies of precisely characterized strains demonstrating well-defined and beneficial engagements with the host. Zmora and colleagues and Suez and colleagues are to be congratulated on their attempts to characterize in detail the impact of one probiotic product on a perturbed, human microbiome. We look forward to further such studies employing well-characterized strains with demonstrated clinical benefits and including relevant clinical endpoints.

Additional reading:

ISAPP comments: International Group of Probiotic Scientists Weighs in on Flawed Conclusions From New Scientific Papers

American Gastroenterological Association response: AGA’s Interpretation of the Latest Probiotics Research

Response by Prof. Gregor Reid:  Trying to Close the Stable Door After the Horse Has Bolted

Role of citizen science in research on fermented foods

By Prof. Sarah Lebeer, Universiteit Antwerpen

Spontaneous vegetable fermentations, with their rich flavors and potential health benefits, are regaining popularity among chefs and the general public. Famous Michelin star chefs, such as Belgium’s Kobe Desramaults, have implemented fermented vegetables in their recipes and offer fermented vegetable juices as non-alcoholic alternatives to wine. Serendipity was surely at play when I made contact with Kobe and his team, and had the opportunity to explore the microbial life of many of his fermented food and beverages.

Thanks to this spontaneous collaboration, I became intrigued by fermented vegetables as a promising alternative to dairy probiotic matrices. They have several benefits:

  • they are lactose-free
  • they contain no milk allergens
  • they are naturally vitamin-, antioxidant- and fiber-rich
  • they are vegan, satisfying the growing dietary trend

 

Together with prof. L. De Vuyst – a fermented food specialist from the VUB University in Brussels – we attracted a talented PhD student Sander Wuyts to study Lactobacillus’ role in the spontaneous fermentation process of carrot juices. I admit that fermented carrot juice is not the tastiest beverage I ever drank, but the fermentation process turned out to be scientifically intriguing: it appeared to be a robust, man-made microbial ecosystem dominated by lactic acid bacteria. We now often use this fermentation process in my lab as a model to study various aspects of niche-adaptation and niche-flexibility of lactic acid bacteria (LAB). And if you mix carrot juice with another fresh vegetable juice, such as cucumber, you’ll be surprised by its interesting light acidic flavor!

But perhaps the most rewarding part about our fermented-vegetables project was that we managed to carry out a Citizen Science project with the Flemish name, Ferme Pekes. You could translate it as ‘Fantastic Carrots’ 😊. Forty citizens volunteered to set up their own carrot juice fermentations at home and delivered with great enthusiasm samples of different time points. The carrots originated from their own garden, the supermarket or organic stores. Our analysis indicated that origin or organic compared to conventional product did not impact the microbial community composition. But we also could show that the LABs – first Leuconostoc then Lactobacillus – out-competed the undesirable Enterobacteriaceae after 3 to 13 days of fermentation. Longer times were needed for carrots derived from winter storage.

Our analyses (phylogenetic placement and comparative genomics, which was recently published in Applied and Environmental Microbiology) also indicated that a high LAB diversity was achieved in the different spontaneous fermentations. This is of interest if you believe it is important to let our immune system come into contact with a large and naturally diverse dose of beneficial bacteria. This idea has been promoted through the years as the hygiene hypothesis or microbial deprivation theory and aligns perfectly with the surge of interest in the health benefits of naturally fermented foods. See the recent ISAPP blog from Prof. Colin Hill, who advocates for the idea of a recommended daily allowance of consumption of live microbes.  (See also a related ISAPP blog here.) Such guidelines should be taken with precaution: the fermentations must be done properly with regard to food safety (see ISAPP blog on Making Safe Fermented Foods at Home).

Citizen Science refers to projects where citizens are actively involved in scientific studies, although it has various definitions and descriptions. In our case, it allowed us to obtain a much larger and more diverse set of samples than we could have created in the lab. Furthermore, the opportunity to directly (on e.g. workshops for adults and kids or at delivery of their samples) or indirectly (as a response to articles in the popular press) communicate with citizens helped us greatly in identifying which other research questions might be of importance for the general public. This approach is increasingly implemented in the fermented food and microbiome field. There are examples of fantastic projects such as on sourdough from Rob Dunn, Benjamin Wolfe and colleagues, the Global FoodOmics initiative and the Flemish Gut Flora project, which will also be presented by Dr. Gwen Falony at our next ISAPP meeting in Antwerp. I am not aware of a Citizen Science project in the probiotic or prebiotic area, but it might be a good idea for a joint ISAPP initiative, for science communication, the creation of richer datasets, validation/confirmation of probiotic efficacy, inspiration for future research questions, for example.

‘Brain fogginess’ and D-lactic acidosis: probiotics are not the cause

Mary Ellen Sanders PhD, Executive Science Officer, International Scientific Association for Probiotics and Prebiotics

Bruno Pot PhD, Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

See here for ISAPP letter to the Clinical and Translational Gastroenterology editor regarding this paper.

See related post Probiotics and D-Lactic Acid Acidosis in Children

Rao and colleagues incriminated probiotics in the induction of D-lactic acidosis in their paper titled “Brain fogginess, gas and bloating: a link between small intestinal bacterial overgrowth (SIBO), probiotics and metabolic acidosis” (Rao et al. 2018). Eamonn Quigley MD, Bruno Pot, microbiologist and I on behalf of ISAPP authored a letter to the editor of Clinical and Translational Gastroenterology (currently In Press), summarizing many medical and other concerns with the study design, execution and conclusions.

It is regrettable that one poorly controlled paper can lead to such negative backlash on the probiotic field. Respectable media outlets including Newsweek, Science Daily, Psychology Today, the Daily Mail, MSN.com, and others blindly reported the results of this study without critical analysis of the paper. These stories advance the opinion that probiotics are potentially harmful and should be sold only as drugs. This flies in the face of many scientific studies that document safety compounded with safe, worldwide consumption for decades of probiotic foods and supplements.

Bifidobacterium as a genus does not yield D-lactate as a metabolic end product. Some Lactobacillus species do (Table 19.1 in Pot 2014). Among common probiotic Lactobacillus species, the following are classified as species that can produce D-lactic acid: L. acidophilus, L. gasseri, L. delbrueckii subsp. bulgaricus (one of the 2 yogurt starter culture bacteria), L. fermentum, L. lactis, L. brevis, L. helveticus, L. plantarum and L. reuteri.  Individual strains within each species may vary with regard to levels of D-lactic acid produced.

The observational nature of the Rao et al. paper precludes any conclusive link between probiotic consumption and symptoms observed.  The authors acknowledge that they have only established an association between probiotic use and the symptoms, but the misleading paper title suggests an intention to indict probiotics, even in the absence of evidence.  It is much more likely that the patient population with underlying SIBO in this study sought relief from their gut symptoms by use of probiotics rather than the probiotics being the cause of their symptoms.

D-lactic acidosis is a rare but serious condition, typically occurring in people with short bowel syndrome. These patients should know that D-lactate-producing probiotics are not recommended for them. In people with a normal gut, D-lactate produced by members of the gut microbiota – including some probiotics – is metabolized by other members of the gut microbiota and does not accumulate. Thus, under normal circumstances, D-lactic acidosis does not result from consumption of D-lactic acid-producing probiotics. The patients in the Rao et al. study showed very low levels of D-lactic acid, calling into question if these SIBO patients were even acidotic.  Moreover, the D-lactic acid that was present was not proven to be a result of probiotic growth. This is important, as intestinal bacteria including Escherichia coli also produce D-lactic acid. In cases of SIBO, numerous metabolites are produced in the small intestine (including alcohol), leading to a variety of SIBO symptoms, possibly including the poorly defined phenomenon of “brain fogginess”.  Many issues that should have been were not addressed in the Rao et al. paper.

The real tragedy with the publication of this paper is that – similar to many such media scares in the past – it is may cause harm.  The sensationalist headlines may dissuade safe probiotic use in people who can truly benefit from them. Scientists and clinical researchers – both academic and from industry – must remain diligent in assessment and reporting of any probiotic harms. However, the Rao et al. paper is not an example of this.

Reference:

Pot B. 2014. The genus Lactobacillus. Chapter 19. In Lactic Acid Bacteria: Biodiversity and Taxonomy, First Edition. Edited by Wilhelm H. Holzapfel and Brian J.B. Wood. 2014 John Wiley & Sons, Ltd.

Other reading:

Mack D. 2004. D(-)-lactic acid producing probiotics, d(-)-lactic acidosis and infants. Canadian J Gastroenterol. 18:671-5. (ISAPP-commissioned paper)

Łukasik J, Salminen S, Szajewska H. Rapid review shows that probiotics and fermented infant formulas do not cause d-lactic acidosis in  healthy childrenActa Paediatr. 2018 Aug;107(8):1322-1326. doi: 10.1111/apa.14338. Epub 2018 Apr 24.