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Episode 11: How to build a satisfying scientific career and make a difference

The Science, Microbes & Health Podcast 

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

How to build a satisfying scientific career and make a difference, with Prof. Gregor Reid

Episode summary:

In this episode, the ISAPP podcast hosts talk about how to succeed as a scientist in the fields of probiotics and prebiotics with Gregor Reid, professor emeritus at Western University, Canada. Prof. Reid, who is ISAPP’s former president and host of the first ISAPP meeting 20 years ago, tells about his career path and shares ways to make a difference outside of the scientific laboratory.

Key topics from this episode:

  • The importance of keeping a sense of humor as a scientist
  • Sometimes it pays to do something unconventional: early in his career, Reid decided to work with a urologist who had a hunch that lactobacilli were important in women’s health; they had difficulty getting funding to investigate further but they persisted over a number of years and eventually published some landmark work
  • Reid (with others) investigated on how biofilms impacted clinical antibiotic treatments
  • When clinical problems drive the research, it can have great impact on people’s lives, yet it can take many years to progress from observation to mechanism to better clinical treatments
  • Probiotics are “an ecological approach to an ecological problem” but often the structures (funding, regulatory, etc.)  are not in place for scientists to study them or pursue them as interventions in industry
  • Prof. Reid has worked in South Africa, led by local people, helping them obtain tools for making fermented yogurt (Yoba-For-Life)
  • For early career scientists who want to make a difference in science beyond publishing papers, it’s important to be proactive and go after what you want
  • The right lab and the right environment are essential
  • Reflect on the personal connection to your work that “makes you almost unstoppable”
  • Partnerships are key for international impacts
  • Those involved in ISAPP can champion a cause that’s important to them within the organization
  • Flexibility will be key for probiotics (and other ‘biotics’) companies in the future
  • The field is poised to expand; all kinds of organisms will benefit from probiotics in the future

 

Episode abbreviations and links:

Landmark papers related to vaginal lactobacilli, biofilms and health:

Recurrent urethritis in women

Bacterial biofilm formation in the urinary bladder of spinal cord injured patients

Bacterial biofilms: influence on the pathogenesis, diagnosis and treatment of urinary tract infections

Ultrastructural study of microbiologic colonization of urinary catheters

Additional resources:

Reflections on a career in probiotic science, from ISAPP founding board member Prof. Gregor Reid. ISAPP blog
The Children of Masiphumelele Township. ISAPP blog

 

About Prof. Gregor Reid:

Gregor Reid is a Fellow of the Royal Society of Canada and Canadian Academy of Health Sciences, and Distinguished Professor Emeritus at Western University. 

Born and raised in Scotland, he did his PhD in New Zealand and immigrated to Canada in 1982. His research, most recently at Lawson Health Research Institute, has focused on the role of beneficial microbes in the health of humans and other life forms. He has produced 32 patents, 586 peer-reviewed publications cited over 50,000 times, has a Google Scholar H index of 116 and has given over 650 talks in 54 countries. He is ranked #3 in Canada and #59 in the world for  Microbiology Scientists by research.com. In 2001, he chaired the UN/WHO Expert Panel that defined the term probiotic. In 2004, he helped introduce probiotic yoghurt to East Africa as a means for women to create microenterprises that by 2019 reached 260,000 adults and children. 

He has received an Honorary Doctorate from Orebro University, Sweden, a Distinguished Alumni award from Massey University, New Zealand, a Canadian Society for Microbiologists Career Award and Western University’s highest accolade of Distinguished Professor. He is Chief Scientific Officer for Seed, a Californian start-up. 

Are probiotics effective in improving symptoms of constipation?

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?

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.

Episode 7: Evidence for probiotic use in pediatric populations

The Science, Microbes & Health Podcast 

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

Evidence for probiotic use in pediatric populations, with Prof. Michael Cabana

Episode summary:

In this episode, the ISAPP hosts discuss probiotics for pediatric populations with Prof. Michael Cabana, MD, MPH, from Albert Einstein College of Medicine and The Children’s Hospital at Montefiore. Prof. Cabana starts by acknowledging the gap between the demand for probiotic interventions and the evidence that currently exists for their efficacy. He gives an overview of the challenges in designing trials on probiotic interventions for children, and summarizes what the evidence shows to date.

Key topics from this episode:

  • A concerning gap exists between the strength of evidence for probiotic interventions in children and the high demand by parents for these interventions.
  • A clinician supports clinical recommendations if they are based on multiple high-quality randomized, controlled trials in different settings. Unfortunately, this level of evidence is often missing for pediatric interventions, including for probiotics.
  • A clinician is less concerned about what regulatory category (drug, supplement) a recommendation falls into, and more about the level of evidence supporting its use.
  • At a minimum, a clinician looks first for evidence of no harm.
  • Conducting clinical trials in children presents many challenges. What are the “5 Ds”, which make clinical studies in children different from those in adults, as described by Forrest in 1997?
  • A lot of adult diseases have roots in childhood, so understanding pediatric health is important not just for kids, but also for adults. Preventing adult disease starts at an early age. This is the ‘delayed payoff’ that Forrest refers to.
  • Compelling evidence exists for probiotic efficacy in children for a few endpoints: colic, antibiotic-associated diarrhea, and necrotizing enterocolitis.
  • When it comes to necrotizing enterocolitis, strong evidence, reproducibility, dose-response, and biological mechanisms are what give clinicians the confidence to use probiotics.
  • An individual patient data meta-analysis (a project that emerged from an ISAPP meeting), enabled combining data from numerous studies that looked at the probiotic L. reuteri DSM17938 given to babies with colic. An overall positive effect was seen. Factors that predicted success in infants were being formula-fed, being younger, and not being on proton pump inhibitors.

 

Episode links:

  • One of many reports on the growing market for probiotic products for infants and children 
  • Information about the 2003 Pediatric Research Equity Act (PREA) and the Best Pharmaceuticals for Children Act (BPCA) mentioned in the podcast 
  • The 5 “Ds” in Pediatrics mentioned in the podcast
  • The TIPS study mentioned in the podcast is described here and here
  • The meta-analysis on colic mentioned in the podcast

 

Additional resources:

ISAPP Digs Deeper into Evidence on Probiotics for Colic with New Meta-Analysis. ISAPP blog
Probiotics to Prevent Necrotizing Enterocolitis: Moving to Evidence-Based Use. ISAPP blog

 

About Prof. Michael Cabana, MD:

Prof. Michael Cabana, MD, MPH, is a Professor of Pediatrics & the Michael I. Cohen University Chair of Pediatrics at Albert Einstein College of Medicine, as well as Physician-in-Chief, The Children’s Hospital at Montefiore (CHAM). He is also a member of the United States Preventive Services Task Force USPSTF (here), a prestigious appointment for medical personnel to weigh evidence (risk vs. harms) on prevention interventions recommended in the United States. He is a clinical trialist (see the trials listed here), with a focus on allergy in children. He has also conducted trials using probiotic interventions. Prof. Cabana served on the ISAPP board of directors from 2008 to 2018. He has an MD from University of Pennsylvania, an MPH from Johns Hopkins, and an MA in business from Wharton Business School.

Dr. Cabana’s comments do not necessarily reflect the views of the USPSTF.

Can Probiotics Cause Harm? The example of pregnancy

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.

Episode 6: Mechanisms of action for probiotics

The Science, Microbes & Health Podcast 

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

Mechanisms of action for probiotics, with Prof. Sarah Lebeer

Episode summary:

In this episode, the ISAPP hosts speak with Prof. Sarah Lebeer of University of Antwerp, Belgium, to bring clarity to a commonly misunderstood topic: probiotic mechanisms of action. They discuss how probiotic mechanisms are often multi-factorial and difficult to unravel scientifically. Nevertheless, Prof. Lebeer describes five distinct mechanisms of action by which a probiotic may benefit a host.

See ISAPP’s other podcast episode on mechanisms of action, with Prof. Maria Marco: Why mechanistic research on probiotics is captivating and important.

Key topics from this episode:

  • Probiotics are live microorganisms with documented health benefits; their mode of action is multifactorial.
  • Mechanism of action is crucial in the probiotic field. This knowledge helps scientists understand how probiotics interact with the human host and the microbiota. However, even if mode of action for a strain is known it can be difficult to translate it into measurable outcomes for the host.
  • Probiotics can benefit health by five main mechanisms of action applicable at different body sites (gut, vagina, skin, nose, etc.):
    1. Modulation of microbe-microbe interactions by inhibiting pathogens and promoting beneficial microbes. This is based on the production of metabolically active molecules, such as lactic acid. 
    2. Enhancement of mucosal barrier function through interaction with epithelial cells, promoting the integrity of the barrier. 
    3. Modulation of immune responses by interacting with various immune cells. Interaction with the immune cells is also the clearest strain-specific capacity of probiotics.
    4. Modulation of metabolic responses by modulating insulin resistance or cholesterol metabolism. This mode of action is novel, and research is emerging.
    5. Modulation of neurological signaling pathways. This is also a novel mode of action with new evidence building up.
  • Postbiotics can have similar modes of action, provided the active molecules are not inactivated.

 

Episode links:

The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic

 

Graphical summary of 5 main mechanisms of action for probiotics

(Image by Sarah Lebeer. Image copyright.)

 

Additional resources:

Current status of research on probiotic and prebiotic mechanisms of action. ISAPP blog
Importance of understanding probiotic mechanisms of action. ISAPP blog

 

About Prof. Sarah Lebeer:

Sarah Lebeer is a research professor at the Department of Bioscience Engineering of the University of Antwerp, Belgium. She has studied bioscience engineering, with a specialisation in cell and gene technology/food & health and obtained her Master at KU Leuven (Belgium). In 2008, she obtained a PhD degree with a topic on the mode of action of gastro-intestinal probiotics in inflammatory bowel diseases and a scholarship in the team of Prof. Jos Vanderleyden (KU Leuven). After a postdoc on the interaction between lactobacilli, viruses and mucosal immunology, in November 2011, she was offered a tenure track position at the University of Antwerp. Since then, she is leading the Laboratory for Applied Microbiology and Biotechnology of the ENdEMIC research group.

In 2020, she was awarded with an ERC Starting Grant that enables her to gain more in-depth knowledge of the evolutionary history and ecology of lactobacilli (https://www.lebeerlab.com). This rationale was also an important driving force to revise the Lactobacillus genus taxonomy with  a large international consortium. Within the ERC project, Sarah has also launched the Isala citizen-science project to gain new insights in the role of vaginal lactobacilli for women’s health (https://isala.be). Since 2018, Sarah is an academic board member of the International Scientific Association on Probiotics and Prebiotics (www.isappscience.org). Communicating about beneficial microbes and probiotics for experts and laymen is an important inspiration for her daily work. 

Episode 4: Weighing evidence for probiotic interventions: Perspectives of a primary care physician

The Science, Microbes & Health Podcast 

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

Weighing evidence for probiotic interventions: Perspectives of a primary care physician, with Prof. Dan Merenstein, MD

Episode summary:

In this episode, the ISAPP host Prof. Dan Tancredi discusses evidence for probiotic interventions with Prof. Dan Merenstein, MD, a family medicine researcher based at Georgetown University. They discuss what it means to practice evidence-based medicine, and what kind of evidence clinicians should look for when deciding whether an intervention is appropriate. Prof. Merenstein shares how probiotic evidence has strengthened in the past few decades, and gives tips on what to look for in a probiotic intervention study.

Key topics from this episode:

  • What is evidence-based medicine, and why is it important to practice it? Evidence-based medicine provides proper care for patients at the appropriate time by focusing on the existing evidence provided by clinical studies.
  • What is evidence, and how do we obtain a high level of evidence for probiotics? Evidence should come from well-designed clinical studies. Animal and in vitro studies provide supportive preclinical information and mechanistic insights.
  • The quality of probiotic research has improved in recent years. Compared with decades ago, studies are more likely to be high quality and more likely to be properly-powered. Such studies have provided a clearer sense of the circumstances under which probiotics may – or may not – provide health benefits.
  • What is the proper way to report clinical trials in the probiotic field? Focus on the rules for conducting a clinical trial: for example, CONSORT (see below). Compliance is also important for good outcomes.
  • How can a clinician evaluate if a patient should use an intervention? The focus should be on the evidence for each intervention, the potential harm of the intervention, and the positive and negative outcomes.
  • Evidence-based medicine also needs to account for regional differences and where the clinical studies have been performed. Evidence about probiotic effects needs to account for microbiome differences and lifestyle differences between countries.
  • For which indications do we now have actionable evidence for probiotic use?
    • Use of certain probiotics together with antibiotic treatment to prevent antibiotic-associated diarrhea
    • Use of an L. reuteri strain to reduce crying time for infants suffering from colic
    • Certain probiotics can help prevent traveler’s diarrhea or treat acute pediatric infectious diarrhea.
    • Probiotics to help with endpoints such as weight loss and metabolic syndrome, for autism and gut-brain indications, for skin conditions such as eczema and acne, and inflammatory bowel disease are active areas of research and soon scientists will have more answers.
  • Probiotic research is improving. Patients are using probiotics, and they are aware of the benefits of probiotics – Prof. Merenstein estimates that 80% of his patients are taking a probiotic. The job of the doctor is to get them to use probiotics in an evidence-based manner.

 

Episode links:

CONSORT (Consolidated Standards of Reporting Trials) establishes a well-accepted, evidence-based, minimum set of recommendations for reporting randomized trials.
IPDMA (individual patient data meta-analysis) – see this Sung et al. paper on infantile colic and L. reuteri
BB12: Bifidobacterium animalis subsp. lactis BB-12, a well-studied probiotic. See this paper.
A priori: without prior knowledge

 

Additional resources:

The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic

 

About Prof. Dan Merenstein:

Dr. Daniel Merenstein is a Professor with tenure of Family Medicine at Georgetown University, where he also directs Family Medicine research. Dr. Merenstein has a secondary appointment in the undergraduate Department of Human Science, in the School of Nursing and Health Studies. Dr. Merenstein teaches two undergraduate classes, a research capstone and a seminar class on evaluating evidence based medical decisions. He has been funded by the NIH, USDA, Foundations and Industry, for grants over $100 million. Dr. Merenstein is the President of the board of directors of the International Scientific Association of Probiotics and Prebiotics.

The primary goal of Dr. Merenstein’s research is to provide answers to common clinical questions that lack evidence and improve patient care. Dr. Merenstein is a clinical trialist who has recruited over 2,100 participants for 10 probiotic trials since 2006. He is an expert on probiotics, antibiotic stewardship in outpatient settings and also conducts HIV research in a large women’s cohort. He sees patients in clinic one day a week.

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

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.

 

Episode 3: The science of fermented foods, part 2

The Science, Microbes & Health Podcast 

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

The science of fermented foods, part 2, with Prof. Bob Hutkins

Episode summary:

Before listening to this episode, it’s recommended that you check out episode #1, The science of fermented foods, Part 1. In this episode, the hosts continue their discussion of fermented foods with Prof. Bob Hutkins, University of Nebraska – Lincoln. Prof. Hutkins elaborates on how the microbes associated with fermented foods may confer health benefits, as well as how food scientists choose strains for fermentation. He emphasizes how the live microbes in fermented foods differ from probiotics.

Key topics from this episode:

  • Why working in the field of fermented foods is exciting and rewarding
  • The challenges for scientists, especially when it comes to designing clinical studies with various fermented foods
  • The benefits of fermented foods – from being safe as well as nutritious, to the health benefits that live microbes present in the foods can provide
  • How microbes are selected for fermentation; companies focus on strain performance – i.e., good growth and survival to preserve the food and provide a desired flavor and texture
  • The activities of live microbes present in fermented foods, from initiating the fermentation process to benefiting human health
  • The differences between probiotics and live microbes in fermented foods
  • How live microbes in fermented foods might affect your gut microbiota and why some scientists believe that fermented foods are important for getting regular doses of live microbes

 

Episode links:

Microbiology and Technology of Fermented Foods, 2nd Ed., by Robert W. Hutkins
The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on fermented foods
The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic
Gut-microbiota-targeted diets modulate human immune status, study by Stanford researchers

 

Additional resources:

Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics.
Postbiotics. ISAPP infographic
Fermented foods. ISAPP infographic
What are fermented foods? ISAPP video
Do fermented foods contain probiotics? ISAPP blog post
How are probiotic foods and fermented foods different? ISAPP infographic
Are fermented foods probiotics? Webinar by Mary Ellen Sanders, PhD

 

About Prof. Bob Hutkins:

Bob Hutkins is the Khem Shahani Professor of Food Microbiology at the University of Nebraska. He received his Ph.D. from the University of Minnesota and was a postdoctoral fellow at Boston University School of Medicine. Prior to joining the University of Nebraska, he was a research scientist at Sanofi Bio Ingredients.

The Hutkins Lab studies bacteria important in human health and in fermented foods. His group is particularly interested in understanding factors affecting persistence and colonization of probiotic bacteria in the gastrointestinal tract and how prebiotics shift the intestinal microbiota and metabolic activities. The lab also conducts clinical studies using combinations of pro- and prebiotics (synbiotics) to enhance health outcomes. More recently we have developed metagenome-based models that can be used in personalized nutrition.

Professor Hutkins has published widely on probiotics, prebiotics, and fermented foods and is the author of the recently published 2nd edition of Microbiology and Technology of Fermented Foods.

Episode 2: Why mechanistic research on probiotics is captivating and important

The Science, Microbes & Health Podcast 

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

Why mechanistic research on probiotics is captivating and important, with Prof. Maria Marco

Episode summary:

In this episode, the ISAPP hosts discuss probiotic mechanisms of action with Prof. Maria Marco, University of California, Davis. Prof. Marco is a well-known probiotic researcher with special expertise in food-associated lactobacilli. Here she explains how studying probiotics in food science can lead to fundamental insights in biology. She shares why it’s important to understand probiotic mechanisms of action, and describes how scientists go about identifying which compounds or pathways are important for probiotic health effects.

Key topics from this episode:

  • The search for probiotic mechanisms of action: why this research is essential and the added value of this type of research for the end consumer.
  • What we now understand about probiotic mode of action: probiotic mode of action for different strains is mediated by multiple working mechanisms, from cell-wall-associated molecules to bacteriocin production and metabolite synthesis.
  • How researchers set the stage for studying probiotics’ mode of action, from large scale screening, to molecular techniques focusing on single molecules and genome comparisons between strains.
  • Whether we need to apply something similar to Koch’s postulates when talking about the effects of probiotics.
  • The potential effects of food or delivery matrix on a probiotic mechanism of action.  
  • What we can learn from the postbiotic research, which can help inform probiotic mechanisms of action.
  • The most exciting developments in probiotic mode of action research in the past 10 years and the future of this area of research.

 

Episode links:

The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic
Prof. Marco refers to two of her mentors, Willem De Vos and Michiel Kleerebezem
See this overview of Koch’s postulates

 

Additional resources:

Bacterial genes lead researchers to discover a new way that lactic acid bacteria can make energy and thrive in their environments, ISAPP blog post featuring recent work from Prof. Marco’s lab

 

About Prof. Maria Marco:

Maria Marco is a Professor in the Department of Food Science and Technology and Chair of the Food Science Graduate Group at the University of California, Davis. She received her PhD in microbiology from the University of California, Berkeley and then was a postdoc and project leader at NIZO Food Research, The Netherlands. Dr. Marco has 20 years’ experience investigating fermented foods, probiotics, and diet-dependent, host-microbe interactions in digestive tract. Her laboratory at UC Davis is broadly engaged in the study of food and intestinal microbiomes and the ecology and genetics of lactic acid bacteria. 

Episode 1: The science of fermented foods, part 1

The Science, Microbes & Health Podcast 

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

The science of fermented foods, part 1, with Prof. Bob Hutkins

Episode summary:

The hosts discuss fermented foods with Prof. Bob Hutkins, University of Nebraska – Lincoln. Prof. Hutkins wrote a popular textbook on fermented foods and has had a 40-year career in fermentation science. He shares why he ended up in fermentation science, as well as how fermented foods are made and how important live microbes are for their health benefits.

Key topics from this episode:

  • What fermented foods are
  • The scientific consensus definition published by ISAPP
  • Fermentation processes and practices used in early times and still used today
  • The benefits and safety of fermented foods, as well as the difference between fermentation and food spoilage
  • The live microbes present in fermented foods, how many are present, and their potential health benefits
  • Why some fermented foods have live microbes and others do not; and how even when live microbes are absent due to heat treatment, for example, these products may still be classified as fermented 
  • The differences between fermented foods, probiotics, and probiotic fermented foods

 

Episode links:

Microbiology and Technology of Fermented Foods, 2nd Ed., by Robert W. Hutkins
The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on fermented foods
The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic
Synbiotics: Definitions, Characterization, and Assessment – ISAPP webinar featuring Prof. Bob Hutkins and Prof. Kelly Swanson

 

Additional resources:

Fermented foods. ISAPP infographic
What are fermented foods? ISAPP video
Do fermented foods contain probiotics? ISAPP blog post
How are probiotic foods and fermented foods different? ISAPP infographic
Are fermented foods probiotics? Webinar by Mary Ellen Sanders, PhD

 

About Prof. Bob Hutkins:

Bob Hutkins is the Khem Shahani Professor of Food Microbiology at the University of Nebraska. He received his Ph.D. from the University of Minnesota and was a postdoctoral fellow at Boston University School of Medicine. Prior to joining the University of Nebraska, he was a research scientist at Sanofi Bio Ingredients.

The Hutkins Lab studies bacteria important in human health and in fermented foods. His group is particularly interested in understanding factors affecting persistence and colonization of probiotic bacteria in the gastrointestinal tract and how prebiotics shift the intestinal microbiota and metabolic activities. The lab also conducts clinical studies using combinations of pro- and prebiotics (synbiotics) to enhance health outcomes. More recently we have developed metagenome-based models that can be used in personalized nutrition.

Professor Hutkins has published widely on probiotics, prebiotics, and fermented foods and is the author of the recently published 2nd edition of Microbiology and Technology of Fermented Foods.

New ISAPP Webinar: Fermented Foods and Health — Continuing Education Credit Available for Dietitians

Dietitians – along with many other nutritional professionals – often receive questions about consuming fermented foods for digestive health. But how strong is the evidence that fermented foods can improve digestive health?

ISAPP is pleased to work with Today’s Dietitian to offer a free webinar in which Hannah Holscher, PhD, RD, and Jennifer Burton, MS, RD, LDN will discuss the foundational elements of fermented foods, the role of microbes in fermentation, how they differ from probiotics and prebiotics, and how to incorporate fermented foods into client diets in an evidence-based manner. Participants will come away with a grasp of the scientific evidence that supports fermented food consumption. This activity is accredited by the Academy of Nutrition and Dietetics Commission on Dietetic Registration (CDR) for 1.0 CPEUs for dietitians.

The one-hour virtual event, titled “Fermented Foods and Health — Does Today’s Science Support Yesterday’s Tradition?”, was held April 20th, 2022, at 2:00 pm Eastern Time.

See the webinar recording here.

ISAPP and Today’s Dietitian also collaborated on a self-study activity titled “Evidence-based use of probiotics, prebiotics and fermented foods for digestive health”. This free activity, which provides more detail on the topic that the 1-hour webinar above, was approved by CDR to offer 2.0 CPEUs for dietitians and is available here through November 2023.

Improving the quality of microbiome studies – STORMS

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

 

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

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.

ISAPP awards the Glenn Gibson Early Career Research Prize to two diet and gut health researchers

The ISAPP board of directors is pleased to announce that the 2022 Glenn Gibson Early Career Research Prize has been awarded to two promising researchers in the field of probiotics, prebiotics and related substances.

Dr. Martin Laursen, Senior Researcher at the National Food Institute, Technical University of Denmark, has demonstrated excellence in his work on the impact of probiotics and human milk oligosaccharides on infant gut microbiota and health. Dr. Eirini Dimidi, Lecturer at King’s College in London, UK, has carried out meaningful work on probiotics, prebiotics, and fermented foods and their impact on constipation.

The award criteria stipulated that the researchers must be fewer than five years from their terminal degree, and their scope of research must be basic or clinical research disciplines in the fields of probiotics, prebiotics, synbiotics, postbiotics or fermented foods. In addition, the researchers were required to show evidence of a significant research finding and its publication(s), new ideas that advance the field, and / or evidence of impact through citizenship, general outreach, social media or other means.

The prize committee chose the two recipients from among dozens of applicants and identified each of them as having made important contributions to the field at this early stage in their scientific careers. Each winner will receive a cash prize and an opportunity to speak at the ISAPP annual meeting, to be held in Spain in June, 2022.

Stay tuned to learn more about these rising star researchers!

See here for details about the 2022 Glenn Gibson Early Career Research Prize

Do fermented foods contain probiotics?

By Prof. Maria Marco, PhD, Department of Food Science & Technology, University of California, Davis

We frequently hear that “fermented foods are rich in beneficial probiotics.” But is this actually true? Do fermented foods contain probiotics?

The quick answer to this question is no – fermented foods are generally not sources of probiotics. Despite the popular assertion to the contrary, very few fermented foods contain microbes that fit the criteria to be called probiotic. But this fact does not mean that fermented foods are bad for you. To uphold the intent of the word probiotic and to explain how fermented foods actually are healthy, we need to find better ways to describe the benefits of fermented foods.

Probiotics are living microorganisms, that when administered in adequate amounts, confer a health benefit on the host (Hill et al 2014 Nat Rev Gastroenterol Hepatol). This current definition reflects minor updates to a definition offered by an expert consultation of scientists in 2001 convened by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization. Evident from the definition, a microbial strain is not a probiotic unless a health benefit has been found with its use. At a minimum, the strain should be proven to be beneficial in at least one randomized controlled trial (RCT). Probiotics must also be defined at the strain level through genome sequencing (a strain is a single genotype of a species).

Fermented foods, on the other hand, have no requirement to improve health. Fermented foods are foods and beverages made through desired microbial growth and enzymatic conversion of food components. This definition was recently formulated by an ISAPP consensus panel of scientific experts to affirm the common properties of all foods of this type and to differentiate foods that may look or taste similar but are not made using microbes (Marco et al 2021 Nat Rev Gastroenterol Hepatol). Fermented foods encompass an expansive variety of foods made from animal and plant sourced ingredients and produced from all types of microbial metabolism. The desired characteristics of these foods are frequently how they look, smell, and taste. There no expectation in this definition that fermented foods alter health in any way.

There is also no requirement for fermented foods contain living microbes at the time they are ingested. Foods such as bread, chocolate, and beer are fermented but then are baked, roasted, and/or filtered. This means those fermented foods cannot be probiotic.

Some fermented foods, such as kimchi and kombucha, are typically eaten with living microbes present. However, the microbes in those foods usually do not meet the criteria to be called probiotic. Whether the fermented food was made at home or purchased from the supermarket, studies investigating whether the microbes in those fermented foods are specifically responsible for a health benefit remain to be done. Those foods also do not contain microbes defined to the strain level, nor is the number of living microbes typically known. An exception to this is if specific strains previously shown to provide a health benefit in one or more RCT are intentionally used in the production of the food and remain viable at expected numbers over the shelf-life of that fermented food product. An example of this would be a commercial fermented yogurt that has an added probiotic strain remaining viable at the time of consumption, beyond the strains that carried out the fermentation.

Despite these distinctions between probiotics an fermented foods, the probiotics term has pervaded common lexicon to mean “beneficial microbes”. In contrast to pathogenic or harmful microbes, beneficial microbes are those that are understood to help rather than hurt bodily functions. However, just as we do not assume that all pathogens cause the same disease or result in the same severity of symptoms, we should also not expect that beneficial microbes all serve the same purpose. By analogy, automobiles are useful vehicles which help us to get from place to place. We do not expect that all automobiles perform like those used for Formula 1 racing. Microbes are needed to make fermented foods and may be beneficial for us, but we should not assume that those drive health benefits like established probiotic strains.

What are the consequences of calling fermented foods probiotic when they include undefined numbers of living microbes for which strain identities are not known? One can suppose that there is no harm in labeling or describing those products as “probiotic” or “containing probiotics”. However, by doing so, confusion and misunderstanding is created and too often, spread by journalists, nutritionists, scientists, and medical professionals. For example, news articles in reputable sources have written that foods like kefir, kimchi, sauerkraut made from beets or cabbage, pickles, cottage cheese, olives, bread and chocolate are rich in probiotics. As misuse perpetuates, what becomes of bona fide probiotics shown with rigorous study to benefit health, such as reducing the incidence and duration of diarrhea or respiratory infections? It becomes difficult to know which strains have scientific proof of benefit. Just as there are laws for standards of food identity, we should strive to do the same when describing microbes in fermented foods.

Avoiding the term probiotic when describing fermented foods should not stop us from espousing the myriad of positive attributes of those foods. Besides their favorable sensory qualities, fermented foods are frequently safer and better tolerated in the digestive tract than the foods they are made from. During the production of fermented foods, microbes remove or reduce toxins in the ingredients and produce bioactive compounds that persist long after the microbes that make them are gone.

Even though the living microbes in fermented foods may not rise to the standard of a probiotic, they may provide health benefits. We just don’t have the studies to prove that they do. With more study, we may find that viable microbes in fermented foods work similarly to probiotics in the digestive tract through shared mechanisms. This is already known for yogurts. Yogurt cultures share the ability to deliver lactase to the intestine, thereby improving tolerance of lactose by intolerant individuals. Clinical and epidemiological studies performed on fermented foods already suggest an association between them and different health benefits but as we recently explained (Marco et al 2021 J Nutrition), more work is needed in order to understand if and what benefits these microbes provide.

For now, we should simply continue enjoying the making and eating of fermented foods and reserve the term probiotics for those specific microbial strains which have been shown to improve our health. Marketers should resist labeling products as containing probiotics if their products do not meet the criteria for a probiotic. Indeed, the descriptor “live and active cultures” more accurately reflects the microbial composition of many fermented foods, and should be used until controlled human trials demonstrating health benefits are conducted.

 

Additional resources:

How are probiotic foods and fermented foods different? ISAPP infographic.

Fermented foods. ISAPP infographic.

What are fermented foods? ISAPP video.

Are fermented foods probiotics? Webinar by Mary Ellen Sanders, PhD.

 

ISAPP’s 2021 year in review

By Mary Ellen Sanders, PhD, ISAPP Executive Science Officer

The upcoming year-end naturally leads us to reflect about what has transpired over the past 12 months. From my perspective working with ISAPP, I witnessed ISAPP board members and the broader ISAPP community working creatively and diligently to find solutions to scientific challenges in probiotics, prebiotics and related fields. Let’s look back together at some of the key developments of 2021.

ISAPP published outcomes from two consensus panels this year, one on fermented foods and one on postbiotics. The popularity of these articles astounds me, with 49K and 29K accesses respectively, as of this writing. I think this reflects recognition on the part of the scientific community of the value – for all stakeholders – of concise, well-considered scientific definitions of terms that we deal with on a daily basis. If we can all agree on what we mean when we use a term, confusion is abated and progress is facilitated. The postbiotics definition was greeted with some resistance, however, and it will remain to be seen how this is resolved. But I think ISAPP’s response about this objection makes it clear that productive definitions are difficult to generate. Even if the field ultimately embraces another definition, it is heartening to engage in scientific debate about ideas and try to find alignment.

Keeping with the idea of postbiotics, a noteworthy development this year was the opinion from the European Food Safety Authority that the postbiotic made from heat-treated Akkermansia muciniphila is safe for use as a novel food in the EU. Undoubtedly, this development is a bellwether for likely future developments in this emerging area as some technological advantages to postbiotics will make these substances an attractive alternative to probiotics IF the scientific evidence for health benefits becomes available.

Recognizing the existing need for translational information for clinicians, ISAPP developed a continuing education course for dietitians. Published in March, it has currently reached close to 6000 dietitians. This course focused on probiotics, prebiotics and fermented foods: what they and how they might be applied in dietetic practice. It is a freely available, self-study course and completion provides two continuing education credits for dietitians.

On a sad note, in March of this year, ISAPP suffered the loss of Prof. Todd Klaenhammer. Todd was a founding ISAPP board member, and directed many of our activities over the course of his 18-year term on the board. He was also my dear friend and major advisor for my graduate degrees at NC State many years ago.  As one former collaborator put it, “I was not prepared to finish enjoying his friendship and mentorship.” See here for a tribute to Prof. Klaenhammer on the ISAPP blog: In Memoriam: Todd Klaenhammer.

So where will 2022 lead ISAPP? The organization has now published five consensus definitions: probiotics, prebiotics, synbiotics, postbiotics and fermented foods – extending its purview beyond where it started, with probiotics and prebiotics. Through the year ahead, ISAPP is committed to providing science-based information on the whole ‘biotics’ family of substances as well as fermented foods. Our Students and Fellows Association is growing, supported by the opportunity for young scientists to compete for the Glenn Gibson Early Career Researcher Prize. We continue to see our industry membership expand. Through our new Instagram account and other online platforms, our overall community is increasing. The ISAPP board of directors continues to evolve as well, with several long-term members leaving the board to make room for younger leaders in the field who will direct the future of the organization. This applies to me as well, as I have made the difficult decision to depart ISAPP in June of 2023. Thus, hiring a new executive director/executive science officer is an important priority for ISAPP in 2022. My 20 years with ISAPP have seen the organization evolve tremendously, through the hard work of incredible board members as well as many external contributors. We will strive to make 2022 – our 20th anniversary – ISAPP’s best year yet.

Scientists looking at a bottle of probiotic supplements.

Current issues in probiotic quality: An update for industry

By Dr. Mary Ellen Sanders, ISAPP, Dr. Kit Goldman, USP, Dr. Amy Roe, P&G, Dr. Christina Vegge, Dr. Jean Schoeni, Eurofins

With probiotic dietary supplement use growing globally and an increasing array of products on the market, probiotic quality is an issue of perpetual relevance to industry. Best practices for producing high-quality probiotics change frequently, making it important for companies to stay informed.

ISAPP convened a webinar on this topic, available to ISAPP members only. The webinar took place November 16, 2021, and was hosted by Executive Science Officer, Dr. Mary Ellen Sanders. Speakers focused on the activities of the United States Pharmacopoeia (USP), a non-profit organization based in the US and operating globally, which for the past 200 years has worked to improve public health through development of quality standards for medicines, dietary supplements and foods. In 2017 USP formed an Expert Panel on probiotics.

Dr. Kit Goldman, Sr Director, Dietary Supplements and Herbal Medicines, USP, spoke about the origin of USP and the USP activities related to probiotic quality. USPs expert volunteers have determined the necessary parameters for probiotic quality standards, which include tests for identification, assay/enumeration and contaminants, and have created standards for a number of probiotic species/strains. In the course of doing so, the Probiotics Expert Panel identified specific areas where more information was needed to fully understand issues related to probiotic quality. This led to the formation of sub-teams to consider aspects of probiotic identification, enumeration and safety.

Dr. Amy Roe, Principal Scientist at P&G, spoke on appropriate regulatory requirements for probiotic safety. Currently, there is no global harmonization on the requirements for establishing probiotic safety for use in foods and supplements. Although ‘history of safe use’ has been central to safety assessments for many current probiotic species, probiotic manufacturers are increasingly seeking to use new strains, species, and next-generation probiotics; justification of safety based on a significant history of use may be challenged. USP and other stakeholders are looking to develop best practices guidelines for assessing the quality and safety of probiotics. A current initiative of the USP seeks to provide expert advice specific to safety considerations for probiotics through reviewing global regulatory guidelines, evaluating appropriateness of traditional animal toxicology studies for studying the safety of probiotics, highlighting the importance of proper manufacturing practices with regard to final product safety, and outlining of essential parameters of a comprehensive safety assessment for a probiotic.

Dr. Jean Schoeni, Fellow at Eurofins, spoke on comparing probiotic enumeration methods. One challenge faced by the USP Probiotics Expert Panel is how to compare the increasing number of probiotic enumeration methods appearing in monograph submissions. A sub-team of the panel developed a solution that combines APLM (Analytical Procedures Lifecycle Management – a streamlined approach for determining the method’s fitness for intended use) with TI (tolerance interval) calculations. Schoeni encouraged companies to adopt this solution, highlighting tools that have been provided to the probiotics industry through publication of the sub-team’s work.

Dr. Christina Vegge spoke on quantification of multi-strain blends. For probiotic products comprising multiple strains, the viable numbers of each strain in these products would ideally be quantified. However, reliance on plate count methods creates analytical challenges regardless of whether the quantification of viable numbers of each strain in the blend is conducted prior to or after blending. Further challenges arise when addressing the reductions in potency over shelf life of the product. For multi-strain products, plate count procedures are insufficient—and currently no official guideline or general best practice exists to resolve this situation. Therefore, the USP Probiotics Expert Panel wants to conduct an explorative study to examine non-culture based technologies to quantify the viable composition of multi-strain blends.

A recording of this webinar is available for ISAPP industry members only. Please see here and email info@nullisappscience.org for the password to access this page.

Publications (open access) from USP Probiotics Expert Panel:

Jackson et al. Improving End-User Trust in the Quality of Commercial Probiotic Products. Front Microbiol. 2019 Apr 17;10:739.  doi: 10.3389/fmicb.2019.00739.

Weitzel MLJ, et al. Improving and Comparing Probiotic Plate Count Methods by Analytical Procedure Lifecycle Management. Front Microbiol. 2021 Jul 12;12: 693066. doi: 10.3389/fmicb.2021.693066.

 

 

The American College of Gastroenterology recommends against use of probiotics for primary or secondary prevention of C. difficile

By Prof. Daniel Merenstein MD, Georgetown University School of Medicine and Prof. Eamonn Quigley MD FRCP FACP MACG FRCPI,  Houston Methodist Hospital and Weill Cornell Medical College

The American College of Gastroenterology (ACG) recently published ACG Clinical Guidelines: Prevention, Diagnosis, and Treatment of Clostridioides difficile Infections. This review considers probiotics for prevention of CDI. The ACG’s recommendations regarding probiotics and C. difficile infection (CDI) are:

  1. We recommend against probiotics for the prevention of CDI in patients being treated with antibiotics (primary prevention) (conditional recommendation, moderate quality of evidence).
  2. We recommend against probiotics for the prevention of CDI recurrence (secondary prevention) (strong recommendation, very low quality of evidence).

The ACG guidelines take a different approach to the evidence relating to probiotics than that of the American Gastroenterological Association (AGA) or the Cochrane Collaboration. The most recent Cochrane review on prevention of C. difficile-associated diarrhea (CDAD) concluded in brief, “moderate certainty evidence suggests that probiotics are effective for preventing CDAD”. In the AGA Clinical Practice Guidelines on the Role of Probiotics in the Management of Gastrointestinal Disorders, the recommendation was:

In adults and children on antibiotic treatment, we suggest the use of S. boulardii; or the 2-strain combination of L. acidophilus CL1285 and Lactobacillus casei LBC80R; or the 3-strain combination of L acidophilus, Lactobacillus delbrueckii subsp bulgaricus, and Bifidobacterium bifidum; or the 4-strain combination of L. acidophilus, L. delbrueckii subsp bulgaricus, B. bifidum, and Streptococcus salivarius subsp thermophilus over no or other probiotics for prevention of C difficile infection. (Conditional recommendation, low quality of evidence.)

In both the AGA and ACG guidelines the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system was used. How, then, did they come to such different conclusions and recommendations?

The ACG guideline stated,  “a meta-analysis of 19 RCTs that concluded that probiotics were helpful at prevention of CDI in hospitalized patients if given close to start of antibiotics, with a 70% lower risk if probiotics were started within 2 days but falling to a 30% risk reduction if probiotics were started after 2 days of antibiotic therapy”. But then they did not take timing of probiotic administration into account as they assessed the evidence. Instead, they use the negative PLACIDE trial to override all other evidence for primary prevention. The PLACIDE trial was a well-done trial, but participants started their probiotic treatment 3- 7 days after antibiotics. Thus, it would seem that the ACG guideline’s conclusion could favor probiotics as long as they can be started within 2 days of the antibiotic and not recommend against probiotic use.

The ACG guideline objects to combining data on different probiotic strains in meta-analyses in order to provide evidence in favor of probiotics: “Evidence to support probiotics for this indication comes mainly from meta-analyses that pool data from small trials of different probiotic formulations and methodologies.” This is true, but the Cochrane review found thirty-nine studies (8,672 participants) that met their eligibility criteria and it is noteworthy that several different probiotics were found to be effective. The Cochrane number needed to treat (NNT) to prevent CDI is 42. However, if the ACG thought this was driven by too many negative trials, they could have qualified their recommendation. The Cochrane review found in subgroup analyses that probiotics are most effective (NNT=12) among trials with a CDI baseline risk >5%. But to conclude there is no benefit of probiotics for primary CDI is not supported by the evidence.

It is puzzling that ACG insists that the probiotic literature be pooled in a strain-specific manner, yet they support conclusions on fecal microbial transplant (FMT) even though FMT interventions are much more heterogeneous than probiotics in regard to composition and mode of administration. They recommend FMT for treatment of C. difficile based on only three double-blinded randomized clinical trials (here, here and here), only one of which was positive. The positive FMT study was conducted at two sites and compared donor stool (heterologous) versus patient’s own stool (autologous) administered by colonoscopy. Overall, 91% of patients in the FMT group achieved clinical cure compared with 63% in the control group. At site #1, the cure rate with donor FMT was 90.0% (CI, 51.8% to 98.7%) versus 42.9% with autologous FMT, whereas in site #2 the cure rate was essentially identical between the two groups. At site #2, donor FMT cure rate was 91.7% (CI, 57.2% to 98.9%) compared with 90.0% (CI, 51.8% to 98.7%) after autologous FMT. We mention this to question the consistency of evidence standards that the ACG guideline authors impose. They admonish pooled data from small trials of different probiotic formulations and methodologies yet ignore heterogeneity in FMT interventions. The data reviewed for probiotics was primarily from double-blinded randomized trials, while for FMT they rely on case series, uncontrolled studies or retrospective studies.

The authors go on to state, “… high quality evidence to support probiotics for most conditions is scarce.” How do they define “scarce” and “most conditions”? As mentioned, Cochrane found thirty-nine studies (8,672 participants) for prevention of CDAD. Under “summary of evidence”, the authors address issues such as size of the market, regulatory oversight, product cost and quality control problems with commercial products, all of which may reflect practical concerns with some probiotic products in the marketplace, but have nothing to do with available evidence. Furthermore, it is the only intervention where the financial value of the industry and cost of interventions is mentioned. Why are the size of the market or costs for FMT or drugs not just as relevant to this review? Cost is discussed throughout the recommendation but without performing or citing a formal cost analysis or cost-effectiveness analysis, even though there are approaches for doing so to inform evidence-based decision-making (here).

The authors indict probiotics for concerns about safety, citing not the thorough review sponsored by AHRQ and conducted by the RAND corporation that looked at 622 studies and found no statistically significant increased relative risk of the overall number of experienced adverse events (RR 1.00; 95% confidence interval [CI]: 0.93, 1.07, p=0.999), but by referring to a review article that cites case reports of blood infections and refers to one study with microbiota, not clinical, endpoints done in Israel on one commercial product. The data actually show that for well characterized, clinically tested probiotics with high levels of quality control the evidence for infectious complications in non-vulnerable populations is virtually nil. ACG does not mention that FMT was shut down due to safety concerns as soon as the pandemic started.

In summary, we are not convinced that the authors have justified their recommendation against the use of probiotics in relation to CDI prevention. They fail to clarify why the results of their GRADE evaluation of probiotic evidence for prevention of C. difficile resulted in totally different conclusions compared to the AGA document, which found evidence sufficient for conditional recommendation of four probiotic preparations. Further,  the review of evidence for probiotics, whether in terms of efficacy or safety, should be addressed in a manner consistent with other interventions considered and editorializing on issues such as market size, profits and product cost, in the absence of an objective approach using appropriate instruments, should be avoided.

The USDA Global Branded Food Products Database is Now Accepting Data on Live Microbes – Call for Data Submission

Marie E. Latulippe, MS, RDN, Director of Science Programs and Brienna Larrick, PhD, PMP Scientific Program Manager, Institute for the Advancement of Food and Nutrition Sciences (IAFNS), Washington DC

As noted by Marco et al. (2020), evidence from observational studies and randomized controlled trials suggests that the consumption of safe, live microbes can support health. However, more data are needed to accept or refute this hypothesis, and to develop a full understanding of population exposure. As of October 2021, the USDA Global Branded Food Products Database is accepting information on live microbes in foods and beverages. With participation from manufacturers, this initiative will eventually enhance our understanding of the numbers of live microbes that populations consume from food.

The USDA Global Branded Food Products Database contains ingredient and nutrition composition data on over 368,000 branded and private label (i.e., store brand) foods and beverages. This information is provided voluntarily by the food industry. The impact of industry providing these data is substantial; it means these data are available to inform agricultural and food policy decisions by federal agencies, and to support research and regulatory queries by the public and private sectors. By supplying information on live microbes in foods, the food industry can provide researchers with useful data on quantities of live microbes in foods and enable them to link these data to associated health outcomes. Ultimately, this could contribute to determining if a recommended intake level for the consumption of safe, live microbes from foods (e.g., yogurts) is supported by evidence.

The food industry is encouraged to contribute to this initiative by providing the following data on the food and beverage products they produce:

  • Quantity (range) of live microbes
  • Method of analysis used to determine quantity of live microbes
  • Type(s) of live microbes present in the product

Data on live microbes can be submitted via 1WorldSync, a Global Data Synchronization Network (GDSN) data pool provider. For more information, including guidance for submitting data and technical support, visit here.

The partners in this public-private partnership are USDA, IAFNS, GS1 US, 1WorldSync, NielsenIQ Label Insight, and the University of Maryland. For more information on the USDA Global Branded Food Products Database, visit here.

ISAPP and IAFNS collaborate on a project focused on live dietary microbes through the IAFNS gut microbiome committee. Mary Ellen Sanders, Executive Science Officer for ISAPP, represents ISAPP on this committee.

Related links:

New ISAPP-led paper calls for investigation of evidence for links between live dietary microbes and health

Gut Microbiome Webinar Series sponsored by IAFNS

 

Should the concept of postbiotics make us see probiotics from a new perspective?

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

In early May 2021 an ISAPP consensus panel  defined postbiotics as “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host“. The fact that non-viable microbes may still deliver health benefits is not new for the scientific community and was reviewed more than 20 years ago. More recent studies demonstrating health effects of non-viable microbes spurred interest in this topic, leading ISAPP to carefully consider the emerging use of the term ‘postbiotic’ and provide a clear, modern, concise definition.

Postbiotics can be contrasted with probiotics: live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. In practice, probiotics  have likely always coexisted with inanimate microbes, as live microbes will die at all phases of production of a product. In the past, it seems the presence of inanimate microbes as part of probiotic products was not really considered. We all knew they were there, but made a default assumption that they had limited significance. As we consider postbiotics, though, we should perhaps look again at how to address the inanimate components of probiotic products.

The presence of inanimate cells in probiotic preparations: from lab to product

A loop of a fresh, overnight, live culture of a probiotic strain may still contain non-viable cells (Fig. 1). During the biomass production of a probiotic culture, an abundance of live cells can be observed in the exponential growth phase, but as the culture enters the stationary phase, a significant increase in the proportion of non-viable cells occurs (Fig. 2). Yet the culture may display a satisfactory high number of viable cells as verified by traditional plating on agar media. Some years ago, it was reported that a fresh culture of a lactobacilli strain may display a live/dead cells ratio of ca. 100/1. However, this ratio may change to 1:1 after freeze-drying, as studied using flow cytometry, a technology that allows the quantification of both live and dead cells in a culture-independent way. Therefore, a recently freeze-dried culture of a probiotic strain may contain 1010 log CFU/g of live cells, but also the same amount of non-viable cells.

Food supplements may have a shelf life between 12 and 24 months at room temperature and over this time, a proportion of cells will likely lose viability along the shelf life. This depends on the intrinsic resistance of the strain, the nature of the matrix used for freeze-drying, the water activity remaining after lyophilization, the package and the storage conditions. Taking this into consideration, the probiotic supplements industry overfills probiotic capsules or sachets with 1.5 to 4 times more live cells, in order to warrant the delivery at the end of shelf life of the minimum amount of live cells to be able to deliver the expected health benefit. Considering that both freeze-drying and long-term storage may significantly increase the proportion of inanimate microorganisms in a probiotic supplement, a probiotic supplement could easily consist of more inanimate microorganisms than live ones. Yet if the products delivers the minimum amount of live cells to confer a health benefit, this makes the product fit the definition of probiotics so it must be considered a probiotic product. The probiotic focus has been prevalent during previous clinical trials and also during the shelf life of a probiotic product. Maybe we were just overlooking what was going on beyond the information obtained by CFU. These new insights do not change the status of a probiotic, but with due attention given to postbiotic components, offers the possibility to have better and better characterized products in the future.

Figure 1, above – Fluorescence microscopy images of an overnight (18h) culture of bifidobacteria (left) and lactobacilli (right) showing live (green) and non-viable cells (red). The Live/dead BackLight Invitrogen® kit was used for staining cells.

Figure 2, above – Fluorescence microscopy images of a culture of lactobacilli in the exponential (left) and late stationary (right) growth phase showing live (green) and non-viable cells (red). The Live/dead BackLight Invitrogen® kit was used for staining cells.

Are dead probiotics ‘postbiotics’?

What is the contribution of these inanimate cells to the overall health benefit observed for the probiotic culture? In most cases, no evidence exists documenting health benefits of inanimate probiotics. But we may have reason to suspect it may be relevant. For example, a live culture of Bifidobacterium bifidum MIMBb75 significantly alleviated irritable bowel syndrome symptoms and improved quality of life in a double-blind, placebo-controlled study when delivered at 109 CFU, but also the same strain performed equally well for the same end-point when delivered as a heat-inactivated culture. Also, a novel next-generation probiotic strain of Akkermansia muciniphila performed equally well in its live and pasteurized form for improving several metabolic parameters in overweight and obese volunteers. In these cases, it can be said that both strains fit simultaneously the probiotic and the postbiotic definitions.

However, does this mean that as the strain gradually loses viability during storage it gradually becomes a postbiotic? No! This is because method of strain inactivation may play an important role in the health benefit observed. For example, the health benefit delivered by a strain that underwent a heat inactivation can not be assumed to have the same functionality if it is left to die on its own on the shelves. A heat treatment may, for instance, modifiy the spatial display of surface proteins and this may lead to a different immunomodulating capacity of the strain when compared to spontaneous and gradual cell viability lost along storage.

Characterizing probiotic products with an eye to the presence of non-viable cells

By definition, probiotics must be quantified. In the past, this quantification has been limited to numbers of viable cells, typically using a colony count method. This is wholly appropriate, as probiotics must be alive. Yet for the future, will it become necessary to quantify the numbers of non-viable microbial cells as well? With evidence emerging that these non-viable cells may be functional components, then a reasonable argument can be made that this component of a probiotic product should also be quantified. This has implications for characterizing products for use in intervention trials and for the marketplace. The challenge for the marketplace is that probiotic products should deliver the functionality observed in intervention trials.

Reports of trials typically indicate a viable count of the probiotic being tested, but these can be reported in different ways. For example, the statement may indicate delivery of 1.9 × 107 CFU/day of the strain XXX, or delivery of > 1.9 × 107 CFU/day of the strain XXX. These are very different and neither gives any indication of the level of non-viable microbes. The first expression is a specific measure of the viable count at a particular point in time. The second indicates a target minimum and the actual count of viable cells could be much greater. Counts all along the intervention are rarely reported, even though that count could change substantively over time. Papers rarely report if the same batch or different batches of the probiotic preparation were used. The potentially increasing proportion of inanimate microbes is never reported.

In light of postbiotics, future studies should report quantifications of both live and inanimate microbes. Although it is not clear at this time what role inanimate microbes may play in probiotic efficacy, a first step is understanding the composition of probiotic products.

 

ISAPP members can access Dr. Vinderola’s webinar on this topic here. Email info@nullisappscience.org if you require the password.

Hands holding mobile phone

Virtual events continue to fill gaps as in-person meetings are being planned

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

For scientists, annual meetings provide coveted opportunities to hear about the latest scientific advances from expert researchers, and they are where students and trainees get to present their research, often for the first time. Of course, meeting and socializing with colleagues, both new and old, during breaks and evening sessions is also an important part of these conferences.

Yet over the past two years, most occasions to meet face-to-face were canceled. Virtual meetings became the new normal and, even though a poor substitute for in-person gatherings, provided opportunities to connect and share emerging science. As we anticipate being together again in person – hopefully for 2022 meetings – take note of three upcoming conferences to fill the gap. Each of these feature meetings are related to the gut microbiome, diet, and health.

(1) In October, the Agriculture and Health Summit: Cultivating Gut Health at the Crossroads of Food & Medicine is a FREE three-day virtual conference that brings together a unique combination of researchers, industry leaders and thought leaders from the biomedical and agricultural sectors for important conversations about the future of human health. The event will provide a rare opportunity for individuals with diverse areas of expertise to discuss opportunities and challenges in creating ‘foods for health’ through the gut microbiome, working toward solutions in nutrition and medicine. More information can be found here. Among the presenters are ISAPP Executive Science Officer, Mary Ellen Sanders, and board members, Dan Merenstein and Bob Hutkins.

 

(2) Then in November, a Nature-sponsored online conference called Reshaping the Microbiome through Nutrition will be held. According to the website, “this conference will bring together researchers working on the microbiome and nutrition to discuss how our microbiota use and transform dietary components, and how these nutrients and their products influence host health throughout life, including effects on development and infectious and chronic diseases. A central theme of the meeting will be how diet and dietary supplements could be harnessed to manipulate the microbiome with the aim of maintaining health and treating disease”More information is found here.

(3) Another meeting in November is organized across ten centers/institutes at the NIH and the Office of Dietary Supplements and the Office of Nutrition Research. This two-day conference November 5 and 8, titled Precision Probiotic Therapies—Challenges and Opportunities, features a Keynote address by Prof. Jeff Gordon, from the Washington University School of Medicine. ISAPP president Prof. Dan Merenstein, Georgetown University School of Medicine, is also presenting. To register for this FREE meeting, see here.

 

In this current era, interest in how diet (including probiotics, prebiotics, and fermented foods) influences the microbiome and affects human and animal health has never been greater, as is evident by these and other similarly-themed conferences.

ISAPP is planning its next annual by-invitation meeting, to be held in person.

 

Using probiotics to support digestive health for dogs

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

Because dogs are considered to be members of the family by most pet owners today, their health and well-being is a top priority. As with humans, nutritional products supporting gastrointestinal health are some of the most popular. Many pets are healthy, but loose stools, constipation, and various gastrointestinal disorders and diseases such as inflammatory bowel disease and irritable bowel syndrome are common. In fact, within the pet food conversation, digestive health improvements have been the most discussed health benefits among social media discussion posts over the past 2 years (see here). Given the high interest in digestive health, it is not surprising that the canine microbiome has been of great interest over the past decade, with many recent reviews reporting on the overall composition of the gastrointestinal microbiota and how it is impacted by diet (Barko et al., 2018; Alessandri et al., 2020; Wernimont et al., 2020). Gastrointestinal microbiome changes contributing to or resulting from digestive diseases have also been documented in dogs (Redfern et al., 2017; Ziese and Suchodolski, 2021). Animals under high levels of stress or undergoing antibiotic therapy are also known to have poor stool quality and an altered gut microbiota (i.e., dysbiosis) (Pilla et al., 2020).

Dietary fibers and prebiotics are commonly used in complete and balanced diets to improve or maintain stool quality, provide laxation, and positively manipulate the microbiota of healthy animals. The use of probiotics is also popular in dogs, but the route of administration, efficacy, and reason for use is usually different than that of fiber and prebiotics. Probiotics are usually provided in the form of supplements (e.g., powders, capsules, pastes) and are most commonly used to treat animals with gastrointestinal disease rather than support the healthy condition. Live microbes are added to many dry extruded foods as ‘probiotics’, but in many cases, maintaining viability and evidence for a health benefit for dogs is lacking for these products. Such microbes would not meet the minimum criteria to be called a ‘probiotic.’ Viability is a challenge because most HACCP plans for producing complete and balanced pet foods include a kill step that inactivates all microorganisms. Therefore, inclusion must be applied post-extrusion on the outside of the kibble. Even if applied in this way, low numbers of viable organisms are common (Weese and Arroyo, 2003). Post-production inclusion is not possible for other diet formats (e.g., cans, pouches, trays). Although spore-forming bacteria that may survive the extrusion process have been of interest lately, evidence of efficacy is lacking thus far.

Picture of Simka (a Samoyed) courtesy of ISAPP board member Dr. Daniel Tancredi

Even though health benefits coming from the inclusion of live microorganisms in dog foods is not supported by the peer-reviewed literature, such evidence exists for many probiotic supplements. The clinical effects of probiotics in the prevention or treatment of gastrointestinal diseases in dogs have been reviewed recently (Schmitz and Suchodolski, 2016; Suchodolski and Jergens, 2016; Jensen and Bjornvad, 2018; Schmitz, 2021). Although some similarities exist, recent research has shown that distinct dysbiosis networks exist in dogs compared to humans (Vazquez-Baeza et al., 2016), justifying unique prevention and/or treatment strategies for dogs.

One population of dogs shown to benefit from probiotics has been those with acute idiopathic diarrhea and gastroenteritis, with a shorter time to resolution and reduced percentage of dogs requiring antibiotic administration being reported (Kelley et al., 2009; Herstad et al., 2010; Nixon et al., 2019). Probiotic administration has also been shown to benefit dogs undergoing antibiotic therapy and those engaged in endurance exercise – two conditions that alter the gastrointestinal microbiota and often lead to loose stools. In those studies, consumption of a probiotic helped to minimize gastrointestinal microbiome shifts and reduced the incidence and/or shortened the length of diarrhea (Gagne et al., 2013; Fenimore et al., 2017). Dogs diagnosed with inflammatory bowel disease have also been shown to benefit from probiotic consumption (Rossi et al., 2014; White et al., 2017). In these chronic conditions, drug therapy is almost always required, but probiotics have been shown to help normalize intestinal dysbiosis, increase tight junction protein expression, and reduce clinical and histological scores.

So what is the bottom line? Well, for dogs with a sensitive stomach and/or digestive health issues, probiotics may certainly help. Rather than relying on live microbes present in the dog’s food or adding a couple spoonfuls of yogurt to the food bowl each day, it is recommended that owners work with their veterinarian to identify a probiotic that has the best chance for success. The probiotic selected should provide an effective dose, be designed for dogs, target the specific condition in mind, and be backed by science. As summarized here, it is important to remember that all probiotics are different so the specific microorganism(s), supplement form, storage conditions, and dosage are all important details to consider.

 

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.

ISAPP board member Prof. Dan Tancredi kindly provided pictures of Simka, pet Samoyed, for the post.

 

Bacterial vesicles: Emerging potential postbiotics

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

The recently published ISAPP consensus paper defines a postbiotic as “a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host“. Such a definition quickly brings to mind that a postbiotic is not equivalent to microbial metabolites. A postbiotic should also contain inanimate microbial cells or cell fragments. Metabolites or fermentation products may be present, but they are not required.

Because microbes are complex entities, we must be open to innovative understandings of what a postbiotic might entail. Indeed, although not explicitly mentioned in the ISAPP consensus paper, extracellular membrane vesicles may comprise an innovative conceptualization of a postbiotic, falling within the ‘cell component’ part of the postbiotic definition.

Bacterial vesicles

Extracellular membrane vesicles (EMV) are universal carriers of biological information produced in all domains of life. Bacterial EMV are small, spheroidal, membrane-derived proteoliposomal nanostructures, typically ranging from 25 – 250 nm in diameter, containing proteins, lipids, nucleic acids, metabolites, numerous surface molecules and many other biomolecules derived from their progenitor bacteria (Figure 1). Bacterial vesicles have been known for more than 50 years as structures able to carry cellular material (Ñahui Palomino et al. 2021).  However, studies on membrane vesicles derived from Gram-positive bacteria are more recent as it was for a time believed they were incapable of producing vesicles due to their thick and complex cell walls, and the lack of an outer membrane. Today, EMVs have been isolated from Gram-positive probiotic bacteria, including those belonging to the Lactobacillaceae family (under which Lactobacillus was recently split into many new genera) and the Bifidobacterium genus. In probiotic bacteria, vesicles may mediate quorum sensing and material exchange. Perhaps even more important, they can act as mediators of bacteria-to-cell and bacteria-to-bacteria interactions. As bacterial EMV are inanimate structures that cannot replicate, they fit the postbiotic definiton as cell components as long as other criteria stipulated by the definition are met.

Figure 1. Membrane vesicles budding on the surface of L. reuteri DSM 17938 and released into the surrounding medium. These vesicles were described in by Grande et al. 2017. Photo used with permission of BioGaia.

Functions of bacterial vesicles related to potential health benefits

Underlying mechanisms and corresponding molecules driving health effects of bacterial vesicles are not well understood, in part due to reliance on in vitro models. Bacterial EMV derived from Lactobacillaceae spp., Bifidobacterium spp., and Akkermansia spp. have been reported to alleviate metabolic syndrome and allergy symptoms, promote T-cell activation and IgA production, strengthen gut barrier function, and exhibit anti-viral and immunomodulatory properties (Kim et al. 2016; Tan et al. 2018; Ashrafian et al. 2019; Molina-Tijeras et al. 2019; Palomino et al. 2019; Shehata et al. 2019; Bäuerl et al. 2020). Interestingly, vesicles from Limosilactobacillus reuteri DSM 17938 (West et al. 2020) and Lacticaseibacillus casei BL23 (Domínguez Rubio et al. 2017) may accomplish some of the the effects of these probiotic bacteria. In fact it is not unreasonable to think that EMVs may be already present and active in probiotic products.

Challenges for bacterial vesicle production

To develop a postbiotic from microbial EMVs, many challenges need to be overcome.  Defining optimal cultivation conditions, and methods for vesicle release, isolation and scaling up are some of the challenges of bacterial vesicle production. There are several studies showing that altering the cultivation parameters can impact vesicle production. Examples of treatments shown to increase vesicle release include exposure to UV radiation and antibiotic pressure (Gamalier et al. 2017; Gill et al. 2019). Exposure to glycine has also been shown to increase vesicle production (Hirayama & Nakao 2020). Interventions during culture, for example by introducing agitation and varying pH, can possibly be ways to potentiate vesicle release and increase their bioactivity (Müller et al. 2021). A recent report also revealed that B. longum NCC2705 released a myriad of vesicles when cultured in human fecal fermentation broth, but not in basal GAM anaerobic medium (Figure 1). Moreover, the B. longum vesicle production pattern differed among individual fecal samples suggesting that metabolites derived from symbiotic microbiota stimulate the active production of vesicles in a different manner (Nishiyama et al. 2020). Whether any of these treatments and culture conditions are general or strain specific remains to be elucidated. Large differences in the number of vesicles that may be obtained by different extraction methods can occur (Tian et al. 2020). Tangential flow filtration or the use of antibodies targeting specific epitopes of the vesicles are some of the options proposed for the large scale isolation of EMV (Klimentová & Stulík 2015).

Figure 2. Left: Bifidobacterium longum NCC2705 grown on GAM broth. Right: secretion of membrane vesicles by Bifidobacterium longum NCC2705: the strain was cultured in bacterial-free human fecal fermentation broth and secreted a myriad of membrane vesicles. Reported and adapted from Nishiyama et al. 2020.

Progress has been made on the production of bacterial vesicles in recent years, yet several issues remain to be clarified including how vesicles are generated from the progenitor microbe, how the composition of vesicles changes according to the culture conditions, how to target specific bacterial vesicle purification from a pool of vesicles derived from other organisms (for example, bacterial vesicles produced in milky media can be accompanied by vesicles from eukaryotic cells present in the milk), safety aspects, quantification methods and the regulation of their use by the corresponding authority.

Their future as potential postbiotics

Membrane vesicles are an exciting opportunity for the development of postbiotics. A potential benefit of vesicles is that their small size compared to whole cells may enable them to more readily migrate to host tissues that could not be otherwise reached by a whole cell (Kulp & Kuehn 2010). Their nanostructure enables them to penetrate through the gut barrier and to be delivered to previously unreachable sites through the bloodstream or lymphatic vessels, and to interact with different cell types (Jones et al. 2020). For example, bacterial rRNA and rDNA found in the bloodstream and the brain of Alzheimer’s patients were postulated to have originated from bacteria vesicles (Park et al. 2017). Safety of EMVs must be carefully considered and assessed, even if they are derived from microbes generally recognized as safe, as their small size may increase penetration capacity with potential and yet unknown systemic effects. Novel postbiotics derived from microbial membrane vesicles is an intriguing area for future research to better understand production parameters, safety and functionality.

Thanks to Cheng Chung Yong, postdoctoral researcher at Morinaga Milk Industry Co., LTD (Japan) and Ludwig Lundqvist, industrial PhD student at BioGaia AB (Sweden) for their contributions to this blog, and Mary Ellen Sanders and Sarah Lebeer from ISAPP for fruitful discussions.

References

Ashrafian, F., Shahriary, A., Behrouzi, A., Moradi, H.R., Keshavarz Azizi Raftar, S., Lari, A., Hadifar, S., Yaghoubfar, R., Ahmadi Badi, S., Khatami, S. and Vaziri, F., 2019. Akkermansia muciniphila-derived extracellular vesicles as a mucosal delivery vector for amelioration of obesity in mice. Frontiers in microbiology10, p.2155.

Bäuerl, C., Coll-Marqués, J.M., Tarazona-González, C. and Pérez-Martínez, G., 2020. Lactobacillus casei extracellular vesicles stimulate EGFR pathway likely due to the presence of proteins P40 and P75 bound to their surface. Scientific reports10(1), pp.1-12.

Domínguez Rubio, A.P., Martínez, J.H., Martínez Casillas, D.C., Coluccio Leskow, F., Piuri, M. and Pérez, O.E., 2017. Lactobacillus casei BL23 produces microvesicles carrying proteins that have been associated with its probiotic effect. Frontiers in microbiology8, p.1783.

Gamalier, J.P., Silva, T.P., Zarantonello, V., Dias, F.F. and Melo, R.C., 2017. Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation. Microbiological research194, pp.38-46.

Grande, R., Celia, C., Mincione, G., Stringaro, A., Di Marzio, L., Colone, M., Di Marcantonio, M.C., Savino, L., Puca, V., Santoliquido, R. and Locatelli, M., 2017. Detection and physicochemical characterization of membrane vesicles (MVs) of Lactobacillus reuteri DSM 17938. Frontiers in microbiology8, p.1040.

Gill, S., Catchpole, R. & Forterre, P., 2019. Extracellular membrane vesicles in the three domains of life and beyond. FEMS microbiology reviews, 43(3), pp.273–303.

Hirayama, S. & Nakao, R., 2020. Glycine significantly enhances bacterial membrane vesicle production: a powerful approach for isolation of LPS-reduced membrane vesicles of probiotic Escherichia coli. Microbial biotechnology, 13(4), pp.1162–1178.

Jones, E.J., Booth, C., Fonseca, S., Parker, A., Cross, K., Miquel-Clopés, A., Hautefort, I., Mayer, U., Wileman, T., Stentz, R. and Carding, S.R., 2020. The uptake, trafficking, and biodistribution of Bacteroides thetaiotaomicron generated outer membrane vesicles. Frontiers in microbiology11, p.57.

Kim, J.H., Jeun, E.J., Hong, C.P., Kim, S.H., Jang, M.S., Lee, E.J., Moon, S.J., Yun, C.H., Im, S.H., Jeong, S.G. and Park, B.Y., 2016. Extracellular vesicle–derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression. Journal of Allergy and Clinical Immunology137(2), pp.507-516.

Kulp, A. & Kuehn, M.J., 2010. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annual review of microbiology, 64, pp.163–184.

Molina-Tijeras, J.A., Gálvez, J. & Rodríguez-Cabezas, M.E., 2019. The immunomodulatory properties of extracellular vesicles derived from probiotics: a novel approach for the management of gastrointestinal diseases. Nutrients, 11(5), p.1038.

Müller, L., Kuhn, T., Koch, M. and Fuhrmann, G., 2021. Stimulation of probiotic bacteria induces release of membrane vesicles with augmented anti-inflammatory activity. ACS Applied Bio Materials4(5), pp.3739-3748.

Ñahui Palomino, R.A., Vanpouille, C., Costantini, P.E. and Margolis, L., 2021. Microbiota–host communications: Bacterial extracellular vesicles as a common language. PLoS Pathogens17(5), p.e1009508.

Nishiyama, K., Takaki, T., Sugiyama, M., Fukuda, I., Aiso, M., Mukai, T., Odamaki, T., Xiao, J. Z., Osawa, R., & Okada, N. 2020. Extracellular vesicles produced by Bifidobacterium longum export mucin-binding proteins. Applied and Environmental Microbiology, 86(19), e01464-20.

Palomino, R.A.Ñ., Vanpouille, C., Laghi, L., Parolin, C., Melikov, K., Backlund, P., Vitali, B. and Margolis, L., 2019. Extracellular vesicles from symbiotic vaginal lactobacilli inhibit HIV-1 infection of human tissues. Nature communications10(1), pp.1-14.

Park, J.Y., Choi, J., Lee, Y., Lee, J.E., Lee, E.H., Kwon, H.J., Yang, J., Jeong, B.R., Kim, Y.K. and Han, P.L., 2017. Metagenome analysis of bodily microbiota in a mouse model of Alzheimer disease using bacteria-derived membrane vesicles in blood. Experimental neurobiology26(6), p.369.

Shehata, M.M., Mostafa, A., Teubner, L., Mahmoud, S.H., Kandeil, A., Elshesheny, R., Boubak, T.A., Frantz, R., Pietra, L.L., Pleschka, S. and Osman, A., 2019. Bacterial outer membrane vesicles (omvs)-based dual vaccine for influenza a h1n1 virus and mers-cov. Vaccines7(2), p.46.

Tan, K., Li, R., Huang, X. and Liu, Q., 2018. Outer membrane vesicles: current status and future direction of these novel vaccine adjuvants. Frontiers in microbiology9, p.783.

Tian, Y., Gong, M., Hu, Y., Liu, H., Zhang, W., Zhang, M., Hu, X., Aubert, D., Zhu, S., Wu, L. and Yan, X., 2020. Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry. Journal of extracellular vesicles9(1), p.1697028.

West, C.L., Stanisz, A.M., Mao, Y.K., Champagne-Jorgensen, K., Bienenstock, J. and Kunze, W.A., 2020. Microvesicles from Lactobacillus reuteri (DSM-17938) completely reproduce modulation of gut motility by bacteria in mice. PloS one15(1

Can dietary supplements be used safely and reliably in vulnerable populations?

By Dr. Greg Leyer, Sr. Director – Scientific Affairs, Chr. Hansen, Inc., Madison, WI and Prof. Dan Merenstein, Department of Family Medicine, Georgetown University Medical Center, Washington DC

What is it that doctors look for when recommending or prescribing therapies to patients? If it is a drug, a supplement, a new diet, or even a new exercise regimen, they look for safety and efficacy. There are of course other things to consider, including cost, ease of administration, and patient compliance, among others. But safety and efficacy are their foremost concerns.

A recently published clinical report from the American Academy of Pediatrics (AAP) (Poindexter 2021) examined the evidence for probiotics to prevent morbidity and mortality in preterm infants. They concluded that probiotics could not be recommended. This differs from conclusions of the American Gastroenterological Association (AGA) (Su et al. 2020), which recommended specific probiotic strains for preterm (less than 37 weeks gestational age) and low birth weight infants. The AAP report also differs from the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) (Van den Akker et al. 2020), which recommends specific strains for this use, although their recommendations are not fully aligned with AGA’s (see What’s a Clinician to do When the Probiotic Recommendations from Medical Organizations Do Not Agree?).

The AAP report does a thorough job of reviewing data on use of probiotics in the NICU, including conflicting studies, lack of confirmatory studies of efficacious strains, and safety and cross contamination inside the NICU. However, the overriding theme of the report is “clinicians must be aware of the lack of regulatory standards for commercially available probiotic preparations manufactured as dietary supplements and the potential for contamination with pathogenic species.” Therefore, at the heart of the AAP failure to recommend probiotics is the concern that the quality of available products is insufficient. Because of the absence of a pharmaceutical-grade probiotic product for use in the United States, they posit, they cannot recommend usage. It is noteworthy that the trials performed on premature infants resulting in multiple conclusions of safety and efficacy have thus far utilized probiotic products manufactured as dietary supplements.

Probiotics can be marketed as drugs if they follow that regulatory pathway, but generally in the US they are sold under the regulatory classification of dietary supplements. Is the AAP correct that no dietary supplement is of sufficient quality to recommend for use in preterm infants?

Quality of probiotic dietary supplement probiotics. Dietary supplements were a category of product developed to supplement the diet of the generally healthy population, not to treat or prevent disease. In practice this is an important distinction, because while the safety standard is high for dietary supplements for healthy individuals (see comments by food safety expert Jim Heimbach here), such supplements do not need to be established as safe for patient populations. But in the case of probiotics, many clinical trials have evaluated safety and efficacy for prevention or treatment of disease, more aligned with drug uses. Yet probiotic products supported by these data are not marketed in the US as drugs.

It is a common misperception that dietary supplements are “not regulated”. However, the FDA has clear good manufacturing practices (GMP’s) and regulations dedicated to dietary supplement manufacturing.  The onus is on manufacturers to establish appropriate product specifications based on intended use and risk. Reputable manufactures establish rigorous purity, strength, and identity quality standards consistent with the intended population and sufficient for that use. Products intended for infants, including premature infants, should be manufactured under quality standards more rigorous than those intended for a healthy adult population. For example, Chr. Hansen bases the enhanced specifications for products aimed at infants, and preterm infants, on elements of Codex standards for infant formula, amongst other stringent microbiological criteria. This would include manufacturing the probiotic strain to an “infant” grade, employing stricter environmental monitoring, sanitation, and airflow control throughout the process, careful selection of raw ingredients for infant compatibility, and enhanced testing and purity standards using validated methods at every step. The internal manufacturing standards that Chr. Hansen applies for products intended for infants, and preterm infants, are much stricter than typical dietary supplement standards, and are appropriate for their intended use.

Therefore, there are high quality, safe probiotic products produced under dietary supplement regulations even though such products do not carry any label statement claiming this added level of quality. However, products sourced for hospitals to stock in formularies could work with the supplier to demand this extra level of product testing specifications. Pharmacies can institute quality agreements with vendors that would delineate their expectations for the strains present, the levels of live microbes acceptable in the final product, etc. This agreement could also mandate that any product change – as defined in the agreement – would require the vendor to notify the customer. Such an agreement might be burdensome for a hospital pharmacist, but a sophisticated dietary supplement company should be able to assure the hospital formulary of their quality.

Products made using strict specifications, geared towards infant and premature infant applications, are on the market and are safely being used in this patient population in many NICUs and as part of infant formulas. We disagree with AAP’s position that a pharmaceutical approach is needed, as long as a product of sufficient quality can be provided. To deny preterm infants probiotics, which have a significant chance of improving their clinical outcomes, is not in line with other medical recommendations. Instead, the hospital formularies should stock products that have been scrutinized for sufficient evidence of safety and efficacy. Suppliers of stocked products should provide product testing results, a description of the quality standards employed during production, and a rationale for the suitability of the standards for preterm infants. Third party verification of adherence to these quality standards would assure medical professionals regarding the safety of these products for use.

References

CAC/RCP 66-2008. Code of hygienic practice for powdered formulae for infants and young children. Codex.

Poindexter, B. 2021. Use of Probiotics in Preterm Infants. Pediatrics 147 (6): e202 1051485.

Su et al. 2020. AGA Clinical Practice Guidelines on the Role of Probiotics in the Management of Gastrointestinal Disorders. Gastroenterology 159:697-705.

Van den Akker et al.  2020. Probiotics and Preterm Infants: A Position Paper by the European Society for Paediatric Gastroenterology Hepatology and Nutrition Committee on Nutrition and the European Society for Paediatric Gastroenterology Hepatology and Nutrition Working Group for Probiotics and Prebiotics. Journal of Pediatric Gastroenterology and Nutrition. 70(5):664-680.