New global guidelines for probiotics and prebiotics for gut health and disease

/in ISAPP Science Blog /by KC

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

The use of probiotics and prebiotics in the practice of gastroenterology must be guided by evidence – and with new evidence continually emerging, clinicians can benefit from efforts to summarize this evidence and determine how it applies in clinical practice.

In February 2023, the World Gastroenterology Organisation provided an updated resource in this area, titled “WGO Practice Guideline. Probiotics and Prebiotics”. This project was led by Prof. Francisco Guarner MD PhD, a clinical gastroenterologist and clinical researcher in probiotics and prebiotics, and brought together experts in gastroenterology, pediatrics, family medicine, probiotics, and prebiotics. Prof. Hania Szajewska MD PhD, a clinical pediatrician and clinical researcher in probiotics from the Medical University of Warsaw, was integral to assessing evidence for pediatric populations for the guidelines. Mary Ellen Sanders PhD co-chaired the project.

For 2023 update, 800 bibliographical entries of papers published in the 2017-2021 period were scrutinized. The review team adopted the guidelines for evaluation of probiotics established by FAO/WHO experts in 2002, where at least one double blind, randomized, placebo-controlled human trial with appropriate sample size and primary outcome is required to determine if the tested product is efficacious, and qualifies as a probiotic.

ISAPP was well-represented among the experts involved on the project, as four current board members contributed. In addition to Sanders and Szajewska, Prof. Dan Merenstein MD (current ISAPP president) and Prof. Seppo Salminen PhD (current past president) populated the team.

The Guideline is intended to provide specific information on interventions that may have benefit for indicated conditions. Recommendations included probiotics or prebiotics found in at least one randomized, controlled trial showing benefit. Trials that did not show benefit were not included. The Guideline serves an important role in informing gastroenterologists around the world, especially in regions where product availability might be limited. Especially useful are Tables 8 and 9, which summarize evidence for adult and pediatric uses, respectively.

Guarner states, “We hope our WGO guideline will assist doctors, pharmacists, dietitians and other healthcare professionals all around the world to integrate probiotics and prebiotics in an evidence-based manner into their daily work of patient care.”

The Guideline provides text that introduces current understanding of probiotics and prebiotics and then comprehensively evaluates the evidence for gastrointestinal conditions. Evidence is graded from 1-3, with Level 1 referring to evidence supported by systematic review of randomized trials, Level 2 supported by randomized trials with consistent effect, without systematic review, and Level 3, supported by a single randomized controlled trial, as per the Oxford Centre for Evidence-Based Medicine.

The 2017 iteration of these guidelines was available in six languages (English, French, Portuguese, Mandarin, Russian and Spanish). This guideline is the most accessed guideline title on the WGO website,  accounting for nearly one-quarter of all visits to the site. The 2023 version is only available in English so far, but translations are underway.

Clinical conditions for which some evidence was found include:

  • Diarrheal conditions: acute, antibiotic-associated, difficile-associated, radiotherapy-associated, enteral nutrition-associated, nosocomial,
  • Diverticular disease
  • Functional abdominal pain
  • Functional constipation
  • Insulin resistance
  • Health-related quality of life
  • Helicobacter pylori infection
  • Hepatic encephalopathy
  • Infantile colic
  • Inflammatory bowel disease
  • Irritable bowel syndrome
  • Lactose maldigestion
  • Nonalcoholic fatty liver disease
  • Nonalcoholic steatohepatitis
  • Necrotizing enterocolitis

 

About WGO:
World Gastroenterology Organisation (WGO) is a federation of over 100 Member Societies and four Regional Associations of gastroenterology representing over 60,000 individual members worldwide.  The WGO Guidelines Library contains practice guidelines written from a viewpoint of global applicability. The Guidelines go through a rigorous process of authoring, editing, and peer review and are as evidence based as possible.

Are the microbes in fermented foods safe? A microbiologist helps demystify live microbes in foods for consumers

/in Consumer Blog, ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola, PhD,  Associate Professor of Microbiology at the Faculty of Chemical Engineering from the National University of Litoral and Principal Researcher from CONICET at the Dairy Products Institute (CONICET-UNL), Santa Fe, Argentina.

Since very early in my career I was drawn to science communication. I feel that rather than just producing my own results, silently in my lab, I can extend the reach of the science by amplifying other people’s work. At least in the southern cone where budgets for research have been always limited, science communication is a way to be active in science.

Before the pandemic I used my Instagram account mostly to share personal moments with my circle of family and friends. But when the COVID-19 pandemic hit, I saw interest in fermented foods skyrocket. I started sharing tips about how to prepare fermented foods, telling the science behind them, separating myths from facts, making Instagram Live videos with fermentationists, nutritionists, pediatricians and gastroenterologists, and I turned my personal Instagram account into a public one with an outreach of more than 100,000 followers (@gvinde), from Mexico down to Argentina.

During the pandemic, people were largely homebound and concerned about staying healthy.  The idea of healthy food to keep a diverse gut microbiome that had the potential to enhance our gut and respiratory immune systems against coronavirus really resonated with people. I even had the chance to participate in several radio and TV programs discussing these topics as well as making yoghurt, kefir, kombucha, sauerkraut and sourdough bread at home. I saw that people had the time to devote part of their days at home to keep these communities of microbes “cooking” for them. But these activities revealed to me that more people than I realized did not know that we can eat microbes in a safe way and that they may actually be good for us.

In my encounters, I found much confusion about fermented dairy products. People believe that dairy products must be kept refrigerated, but at the same time they see ultrapasteurized milk, powdered milk or hard cheeses marketed at room temperature. People find it difficult to understand why pasteurized milk should go in the refrigerator but not unopened ultrapasteurized milk.

Some hesitancy around bacterial safety exists because Argentina leads the world in annual cases of Uremic Hemolitic Syndrome (UHS), a life-threatening condition for children, especially those under the age of 5 years, caused by shiga-toxin producing Escherichia coli. Almost 400 children get sick in Argentina every year due to UHS. Among other recommendations, pediatricians tell parents not to offer their children unpasteurized dairy products. This leads to the the most common question I receive on Instagram from parents worried about yoghurt safety: Is yoghurt pasteurized?  “No!” I emphasize. “Yoghurt is not pasteurized, but it is made out of pasteurized milk. In fact, yoghurt has viable bacteria.” And this is when the panic begins.

“If yoghurt has live bacteria, then can’t any bacteria grow there, even the bacteria responsible for UHS? If I leave yoghurt outside the refrigerator or in my car too long, won’t this make it more likely that the UHS bacteria will grow?” This is where I try to use an army of arguments to communicate science in the simplest possible way, from more philosophical to more science-based facts.

The first thing I share is that fermentation was invented well before refrigerators. Fermentation was used by people to preserve foods, for periods well longer than the time it takes to take the yoghurt from the supermarket to make it home or than the time a yoghurt sits in the backpack of my child waiting for school lunchtime. I once posted that I ate a yoghurt that was left in my car for one whole day. That generated a lot of debate on social media!

Then I inform them that the fermentation process to make yoghurt causes the pH to drop well below values needed for pathogens to grow. That it is highly unlikely that a pathogen can enter a well-sealed yoghurt, and in the event that it would be possible, the acidic conditions would impair the pathogen from growing to a level that could be life-threating.

People not only worried about yoghurts bought in the supermarket, well-sealed and made under the strictest safety conditions in industry. In the pandemic many parents learned how to make yoghurt at home, and they wanted to know how safe it is. In these cases, I advised the following to assure their homemade yogurt was safe: use a yoghurt from the supermarket to launch your own fermentation, use pasteurized milk, use good quality water to wash your kitchen devices, and wash your hands properly. In addition you can use a domestic pHmeter or pH indicators to make sure pH dropped below 4.5. In a successful fermentation – after about 1 gallon sitting 8-12 hours at a warm temperature – the fluid milk will transform into a gel. If not, you should discard it.

If these arguments are not enough, then I draw their attention to the well-respected product milk kefir. At least in this region, kefir is surrounded by a halo of “something that is good, no matter what”. People are familiar with the process of fermenting milk kefir at room temperature for a full day. So I make this comparison: commercial yoghurt is fermented for 6 hours, then it is refrigerated and taken to the supermarket. If you are OK letting milk kefir ferment for a whole day, shouldn’t yogurt sitting without refrigeration for a few more hours be harmless enough? It likely would only get more acidic because bacteria will resume fermentation. This fermented food would not become a life-threatening food in just a couple of hours. If milk kefir does not in 24 hours, why should yoghurt?

To further argue, I comment that kombucha is fermented at room temperature for 10 days, sauerkraut for 2 weeks and kimchi for several months. And they are all consumed with their microbes alive. They key is that the microbes that flourish make the environment inhospitable to pathogens.

Still I feel that there is a lot of uncertainty among consumers about the safety of fermented foods and this is may be an obstacle to making them more popular. Scientists must meet the challenge to communicate to lay audiences about how to make fermented foods safely at home and how to store them so they are safe. Nothing is ever 100% safe, but the small risks associated with fermented foods are greatly outweighed by the enjoyment of making and consuming fermented foods.

 

Additional reading:

Suggestions for Making Safe Fermented Foods at Home

2022 TEDx talk

2021 Teaching how to make kefir on TV during the pandemic

2019 participation in Argentina’s most famous TV show, featuring the same host for more than 50 years non-stop

Horse with rider.

Probiotic Use in Horses: What is the Evidence?

/in ISAPP Science Blog /by KC

By Kelly S. Swanson, PhD, The Kraft Heinz Company Endowed Professor in Human Nutrition, University of Illinois at Urbana-Champaign, USA

Horses play a special role in many people’s lives, serving as a partner in leisure activities, therapy, various forms of work, and athletic competitions. Being large herbivores, they are adapted to a diet rich in grasses and other high-fiber forages. The complex community of microbes inhabiting the hindgut (cecum and colon) is necessary for the efficient breakdown of these fibers as well as maintaining the gastrointestinal health and overall health of horses. In recent years, a lot has been learned about the composition and activity of the gastrointestinal microbiota of horses and their role in health and disease (Kauter et al, 2019). There has also been interest in testing whether yeast- or bacteria-based probiotics may help manage equine health and disease.

Is there evidence supporting probiotic use in horses? The answer depends on the animal’s life stage, dietary and exercise strategy, and health status.

Probiotics for foals

A common target of probiotic use has been young growing foals. Similar to other host species, the gastrointestinal microbiota population of foals has a lower diversity and stability than that of adult horses (Earing et al., 2012; De La Torre et al., 2019). This instability makes foals more susceptible to pathogen-induced microbiota alterations, diarrhea, dehydration, and intestinal inflammation (Frederick et al., 2009; Schoster et al., 2017; Oliver-Espinosa, 2018). But probiotic use in foals has had both helpful and harmful outcomes. Positive results were obtained with a probiotic containing 5 Lactobacillus strains (L. salivarius YIT 0479, L. reuteri YIT 0480, L. crispatus YIT 0481, L. johnsonii YIT 0482, L. equi YIT 0483), which were shown to increase body weight and reduce diarrhea incidence in 3-4 week old foals (Yuyama et al., 2004). Similarly, a probiotic composed of 4 Lactobacillus strains (L. reuteri KK18, L. ruminis KK14, L. equi KK15, L. johnsonii KK21) and 1 Bifidobacterium strain (B. boum HU) was reported to reduce the incidence and duration of diarrhea in foals during their first 5 months of life (Tanabe et al., 2014). However, administration of a different probiotic (L. pentosus WE7) was associated with anorexia, development of diarrhea, and greater need for veterinary examination and treatment (Weese and Rousseau, 2005). Based on the evidence thus far, caution should be used when considering probiotic use in foals.

Probiotics for adult horses

Even though adult horses have a more stable and rich gastrointestinal microbiota than young animals, microbiota disruptions can occur with rapid changes in diet, transportation stress, the onset of gastrointestinal disease, or other diseases such as laminitis or grass sickness (Garrett et al., 2002; Costa et al., 2012; Moreau et al., 2014). Horses are susceptible to gastrointestinal disorders such as enterocolitis that may be due to antibiotic use, stressful conditions, or pathogen infection (e.g., Clostridioides difficile; Salmonella). Not all probiotic interventions have led to improvements, but there are examples of success. In one study, a Saccharomyces boulardii treatment reduced the severity and duration of illness in horses with acute enterocolitis (Desrochers et al., 2005). In another study, a probiotic mixture of 3 Lactobacillus strains (of the species L. plantarum, L. casei, L. acidophilus) and 1 Enterococcus strain (E. faecium) reduced the incidence of Salmonella shedding in horses admitted for routine medical and surgical treatments (Ward et al., 2004). Overall, there is weak evidence for probiotic use in horses with enterocolitis at this time.

In healthy adult horses, the reasons for using probiotics may differ depending on the fiber and starch content of the diet being fed. In horses fed a high-fiber diet composed of grasses and hay, live yeast cultures (Saccharomyces cerevisiae) have increased nutrient breakdown and energy extraction (Medina et al., 2002; Jouany et al., 2008; Garber et al., 2020). Such increased efficiency may be helpful for horses eating low-quality forages or performance animals that have higher energy requirements. To meet the energy needs of many high-energy or performance animals, grains that are rich in starch and have a higher energy content are often fed. A high-starch diet helps meet the energy requirement, but if not managed properly, it can exceed the capabilities of the horse’s small intestine, resulting in significant starch loads entering the hindgut. These starches are highly fermentable by hindgut microbiota, resulting in the rapid production of lactic acid and short-chain fatty acids. The accumulation of these acids can lead to hindgut acidosis and diseases such as colic or laminitis. Lactobacilli have been shown to modify equine microbiota populations, decreasing amylolytic bacteria and increasing lactic-acid utilizers, and ultimately attenuating starch breakdown and pH decline ex vivo (Harlow et al., 2017). Live yeast cultures have also been shown to help attenuate the hindgut lactic acid concentrations and maintain the hindgut pH of horses fed high-starch diets (Medina et al., 2002). These studies suggest that probiotics may be useful in increasing the digestive efficiency and/or maintaining the hindgut homeostasis of healthy adult horses.

Probiotics for horse athletic performance

Because probiotics have been used to support exercise performance in humans (Pyne et al., 2015), similar interventions have been tested in performance horses recently. In one study a probiotic mixture of 5 Lactobacillus strains (L. acidophilus DSM 32241, L. plantarum DSM 32244, L. casei DSM 32243, L. helveticus DSM 32242, L. brevis DSM 27961), 2 Bifidobacterium strains (B. lactis DSM 32246, B. lactis DSM 32247)), and 1 Streptococcus strain (S. thermophilus DSM 32245) reduced post-exercise blood lactate concentrations and modified blood and urinary metabolite profiles (Laghi et al., 2018). In another study, a probiotic mixture of 2 Lactobacillus strains (from the species L. plantarum and L. paracasei) increased blood oxygen saturation and reduced blood lactic acid concentrations (Zavistanaviciute et al., 2019). Because lactic acid production and accumulation results in fatigue and reduced performance, these studies suggest that probiotics may support athletic performance in horses. The results of these studies are promising, but more research is necessary.

State of the science

Data to support use of probiotics in horses is emerging, but the occurrence of harmful outcomes in at least one study reinforces the need for high quality studies that can precisely establish efficacious conditions and formulations for use. Similar to recommendations for other host species, equine probiotics should provide an effective dose, be designed for horses, target a specific life stage and condition, and be supported by evidence. It is important to remember that probiotic efficacy can depend on specific microbial strains, supplement form, storage conditions, and dosage  – see ISAPP’s infographic ‘What Qualifies as a Probiotic’ for more details on probiotics.

Kelly Swanson joined the ISAPP board of directors in June, 2020, providing valuable expertise in animal gut health and overall health. Swanson also chaired the 2019 ISAPP-led international consensus panel on the definition of synbiotics.

How metabolites help us to understand the effect of gut microbes on health

/in ISAPP Science Blog /by KC

By Dr. Anisha Wijeyesekera, University of Reading, UK

Much literature relating to the gut microbiota has focused on microbial composition (for example, using culture-dependent and -independent molecular biology approaches). Composition is important; knowing which microbes are present in a community enables us to gain insight into population dynamics and how these may be affected by disease, lifestyle and environmental factors (including diet). However, composition does not provide information on microbial function, and considering the gut contains the most metabolically active microbial community in the whole body, it is thus equally as important to be able to answer the question “what are the microorganisms doing”? This is of particular importance with respect to better understanding the impact of dietary interventions such as prebiotics, probiotics and other ‘biotics on health, where health benefits conferred on the host are mediated via the gut microbiota.

Investigating microbial function

Advances in phenotypic analytical technologies (for example, high-throughput sequencing, biochemical analysis, as well as bioinformatics and other multivariate data analysis approaches), have resulted in a stepwise change in our understanding of microbial function. Metabolic phenotyping (also referred to as metabolomics, metabonomics or metabolic profiling) is an exciting field in systems biology that provides information on the multiple metabolic mechanisms taking place in a system, at a given moment in time (see here). This top-down approach enables high-throughput detection and quantification of low molecular weight molecules present in a biological sample at the time of sampling, without a priori knowledge of metabolites present. Hence, it is ideally suited to augment and complement information obtained from microbial profiling approaches such as metataxonomics, to gain deeper insight into microbial function.

Metabolic phenotyping is conducted by applying analytical chemistry technologies (typically, 1H-nuclear magnetic resonance spectroscopy, and/or mass spectrometry often with chromatographic separation techniques such as gas chromatography and liquid chromatography (for prior separation of molecules followed by detection)) to capture a biochemical snapshot of a sample. In human samples (e.g. urine, blood plasma/serum and stool), metabolites detected using metabolic phenotyping are low molecular weight molecules and include intermediate and end by-products of endogenous host metabolic pathways (e.g. TCA cycle, amino acid metabolism), but also exogenous signals arising from diet, drugs and other lifestyle and environmental stimuli, including products of microbe-host co-metabolism, which provide insight into host-gut microbiota interactions. These include short-chain fatty acids (predominantly acetate, butyrate and propionate, which have a key role in host energy metabolism), bile acids (involved in the gut-liver axis), biogenic amines (involved in the gut-brain axis) and vitamins. Metabolic phenotyping, which provides functional assessment of the gut microbiota and captures information on microbial metabolic activity following ‘biotics intervention, can aid in forming hypotheses about microbial activity that may lead to health benefits.

Challenges in determining the functions of microbes

Nevertheless, functional assessment of the microbiota remains analytically challenging. For example, human metabolic phenotypes contain information relating to different forms of optically active isomers, such as lactate and amino acids (where D- forms originate from bacteria). These enantiomers cannot be differentiated using standard metabolic phenotyping experiments, and it would be important particularly in studies identifying potential biomarkers of disease, to understand the origin of these compounds. Hence, we and others have also conducted mechanistic studies using in vitro human gut model systems (e.g. the model developed by Macfarlane et al., 1998, which  has been validated against gut contents from sudden death victims and give a very close analogy to bacterial activities and composition in different areas of the hindgut), Metabolic screening of fermentation samples using metabolic phenotyping approaches provides a unique opportunity to capture dynamic microbial metabolism that is reflective of the gut microbiome in vivo, and removes contributions derived from host physiological processes (see here).

Unravelling the close interplay between microbes and host, using approaches such as metabolic phenotyping, not only provides insight into host-gut interactions, but aids our understanding of the alterations in gut microbiota mediated mechanisms that result in disease, and which demonstrate potential as therapeutic targets. More research in this area will aid in deepening understanding of the role of the gut microbiota in health and disease, and aid in the design of interventions targeting the gut microbiota (for example, the development of functional foods) for therapeutic benefit.

Food of the future: Fermented and sustainable

/in ISAPP Science Blog /by KC

By Dr. Mary Ellen Sanders, ISAPP Executive Science Officer

An exciting research initiative at the crossroads of fermented foods and sustainable diets is underway. Funded by the EU and Switzerland, and coordinated by KU Leuven in Belgium, HealthFerm is a 4-year, 13.1 MM € project involving 23 partners from 10 countries. Prof. Christophe Courtin, KU Leuven, serves as the overall project coordinator.

HealthFerm seeks to understand how to transition toward more sustainable, healthy diets through leveraging fermented foods and technologies. Its overall aim is to understand the interaction between food fermentation microbiomes, fermented plant-based foods, the human gut microbiome and human health. Many information gaps will be addressed by the project, which is organized around six work projects that are designed to integrate basic research, intervention studies, fermentation technology, consumer behavior and communication strategies.

Scientific perspectives on fermented food is at the heart of HealthFerm. Fermented foods were defined in an ISAPP consensus paper as ‘foods made through desired microbial growth and enzymatic conversions of food components’. Predating ancient Egyptian society, fermented foods and beverages are thought to have originated over 8000 years ago, and today over 5000 varieties are enjoyed around the globe, contributing substantially to human nutrition. Fermented foods have many advantages over the raw materials from which they are made, including improved sensory characteristics, safety and stability as well as potential health benefits. How the live microbial components of fermented foods may drive the health benefits of fermented foods is an active area of research.

Prof. Courtin shares some of his thoughts about HealthFerm.

Why focus on fermentation as a means of attaining more sustainable diets?

Courtin: When considering a sustainable diet, we automatically look at replacing part of our animal-based foods with plant-based foods. But plant biomass is often less functional and more recalcitrant than animal-based materials. Look for example at the whipping behavior of egg proteins or the availability of iron. Getting more out of plant fiber through fermentation is also a point of attention. In short, we believe that fermentation can help us functionalize plant materials and make it more nutritious.

 

Fermented foods have been around a long time. Why do you think now is the time to leverage their benefits?

Courtin: Societies are increasingly interested in fermented foods for a large number of reasons. We want to leverage that. From a scientific point of view, state-of-the-art omics-technologies coupled with bioinformatics allow us to look in depth into food microbiomes better than ever before and use them in a targeted way to functionalize plant materials. In addition, they also allow performing human intervention trials and doing relevant analyses to understand if and how fermented foods can improve human health, focusing on the gut microbiome and cardiometabolic health.

 

Looking ahead, what is your greatest hope for the project?

Courtin: I hope we can come to a rational design of new fermentation processes and products for the crops we target (faba bean, yellow pea, wheat, oats), using microbial resources we mobilize in collaboration with citizens and companies through community science projects. I also hope we get clear results and mechanistic insights from the intervention trials on the effects of consuming fermented foods and diets.

 

ISAPP is represented on the HealthFerm Stakeholder Board, which convened its first meeting January 20, 2023.

 

Looking back and looking ahead: ISAPP session focuses on the past, present, and future of the biotics field

/in ISAPP Science Blog /by KC

Kristina Campbell, MSc, and Prof. Dan Tancredi, PhD, Professor of Pediatrics, UC Davis School of Medicine and Center for Healthcare Policy and Research

Twenty years ago, in 2002, the first ISAPP meeting was held in London, Canada. At the time, the field was much less developed: only small human trials on probiotics or prebiotics had been published, no Nutrition and Health Claims legislation existed in the EU, and the human microbiome project hadn’t been conceived.

Now in ISAPP’s 20th year, the scientific landscape of probiotics and prebiotics is vastly different. For one thing, probiotics and prebiotics now form part of the broader field of “biotics”, which also encompasses both synbiotics and postbiotics. Hundreds of trials on biotics have been published, regulations on safety and health claims has evolved tremendously globally, and ”biotics” are go-to interventions (both food and drug) to modulate the microbiota for health.

At the ISAPP annual meeting earlier this year, scientists across academia and industry joined together for an interactive session discussing the past, present and future of the biotics field. Three invited speakers set the stage by covering some important advancements in the field. Then session chair (Prof. Daniel Tancredi) invited the participants to divide into 12 small groups to discuss responses to a set of questions. The session was focused on generating ideas, rather than achieving consensus.

The following is a summary of the main ideas generated about the past, present and future of the biotics field. Many of the ideas, naturally, were future-focused – participants were interested in how to move the field of biotics forward with purpose.

The past 20 years in the biotics field

Prof. Eamonn Quigley had the challenge of opening the discussion about the past by summing up the last 20 years in the biotics field. He covered early microbiological progress in the biotics field, such as the production of antimicrobials and progress in understanding the biology of lactic acid bacteria and their phages. In the modern era, scientists made strides in understanding the role of gut bacteria and metabolites in hepatic encephalopathy; the role of C. difficile in pseudo-membranous colitis; and in the 90s, the concept of bacterial translocation in the intestines. Prof. Quigley summarized the progress and challenges in advancing the underlying science and in developing actionable clinical evidence. He noted that more high-quality clinical trials are being published lately.

The discussion participants noted the following achievements in the field over the past two decades:

Recognition that microbes can be ‘good’. A massive shift in public consciousness has taken place over the past 20 years: the increased recognition that microorganisms are not just pathogens, they have a role to play in the maintenance of health. This added impetus to the idea that consuming beneficial microbes or other biotics is desirable or even necessary.

The high profile of biotics. An increasing number of people are familiar with the basic idea of biotics. Especially for probiotics, there is a strong legacy of use for digestive health; they are also widely available to consumers all around the world.

ISAPP’s published papers. Participants appreciated the papers published as a result of ISAPP’s efforts, including the five scientific consensus definition papers. These have raised the profile of biotics and clarified important issues.

Connections between basic and clinical scientists. Collaborations between biotics scientists and clinicians have been increasing over the past two decades, leading to better questions and higher quality research. ISAPP is one of the leading organizations that provides opportunities for these two groups to interact.

These were among the challenges from the past two decades, as identified by discussion participants:

Lack of understanding among those outside the probiotic/prebiotic field. Although the science has advanced greatly over the past 20 years, some outside the biotics field continue to believe the evidence for probiotic efficacy is thin. It appears some early stereotypes about probiotics and other biotics persist, especially in some clinical settings. This also leads to consumer misunderstandings and affects how they use biotics substances.

Too many studies lacking in quality. In the past, many studies were poorly designed; and sometimes the clinical research did not follow the science. Further, a relative lack of mechanistic research is evident in the literature.

Lack of regulatory harmony. Probiotics and other biotics are regulated in different ways around the world. The lack of harmonized regulations (for example, EFSA and FDA having different regulatory approaches) has led to confusion about how to scientifically substantiate claims in the proper way to satisfy regulators.

Lack of standardized methodologies. Many scientific variables related to biotics, such as microbiome measurements, do not have standardized methodologies, making comparability between studies difficult.

Not having validated biomarkers. The absence of validated biomarkers was noted as a potential impediment to conducting feasible clinical research studies.

The current status of the biotics field

At the moment, the biotics field is more active than ever. The industry has grown to billions of dollars per year and microbial therapeutics are in development all across the globe. The number of published pro/prebiotic papers is over 40K and the consensus definitions alone have been accessed over half a million times.

Prof. Kristin Verbeke spoke at the interactive session about the biotics field at present. She noted that the field has faced the scientific reality that there is no single microbiota configuration exclusively associated with health. The current trajectory is to develop and expand systems biology approaches for understanding the taxonomic and functional composition of microbiomes and how those impact health. Scientists are increasingly making use of bioinformatics tools to improve multi-omic analyses, and working toward proving causation.

The future of the biotics field

Prof. Clara Belzer at the ISAPP 2022 annual meeting

Prof. Clara Belzer spoke on the future of the biotics field, focusing on a so-called “next-generation” bacterium, Akkermansia muciniphila. She covered how nutritional strategies might be based on improved understanding of the interplay between microbes and mucosal health via mucin glycans, and the potential for synthetic microbial communities to lead to scientific discoveries in microbial ecology and health. She also mentioned some notable citizen science education and research projects, which will contribute to overall knowledge in the biotics field.

Participants identified the following future directions in the field of biotics:

Expanding biotics to medical (disease) applications. One group discussed at length the potential of biotics to expand from food applications (for general overall health) to medical applications. The science and regulatory frameworks will drive this shift. They believed this expansion will increase the credibility of biotics among healthcare practitioners, as the health benefits will be medical-condition-specific and will also have much broader applicability.

As for which medical conditions are promising, the group discussed indications for which there are demonstrated mechanistic as well as clinical effects: atopic diseases, irritable bowel syndrome, and stimulating the immune system to boost vaccine efficacy. In general, three different groups of medical conditions could be targeted: (1) common infections, (2) serious infectious diseases, and (3) chronic diseases for which drugs are currently inadequate, such as metabolic disorders, mental health disorders and autoimmune diseases.

Using biotics as adjuncts to medical treatments. An area of huge potential for biotics is in complementing existing medical treatments for chronic disease. There is evidence suggesting in some cases biotics could be used to increase the efficacy of drugs or perhaps reduce side effects, for example with proton pump inhibitors, statins, NSAIDs, metformin, or cancer drugs. Biotics are not going to replace commonly used drugs, but helping manage certain diseases is certainly within reach.

Using real-world data in studies. Participants said more well-conducted studies should be done using real world data. This seems in line with the development of citizen science projects as described by Clara Belzer and others at the ISAPP meeting. Real-world data is particularly important in the research on food patterns/dietary habits as they relate to biotics.

Considering new probiotic formulations. In some cases, a cocktail of many strains (50-60, for example) may be necessary for achieving a certain health effect. Using good models and data from human participants, it may be possible to create these multi-strain formulations with increased effects on the gut microbial ecosystem and increased efficacy.

Embracing omics technology and its advancement. Participants thought the next five years should see a focus on omics data, which allows for stratifying individuals in studies. This will also help increase the quality of RCTs.

More mechanism of action studies. Several groups expressed the importance of investing in understanding mechanisms of action for biotic substances. Such understandings can help drive more targeted clinical studies, providing a rationale for the exact type of intervention that is likely to be effective. Thus, clinical studies can be stronger and have more positive outcomes.

Increased focus on public / consumer engagement. Educational platforms can engage consumers, providing grassroots support for more research resources as well as advancing regulatory frameworks. Diagnostic tools (e.g. microbiome tests with validated recommendations) will help drive engagement of consumers. Further, science bloggers are critical for sharing good-quality information, and other digital channels can have great impact.

Defining and developing “precision biotics”. One group talked about “precision biotics” as solutions that target specific health benefits, which also have a well-defined or unique mechanism of action. At present, this category of biotics is in its very early stages; a prerequisite would be to better define the causes and pathways of gastrointestinal diseases.

Increasing incentives for good science. Participants discussed altering the regulatory and market environments so that good science and proper randomized, controlled trials on biotics are incentivized. Regulators in particular need to change their approaches so that companies are driven primarily by the science.

Precise characterization of responders and non-responders. The responder and non-responder phenomenon is seen with many biotic interventions. Across the field, deep characterization of subjects using multi-omics approaches with a high resolution is needed to determine what factors drive response and non-response to particular biotics substances.

Overall, participants’ ideas centered around the theme of leaning into the science to be able to create better-quality biotics products that support the health of different consumer and patient groups.

 

Special thanks to the table discussion leaders: Irene Lenoir-Wijnkoop, Zac Lewis, Seema Mody, David Obis, Mariya Petrova, Amanda Ramer-Tait, Delphine Saulnier, Marieke Schoemaker, Barry Silkington, Stephen Theis, Elaine Vaughan and Anisha Wijeyesekera.

Picture of panelists on stage with conference participants in the audience

Definition of postbiotics: A panel debate in Amsterdam

/in ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola, PhD,  Associate Professor of Microbiology at the Faculty of Chemical Engineering from the National University of Litoral and Principal Researcher from CONICET at the Dairy Products Institute (CONICET-UNL), Santa Fe, Argentina.

A panel debate titled “Postbiotics, definition and scopes” was convened at the 9th Beneficial Microbes conference in Amsterdam on November 14, 2022. The aim of this panel was to advance the discussion about postbiotics in the aftermath of some published disagreement (see here and here) about the definition of postbiotics produced and published by ISAPP: “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”. The debaters included Prof. Seppo Salminen and myself (Dr. Gabriel Vinderola), both members of the board of directors of ISAPP and co-authors of the ISAPP postbiotics definition, supporting the ISAPP definition, and Prof. Lorenzo Morelli (in attendance virtually) and Dr. Guus Roeselers challenging the ISAPP definition. The debate was attended by around 150 persons, and consisted of 15-minute opening arguments on both sides, followed by a 30 min open discussion guided by the conference chair, Dr. Koen Venema.

I introduced ISAPP as a non-profit organization dedicated to advancing the science on probiotics, prebiotics and related substances. Among many other activities, ISAPP has produced 5 different consensus definitions: probiotics, prebiotics, synbiotics, postbiotics and fermented foods. Each consensus panel was composed of academic scientists with different backgrounds, expertise and perspectives, comprising at least 11 authors from 4 – 10 countries, who came together to incorporate broad perspectives and engage in thoughtful debate. To date, all 5 consensus papers have had almost half a millon accesses at Nature Reviews Gastroentetology and Hepatology, the journal where all of the definitions are published.

The discussion within ISAPP about the need for a postbiotic definition dates back to our 2019 annual meeting. Emerging research on the health benefits conferred by non-viable microbes, their fragments and metabolites was discussed at the meeting, and this planted the seed for a definition that would cover this area. Many different terms such as heat-killed probiotics, heat-treated probiotics, heat-inactivated probiotics, tyndallized probiotics, paraprobiotics, ghost probiotics, cell fragments, cell lysates and postbiotics had been used to encompass these substances.

The panel discussed these different terms and previously published definitions. Those opposed to the ISAPP definition preferred the Tsilingiri and Rescigno (2013)1 definition of postbiotics, which focuses on metabolites produced by probiotics. I reviewed the limitations of that definition, which were outlined in Salminen et al. (2021)2. One concern is that requiring a postbiotic to be derived from a probiotic creates an unnecessary burden of first meeting the criteria for a probiotic before developing a postbiotic.

Morelli emphasized the importance of definitions for regulatory bodies and stated that researchers should provide guidance on criteria to meet a definition. He quoted the first published definition of postbiotic by Tsilingiri and Rescigno in 20131: “any factor resulting from the metabolic activity of a probiotic or any released molecule capable of conferring beneficial effects to the host in a direct or indirect way”. Morelli stated that one value of this definition was that it was clear to regulators; metabolites are measurable and produced by microbes already accepted as food components with a long history of safe use. He considered this of paramount relevance as otherwise, the novel foods path would be required. He challenged the ISAPP approach as defining a substance that was unclear how to measure. Morelli showed pictures depicting the deterioration of the biomass of freeze-dried cultures during storage, to underscore the challenges of controlling the quality of products based on biomass of non-viable microbes. He added, “If we don´t know which are the components responsible for the health benefits, then it is challenging to determine what to measure.” He questioned the ability to establish the shelf life of such a product. The need to be precise in terms of how to quantify the active components of non-viable cells was essential to his criticism of ISAPP’s definition of postbiotics. Prof. Morelli concluded that researchers must address this issue of quantification methods, both to advance research and to provide regulatory bodies needed approaches to regulating non-viable microbes.

Conclusions from the debate were that the flaws of definitions previous to the ISAPP definition are apparent, and that the substance defined by ISAPP was useful to delineate, but that clear approaches to measurement of the active component(s) of non-viable microbes are needed to make the ISAPP definition workable in scientific and regulatory circles. The debate was very worthwhile, since science advances through respectful debates such as this.

It is clear that characterization of postbiotic products may be challenging, especially with increased complexity that arises by use of multiple inanimate strains, inclusion of  metabolic  endproducts, and the presence of whole and fragmented cells. But these challenges are not unique to postbiotics. Probiotic products can comprise complex mixtures of multiple strains as well as metabolic products (as the biomass during industrial production is harvested for freeze-drying, but not washed), along with significant amounts of non-viable microbes, which all may contribute to the overall health benefit. These facts are usually overlooked when relying just on viable cells for quantification.

Many commercial products carrying inanimate microbes and metabolic fermentation products, that potentially fit the ISAPP definition of postbiotics, are already available in the market. These are diverse products such as a mixture of two lactobacilli aimed at treating infant and adult diarrhea3 or a fermented infant formula to support pediatric growth4. Similar products also target animal nutrition5. A tightly controlled manufacturing process may be the path forward to warrant reproducibility of health benefits. Suitable characterization methodologies such as flow cytometry for non-viable microbes and mass spectrometry for metabolites seem to be relevant to sufficient postbiotic product characterization.

In brief, the ISAPP definition itself seemed well accepted by the meeting participants, but concerns were raised about how to quantify postbiotics according to the definition. We intend to address this point through consultations with experts, proposing scientific paths to help conceptualize factors that need to be considered for postbiotic quantification.

Picture of panelists on stage with conference participants in the audience

Panel debate about ISAPP’s definition of postbiotics held at Beneficial Microbes conference in Amsterdam on November 14th, 2022. On the stage, from left to right: Koen Venema (conference chair), Gabriel Vinderola, Seppo Salminen, Guus Roeselers and Lorenzo Morelli (on screen).

References

  1. Tsilingiri, K. & Rescigno, M. Postbiotics: What else? Benef. Microbes (2013) doi:10.3920/BM2012.0046.
  2. Salminen, S. et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. (2021) doi:10.1038/s41575-021-00440-6.
  3. Malagón-Rojas, J. N., Mantziari, A., Salminen, S. & Szajewska, H. Postbiotics for Preventing and Treating Common Infectious Diseases in Children: A Systematic Review. Nutrients 12, (2020).
  4. Béghin, L. et al. Fermented infant formula (with Bifidobacterium breve C50 and Streptococcus thermophilus O65) with prebiotic oligosaccharides is safe and modulates the gut microbiota towards a microbiota closer to that of breastfed infants. Clin. Nutr. 40, 778–787 (2021).
  5. Kaufman, J. D. et al. A postbiotic from Aspergillus oryzae attenuates the impact of heat stress in ectothermic and endothermic organisms. Sci. Rep. 11, 6407 (2021).

Additional reading:

Follow up from ISAPP webinar – Probiotics, prebiotics, synbiotics, postbiotics and fermented foods: how to implement ISAPP consensus definitions

Postbiotics: The concept and their use in healthy populations

 

Watch / listen to the debate here: https://youtu.be/pATNfhQY4P4

 

 

The many functions of human milk oligosaccharides: A Q&A with Prof. Ardythe Morrow

/in ISAPP Science Blog /by KC

Human milk is the ‘gold standard’ of infant nutrition—and some scientists have set their sights on working towards that standard to improve the health of infants who are not breastfed. Among the many important components of human milk are human milk oligosaccharides (HMOs): complex carbohydrates that are 3-32 sugars in length. Over 200 different HMO molecules have been discovered, but a mother typically has between 12 and 20 in her milk. Some types of HMOs are affected by genetic polymorphisms – for example, only those who have the FUT2 (secretor) gene have breast milk containing HMOs called 2′-fucosylated (2’-FL) glycans.

ISAPP held a webinar in October, 2022 featuring Prof. Ardythe Morrow, University of Cincinnati College of Medicine, speaking about the latest research on HMOs and their health effects in both infants and adults.

HMOs as prebiotics

Prof. Morrow emphasized that research to date on HMOs shows they clearly fit the scientific consensus definition for prebiotics: a “substrate that is selectively utilized by host microorganisms conferring a health benefit”. HMOs are utilized by bacteria in the infant gut—mainly bifidobacteria, but also other genera (Yu, Chen & Newburg, 2013)—producing end-products that benefit infant health. B. longum subsp. infantis are the quintessential bacteria that grow on HMOs; pathogens do not typically grow on them.

Within the prebiotic category, HMOs are unique. Unlike other prebiotic substances they are structurally similar to gut oligosaccharides, which populate the surface of mucosal surfaces of the GI tract and are abundant in the mucin layer. They also can function via mechanisms that do not require utilization by gut microbes.

Beyond prebiotic function

Prof. Morrow emphasized that HMOs are multi-functional agents: in addition to their prebiotic functions, they have direct functions in the infant gut that are not mediated by microbes. First, individual HMOs have been shown to bind pathogens and inhibit infections and bind to immune cells to optimize their function (Triantis, Bode & van Neerven, 2018). Further, they can enhance neurodevelopment and brain function (Furness, Kunze & Clerc 1999; Sharon et al, 2016). The latter is a more recent domain of research, but so far it is known that basic neurodevelopmental processes are modulated in animals that are germ-free or have a depleted gut microbiota.

Certain HMOs (notably 2’-FL) can be produced synthetically and are being tested in infant formulas, and more recently for healthy adults (Elison et al., 2016). Prof. Morrow noted HMOs also have potential as novel therapeutics for various indications, such as inflammatory bowel disease (IBD). Determining which specific HMOs are most effective in these outcomes, and the dose needed, is an active area of research.

The webinar participants generated some interesting questions, some of which Prof. Morrow answers below.

Are 2’FL and LNnT (Lacto-N-neotetraose) found in cow’s milk?

2′-FL is not found in cow’s milk. Other oligosaccharides, especially sialyl oligosaccharides, are present but generally at very low levels.

How similar to HMOs are the glycosylation patterns on gut mucin?

Mucin glycosylation is not identical to human milk. But there are structural motifs that recur in both milk and gut mucin.

Do the more abundant HMOs have more potential for health benefit, compared with those at lower abundances in human milk?

We do not know that more abundance means more functionality or importance. But it is a reasonable place to start with the research. Also, several of the most abundant HMOs are trisaccharides (2’FL, 3FL, 3′-SL, and 6′-SL), and these are the most manageable to synthesize and start with.

For non-secretors, HMO complexity in milk is around 30% lower than for secretors. Does this factor affect the beneficial functions of non-secretor HMOs?

Having lower HMO content might be an issue in some circumstances. But we cannot say that it is a general problem. Furthermore, if non-secretors have more sialyloligosaccharides and 3-FL instead of 2′-FL, for example, perhaps this helps protect against viruses that bind to sialic acid epitopes (for example, influenza). Or perhaps this helps with increasing sialic acid to the brain (see Mudd et al., 2017). So, my argument is that at this point in our knowledge, we should avoid any idea of “superior” or “inferior” milk for the general healthy public. More likely, there are situation-specific benefits or disadvantages for different milk oligosaccharide phenotypes.

What do you think is more important for infant formula, more HMO complexity or more structure-function relations?

A set of HMOs for normal infant nutrition will be important, and these include fucosyllactoses, sialyllactoses, and neutral oligosaccharide with neither sialic acid nor fucose. Structure-function orientation is important to guide use in special populations with specific health needs.

Long term, will HMOs replace FOS and GOS in infant formulas?

All of the efforts in making infant formula have the goal of doing the best possible job of mimicking the physiological function of breastmilk, but cost and function are also relevant factors to consider in this process. It’s important that babies get some form of prebiotic. GOS is structurally more similar to HMOs, but it’s not enough on its own. Ideally, we’d hope for a rational mixture of different oligosaccharides backed by research confirming their combined functions.

Can we really replicate HMOs with synthetic formula, given the large number of diverse HMOs present in human milk?

I do not foresee ever achieving full replication, no. But getting closer to mother’s milk, yes, over time.

How is the dosing of HMOs in clinical trials for adults being determined? Should it be based on human milk concentration?

Elison et al. published a dosing study based on tolerance and shift of microbiota. A dosing study is now underway in Cincinnati, too.

Since it is fairly difficult to manufacture HMOs, do you think they provide sufficient advantages compared to GOS to justify their use as prebiotics in adults?

We do not yet know whether HMOs might have enough advantage over GOS in some situations, or whether prebiotic combinations might be best. This is research in progress! The reason for testing 2′-FL in IBD is because of the structure-function evidence. IBD is increased in non-secretors, and is associated with dysbiosis, inflammation, and so on. We will learn from the ongoing research.

Do you think adults will differ in response to HMOs therapeutically, possibly based on genetic differences?

I don’t yet have data on this, but have a study ongoing that I hope will be able to address this very question.

 

Watch the recording of this webinar below:

 

 

 

 

Shaping microbial exposures and the immune system in childhood: Can sandboxes be probiotic?

/in ISAPP Science Blog, Consumer Blog /by KC

By Prof. Seppo Salminen, University of Turku, Finland

Gut microbiota researchers have established that microbial exposures in early life can be influential on health later in life. Children who develop asthma in early childhood, for example, have an altered gut microbiota linked with exposure to less diverse microorganisms in their first year. The ‘biodiversity hypothesis’ has been advanced recently, suggesting that western lifestyles and low biodiversity in urban environments reduce contact with microbes both via food and via the natural environment, presenting fewer opportunities for children to be exposed to a diversity of microbes in their earliest years and increasing the risk of non-communicable diseases. If this is the case, the environments of daycare and kindergarten facilities come under scrutiny as a source of microbial exposures at a crucial time of life. So is it beneficial to intervene in children’s environments to ensure more diverse microbial exposures? Can we enhance gut microbial diversity and richness in children through environmental interventions?

A new study provided proof that shaping children’s microbial exposures may be possible. The study was the first of its kind – a placebo-controlled, double-blinded study on the effect of environmental exposures on gut microbiota diversity and immune parameters in young children. The study used playground sandboxes at daycare facilities as sources of environmental microbial diversity and explored whether these could have effects on the children.

Six day-care centers in southern Finland were enrolled in the study, with two randomly assigned to intervention and four to placebo. Identical-looking playground sandboxes were used. Intervention sandboxes were filled with sand of glacial origin enriched with a known biodiversity powder (including commercial soil, deciduous leaf litter, peat, and Sphagnum moss; described in detail by Hui et al., 2019 ; Grönroos et al, 2018). In control centers the sand was regular sandbox sand and placebo peat material. Altogether, 26 children ages 3-5 participated in supervised play for 20 minutes in the morning and afternoon for two weeks. Researchers measured the composition of gut and skin microbiota, as well as blood immune markers.

The results demonstrated that exposure to diverse environmental microbiota enhanced both the bacterial richness and diversity of the skin bacterial community. The microbiome of the skin changed only in those children who had played in a sandbox enriched with natural materials. The authors also found that the daily exposure to higher microbial biodiversity resulted in positive differences in immune response. For instance, the authors reported shifts in skin microbiota associated with IL-10 and T cell frequencies. This provides the first evidence from a placebo-controlled, double-blinded study in young children showing the differential effects on microbiota and immunity of daily exposure to defined microbial biodiversity.

An interesting follow-up could be using sandboxes to deliver probiotics with a proven health impact to children. Since the sandbox microbes were shown to influence children’s immune systems, could researchers go one step further and modulate children’s microbiota in a targeted manner? A probiotic must be defined, shown to have a health benefit and administered in an efficacious dose. In the case of sandboxes, the health benefit would need to be demonstrated for a certain level or duration of environmental exposure.

Playgrounds and sandboxes require materials that tolerate heavy wear and tear and are safe at the same time. Such materials need to be kept free of unnecessary contamination as sandboxes, for example, can also be good reservoirs of some detrimental bacteria. Therefore, it could be important to have defined natural materials for a positive impact on health. In the future, we may see many creative approaches to ensuring children receive appropriate health-supporting microbial exposures early in life. However, creating probiotic approaches requires identification of specific microbes in the biodiversity powder.

Why researchers need to understand more about the small intestinal microbiome

/in ISAPP Science Blog /by KC

By Prof. Eamonn M. M. Quigley, MD, The Methodist Hospital and Weill Cornell School of Medicine, and Prof. Purna Kashyap, MD, Mayo Clinic

The phrase “gut microbiota” properly refers to the microorganisms living throughout the entire digestive tract, including the mouth and the upper digestive tract, through the length of the small intestine as well as the large intestine. Yet the vast majority of scientific studies on the gut microbiota make conclusions based only on stool samples, meaning that the contributions to health and disease of microorganisms from most of the digestive tract are largely unexplored.

Researchers have established that the microorganisms throughout different parts of the digestive tract vary greatly. In particular, the microorganisms living in the small intestine are fewer in number than those in the colon. They are less diverse, and they change more over time because of their dynamic environment (fluctuations in oxygen, digestive secretions, dietary substrates, among other influences).

The dynamic composition and biologic functions of the small intestinal microbiome in health and disease are mostly unknown. Research has been hampered by the difficulty in obtaining samples from this area of the digestive tract and, in particular, its more distal reaches. Participants in a 2022 ISAPP discussion group argued, however, there are some good reasons to dedicate more effort to investigation of the small intestinal microbiome:

  • The small intestine has critical homeostatic functions in relation to nutrient digestion and absorption, immune engagement and interactions with the enteric and central nervous systems, as well as the neuroendocrine system. Each of these could be influenced by microbiota-host interactions. Important locations for these interactions include the gut barrier and mucosa- or gut-associated lymphoid tissue. The nature of microbiota-host interactions in these particular areas needs to be better understood, as they could have implications for systemic host health.
  • Diet plays a critical role in symptom generation in many gastrointestinal disorders; it is important to better understand diet-microbe interactions in the gut lumen to determine how the small intestinal microbiome may be contributing to diet-triggered symptoms.
  • A disordered small intestinal microbiome is commonly implicated in the pathogenesis of various gastrointestinal and non-gastrointestinal symptoms, from irritable bowel syndrome to Alzheimer’s disease, through the much-disputed concept of small intestinal bacterial overgrowth (SIBO). A precise definition of the normal small intestinal microbiome is a prerequisite to the accurate diagnosis of SIBO and linking it with various disease states.

How can we gain more information on the small intestinal microbiome? Our group tackled the limitations of current definitions and diagnostic methods, noting that this field may be advanced in the near future by new technologies for real-time sampling of intestinal gases and contents. The group discussed optimal methods for the sampling of small intestinal microbes and their metabolic products—noting that a full range of ‘omics technologies applied in well-defined populations could lead to further insights. In the meantime, the gastroenterologists in our group advised restraint in the diagnosis of SIBO and the need to exert caution in identifying it as the cause of symptoms. Clinical progress in this area is best achieved through the application of modern molecular methods to the study of human small intestinal microorganisms.

Are probiotics effective in improving symptoms of constipation?

/in ISAPP Science Blog, Consumer Blog /by KC

By Eirini Dimidi, PhD, Lecturer at King’s College London

Constipation is a common disorder that affects approximately 8% of the general population and is characterised by symptoms of infrequent or difficult bowel movements (1). People who suffer with constipation often report that it negatively affects their quality of life and the majority use some sort of treatment, such as fibre supplements and laxatives, to alleviate their symptoms (2). However, approximately half of those report they are not completely satisfied with the treatment options currently available to them, mainly due to lack of effectiveness in improving their symptoms (2).

Could probiotics offer an effective alternative way to treat constipation symptoms?

Our team at the Department of Nutritional Sciences at King’s College London has investigated the potential benefits of probiotic supplements in chronic constipation. We have extensively reviewed the available evidence on their mechanisms of action in affecting gut motility and their effectiveness in improving symptoms, and we have also conducted a randomised controlled trial of a novel probiotic in 75 people with chronic constipation (3-5).

USE OF PROBIOTICS

Before looking at the evidence on the effectiveness of probiotics in constipation, it is easy to see that some people with constipation already choose to try probiotics for their gut health. A national UK survey of over 2,500 members of the public, which included people with and without constipation, showed that people with constipation have a 5.2 higher chance of currently using probiotics for gut health, compared to people who don’t suffer from it (3).

However, the majority of doctors do not recommend probiotics for the relief of constipation symptoms, nor do they believe there is enough evidence to support their use in this condition (3).

So, what is the current evidence on probiotics and constipation?

MECHANISMS OF ACTION OF PROBIOTICS

Probiotics may impact gut motility and constipation through several mechanisms of action. Depending on the strain, they may affect the number and composition of gut microbes, as well as the compounds they release. The gut microbiota and their released compounds can then interact with our immune and nervous system, with the latter being the primary regulator of gut motility, ultimately improving constipation symptoms. Therefore, there is a rationale to support a potential improvement in constipation. But is this supported by evidence from clinical trials?

EFFECTIVENESS OF PROBIOTICS

A systematic review of the literature showed Bifidobacterium lactis strains appear to improve several symptoms of constipation, such as infrequent bowel movements and hard stools (4). At the same time, other probiotic species did not improve any symptoms. This is an important finding as it highlights that not all probiotics have the same effects in constipation, and that only certain probiotics may improve constipation. Therefore, people with constipation may only benefit from specific probiotic products – but which products would those be? Since the systematic review above showed that several B. lactis strains were effective, does this means that people with constipation may benefit from any B. lactis-containing product?

Unfortunately, it is a bit more complicated. Since the publication of the aforementioned review, new studies have been published showing that, while some probiotic products with B. lactis are effective, various other B. lactis probiotics do not impact constipation (5-6). This may be explained by strain-specific effects, but also other methodological differences among studies (e.g. probiotic dose).

TAKE HOME MESSAGE

Can we recommend probiotics for the management of constipation? At the moment, there is some low quality evidence to support the use of certain Bifidobacterium lactis strains to help manage symptoms of constipation. Further high-quality studies are needed to clarify which specific probiotic strains may be effective. However, given that there is some evidence in this area (albeit limited), along with the fact probiotics are safe for the general population to consume (unless clinically contraindicated), people with constipation could try a probiotic product of their choice for four weeks, should they wish to, bearing in mind the uncertainty in the evidence so far. But scientists continue to work to answer this question because the evidence is promising enough to warrant continued study of probiotics for constipation.

 

    1. Palsson, Gastroenterol 2020;158:1262-1273
    2. Johanson & Kralstein, Aliment Pharmacol Ther 2007;25(5):599-608
    3. Dimidi et al, Nutrition 2019;61:157-163
    4. Dimidi et al, Am J Clin Nutr 2014;100(4):1075-84
    5. Dimidi et al, Aliment Pharmacol Ther 2019;49:251-264
    6. Wang et al, Beneficial Microbes 2021;12:31-42

 

 

Can diet shape the effects of probiotics or prebiotics?

/in Consumer Blog, ISAPP Science Blog /by KC

By Prof. Maria Marco PhD, University of California – Davis and Prof. Kevin Whelan PhD, King’s College London

If you take any probiotic or prebiotic product off the shelf and give it to several different people to consume, you might find that each person experiences a different effect. One person may notice a dramatic reduction in gastrointestinal symptoms, for example, while another person may experience no benefit. On one level this is not surprising, since every person is unique. But as scientists, we are interested in finding out exactly what makes a person respond to a given probiotic or prebiotic to help healthcare providers know which products to recommend to which people.

Among factors that might impact someone’s response to a probiotic or prebiotic – such as baseline microbiota, medications, and host genetics – diet emerges as a top candidate. Ample evidence has emerged over the past ten years that diet has direct and important effects on the structure and function of the gut microbiome. Overall the human gut microbiome is shaped by habitual diet (that is, the types of foods consumed habitually over time), but the microbes can also can fluctuate in response to short-term dietary shifts. Different dietary patterns are associated with distinct gut microbiome capabilities. Since probiotics and prebiotics may then interact with gut microbes when consumed, it is plausible that probiotic activity and prebiotic-mediated gut microbiome modulation may be impacted by host diet.

A discussion group convened at ISAPP’s 2022 annual meeting brought together experts from academia and industry to address whether there is evidence to support the impact of diet on the health effects of probiotics and prebiotics. To answer this question, we looked at how many probiotic or prebiotic studies included data on subjects’ diets.

  • Prebiotics: Our review of the literature showed that only a handful of prebiotic intervention studies actively measured background diet as a potential confounder of the effect of the prebiotic. One such study (Healey, et al., 2018) classified individuals based on habitual fiber intake, and in doing so found that the gut microbiome of individuals consuming high fiber diets exhibited more changes to microbiome composition than individuals with low fiber intake. While both groups consuming prebiotics showed enrichment of Bifidobacterium, those with high fiber intake uniquely were enriched in numerous other taxa, including butyrate-producing groups of microbes. Prebiotics also resulted in improved feelings of satiety, but only among the high fiber diet consumers.
  • Probiotics: We found no evidence of published human RCTs on probiotics that investigated diet as a possible confounding factor. This is a significant gap, since we know from other studies that host diet affects the metabolic and functional activity of probiotic lactobacilli in the digestive tract. Moreover, the food matrix for the probiotic may further shape its effects, via the way in which the probiotic is released in situ.

Our expert group agreed that diet should be included in the development of new human studies on probiotics and prebiotics, as well as other ‘-biotics’ and fermented foods. These data are urgently needed because although diet may be a main factor affecting outcomes of clinical trials for such products, it is currently a “hidden” factor.

We acknowledge there will be challenges in taking diet into account in future trials. For one, should researchers merely record subjects’ habitual dietary intake, or should they provide a prescribed diet for the duration of the trial? The dietary intervention (nutrient, food, or whole diet) must also be clearly defined, and researchers should carefully consider how to measure diet (e.g. using prospective or retrospective methods). In the nutrition field, it is well known that there are challenges and limitations in the ways dietary intake is recorded as well as the selection of dietary exclusion criteria. Hence, it is crucial that dietitians knowledgeable in dietary assessment and microbiome research contribute to the design of such trials.

If more probiotic and prebiotic trials begin to include measures of diet, perhaps we will get closer to understanding the precise factors that shape someone’s response to these products, ultimately allowing people to have more confidence that the product they consume will give them the benefits they expect.

Human milk oligosaccharides as prebiotics to be discussed in upcoming ISAPP webinar

/in Consumer Blog, ISAPP Science Blog /by KC

Human milk oligosaccharides (HMOs), non-digestible carbohydrates found in breast milk, have beneficial effects on infant health by acting as substrates for immune-modulating bacteria in the intestinal tract. The past several years have brought an increase in our understanding of how HMOs confer health benefits, prompting the inclusion of synthetic HMOs in some infant formula products.

These topics will be covered in an upcoming webinar, “Human milk oligosaccharides: Prebiotics in a class of their own?”, with a presentation by Ardythe Morrow PhD, Professor of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine. The webinar will provide an overview of what HMOs are, how they are breaking new ground with the types of health benefits they can provide to infants and the recent technological innovations that will facilitate their translation into new infant formulas.

Dr. Karen Scott, Rowett Institute, University of Aberdeen, and Dr. Margriet Schoterman, FrieslandCampina, will host the webinar. All are welcome to join this webinar, scheduled for Wednesday, Oct 19th, 2022, from 10-11 AM Eastern Daylight Time.

Registration is now closed. Please watch the recording of this webinar below.

Can Probiotics Cause Harm? The example of pregnancy

/in ISAPP Science Blog /by KC

By Prof. Dan Merenstein MD, Georgetown University School of Medicine, Washington DC, USA and Dr. Maria Carmen Collado, Institute of Agrochemistry and Food Technology-National Research Council (IATA-CSIC), Valencia, Spain

Limiting excessive weight gain and controlling blood pressure during pregnancy are important to prevent pre-eclampsia and other complications of pregnancy. Researchers have examined if there is a role for probiotics in maintaining a healthy pregnancy. A recent Cochrane review, which evaluated evidence on probiotics for preventing gestational diabetes (GDM), concluded, “Low-certainty evidence from six trials has not clearly identified the effect of probiotics on the risk of GDM. However, high-certainty evidence suggests there is an increased risk of pre-eclampsia with probiotic administration.” This was an unexpected conclusion, which raised concerns about probiotic safety. A close look at the basis for this statement is warranted to determine if certain strains of probiotics are contraindicated for pregnant women.

Most people familiar with probiotic science understand that giving anyone live bacteria carries some risk. The definition of probiotics is live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. It is not live microorganisms that, when administered in adequate amounts, confer a health benefit on the host that outweighs potential adverse events. But clinicians understand that risk versus benefit must be considered for all interventions.

Many interventions associated with significant positive outcomes also are associated with some adverse events, some quite significant. For example, a recent United States Preventive Services Task Force report found that beta carotene, with or without vitamin A, was significantly associated with an increased risk of lung cancer and cardiovascular disease mortality. Aspirin kills thousands of people each year, with many more hospitalized with significant bleeds. While for an exercise doctors recommend all the time, biking, the CDC reports nearly 1,000 bicyclists die and over 130,000 are injured in crashes every year in the US.

Studies that led to the Cochrane conclusion

But let’s get back to trying to understand what made the Cochrane review come out with this warning about probiotics and pre-eclampsia. Turns out the conclusion was based on four randomized clinical trials which reported pre-eclampsia as an adverse event. All four studies were well done with low risk of bias per the Cochrane report.

Here is a summary of the four studies that collected preeclampsia data, included in the Cochrane review:

Callaway et al.(2019) studied  a mixture of Lactobacillus rhamnosus (LGG) and Bifidobacterium animalis subspecies lactis BB-12 for the prevention of gestational diabetes The reported pre-eclampsia in the probiotic group was 19 (9.2%) participants compared to 10 (4.9%) in the placebo group, p-value=0.09. This was in an obese cohort, with an average BMI of both groups near 32 (kg/m2).

Lindsay et al. (2014) evaluated the effect of Lactobacillus salivarius UCC118 on maternal fasting glucose. They reported preeclampsia in 3/62 in the probiotic group versus 2/74 in the placebo group (p-value >0.366).  Again, this was in an obese cohort with early pregnancy BMIs in the probiotic group, averaging 32.9 versus 34.1 in the placebo group.

Pellonpera et al. (2019) conducted a 4-arm study to determine if fish oil and or Lactobacillus rhamnosus HN001 and Bifidobacterium animalis ssp. lactis 420 could prevent gestational diabetes. In total there were 10 cases of pre-eclampsia among the four groups as shown below, (each group had about 95 total participants) and no significant differences between them, p value=0.80.

  • Fish oil + placebo, 1 of 95 participants (1.1%)
  • Probiotics + placebo, 4 of 96 participants (4.2%)
  • Fish oil + probiotics 3, of 96 participants (3.1%)
  • Placebo + placebo, 2 of 93 participants (2.2%)

Okesene-Gafa et al. (2019) published in the American Journal of Obstetrics and Gynecology in 2019 looking at culturally tailored dietary intervention and or daily probiotic capsules containing lactobacillus rhamnosus GG and Bifidobacterium lactis BB12 impact pregnancy weight-gain and birthweight. (This was also an obese cohort with an average BMI of 38.8.) They found pregnancy induced hypertension in the probiotic group in 4/96 (4.2%) of women versus 2/93 (2.2%) in the placebo group (p value=0.31).

Is there a rationale for the preeclampsia warning?

The increased rate of preeclampsia in probiotic groups was only with studies using obese subjects. Importantly, obesity has been associated with a higher risk of preeclampsia (see here and here). A recent meta-analysis, which included 86 studies representing 20,328,777 pregnant women, showed that higher BMI is associated with adverse pregnancy outcomes, among them, gestational diabetes and preeclampsia. Furthermore, the adjusted risk of preeclampsia is estimated to be double for overweight mothers and almost triple for obese mothers, compared to normal weight mothers.

It has been reported that pro-inflammatory signals (TNF-alpha, IL6) produced in adipose tissue of obese individuals induces a proinflammatory state characterized by insulin resistance and altered endothelial function. The gut microbiota is also disrupted in these individuals, consistent with observations that report an altered gut microbiota composition in obese versus lean individuals (see here, and the effects on offspring here and here). This suggests that obese mothers may have an increased risk of adverse events, but still the evidence supports that the addition of certain strains of probiotics may exacerbate this risk. Furthermore, it is relevant to mention the accumulating data showing that during gestation in parallel to the physiological, immune and metabolic adaptations, gut microbiota changes over the pregnancy (see here, here, here and here) although little is known on the impact of pre-gestational BMI on gut microbiota changes during pregnancy. However, specific microbial shifts have been reported to be predictive of GDM and also, gut microbial differences in women with and without GDM have been reported (here and here) . It has been also reported that the gut microbiota shifts (in composition and activity metabolites) in women with preeclampsia (see here). Thus, it is quite possible that the women in these studies, obese women, react to gut-microbiota-related interventions differently than non-obese women and that their pre-pregnancy weight puts them at an increased risk of complications.

It is worth noting that the total number of cases cited in the Cochrane review supporting their conclusion was 31 cases of preeclampsia in 472 women who took probiotics versus 17 in 483 women in the placebo groups. Thus, 14 more women who experienced preeclampsia, 9 of whom came from one of the studies [“probiotics increase the risk of pre-eclampsia compared to placebo (RR 1.85, 95% CI 1.04 to 3.29; p-value=0.04; 4 studies, 955 women; high-certainty evidence”] This is not a very large number of subjects for such a strong conclusion. The authors don’t mention if this high-certainty evidence is in all women or just obese women. By combining four studies, in which none found a significant increase in preeclampsia, the authors did find significance. Is this a convincing number of subjects? The Cochrane author, Dr. Marloes Dekker Nitert replied to an inquiry from us that she believes that this difference makes it unethical to conduct further studies in pregnant women, stating, “I think that there now is a lack of clinical equipoise to do an RCT on a combination of Lactobacillus/Bifidobacterium.”

This is a strong statement but is consistent with their high-certainty of evidence statement. We acknowledge that something does appear to be going on. It is possible that certain populations react differentially to certain strains. Thus, maybe mild to morbidly obese women are a subgroup that needs closer monitoring during pregnancy and maybe even in non-pregnant settings, as they may react differently to probiotic interventions. Maybe it is just certain strains, as the Cochrane author was very clear in her email to state, “a combination of Lactobacillus/Bifidobacterium” and not generalize to all probiotics. We agree and in fact it is possible that different strains of Lactobacillus/Bifidobacterium will have different outcomes. Pregnancy is also a continuum and to think that giving an intervention during the first trimester is the same as during the third makes little scientific or clinical sense. Along these lines, one study showed the association of probiotic intake with different effects in early versus late pregnancy; an analysis that specifically focused on women in the third trimester of pregnancy found no association between probiotics and adverse fetal outcomes.

Conclusions

In summary, we must recognize that certain strains of probiotics may cause harm in certain populations. This reinforces the importance of diligent collection of adverse event data during all clinical trials. Although Cochrane is renowned to conduct analyses of the highest caliber, we wonder if four studies of 955 mostly obese women, in which 14 more in the probiotic group than the placebo group have a secondary outcome of harm, warrant the conclusion that there is “high-certainty evidence” that probiotics cause harm. This seems overstated based on our review of the literature. Should women and clinicians pay particular attention to this subgroup (obese pregnant women) and this outcome (preeclampsia, hypertension)? We think the answer is yes. But we do not conclude that all women at all stages of pregnancy need to refrain from probiotics. Fortunately, at the time of writing there appear to be 87 trials listed on clinicaltrials.gov looking at probiotics and pregnancy. As in many things the details still need to be further elucidated and we expect more clarification on this issue over the next 5-10 years.

A pediatrician’s perspective on c-section births and the gut microbiome

/in Consumer Blog, ISAPP Science Blog /by KC

By Prof. Hania Szajewska, MD, Medical University of Warsaw, Poland and Kristina Campbell, MSc, ISAPP Consulting Communications Director

The decision to have a Cesarean section (C-section) should always depend on whether this is the best choice for the mother and baby, and it is never made by pediatricians. However, pediatricians are often asked about the consequences of C-section delivery for a child later in life and whether potential C-section-related harms may be reduced.

The data show that delivery by C-section is now more common than ever globally. The World Health Organization estimates the  C-section rate is around 21% of all births, and predicted to continue increasing. Although C-section rates are increasing both in developed and developing countries, Korea, Chile, Mexico, and Turkey have the highest rates in the world, with C-sections constituting 45% to 53% of all births. C-sections outnumber vaginal births in countries that include Dominican Republic, Brazil, Cyprus, Egypt, and Turkey.

Cesarean delivery is a medical procedure that can of course save an infant (or a mother) in a moment of danger, making birth less risky overall. But analyses have shown not all C-sections are initiated for safety reasons—some are driven by convenience and other non-medical factors. In areas with the highest C-section rates, only around half of the time are they required for life-saving reasons. Although the rate of medically necessary C-sections globally is difficult to establish, the WHO estimates it is between 10-15% of all births.

Non-essential C-sections would be perfectly reasonable if the health risks later in life were negligible. But are they? Scientific work in the past decade has shown that, in fact, there may be downsides to being born by C-section—and these health risks may manifest later in a child’s life.

By now, many observational studies have associated Cesarean births with an increased risk of various chronic health conditions that appear long after birth. C-section is associated with a higher risk of asthma and allergy, as well as obesity and type 2 diabetes. A systematic review and meta-analysis (incorporating 61 studies, which together included more than 20 million deliveries) also linked C-sections with autism spectrum disorders and attention deficit hyperactivity disorder (ADHD). Type 1 diabetes is also more prevalent in children born by c-section.

Since association is not the same as causation, scientists have looked at possible biological correlates of C-section and how they could be tied to future health problems. A leading hypothesis is that C-section deliveries cause health problems by disrupting the infant’s normal gut microbiota (i.e. the collection of microorganisms in specific ‘habitats’ on the infant’s body, such as the gut) within a critical time window for immune system development.

An altered microbiota in C-section births

One of the main clues about whether C-section births affect health via the microbiota is the consistent observation that infants born by C-section have a different collection of microorganisms in their digestive tracts and elsewhere on their bodies immediately after birth, compared with vaginally-born controls. Newborns delivered by C-section tend to harbor in their guts disease-causing microbes commonly found in hospitals (e.g. Enterococcus and Klebsiella), and lack strains of gut bacteria found in healthy children (e.g. Bacteroides species). Because it is known that gut microbiota are in close communication with the immune system, this difference in birth microbes may set the immune system up for later dysfunction.

However, an important confounding factor exists. Antibiotic administration is a recommended medical practice for C-section births in order to prevent infections. Antibiotics are potent disruptors of microbial communities – in this case the mother’s, or perhaps the infant’s if antibiotics are administered prior to umbilical cord clamping. It is not yet clear whether the timing of antibiotic administration can prevent such disruptions. (See conflicting evidence here and here; also see here.).

Gut microbiota disruption is associated with C-sections, but since C-section and antibiotics nearly always go together (with potential exposure of the infant to these drugs), it is not clear to what extent C-section and/or antibiotic treatments drive increased risk of chronic disease later in life. Antibiotic treatments within the first 2 years of life are independently associated with an increased risk of several conditions: childhood-onset asthma, allergic rhinitis, atopic dermatitis, celiac disease, overweight / obesity, and ADHD.

Options for microbiota ‘restoration’

If mechanistic studies continue to support the idea that the C-section-disrupted gut microbiota is the trigger for chronic diseases later in life, strategies could be proposed for ‘restoring’ or normalizing the infant gut microbiota after such births. Already some microbiota modifying interventions have been evaluated:

  • Probiotics: Undesired changes in microbiota composition and function caused by antibiotic treatments and/or caesarean birth may be addressed by probiotics—i.e. “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. In one study, a mixture of several probiotic strains along with at least partial breastfeeding shifted the infant gut microbiota toward a more favorable profile. Intriguingly, probiotics (of various strains) prevent IgE-associated allergy until age 5 years, specifically in cesarean-delivered children but not in all children.
  • Synbiotics: A study showed supplementation with scGOS/lcFOS and B. breve M-16V appeared to compensate for delayed Bifidobacterium colonization in the guts of C-section delivered infants.
  • Maternal vaginal microbial transfer (otherwise known as ‘vaginal seeding’ or ‘microbial bath’: This is a procedure in which infants born by C-section, immediately after birth, are swabbed with gauze that contains microbes from the mother’s vaginal tract. After the media attention given to early studies, there is increased demand from parents for this procedure. Although some studies have found it effective for normalizing the infant gut microbiota, safety is not completely established and it is too early for routine use of this procedure. Parents wishing to try this approach are advised to participate in a study as part of an institutional review board-approved research protocol.
  • Maternal fecal microbiota transplantation: This procedure involves fecal microbes from the mother, orally administered to C-section infants after birth. A proof-of-concept study showed that after this intervention the gut microbiota of C-section-born infants looked more similar to that of vaginally born infants. But for this procedure, as above, the risk of transmitting harmful microbes is a concern, making it too early to recommend the procedure unless it is part of an institutional review board-approved research protocol.
  • Breastfeeding: Breastfeeding is the gold standard for infant nutrition, and breast milk contains live microorganisms as well as other components that interact with the gut microbiota. Exclusive breastfeeding for about 6 months of C-section infants helps the gut microbiota shift toward a profile seen in vaginally born infants.

So far, probiotics, synbiotics, and microbiota ‘restoration’ are not sufficiently reliable solutions for correcting the microbiota disruptions that accompany C-section births. Further studies are needed to develop these approaches.

A leading strategy

At present, breastfeeding is the main strategy for supporting the infant gut microbiota after C-section for the greatest chance of avoiding negative health consequences. Breastfeeding has multiple benefits, but may be of increased importance after C-section birth. Mothers should be supported after giving birth by C-section to breastfeed the infant during this critical period of early life and immune system development.

 

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

/in ISAPP Science Blog /by KC

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!

Bifidobacteria in the infant gut use human milk oligosaccharides: how does this lead to health benefits?

/in ISAPP Science Blog /by KC

By Martin Frederik Laursen, Technical University of Denmark, 2022 co-recipient of Glenn Gibson Early Career Research Prize

Breast milk is the ‘gold standard’ of infant nutrition, and recently scientists have zeroed in on human milk oligosaccharides (HMOs) as key components of human milk, which through specific interaction with bifidobacteria, may improve infant health. Clarifying mechanisms by which HMOs act in concert with bifidobacteria in the infant gut may lead to better nutritional products for infants.

Back in early 2016, I was in the middle of my PhD studies working on determinants of the infant gut microbiota composition in the Licht lab at the National Food Institute, Technical University of Denmark. I had been working with fecal samples from a Danish infant cohort study, called SKOT (Danish abbreviation for “Diet and well-being of young children”), investigating how the diet introduced in the complementary feeding period (as recorded by the researchers) influences the gut microbiota development 1,2. Around the same time, Henrik Munch Roager, PostDoc in the lab, was developing a liquid chromatography mass spectrometry (LC-MS)-based method for quantifying the aromatic amino acids (AAA) and their bacterially produced metabolites in fecal samples (the 3 AAAs and 16 derivatives thereof). These bacterially produced AAA metabolites were starting to receive attention because of their role in microbiota-host cross-talk and interaction with various receptors such as the Aryl Hydrocarbon Receptor (AhR) expressed in immune cells and important for controlling immune responses at mucosal surfaces 3,4. However, virtually nothing was known about bacterial metabolism of the AAAs in the gut in an early life context. Further, the fecal samples collected from the SKOT cohort were obtained in a period of life when infants are experiencing rapid dietary changes (e.g. cessation of breastfeeding and introduction of various new foods). Thus, we wondered whether the AAA metabolites would be affected by diet and whether these metabolites might contribute to the development of the infant’s immune system. Our initial results quickly guided us on the track of breastfeeding and bifidobacteria! Here is a summary of the story, published last year in Nature Microbiology5. (See the accompanying News & Views article here.)

We initially looked at the data from a subset of 59 infants, aged 9 months, from the SKOT cohort. Here we found that both the gut microbiome and the AAA metabolome were affected by breastfeeding status (breastfed versus weaned). It is well established that certain bifidobacteria dominate the bacterial gut community in breastfed infants due to their efficient utilization of HMOs – which are abundant components of human breastmilk 6. Our data showed the same, namely enrichment of Bifidobacterium in the breastfed infants, but also indicated that the abundance of specific AAA metabolites were dependent on breastfeeding.

Trying to connect the gut microbiome and AAA metabolome, we found striking correlations between the relative abundance of Bifidobacterium and specifically abundances of three aromatic amino acid catabolites – namely indolelactic acid (ILA), phenyllactic acid (PLA) and 4-hydroxyphenyllactic acid (4-OH-PLA), collectively aromatic lactic acids. These metabolites are formed in two enzymatic reactions (a transamination followed by a hydrogenation) of the aromatic amino acids tryptophan, phenylalanine and tyrosine. However, the genes involved in this pathway were not known for bifidobacteria. Digging deeper we discovered that not all Bifidobacterium species found in the infant’s gut correlated with these metabolites. This was only true for the Bifidobacterium species enriched in the breastfed infants (e.g. B. longum, B. bifidum and B. breve), but not post-weaning/adult type bifidobacteria such as B. adolescentis and B. catenulatum group.

We decided to go back to the lab and investigate these associations by culturing representative strains of the Bifidobacterium species found in the gut of these infants. Indeed, our results confirmed that Bifidobacterium species are able to produce aromatic lactic acids, and importantly that the ability to produce them was much stronger for the HMO-utilizing (e.g. B. longum, B. bifidum and B. breve) compared to the non-HMO utilizing bifidobacteria (e.g. B. adolescentis, B. animalis and B. catenulatum). Next, in a series of experiments we identified the genetic pathway in Bifidobacterium species responsible for production of the aromatic lactic acids and performed enzyme kinetic studies of the key enzyme, an aromatic lactate dehydrogenase (Aldh), catalyzing the last step of the conversion of aromatic amino acids into aromatic lactic acids. Thus, we were able to demonstrate the genetic and enzymatic basis for production of these metabolites in Bifidobacterium species.

To explore the temporal dynamics of Bifidobacteria and aromatic lactic acids and validate our findings in an early infancy context (a critical phase of immune system development), we recruited 25 infants (Copenhagen Infant Gut [CIG] cohort) from which we obtained feces from birth until six months of age. These data were instrumental for demonstrating the tight connection between specific Bifidobacterium species, HMO-utilization and production of aromatic lactic acids in the early infancy gut and further indicated that formula supplementation, pre-term delivery and antibiotics negatively influence the concentrations of these metabolites in early life.

Having established that HMO-utilizing Bifidobacterium species are key producers of aromatic lactic acids in the infant gut, we focused on the potential health implications of this. We were able to show that the capacity of early infancy feces to in vitro activate the AhR, depended on the abundance of aromatic lactic acid producing Bifidobacterium species and the concentrations of ILA (a known AhR agonist) in the fecal samples obtained from the CIG cohort. Further, using isolated human immune cells (ex vivo) we showed that ILA modulates cytokine responses in Th17 polarized cells – namely it increased IL-22 production in a dose and AhR-dependent manner. IL-22 is a cytokine important for protection of mucosal surfaces, e.g. it affects secretion of antimicrobial proteins, permeability and mucus production 7. Further, we tested ILA in LPS/INFγ induced monocytes (ex vivo), and found that ILA was able to decrease the production of the proinflammatory cytokine IL-12p70, in a manner dependent upon both AhR and the Hydroxycarboxylic Acid (HCA3) receptor, a receptor expressed in neutrophils, macrophages and monocytes and involved in mediation of anti-inflammatory processes 8,9. Overall, our data reveal potentially important ways in which bifidobacteria influence the infant’s developing immune system.

Figure 1 – HMO-utilizing Bifidobacterium species produce immuno-regulatory aromatic lactic acids in the infant gut.

Our study provided a novel link between HMO-utilizing Bifidobacterium species, production of aromatic lactic acids and immune-regulation in early life (Figure 1). This may explain previous observations that the relative abundance of bifidobacteria in the infant gut is inversely associated with development of asthma and allergic diseases 10–12 and our results, together with other recent findings13–15 are pointing towards aromatic lactic acids (especially ILA) as potentially important mediators of beneficial immune effects induced by HMO-utilizing Bifidobacterium species.

 

References

  1. Laursen, M. F. et al. Infant Gut Microbiota Development Is Driven by Transition to Family Foods Independent of Maternal Obesity. mSphere 1, e00069-15 (2016).
  2. Laursen, M. F., Bahl, M. I., Michaelsen, K. F. & Licht, T. R. First foods and gut microbes. Front. Microbiol. 8, (2017).
  3. Zelante, T. et al. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity 39, 372–385 (2013).
  4. Sridharan, G. V. et al. Prediction and quantification of bioactive microbiota metabolites in the mouse gut. Nat. Commun. 5, 1–13 (2014).
  5. Laursen, M. F. et al. Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut. Nat. Microbiol. 6, 1367–1382 (2021).
  6. Sakanaka, M. et al. Varied pathways of infant gut-associated Bifidobacterium to assimilate human milk oligosaccharides: Prevalence of the gene set and its correlation with bifidobacteria-rich microbiota formation. Nutrients 12, 71 (2020).
  7. Keir, M. E., Yi, T., Lu, T. T. & Ghilardi, N. The role of IL-22 in intestinal health and disease. J. Exp. Med. 217, (2020).
  8. Peters, A. et al. Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3. PLoS Genet. 15, e1008145 (2019).
  9. Peters, A. et al. Hydroxycarboxylic acid receptor 3 and GPR84 – Two metabolite-sensing G protein-coupled receptors with opposing functions in innate immune cells. Pharmacol. Res. 176, (2022).
  10. Fujimura, K. E. et al. Neonatal gut microbiota associates with childhood multisensitized atopy and T cell differentiation. Nat. Med. 22, 1187–1191 (2016).
  11. Stokholm, J. et al. Maturation of the gut microbiome and risk of asthma in childhood. Nat. Commun. 9, 141 (2018).
  12. Seppo, A. E. et al. Infant gut microbiome is enriched with Bifidobacterium longum ssp. infantis in Old Order Mennonites with traditional farming lifestyle. Allergy Eur. J. Allergy Clin. Immunol. 76, 3489–3503 (2021).
  13. Meng, D. et al. Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine. Pediatr. Res. 88, 209–217 (2020).
  14. Ehrlich, A. M. et al. Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells. BMC Microbiol. 20, 357 (2020).
  15. Henrick, B. M. et al. Bifidobacteria-mediated immune system imprinting early in life. Cell 184, 3884-3898.e11 (2021).

Probiotics vs. prebiotics: Which to choose? And when?

/in Consumer Blog, ISAPP Science Blog /by KC

By Dr. Karen Scott, PhD, Rowett Institute, University of Aberdeen, Scotland

As consumers we are constantly bombarded with information on what we should eat to improve our health. Yet the information changes so fast that it sometimes seems that what was good for us last week should now be avoided at all costs!

Probiotics and prebiotics are not exempt from such confusing recommendations, and one area lacking clarity for many is which of them we should pick, and when. In this blog I will consider the relative merits of probiotics and prebiotics for the gut environment and health.

By definition, both probiotics and prebiotics should ‘confer a health benefit on the host’. Since an improvement in health can be either subjective (simply feeling better) or measurable (e.g. a lowering in blood pressure) it is clear that there is not a single way to define a ‘health benefit’. This was discussed nicely in a previous blog by Prof Colin Hill.

Although consumption of both probiotics and prebiotics should provide a health benefit, this does not mean that both need to act through the gut microbiota. Prebiotics definitively need to be selectively utilised by host microorganisms – they are food for our existing microbiota. However, depending on the site of action, this need not be the gut microbiota, and prebiotics targeting other microbial ecosystems in or on the body are being developed. Traditionally prebiotics have specifically been used to boost numbers of gut bacteria such as Bifidobacterium and the Lactobacilliaceae family, but new prebiotics targeting different members of the gut microbiota are also currently being researched.

Probiotics are live bacteria and despite a wealth of scientific evidence that specific probiotic bacterial strains confer specific health benefits, we often still do not know the exact mechanisms of action. This can make it difficult both to explain how or why they work, and to select new strains conferring similar health benefits. Many probiotics exert their effects within the gut environment, but they may or may not do this by interacting with the resident gut microbiota. For instance probiotics that reduce inflammation do so by interacting directly with cells in the mucosal immune system. Yet strains of lactobacilli (see here for what’s included in this group of bacteria) may do this by modulating cytokine production while Bifidobacterium strains induce tolerance acquisition. These very different mechanisms are one reason why mixtures containing several probiotic species or strains may in the end prove the most effective way to improve health. On the other hand, some probiotics do interact with the resident gut microbes: probiotics that act by inhibiting the growth of pathogenic bacteria clearly interact with other bacteria. Sometimes these may be potential disease-causing members of the resident microbiota, normally kept in check by other commensal microbes that themselves have become depleted due to some external impact, and some may be incoming pathogens. Such interactions can occur in the gut or elsewhere in the body.

This brings me back to the original question, and one I am frequently asked – should I take a probiotic or a prebiotic? The true and quick answer to this question is ‘it depends’! It depends why you are asking the question, and what you want to achieve. Let’s think about a few possible reasons for asking the question.

I want to improve the diversity of my microbiota. Should I take a prebiotic or a probiotic?

My first reaction was that there is an easy answer to this question – a prebiotic. Prebiotics are ‘food’ for your resident bacteria, so it follows that if you want to improve the diversity of your existing microbiota you should take a prebiotic. However, in reality this is too simplistic. Since prebiotics are selectively utilised by a few specific bacteria within the commensal microbiota to provide a health benefit, taking a prebiotic will boost the numbers of those specific bacteria. If the overall bacterial diversity is low, this may indeed improve the diversity. However, if the person asking the question already has a diverse microbiota, although taking one specific prebiotic may boost numbers of a specific bacterium, it may not change the overall diversity in a measurable way. In fact the best way to increase the overall diversity of your microbiota is to consume a diverse fibre-rich diet – in that way you are providing all sorts of different foods for the many different species of bacteria living in the gut, and this will increase the diversity of your microbiota.  Of course, if you already consume a diverse fibre-rich diet your microbiota may already be very diverse, and any increased diversity may not be measurable.

I want to increase numbers of bifidobacteria in my microbiota. Should I take a prebiotic or a probiotic?

Again, I initially thought this was easy to answer – a prebiotic. There is a considerable amount of evidence that prebiotics based on fructo-oligosaccharides (FOS or inulin) boost numbers of bifidobacteria in the human gut. But this is only true as long as there are bifidobacteria present that can be targeted by consuming suitable prebiotics. Some scientific studies have shown that there are people who respond to prebiotic consumption and people who do not (categorised as responders and non-responders). This can be for two very different reasons. If an individual is devoid of all Bifidobacterium species completely, no amount of prebiotic will increase bifidobacteria numbers, so they would be a non-responder. In contrast if someone already has a large, diverse bifidobacteria population, a prebiotic may not make a meaningful impact on numbers – so they may also be a non-responder.

However, for those people who do not have any resident Bifidobacterium species, the only possible way to increase them would indeed be to consume a probiotic- specifically a probiotic containing one or several specific Bifidobacterium species. Consuming a suitable diet, or a prebiotic alongside the probiotic, may help retention of the consumed bifidobacteria, but this also depends on interactions with the host and resident microbiota.

I want to increase numbers of ‘specific bacterium x’ in my microbiota. Should I take a prebiotic or a probiotic?

The answer here overlaps with answer 2, and depends on the specific bacterium, and what products are available commercially, but the answer could be to take either, or a combination of both – i.e. a synbiotic.

If bacterium x is available as a probiotic, consuming that particular product could help. If bacterium x has been widely researched, and the specific compounds it uses for growth have been established, identifying and consuming products containing those compounds could boost numbers of bacterium x within the resident microbiota. Such research may already have identified combination products – synbiotics – that could also be available.

One caveat for the answers to questions 2 and 3 is that probiotics do not need to establish or alter the gut microbiota to have a beneficial effect on health. In fact, a healthy large intestine has a microbial population of around 1011-1012 bacterial cells per ml, or up to 1014 cells in total, while a standard pot of yogurt contains 1010 bacterial cells (108 cells/ml). Assuming every probiotic bacterial cell reaches the large intestine alive, they would be present in a ratio of 1: 10,000. This makes it difficult for them to find a specific niche to colonise, so consuming a probiotic may not “increase numbers of ‘specific bacterium x’ in my microbiota”, but this does not mean that the function of the probiotic within the gut ecosystem would not provide a health benefit. Many probiotics act without establishing in the microbiota.

I’ve been prescribed antibiotics. Should I take a prebiotic or a probiotic?

In this case the answer is clear cut – a probiotic.

There is a lot of evidence that consumption of probiotics can alleviate symptoms of, or reduce the duration of, antibiotic associated diarrhoea. From what we know about mechanisms of action, consumption of antibiotics kills many resident gut bacteria, reducing the overall bacterial population and providing an opportunity for harmful bacteria to become more dominant. Consuming certain probiotics can either help boost bacterial numbers in the large intestine, preventing the increased growth in pathogenic bacteria until the resident population recovers, or can increase production of short chain fatty acids, decreasing the colonic pH, preventing growth of harmful bacteria. Ideally probiotics would be taken alongside antibiotics, from day 1, to avoid the increase in numbers of the potentially harmful bacteria in the first place. This has been shown to be more effective. Consuming the probiotic alongside prebiotics that could help the resident microbiota recover more quickly may be even more effective. Even if you’ve already started the course of antibiotics, it’s not too late to start taking probiotics to reduce any side-effects. Always remember to complete taking the course of antibiotics as prescribed.

 

 

Putting all of this together to answer the initial question of whether it’s better to take probiotics or prebiotics, a better answer may in fact be take both to cover the different effects each has, maximising the benefit to health. There are specific times when probiotics are better, and other times when prebiotics are better, and consuming both together may make each more effective. In any case care has to be taken to consume a product that has been confirmed through robust studies to have the specific benefit that is required.

 

Do polyphenols qualify as prebiotics? The latest scientific perspectives

/in ISAPP Science Blog, Consumer Blog /by KC

Kristina Campbell, Consulting Communications Director, ISAPP

When the ISAPP scientific consensus definition of ‘prebiotic’ was published in 2017, the co-authors on the paper included polyphenols as potential prebiotic substances. At the time, the available data on the effect of polyphenols on the gut microbiota were insufficient to show a true prebiotic effect.

An ISAPP webinar held in April 2022, aimed to give an update on the health effects of polyphenols and their mechanisms of action, along with how well polyphenols fit the prebiotic definition. Prof. Daniele Del Rio from University of Parma, Italy, and Prof. Yves Desjardins from Université Laval, Canada, presented the latest perspectives in the field.

What are polyphenols?

Polyphenols are a group of compounds found in plants, with over 6000 types identified to date. They can be divided into two main categories, flavonoids and non-flavonoids.

Polyphenols are absorbed in two different ways in the body. A very small fraction is absorbed in the small intestine, but 95% of them reach the lower gut and interact with gut microbiota. Although polyphenols have a special capacity to influence the activities of microorganisms, some resident microorganisms, in turn, can change the chemical structure of polyphenols through enzymatic action. These interactions produce a unique array of metabolites, which may be responsible for some of polyphenols’ prebiotic effects.

What are the health effects of polyphenols?

Epidemiological studies show that polyphenols in the diet are associated with many health benefits, including prevention of cardiovascular disease, certain cancers, and metabolic disease. These effects occur through various mechanisms. However, association is not proof of causation. So how good is the evidence that polyphenols can lead to health benefits?

Numerous human studies exist, but the most robust study to date for the health benefits of polyphenols is a randomized, controlled trial of over 20,000 adults, published in 2022, which showed supplementation with cocoa extract reduced death from cardiovascular events (although it did not reduce the number of cardiovascular events).

What are the mechanisms of action for polyphenols?

Polyphenols have multiple mechanisms of action. Del Rio focuses on the metabolites produced from dietary polyphenols called flavan-3-ols, which are found in red wine, grapes, tea, berries, chocolate and other foods. Along with colleagues, he showed that the metabolites produced in response to a polyphenol-rich food occur two ‘waves’: a small wave in the first 2 hours after ingestion, and a larger wave 5-35 hours after ingestion. The second wave is produced when flavan-3-ols reach the colon and interact with gut microbiota.

Work is ongoing to link these metabolites to specific health effects. Along these lines, Del Rio described a study showing how cranberry flavan-3-ol metabolites help defend against infectious Escherichia coli in a model system of bladder epithelial cells. These polyphenols are transformed by the gut microbiota into smaller compounds that are absorbed—so the health benefit comes not from the activity of polyphenols directly, but from the molecule(s) that the gut microbiota has produced from the polyphenols.

How else do polyphenols work? Ample evidence suggests polyphenols interact in different ways with gut microbes: they have direct antimicrobial effects, they affect quorum sensing, they compete with bacteria for some minerals, and/or they modify ecology, thereby affecting biofilm formation. Desjardins explained that these interactions may occur in parallel: for example, polyphenols may exert antimicrobial effects when they reach the colon, and at the same time, microorganisms in the gut begin to degrade them.

The mode of action of polyphenols Desjardins studies is the prebiotic mode of action—or as he describes it, “prebiotic with a twist”. A landmark paper from 2015 showed how cranberry polyphenols had protective effects on metabolism and obesity through the creation of mucin in the intestine, which formed a good niche for Akkermansia muciniphila, a keystone bacterial species for good metabolic health. Other polyphenols have since been shown to work the same way: by stimulating production of mucin, thereby providing ideal conditions for beneficial bacteria to grow. In this way, polyphenols appear to show small-scale effects comparable to the effects of probiotics, by inducing a host response that alters the bacterial niche.

Are the effects of polyphenols individual?

Del Rio offered some evidence that the health effects of polyphenols, via metabolites, is personalized: a study showed the existence of three distinct patterns of metabolite production in response to dietary polyphenols (ellagitannins). These may depend on the particular microbes of the gut and their ability to produce the relevant metabolites—so in essence, in each case the gut microbiota is equipped to produce a certain set of metabolites in response to polyphenols. More work is needed, however, to be able to personalize polyphenol intake.

Do polyphenols qualify as prebiotic substances?

Polyphenols clearly interact with gut microbiota to influence human health. The definition of a prebiotic is “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. Given the available evidence that polyphenols are not metabolized or utilized by bacteria in all cases in the same direct way as carbohydrate prebiotics, Desjardins sees them as having a “prebiotic-like effect”. Rather, polyphenols are transformed into other biologically active molecules that ultimately provide health benefits to the host. These prebiotic-like properties of polyphenols are nicely summarized in a 2021 review paper and include decreasing inflammation, increasing bacteriocins and defensins, increasing gut barrier function (thereby reducing low-grade inflammation), modulating bile acids, and increasing gut immuno-globulins.

Overall, the speakers showed that polyphenols exert their health effects in several ways—and while the gut microbiota are important for their health effects, polyphenols, as a heterogenous group, may not strictly meet the criteria for prebiotics. Clearly, more research on polyphenols may reveal other mechanisms by which these important nutrients influence the gut microbiome and contribute to host health, and they may someday be regarded as prebiotics.

Watch the replay of the ISAPP webinar here.

The gut mycobiome and misinformation about Candida

/in ISAPP Science Blog /by KC

By Prof. Eamonn Quigley, MD, The Methodist Hospital and Weill Cornell School of Medicine, Houston

As a gastroenterologist, I frequently meet with patients who are adamant that a Candida infection is the cause of their ailments. Patients experiencing a range of symptoms, including digestive problems, sometimes believe they have an overgrowth of Candida in their gastrointestinal (GI) tract and want to know what to do about it. Their insistence is perhaps not surprising, given how many many websites and social media ‘gurus’ share lists of symptoms supposedly tied to Candida infections. Even cookbooks exist with recipes specifically tailored to “cure” someone of Candida infection through dietary changes. Some articles aim to counter the hype – for example, an article titled “Is gut Candida overgrowth actually real, and do Candida diets work?” Yet patients are too often confused about the evidence on Candida and other fungi in the GI tract. In a 2021 ISAPP presentation on the gut mycobiome, I provided a clinical perspective on fungal infections and the related evidence base.

Fungal infections do occur

Much of the misinformation I encounter on Candida infections focuses on selling a story that encourages people to blame Candida overgrowth as the cause of their symptoms and undertake expensive or complicated dietary and supplement regimens to “cure” the infection. This is not to say that fungal infections do not take place in the body. Fungal infections, from Candida or other fungi, frequently occur on the nails or skin. Patients taking oral or inhaled steroids may develop Candida infections in the oropharynx and esophagus. Immunocompromised patients also face a greater risk of Candidiasis and Candidemia—these include HIV patients; patients undergoing chemotherapy; transplant patients; and patients suffering from malnutrition.

Fungal infections are rare in the GI tract

Regardless, instances of documented Candida infection in the GI tract remain few in number. One study published in the 90s reported 10 patients hospitalized with severe diarrhea1. These patients suffered from chronic illness, underwent intense antimicrobial treatment or chemotherapy, and faced severe outcomes such as dehydration—and clinicians consistently identified the growth of Candida albicans in the patient fecal samples. Other studies on the matter lack the clinical evidence to conclude that fungal infections drive GI disease. A study examining small intestinal fungal overgrowth identified instances of fungal overgrowth among 150 patients with unexplained symptoms2. However, the lack of documentation of response to an antifungal treatment protocol makes it difficult to attribute the observed symptoms to the presence of fungal organisms.

 The gut mycobiome in IBS

The gut microbiome has taken centre stage in common discourse about gut health. In line with this movement, my colleagues at Cork investigated the fungal members of the gut microbiome – that is, the gut mycobiome – in the guts of patients diagnosed with irritable bowel syndrome (IBS)3 to ascertain whether there was any correlation with symptoms. This effort revealed DNA sequences belonging to many fungal species. However, no significant differences in the number of fungal species were observed between IBS patients and volunteers. A smaller study done on a Dutch cohort, on the other hand, detected significantly reduced total fungal diversity among IBS patients4. So, it’s not yet clear whether mycobiome differences exist across populations with IBS.

Studying the gut mycobiome for further insights

The few studies that have examined the human gut mycobiome expose the need to answer basic questions about the fungal components of the gut microbiome. For instance, what is the gut mycobiome composition among people not suffering from GI-related symptoms? Efforts to answer these questions would require longitudinal sample collection to account for the high turnover of microbes in the GI tract. We would also need to perform stool measures not typically performed in the clinic to better correlate fungal overgrowth with GI-related symptoms. Overall, any gut mycobiome study requires careful and detailed experimental design.

We also have to consider where the gut mycobiome originates. A recent study in mSphere showed that the increased amount of DNA belonging to S. cerevisiae in stool samples coincided with the number of times subjects consumed bread and other fungi-rich foods5. S. cerevisiae also failed to grow in lab conditions mimicking the gut environment after 7 days of incubation. These findings suggest that the fungi identified in gut mycobiome profiles are not persistent gut colonizers, but transient members of the gut microbiome that come from the food we digest or our saliva.

A survey of the literature on the gut mycobiome and fungal infections in the GI tract highlights the need to conduct more studies on the role fungi play in gut and overall health. My clinical approach when I encounter someone claiming to have GI symptoms caused by Candida infection is a skeptical, yet empathetic response. Through proper communication of the evidence, we can investigate the origin of symptoms together and identify the best treatment methods for any GI-related disease, whether caused by fungal infections or not.

ISAPP held a mini-symposium featuring six short lectures that explore different aspects of the human mycobiome, including research, clinical and industry perspectives. See here for the replay, with Dr. Quigley’s talk at 1:12:30.

References

(1)        Gupta, T. P.; Ehrinpreis, M. N. Candida-Associated Diarrhea in Hospitalized Patients. Gastroenterology 1990, 98 (3), 780–785. https://doi.org/10.1016/0016-5085(90)90303-i.

(2)        Jacobs, C.; Coss Adame, E.; Attaluri, A.; Valestin, J.; Rao, S. S. C. Dysmotility and Proton Pump Inhibitor Use Are Independent Risk Factors for Small Intestinal Bacterial and/or Fungal Overgrowth. Aliment Pharmacol Ther 2013, 37 (11), 1103–1111. https://doi.org/10.1111/apt.12304.

(3)        Das, A.; O’Herlihy, E.; Shanahan, F.; O’Toole, P. W.; Jeffery, I. B. The Fecal Mycobiome in Patients with Irritable Bowel Syndrome. Sci Rep 2021, 11 (1), 124. https://doi.org/10.1038/s41598-020-79478-6.

(4)        Botschuijver, S.; Roeselers, G.; Levin, E.; Jonkers, D. M.; Welting, O.; Heinsbroek, S. E. M.; de Weerd, H. H.; Boekhout, T.; Fornai, M.; Masclee, A. A.; Schuren, F. H. J.; de Jonge, W. J.; Seppen, J.; van den Wijngaard, R. M. Intestinal Fungal Dysbiosis Is Associated With Visceral Hypersensitivity in Patients With Irritable Bowel Syndrome and Rats. Gastroenterology 2017, 153 (4), 1026–1039. https://doi.org/10.1053/j.gastro.2017.06.004.

(5)        Auchtung, T. A.; Fofanova, T. Y.; Stewart, C. J.; Nash, A. K.; Wong, M. C.; Gesell, J. R.; Auchtung, J. M.; Ajami, N. J.; Petrosino, J. F. Investigating Colonization of the Healthy Adult Gastrointestinal Tract by Fungi. mSphere 2018, 3 (2), e00092-18. https://doi.org/10.1128/mSphere.00092-18.

 

 

Improving the quality of microbiome studies – STORMS

/in ISAPP Science Blog /by KC

By Mary Ellen Sanders, PhD, ISAPP Executive Science Officer

In mid-March I attended the Gut Microbiota for Health annual meeting. I was fortunate to participate in a short workshop chaired by Dr. Geoff Preidis MD, PhD, a pediatric gastroenterologist from Baylor College of Medicine and Dr. Brendan Kelly MD, MSCE, an infectious disease physician and clinical epidemiologist from University of Pennsylvania. The topic of this workshop was “Designing microbiome trials – unique considerations.”

Dr. Preidis introduced the topic by recounting his effort (Preidis et al. 2020) to review evidence for probiotics for GI endpoints, including for his special interest, necrotizing enterocolitis (NEC). After a thorough review of available studies testing the ability of probiotics to prevent morbidity and mortality outcomes for premature neonates, he and the team found 63 randomized controlled trials that assessed close to 16,000 premature babies. Although the effect size for the different clinical endpoints was impressive and clinically meaningful, AGA was only able to give a conditional recommendation for probiotic use in this population.

Why? In part, because inadequate conduct or reporting of these studies led to reduced confidence in their conclusions. For example, proper approaches to mitigate selection bias must be reported. Some examples of selection bias include survival bias (where part of the target study population is more likely to die before they can be studied), convenience sampling (where members of the target study population are not selected at random), and loss to follow-up (when probability of dropping out is related to one of the factors being studied). These are important considerations that might influence microbiome results. If the publication on the trial does not clearly indicate how these potential biases were addressed, then the study cannot be judged as low risk of bias. It’s possible in such a study that bias is addressed correctly but reported incompletely. But the reader cannot ascertain this.

With an eye toward improving the quality and transparency of future studies that include microbiome endpoints, Dr. Preidis shared a paper by a multidisciplinary team of bioinformaticians, epidemiologists, biostatisticians, and microbiologists titled Strengthening The Organization and Reporting of Microbiome Studies (STORMS): A Reporting Checklist for Human Microbiome Research.

Dr. Preidis kindly agree to help the ISAPP community by answering a few questions about STORMS:

Dr. Preidis, why is the STORMS approach so important?

Before STORMS, we lacked consistent recommendations for how methods and results of human microbiome research should be reported. Part of the problem was the complex, multi-disciplinary nature of these studies (e.g., epidemiology, microbiology, genomics, bioinformatics). Inconsistent reporting negatively impacts the field because it renders studies difficult to replicate or compare to similar studies. STORMS is an important step toward gaining more useful information from human microbiome research.

One very practical outcome of this paper is a STORMS checklist, which is intended to help authors provide a complete and concise description of their study. How can we get journal editors and reviewers to request this checklist be submitted along with manuscripts for publication?

We can reach out to colleagues who serve on editorial boards to initiate discussions among the editors regarding how the STORMS checklist might benefit reviewers and readers of a specific journal.

How does this checklist differ from or augment the well-known CONSORT checklist?

Whereas the CONSORT checklist presents an evidence-based, minimum set of recommendations for reporting randomized trials, the STORMS checklist facilitates the reporting of a comprehensive array of observational and experimental study designs including cross-sectional, case-control, cohort studies, and randomized controlled trials. In addition to standard elements of study design, the STORMS checklist also addresses critical components that are unique to microbiome studies. These include details on the collection, handling, and preservation of specimens; laboratory efforts to mitigate batch effects; bioinformatics processing; handling of sparse, unusually distributed multi-dimensional data; and reporting of results containing very large numbers of microbial features.

How will papers reported using STORMS facilitate subsequent meta-analyses?

When included as a supplemental table to a manuscript, the STORMS checklist will facilitate comparative analysis of published results by ensuring that all key elements are reported completely and organized in a way that makes the work of systematic reviewers more efficient and more accurate.

I have been struck through the years of reading microbiome research that primary and secondary outcomes seem to be rarely stated up front. Or if such trials are registered, for example on clinicaltrials.gov, the paper does not necessarily focus on the pre-stated primary objectives. This approach runs the risk of researchers finding the one positive story to tell out of the plethora of data generated in microbiome studies. Will STORMS help researchers design more hypothesis driven studies?

Not necessarily. The STORMS checklist was not created to assess study or methodological rigor; rather, it aims to aid authors’ organization and ease the process of reviewer and reader assessment of how studies are conducted and analyzed.  However, if investigators use this checklist in the planning phases of a study in conjunction with sound principles of study design, I believe it can help improve the quality of human microbiome studies – not just the writing and reporting of results.

Do you have any additional comments?

One of the strengths of the STORMS checklist is that it was developed by a multi-disciplinary team representing a consensus across a broad cross-section of the microbiome research community. Importantly, it remains a work in progress, with planned updates that will address evolving standards and technological processes.  Anyone interested in joining the STORMS Consortium should visit the consortium website (www.stormsmicrobiome.org).

See related blog:   ISAPP take-home points from American Gastroenterological Association guidelines on probiotic use for gastrointestinal disorders

 

ISAPP’s Guiding Principles for the Definitions of ‘Biotics’

/in ISAPP Science Blog /by KC

By Mary Ellen Sanders, PhD, ISAPP Executive Science Officer

Articulating a definition for a scientific concept is a significant challenge. Inevitably, scientists have different perspectives on what falls inside and outside the bounds of a term. Prof. Glenn Gibson, ISAPP co-founder and longtime board member, recently published a paper that describes his path to coining the word ‘prebiotic’, with this observation: “One thing I have learned about definitions is that if you propose one, then be ready for it to be changed, dismissed or ignored!”

Mary Ellen Sanders with Glenn Gibson

Members of the ISAPP board, however, have remained steadfast in their belief that such definitions are worth creating. They are the basis for shared understanding and coordinated progress across a scientific field.

Developing the consensus definition papers on probiotics, prebiotics, synbiotics, postbiotics and fermented foods was demanding on the part of all involved. The objective of the panels that met to discuss these definitions was clear – to provide common ground for consistent use of this growing body of terms for all stakeholders. Although some disagreement among the broader scientific community exists about some of the definitions, ISAPP’s approach relied on important, underlying principles:

  • Don’t unnecessarily limit future innovation
  • Don’t unnecessarily limit mechanisms of action
  • Don’t unnecessarily limit scope (host, regulatory category, mechanism, site of action, etc.)
  • Require a health benefit on a target host to be demonstrated – otherwise, what is the value of these biotic substances? (Of course, fermented foods were the exception in this criterion, because the value of consuming fermented foods even in the absence of an established health benefit is evident.)
  • Limit to preparations that are administered, not substances produced by in situ activities

In my opinion, many published definitions, including previous ones for postbiotics (see supplementary table here), are untenable because they don’t recognize these principles. There may also be a tendency to rely on historical use of terms, rather than to describe what is justified by current scientific knowledge. A good example of this is provided by the first definition of probiotics, published in 1965. It was “substances secreted by one microorganism that stimulate another microorganism” (Lily and Stillwell, 1965), which is far from the current definition of “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hill et al. 2014).

If you’re looking for a concise summary of the five published ISAPP definitions, see here for our definitions infographic.

Additional reflections: I noted with a smile Glenn’s views on ISAPP, specifically on the appropriate pronunciation of the abbreviation ‘ISAPP’. “My only negative is that everyone involved in the organisation aside from 2 or 3 of us pronounce its acronym wrongly.” Most board members, including myself, have always pronounced this as ‘eye-sap’. Glenn opines, “The abbreviation is not eye-SAPP, it is ISAPP (with the ‘I’ – remarkably enough – being spoken as it is in the word ‘International’).” I wonder how he pronounces IBM?

 

 

 

 

Domestic horses from different geographical locations harbor antibiotic resistant gut bacteria, unlike their wild counterparts

/in ISAPP Science Blog /by KC

By Dr. Gabriel Vinderola, PhD,  Associate Professor of Microbiology at the Faculty of Chemical Engineering from the National University of Litoral and Principal Researcher from CONICET at Dairy Products Institute (CONICET-UNL), Santa Fe, Argentina

It all started on the 12-hour ferry trip that links Turku with Stockholm during one of the last still warm- summer days of September 2016, when a group of scientists met: Seppo Salminen, Miguel Gueimonde, Carlos Gómez- Galllego and Akihito Endo (joining us virtually from Japan well before the pandemic made these virtual meetings so popular). One of the topics was the possibility of conducting a study comparing the gut microbiome of feral and domestic horses. We had no specific funding for the project but we agreed it would be worthwhile and all agreed to participate.

Misaki wild horses from Cape Toi’s Reserve, Japan. Photo courtesy of Seppo Salminen.

Domesticated horses live under different conditions compared with their wild ancestors. We hypothesized that the animals’ housing, regular veterinary care and feeds would lead to an altered microbiota compared to wild horses. The project was ambitious and challenging in several ways: we aimed at sequencing all microbes, not just bacteria, by using whole genome sequencing; sampling droppings from feral horses needed special permission from the parks or reserves where these horses were held; the project required shipping samples from different parts of the world to the same place where they would be processed; and this all had to be managed without specific financial support to cover the expenses. Curiosity, personal dedication and funding from each end fueled this project.

Little by little, samples of feces of feral and domestic horses were collected in Argentina, Finland, Spain, Russia and Japan. Fecal DNA was extracted in every sampling location and sent to Prof. Li Ang in China for whole genome sequencing and data analysis.  A remarkable contribution was made by Prof. Ang and his team from the Zhengzhou University in China. In his words:

The biggest challenge was that very few sequences (less than 5%) were from known species hosted in the gut of horses. This number is usually 50-60% or 80-90% in human adults or infants gut microbiota, respectively. Thus we had to use ‘old school methods’ to get microbiota profiles, by constructing a custom reference database with whole genomes and then choosing specific alignments, a process that required thousands of computing hours. Interestingly, we found some specific species in horses from different locations. For example, we found shiitake mushroom in Japanese horses, a common food in East Asia families, but not in horses from other locations.

Cimarron wild horses from the State Park Ernesto Tornquist, Argentina. Photo courtesy of Seppo Salminen.

The fecal microbiome of 57 domestic and feral horses from five different locations on three continents were analyzed, observing geographical differences. A higher abundance of eukaryota (p < 0.05) and viruses (p < 0.05) and lower abundance of archaea (p < 0.05) were found in feral animals when compared with domestic ones. The abundance of genes coding for microbe-produced enzymes involved in the metabolism of carbohydrates was significantly higher (p < 0.05) in feral animals regardless of the geographic origin, which may reflect the fact that feral horses are exposed to a much more diverse natural vegetal diet than their domesticated counterparts. Differences in the fecal resistomes between both groups of animals were also observed. The domestic/captive horse microbiomes were enriched in genes conferring resistance to tetracycline, likely reflecting the use of this antibiotic in the management of these animals. Our data also showed an impoverishment of the fecal microbiome in domestic horses with diet, antibiotic exposure and hygiene being likely drivers, a fact that has been also reported for us, humans.

Almost 6 years passed since the results of those ideas discussed on board a ferry slowly galloped into the cover of the February edition of Nature Communications Biology. We hope this will be a starting point for more work that can help uncover the best ways to support equine health.

Mini-tutorial on statistical analysis: Correcting a common misinterpretation of p-values

/in ISAPP Science Blog /by KC

Daniel Tancredi PhD, Professor of Pediatrics, UC Davis School of Medicine and Center for Healthcare Policy and Research, Sacramento, CA.

Decision makers frequently rely on p-values to decide whether and how to use a study to inform their decisions. Many misinterpret what a low p-value actually means, however. I will attempt to correct this common misinterpretation and explain how to use small p-values to evaluate whether a null hypothesis is plausible. I will show that a low p-value should be used in the same way that a clinician should use a valuable but imperfect clinical diagnostic test result; as one factor, but not the only one, on which to base a decision.

Typically, readers assume that if a p value is low, such as less than 0.05, and thus the test statistic is statistically significant at the conventional level, that there is a good chance that the study results are not “due to chance”. But it is not as simple as that. Let’s suppose that one has a p-value that was generated in a well-designed placebo-controlled randomized clinical trial. We will assume that the trial had a sample size that would provide 80% power to detect what the investigators considered to be the minimum clinical significant difference. The null hypothesis in a trial like this would predict that there is no difference between the placebo and intervention arms for the primary outcome. Once the data were analyzed and reported properly, the p-value was estimated to be just under 0.05. Does this p value less than 0.05 mean that the null hypothesis has no more than a 5% probability of being true? Does it even ensure that the null hypothesis is unlikely? If the calculated p-value was greater than 0.05 (let’s say p=0.10) would that be enough to ensure that the probability of the null hypothesis being true is definitely greater than 5%?

The answer to each of these questions is “no”! This is where, unfortunately, it begins to get (a little) complicated for many users of statistics. The p-value is calculated under the assumption that the null hypothesis is true, and so it does not and cannot measure the probability of that assumption being correct.1 Even though it is common to interpret a p-value as though it is an objective and sufficient statement about the probability that the null hypothesis is true, that is not the case. Statisticians have been trying to communicate this nuance for decades, including the issuance of a statement in 2016 on p-values by the American Statistical Association, a rare statement on statistical practice in that prominent organization’s long history1.

Using a p-value to calculate the probability that the null hypothesis is true

If one wants to use a p-value as one factor in a procedure that can produce a statement about the probability that the null hypothesis is true, one needs to supply an additional input that can be very difficult to obtain. This is a prior (or pre-study) probability, a quantitative estimate of the probability that the null hypothesis is true. This is based on a considered judgment of the state of existing knowledge, what is already known (outside the study results) about how and by how much the intervention may affect the outcomes being assessed.2 Of course, such a judgment can vary a great deal from one individual to the next, according to his/her ability to gather and appraise that knowledge. These judgments can also be influenced by other interests, including financial and ideological, how much of a stake one has in each of the various competing scientific explanations.  Depending on the prior (or pre-experiment) probability for the null hypothesis, a p-value of 0.01 may not be enough to ensure that the null hypothesis has less than a 50% posterior (or after-experiment) probability of being true, whereas a p-value of 0.10 may be enough to make the posterior probability of the null hypothesis be comfortably under 5% (for those interested in taking this discussion further, the final section of this post illustrates this in more detail and with examples).

Determining whether an intervention works

Fundamentally, p-values cannot be used by themselves as though they are objective and reliable ways to make prudent decisions about whether an intervention works. Statisticians emphasize the necessity for the results of individual studies to be interpreted in a broader context, one that involves both statistical judgment and judgment on the underlying scientific plausibility of the hypothesized effects. It is well known that when sample sizes are very large, such as in many observational studies involving tens or even hundreds of thousands of observations, p-values can be very low, even for effect sizes whose confidence intervals are relatively narrow yet do not include any effects that would be of practical importance. In evidence-based medicine we typically face the opposite challenge, where small sample sizes and/or relatively infrequent outcome events result in p-values greater than 0.05 and 95% confidence intervals that are ambiguous because they include the null value (as is implied by p>0.05), but with outcomes that would be very important clinically. Thankfully, in my own career, it does seem to me to have become better appreciated that simply describing studies as positive or negative depending on which side of 0.05 the p-value falls is an unreliable method for evaluating evidence.

p-values and meta-analysis

Another thing to keep in mind is that even when a majority of individual studies that address the same research question may have p-values above 0.05, the meta-analysis of those study results can still indicate a statistically and clinically significant effect. As an example I will use a 2017 Cochrane review of the use of probiotics for the prevention of Clostridioides difficile‐associated diarrhea (CDAD) in adults and children.3 The overwhelming majority of studies, 17 of 21, were supposedly “negative” in that they have confidence intervals that include the null value, but the overall pooled estimate reports a statistically significant and clinically important range of effects. Also note that the overwhelming majority of the studies report confidence intervals that are consistent with the confidence interval for the overall pooled estimate, when one considers the degree of overlap. See Figure 1 below.

Figure 1. Forest plot summarizing complete-case analyses from systematically reviewed clinical trials of probiotics for the prevention of Clostridium difficile‐associated diarrhea (CDAD) in adults and children. Although only 4 of the 31 individual trials had statistically significant results, the pooled estimate shows a statistically and clinically signficant reduction in risk of CDAD for the studied probiotics, without statistically significant heterogeneity among the individual trials’ relative risk estimates. Note that the confidence inferval for the pooled estimate is entirely contained by all but two of the confidence intervals from the individual trials and that even the confidence intervals from these two exceptions largely contain the pooled estimate.

Reprint of Figure 3 from Joshua Z Goldenberg, Christina Yap, Lyubov Lytvyn, et al’s “ Probiotics for the prevention of Clostridium difficile‐associated diarrhea in adults and children”, published December 12, 2017 in “Cochrane Database of Systematic Reviews” by John Wiley and Sons. Copyright by John Wiley and Sons. Reprinted under one-time use license from John Wiley.

Summary

In conclusion, p values are an important component of determining whether an outcome can be deemed to be statistically significant, but this depends on the question under investigation, and is only one part of a more complete analysis. When appraising evidence for whether an intervention works, it is important to keep in mind that if one relies only on statistical inferences from individual studies, one is vulnerable to making unreliable assessments that substantially misstate the plausibility that an intervention does (or does not) have an effect. Statistical analysis cannot replace context-specific scientific judgment; both are needed to make reliable evidence appraisals.

A deeper dive into how to use p-values to assess the probability that the null hypothesis is true

A common misinterpretation of p-values is that they measure the probability that the null hypothesis is true, given the sample data. As stated above, the p-value, by itself, cannot speak to this probability, but if one is willing to supply a judgment on the prior probability that the null hypothesis is true, one can use that and the p-value to get a lower bound on the probability of interest. The compelling figure that accompanies Regina Nuzzo’s terrific Nature article on p-values and their shortcomings nicely illustrates such results for six combinations involving three example prior probabilities and p-values of 0.05 and 0.01.4 Table 1 shows posterior probabilities for those and other input combinations.

 

Table 1. Plausible lower bound for the posterior (post-study) probability of the null hypothesis being true for a given prior (pre-study) probability and study p-value. Note that low p-values do not necessarily imply that the null hypothesis is unlikely to be true!
P-value
Prior Probability for Null 0.1000 0.0500 0.0100 0.0050 0.0010 0.0005 0.0001
5% 3.2% 2.1% 0.7% 0.4% 0.1% 0.1% 0.0%
10% 6.5% 4.3% 1.4% 0.8% 0.2% 0.1% 0.0%
25% 17.3% 12.0% 4.0% 2.3% 0.6% 0.3% 0.1%
50% 38.5% 28.9% 11.1% 6.7% 1.8% 1.0% 0.2%
75% 65.3% 55.0% 27.3% 17.8% 5.3% 3.0% 0.7%
90% 84.9% 78.6% 53.0% 39.3% 14.5% 8.5% 2.2%
95% 92.2% 88.6% 70.4% 57.8% 26.3% 16.4% 4.5%
Note: Calculations use the Bayes Factor – e p ln(p), which is shown to be a lower bound for the Bayes Factor among an appealing set of candidates, thus resulting in a plausible “lower bound” for the posterior probability that the null hypothesis is true. For example, when the prior probability is 50%, a p-value of 0.05 implies that the null hypothesis retains at least a 28.9% probability of being true.

 

The calculations used in that figure and in Table 1 for converting the two inputs, a prior probability for the null hypothesis and a p-value, into a posterior probability for the null hypothesis is simply an application of a much more general formula, one that has been known for over 200 years. This formula is simple to state and remember when expressed as odds. According to Bayes Theorem, Posteriors Odds equals Prior Odds multiplied by a term we call the Likelihood Ratio. The likelihood ratio is a ratio of two conditional probabilities for the observed data, with each computed under differing hypotheses.5 [Another very widely-used application of this general formula is when physicians use the results and the operational characteristics (e.g. the sensitivity and specificity) of clinical tests to inform medical diagnoses.6] The formula uses odds not in the way that they are defined in horse racing where long-shots have high odds, but in the way that statisticians define it, as the ratio of the probability of an event to the probability of the absence of that event. To a statistician, high odds mean high probability for the event. When the probability of an event P is greater than 0, the odds are P / (1 – P). For example, if the probability of an event is 0.75 (or 75%), then the odds would be 0.75 / ( 1 – 0.75 ) = 3. If one knows the odds O, then one can find the probability P, using the equation P = O / ( 1 + O ). For example, if the odds are 4:1, or 4, then the probability is 4/5 = 0.80, and if the odds are 1:4, or 0.25, then the probability is 0.25 / 1.25 = 0.20.

In order to use this long-known formula, one has to have a way to convert the p-value into a value to use for the “Likelihood Ratio” term, which in this context is called a Bayes Factor. For the Nature article, Nuzzo used a conversion proposed in the 1990s by Thomas Sellke, M. J. Bayarri, and James O. Berger and that they eventually published in the widely read American Statistician. That conversion has an appealing statistical motivation as the minimum possible value for the Bayesian Factor among a realistic set of candidates and thus it provides a useful plausible lower bound on the Bayesian Factor for p < 1 /e ≈ 0.368, where e is the Euler number, exp(1) ≈ 2.718,7 BayesFactor = – e * p * ln(p), where ln(p) is the natural logarithm of p. (For p ≥1/e, one can use BayesFactor=1.)  For example, p=0.04 would result in a BayesFactor of -exp(1) * 0.04 * ln( 0.04 ), approximately 0.35. So, if one specified that the prior probability for the null hypothesis is 50%, a toss-up, that corresponds to a prior odds of 1, then the BayesFactor for a p-value of 0.04 converts that prior odds of 1 into a posterior odds of 0.35, which corresponds to a posterior probability of 26% for the null hypothesis, substantially higher than 4%. In the analogous setting of diagnostic medicine, consider a test result that moves a physician’s suspicion for whether the patient has a disease from a pre-test value of 50% up to a post-test value of 74%. Such a result would be considered useful, but it would not be considered definitive, something for clinicians to keep in mind when they see that a study’s p-value was just under 0.05!

Another notable conversion of the p-value into a Bayes Factor was, as far as I can tell, first reported in a pioneering 1963 article in the social sciences literature that was authored by illustrious Bayesian statisticians. 8 That same Bayes Factor formula can be found clearly presented in the second5 of Steven N. Goodman’s excellent two-part set of Annals of Internal Medicine articles concerning fallacious use of p-values in evidence-based medicine. That conversion involves statistics that have an approximately normal distribution and is thus applicable to most statistics in the medical literature. That conversion reports the minimum theoretically possible value for the Bayes Factor, BayesFactormin = exp( – Z2 / 2 ), where Z is the number of standard errors the test statistic is from the null value.  (Z can be estimated in Microsoft Excel by using the formula Z = NORMSINV( p ) or Z = NORMSINV( p / 2 ). For example, a two-sided p-value of 0.04 corresponds to Z ≈ -2.054 and a BayesFactormin of exp( – (-2.054 * -2.054) / 2 ) ≈ 0.121. So, if the prior probability for the null hypothesis is 50%, a p-value of 0.04 would mean that, at the minimum, the null hypothesis has a posterior probability of 0.121 / 1.21 = 10.8% of being true, substantively higher than the 4% probability that the popular misinterpretation of p-values would yield. When that factor was introduced in the 1963 article, it was noted by the authors as not being one that would be realistically attained by any study, as it would involve an impossibly lucky guess for the best possible prior probability to use, but it is still useful mathematically because it results in a theoretical minimum for the posterior probability that the null hypothesis is true. In mathematics, we routinely use well-chose impractical scenarios to define the limits for what is practically possible. Given that decisionmakers want to know how probable the null hypothesis remains in light of the study data, it is helpful to know the minimum possible theoretical value for it. Table 2 shows these posterior probabilities for the same inputs used above in Table 1. Notably, a p-value of 0.05 may not even be enough to make the null hypothesis less likely than not!

P-value
Prior Probability for Null 0.1000 0.0500 0.0100 0.0050 0.0010 0.0005 0.0001
5% 1.3% 0.8% 0.2% 0.1% 0.0% 0.0% 0.0%
10% 2.8% 1.6% 0.4% 0.2% 0.0% 0.0% 0.0%
25% 7.9% 4.7% 1.2% 0.6% 0.1% 0.1% 0.0%
50% 20.5% 12.8% 3.5% 1.9% 0.4% 0.2% 0.1%
75% 43.7% 30.5% 9.8% 5.5% 1.3% 0.7% 0.2%
90% 69.9% 56.9% 24.6% 14.9% 3.9% 2.1% 0.5%
95% 83.1% 73.6% 40.8% 27.0% 7.8% 4.3% 1.0%
Note: For 2-sided p-values based on approximately normally distributed test statistics, using the mathematically lowest theoretically possible Bayesian Factor,5,8 thus ensuring the lowest possible value for the posterior probability for the null hypothesis. Although these lower bonds would never be attained in any realistic application, this table is useful in showing the smallest null hypothesis probability that is even theoretically possible. Note that even with a p-value of 0.05, the posterior probability for the null hypothesis may still be high.

References

  1. Wasserstein RL, Lazar NA. The ASA Statement on p-Values: Context, Process, and Purpose. The American Statistician. 2016;70(2):129-133.
  2. Goodman SN. Toward evidence-based medical statistics. 1: The P value fallacy. Annals of Internal Medicine. 1999;130(12):995-1004.
  3. Goldenberg JZ, Yap C, Lytvyn L, et al. Probiotics for the prevention of Clostridium difficile-associated diarrhea in adults and children. Cochrane Database Syst Rev. 2017;12:CD006095.
  4. Nuzzo R. Statistical Errors. Nature. 2014;506(7487):150-152.
  5. Goodman SN. Toward evidence-based medical statistics. 2: The Bayes factor. Annals of Internal Medicine. 1999;130(12):1005-1013.
  6. Deeks JJ, Altman DG. Diagnostic tests 4: likelihood ratios. Bmj. 2004;329(7458):168-169.
  7. Sellke T, Bayarri MJ, Berger JO. Calibration of p values for testing precise null hypotheses. Am Stat. 2001;55(1):62-71.
  8. Edwards W, Lindman H, Savage LJ. Bayesian Statistical-Inference for Psychological-Research. Psychol Rev. 1963;70(3):193-242.

Bacterial genes lead researchers to discover a new way that lactic acid bacteria can make energy and thrive in their environments

/in ISAPP Science Blog /by KC

Lactic acid bacteria are an important group of bacteria associated with the human microbiome. Notably, they are also responsible for creating fermented foods such as sauerkraut, yogurt, and kefir. In the past two decades, culture-independent techniques have allowed scientists to sequence the genomes of these bacteria and discover more about their capabilities.

Researchers studying a type of lactic acid bacteria called Lactiplantibacillus plantarum found something unexpected: they contained genes for making energy in a way that had not been previously documented. Generally, living organisms obtain energy from their surroundings either by fermentation or respiration. L. plantarum have long been understood to obtain energy using fermentation, but the new genetic analysis found they had additional genes that were suited to respiration. Could they be using both fermentation and respiration?

ISAPP board member Prof. Maria Marco is a leading expert on lactic acid bacteria and their role in fermented foods and in human health. In her lab at University of California Davis, she decided to investigate why L. plantarum had genes equipping it for respiration. Her group recently published findings that show a new type of “hybrid” metabolism used by these lactobacilli.

Here is a Q&A with Prof. Marco about these exciting new findings.

What indicated to you that some of the genes in L. plantarum didn’t ‘belong’?

Organisms that use respiration normally require an external molecule that can accept electrons, such as oxygen. Interestingly, some microorganisms can also use solid electron acceptors located outside the cell, such as iron. This ability, called extracellular electron transfer, has been linked to proteins encoded by specific genes. L. plantarum had these genes, even though this species is known to use fermentation. We first learned about their potential function from Dr. Sam Light, now at the University of Chicago. Sam discovered a related pathway in the foodborne pathogen Listeria monocytogenes. Sam came across our research on L. plantarum because we previously published a paper showing that a couple of genes in this pathway are switched on in the mammalian digestive tract. We wondered what the proteins encoded by these genes were doing.

How did you set out to investigate the metabolism of these bacteria?

We investigated this hybrid metabolism in a variety of ways. Using genetic and biochemical approaches we studied the extent to which L. plantarum and other lactic acid bacteria are able to use terminal electron acceptors like iron. Our collaborators at Lawrence Berkeley National Lab and Rice University contributed vital expertise with their electrochemistry experiments, including making fermented kale juice in a bioelectrochemical reactor.

What did you find out?

We discovered a previously unknown method of energy metabolism in Lactiplantibacillus plantarum. This hybrid strategy blends features of respiration (a high NAD+/NADH ratio and use of a respiratory protein) with features of fermentation (use of endogenous electron acceptors and substrate-level phosphorylation).

We verified that this hybrid metabolism happens in different laboratory media and in kale juice fermentations. We also found that, in the complex nutritive environment of a kale juice fermentation, this hybrid metabolism increases the rate and extent of fermentation and increases acidification. Within the ecological context of the fermented food, this could give L. plantarum a fitness advantage in outcompeting other microorganisms. This could potentially be used to change the flavor and texture of fermented foods.

This discovery gives us a new understanding of the physiology and ecology of lactic acid bacteria.

Are there any indications about whether this energy-making strategy is shared by other lactic acid bacteria?

Some other fermentative lactic acid bacteria also contain the same genetic pathway. It is likely that we are just at the tip of the iceberg learning about the extent of this hybrid metabolism in lactobacilli and related bacteria.

Your finding means there is electron transfer during lactic acid bacteria metabolism. What does this add to previous knowledge about bacterially-produced ‘electricity’?

Certain soil and aquatic microbes have been the focus of research on bacterially-produced electricity. We found that by giving L. plantarum the right nutritive environment, it can produce current to the same level as some of those microbes. We believe there is potential to apply the findings from our studies to better inform food fermentation processes and to guide fermentations to generate new or improved products. Because strains of L. plantarum and related bacteria are also used as probiotics, this information may also be useful for understanding their molecular mechanisms of action in the human digestive tract.

How might this knowledge be applied in practice?

Our findings can lead to new technologies which use lactic acid bacteria to produce healthier and tastier fermented foods and beverages. Because this hybrid metabolism leads to efficient fermentation and a larger yield, it could also help minimize food waste. We plan to continue studying the diversity, expression, and regulation of this hybrid metabolism in the environments in which these bacteria are found.