Revealing Matter from Niv Zmora

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A few years ago, I met Niv Zmora at a conference on the gut microbiome. As a scientist working at the Weizmann institute in Israel, he presented on his recent research demonstrating humans’ very individualised response to certain foods (bread, as I recall). We got talking over lunch and found that we both shared a common passion for promulgating the truth, even if it may sometimes be commercially inconvenient.

Research into the gut microbiome is a rapidly expanding area. Of course, where there is scientific development, there is commercial interest. And where there is commercial interest, inevitably, there are products for sale and ill people who want to be well and healthy people who want to be healthier. In a scientific area that is yet so young and about which we still know so little, commercial pressure and human desire for solutions to complicated issues can risk premature assumptions being made. And, of course, scientists are no more protected from these commercial pressures than the rest of us.

In Niv, I feel I had met a kindred spirit. A truth teller. Here, then, is the truth from Niv about his work at the Weizmann institute in general and about two very interesting studies, in particular.


Interview


WHAT, IN YOUR OPINION, IS THE BEST THING ABOUT WORKING AT THE WEIZMANN INSTITUTE?

The Weizmann Institute is a rare gem, where great people come together from all over the world to contribute to science. The best thing, in my experience, has been getting to know my colleagues - a group of friendly, insightful and intelligent people, led by an astute primary investigator, Prof Elinav. Together, we brainstorm and endeavour to uncover scientific truths in the field of the human gut microbiome.

DO YOU HAVE A CANTEEN AT WORK? DOES A CANTEEN FOR GUT MICROBIOME SCIENTISTS SERVE VERY HEALTHY FOOD?

I practiced as a doctor for many years and I often compare the canteen at the Weizmann Institute to the one at the hospital. And the hospital doesn’t fare well in the comparison, which is quite ironic really! The food is served as a buffet, so whether we make healthy choices is up to us. Unintentionally, the Institute is supporting the concept of personalised nutrition!

WHAT IS THE GREATEST CHALLENGE YOU FACE AS A SCIENTIST WORKING IN THIS FIELD TODAY?

There are lots of challenges. Microbiome research has increased exponentially in the last 10 years. There is an overabundance of papers of very variable quality. I believe that in order to find practical health-promoting innovations, scientists need to move beyond associative studies to causal ones.

TELL US ABOUT THE FIRST STUDY WE DISCUSSED – WHAT WERE THE MAIN QUESTIONS YOU WERE TRYING TO ANSWER?

Our first study involved healthy humans (and mice) who were not already consuming probiotics. We wanted to answer these elementary questions about probiotics:

1. Are probiotic bacteria viable during the passage through the gut?

2. Do they colonize and persist in the gut mucous or are they merely washed out with stool (poo) when they reach the end of the gut?

3. Do probiotics change the composition and function of the resident gut bacteria or the host cells they encounter in the gastrointestinal tract (or both)?

We did not, however, choose to focus on the clinical aspects of probiotics – the practical effects the participants experienced. And we did not design the study to prove that probiotics have health-promoting effects.

WHAT WERE YOUR KEY FINDINGS?

1. The gut mucosal microbiome only partially correlates with the microbes we find in the stool – what goes on at our gut mucosa is not necessarily wholly reflected in what we see in our poo.

2. Humans have person-specific approaches to probiotics. Some people are “permissives” and in these people, probiotics are more likely to influence the gut mucosal microbiome and host cell function in the gut. Other people are “resisters” and, as the name suggests, they successfully minimise the foothold probiotics obtain at the gut mucosa and their influence over host cell function. Whether you are a permissive or a resister depends upon both your genetic and microbiome characteristics.

3. The effect probiotics have differs in various sites along the gut.

4. The caecum (the cul-de-sac as the small intestine turns into the large intestine) emerged as a distinguishing region between permissive and resister groups in terms of the activity there.

These differing levels of colonisation resistance between permissives and resisters significantly influenced the probiotics’ effect on participants’ resident microbiome composition and host cell function (especially in immunity and metabolism).

WHAT DOES THIS MEAN IN TERMS OF THE NEXT RESEARCH STEPS?

It means we need to revisit the probiotic paradigm ‘one-pill-fits-all’ and contemplate a personalised approach, where probiotic preparations are composed of bacterial strains tailor-made for the consumer’s medical condition, their age, indigenous microbiome composition and gut characteristics. After a decade of microbiome research, new beneficial strains beyond the usual century-old probiotics (the ones we discovered because they were easy to grow on petri dishes), should be used. And unbiased randomized controlled trials, supervised by health regulatory authorities, should be conducted to assess their efficacy. This will avoid the public being misled by commercial interests.

AND WHAT DOES IT MEAN FOR THE PROBIOTIC POPPERS AMONGST US? HOW DO WE KNOW IF WE ARE RESISTERS OR PERMISSIVES?

In our study, there were some key findings that separated resisters from permissives. Permissives tended to have a lower initial abundance of some of the probiotic strains within their indigenous microbiome compared to resisters. Also, their baseline expression of immune-related and metabolic pathways in various parts of the gut differed.

Unfortunately, routine lab tests performed in common medical practice nowadays are currently unable to identify these factors. We showed that a small cohort of healthy humans featured various degrees of probiotics “persistence”, but whether the presence of probiotics in the gut is linked to clinical health benefits remains to be determined in future studies. We believe that if an evidence-based health-promoting effect is found to be induced by probiotics, tools designed to predict probiotics persistence in individuals should be incorporated into routine lab tests.

STOOL SAMPLES ARE A SIMPLE, NON-INVASIVE AND INCREASINGLY COMMON WAY OF INVESTIGATING OUR MICROBIOTA. ENDOSCOPIES ARE NOT. BUT IF STOOL SAMPLES MAY NOT ACCURATELY REFLECT WHAT IS GOING ON AT THE GUT MUCOUS LINING, WHERE DOES THAT LEAVE US IN TERMS OF INTERPRETING STOOL SAMPLE RESULTS?

This is absolutely right. The stool (poo) microbiome is a limited representation of the mucosal microbiome, and it less reliably represents gut mucosa further away from the anus than it does those areas closer to it. Since it is impossible to invasively acquire mucosal microbiome samples from every patient (via endoscopy and biopsy), we believe that future studies need to harness a way to extrapolate from stool microbiome to the mucosal niche. To be able to do this will first require work on much larger cohorts than we recruited. However, it is important to note that some of the differences that emerged between resisters and permissives could be detected in stool samples. Focusing on these attributes could make non-invasive stool testing appropriate to predict response to probiotics to a limited extent.

COULD YOU RUN THE SAME EXPERIMENT ON FERMENTED VEGETABLES AND DRINKS PLEASE?

It would be incredibly interesting to do this.There are differences – in fermented foods and drinks, the communities of bacteria are naturally-occurring, not put together by scientists in the way they are with commercially formulated probiotics. They also tend to consist of a wider range of bacterial strains, which may increase their opportunity to influence the existing community at the gut mucosa as well as host cell function. However, they are not encapsulated, so they do not get additional protection from our stomach acid on their journey through the gut.

Of course, fermented vegetables contain ranges of naturally-occurring bacteria that we have been eating on our vegetables for millennia. As we ingest the bacteria, we are also ingesting the prebiotic fibre in the veg, which helps feed our resident microbes and we are consuming the vitamins and minerals in the veg too. This is a more complete package, but that would also make it harder to isolate the impact of the microbes, themselves, from the impact of accompanying fibre and other phytochemicals. But having said that, if the complete package had a positive impact, would we need to do that? Maybe we could compare the effect of fermented vegetables to non-fermented vegetables? But we can only speculate. In order to ascertain the effects of probiotic foods and drinks on the gut mucosa and host cell function, we need meticulously planned studies to be conducted.

 
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TELL US ABOUT YOUR SECOND STUDY.

Our second study looked at probiotic supplementation in the context of antibiotic treatment. Probiotics are often recommended by healthcare providers nowadays when antibiotics are prescribed. We were interested find out:

1. If probiotic colonization improved in otherwise healthy people after antibiotic therapy (once some of the resident bacteria have been wiped out by the antibiotics).

2. How probiotics affect the rate and extent of reconstitution of the gut microbiome to its original composition after antibiotic treatment.

We compared a) probiotic treatment to b) watchful waiting (no treatment at all after antibiotics) and c) another bacteria-based therapy – autologous FMT (this is, in short, re-introducing the pre-antibiotic poo of the participants into the gut - fecal microbial transplantation).

WHAT DO THE RESULTS TELL US ABOUT THESE THREE DIFFERENT METHODS OF SUPPORTING OUR GUT TO RECOVER FROM TAKING ANTIBIOTICS?

We found that the administration of autologous FMT hastened gut microbiome recovery to its pre-antibiotic composition more completely and more rapidly than watchful waiting. Surprisingly, probiotics delayed the gut microbiome’s reconstitution to its original condition. In the probiotics group, the gut microbial composition did not return to its untreated state even 5 months following cessation of the probiotic supplement. Autologous FMT was the most successful and probiotics were the least successful. And remember, these were otherwise healthy people. There was no reason for a return to their original microbial constitution to be undesirable.

It should be noted though, that participants in each treatment arm went through two endoscopies - each involved a luminal and mucosal microbiome sample collection and a gut biopsy. Ideally, we would have done 3 of these on each participant – one prior to antibiotic administration to give us a baseline and then the two we did do – immediately post antibiotics (which gave us a baseline for microbiome reconstitution) and 21 days into the reconstitution process. But three would have been too much for participants so, for the gut mucosal pre-antibiotic baseline, we used other participants who were given no antibiotics but underwent just one endoscopy to give us that baseline.

We then assessed reconstitution in 3 ways:

  1. By collection of stool samples and comparing them to participants’ own pre-antibiotics baseline (although we know that the stool sample is an imperfect reflection of what is going on at the mucosa).

  2. Collection of mucosa samples by brushing the gut lining both pre-antibiotics (from the set of participants who were just used to give us that gut mucosal baseline) and 21 days into reconstitution (in the treatment participants).

  3. Collection of host mucosal biopsies to assess changes in host cellular function compared to the other treatment groups and to our non-antibiotic baseline group.

WHY, DESPITE THE FIRST STUDY SHOWING THAT BACTERIAL CONTENT IN OUR STOOL DOESN’T ACCURATELY REPRESENT BACTERIAL POPULATIONS AT OUR GUT MUCOSA, DID PUTTING SOMEONE’S OWN PRE-ANTIBIOTIC POO INTO THEM AFTER ANTIBIOTICS HELP TO REPOPULATE THE GUT MICROBIOME SO RAPDILY AND COMPLETELY?

This is a great question. First, it should be noted that the stool microbiome returned to its original conformation rapidly and completely in the autologous FMT group. As for the microbiome at the gut mucosa, as explained above, we could only compare it to the post-antibiotics state (or to other non-antibiotics treated individuals). We assume that the person’s own faecal matter contained sufficient of their own familiar bacteria and metabolites to promote rapid gut mucosal microbial reconstitution to closer to its original condition because there were sufficient similarities between the stool and the mucosal microbiome to elicit that effect.

DOES BEING A “PERMISSIVE” OR A “RESISTER” HAVE AN IMPACT IN THIS POST-ANTIBIOTIC PROBIOTIC SITUATION TOO?

Absolutely. We saw that some individuals in the probiotics group remained in the post-antibiotics perturbed state longer than others. We can only speculate that there is a continuum of response to probiotics in this scenario as well. However, our sample size was small due to the invasive nature of the study, so we could not meaningfully delve into that.

BOTH STUDIES MENTION CHANGES IN THE CAECUM – THE DEAD END OF THE SMALL INTESTINE AS IT TURNS INTO THE LARGE INTESTINE. WHAT ROLE DO YOU THINK THE CAECUM (AND APPENDIX) MAY BE PLAYING?

This is an interesting question. The appendix is thought to play a role in gut immune function, especially during the early years of life, it also constitutes a reservoir for indigenous gut bacteria. Whether this explains the prominent differences seen in our studies merits more directed research.

IN THE PROBIOTICS GROUP, SEVERAL PROBIOTICS SPECIES REMAINED ELEVATED IN STOOL SAMPLES 5 MONTHS AFTER PARTICIPANTS STOPPED TAKING PROBIOTICS. ISN’T THIS A GOOD SIGN THAT “BENEFICIAL” BACTERIA HAVE “MOVED IN?”

One non-invasive way to prove bacterial colonization in the gut mucosa is sustained shedding of those probiotic strains in poo long after the individuals have stopped taking probiotics. This is definitely a sign that probiotic bacteria moved in. However, I cannot say for certain that this was beneficial for any individual or every individual. It is worth noting that this long-lasting colonization was made possible by the antibiotic treatment that reduced the ability of the remaining resident bacteria to resist colonization by the probiotics.

INTERESTINGLY, PROBIOTICS SPECIES WERE ALSO SIGNIFICANTLY ELEVATED IN THE WATCHFUL WAITING GROUP TOO (EVEN THOUGH THEY WERE NOT GIVEN THE PROBIOTIC CAPSULES), BUT NOT IN THE GROUP THAT GOT THEIR PRE-ANTIBIOTIC POO RETURNED TO THEM. WHY WAS THIS DO YOU THINK?

I think it is important to distinguish between a bloom in species to which the probiotics strains belong to (such as Lactobacillus and Bifidobacterium), which occurred in the watchful waiting group, to a bloom in the specific probiotic strains. Interestingly, after antibiotics, we witnessed an increase in these species due to natural selection in the watchful waiting group – the antibiotic regimen eliminated some bacteria and selected for others, including the ‘probiotic’ species. In advanced analysis (that was not presented in the paper) we verified that the specific probiotic strains that were administered to our subjects were present in long-term follow-up only in the probiotics arm, but not in the watchful waiting arm of the study. In the FMT arm, those long-term antibiotic-mediated shifts in microbiome composition did not occur at all due to a rapid restoration of the microbiome to its original assembly.

WE CURRENTLY UNDERSTAND GUT BACTERIAL DIVERSITY TO BE IMPORTANT FOR HEALTH. WHAT WAS THE EFFECT ON DIVERSITY IN EACH TREATMENT GROUP?

We saw that antibiotics dramatically reduced bacterial diversity, as expected. Based on stool sample analysis, the group which received autologous FMT showed a rapid and near-complete return to gut bacterial richness. while the watchful waiting group reconstituted more slowly. However, the probiotics group showed a delayed and incomplete restoration of their gut bacterial richness. The reconstitution biopsies of the gut mucosa equally showed that the probiotic group fared worst in terms of microbial richness and load. These findings were consistent both in humans and mice.

DID THESE CHANGES AFFECT HOST FUNCTIONAL PATHWAYS VIA GENE EXPRESSION TOO? IF SO, IN WHAT WAY?

In the probiotics group, a similar and statistically significant trend of delayed reconstitution was shown for the host’s functional pathways in the gut.

SO, IF YOU TAKE ANTIBIOTICS AND THEN TAKE PROBIOTICS TO TRY TO REPOPULATE YOUR GUT, DOING SO MAY CAUSE YOUR GUT MICROBIOME TO STAY MORE LIKE A DEPLETED POST-ANTIBIOTIC MICROBIOME THAN IF YOU DIDN’T TAKE PROBIOTICS?

Absolutely. We showed that post-antibiotics probiotics cause delayed gut microbiome reconstitution, which is characterised by reduced bacterial diversity and decreased bacterial load. We conducted test tube experiments and showed that this inhibitory effect by probiotics is mediated by their by-products, mainly from Lactobacillus species.

Some probiotic bacteria possess mechanisms to compete with other bacteria by inhibiting them. We grew the faecal microbiome in the presence of the by-products of the probiotics but not the probiotics, themselves and these by-products inhibited the resident microbes in the faeces. This suggests that there are molecules secreted by probiotic bacteria that account for this inhibitory function.

PROBIOTICS AND THEIR ACTIONS ARE STILL POORLY UNDERSTOOD AND OUR CHOICE OF COMMERCIALLY PRODUCED PROBIOTICS IS BASED ON STRAINS THAT WE STUDIED HISTORCALLY BECAUSE WE COULD GROW THEM ON PETRI DISHES. THIS APPROACH IS DUE AN OVERHAUL. AGREE OR DISAGREE?

I totally agree. Although probiotic formulations have been in use for over a century now and constitute one of the most commonly consumed dietary supplements worldwide, there is still little consensus regarding some important aspects of their biological action. Test tube studies are heavily affected by bacterial concentrations, growth stage, incubation time and growth medium and often do not reliably replicate physiological conditions inside our gut. In these two recently published studies, we showed that empirical use of probiotic products might affect people in an individualized manner and that the use of probiotics could delay gut reconstitution after antibiotics. In light of this evidence, probiotics need objective reassessment.

THIS STUDY SUGGESTS TREATMENT WITH OUR OWN PRE-ANTIBIOTIC POO AFTER ANTIBIOTICS MAY BE THE BEST WAY TO GO. HOW DID YOU ACTUALLY DO THIS IN THE STUDY?

The use of FMT is surprisingly beneficial despite being rather unappealing. It is currently approved by health authorities for the treatment of recurrent infection by the bacterium Clostridium difficile and has been very successful in this area – much more successful than antibiotics. We believe that, in the near future, the exact mechanisms by which FMT confers its health-promoting effects should be clarified. Once a bacterium, a consortium of bacteria or their by-products are identified, it would be more elegant to administer them instead of the whole microbiome stool bulk (also known as crapsules!). In our study, we recruited brave volunteers, who were not intimidated by receiving their own stool (which was acquired and stored in freezers prior to antibiotics therapy). In fact, these individuals had their gut microbiome restored more effectively than the other groups.

CAN WE TRY THIS AT HOME? MAYBE WITH SOME ICE CUBE HOLDERS?!

The processing of stool into a solution for FMT is a laborious protocol that should be performed in lab conditions and requires stringent screening regulations and facilities that are not available at home. I do not advise trying this at home. Wait until treatment is approved and provided by health authorities to minimize the risk of harm. Not least from putting the wrong ice cube in your gin and tonic.

WHAT ARE THE MAIN SHORTCOMINGS OF YOUR STUDIES?

  1. 1.In the antibiotics study, we could not carry out an initial endoscopy to gather baseline lumen and mucosal information in addition to doing the other two endoscopies.

  2. · We did not assess participants’ clinical response to probiotics – for example bowel habits, faecal consistency, bloating, mood.

  3. · We recruited only healthy volunteers. not individuals with medical conditions (although this was a plus too).

  4. · We only looked at one specific probiotic formula (which contained the most commonly consumed strains). It is possible that different combinations affect the gut differently.

  5. · Due to the invasive nature of the studies, the sample sizes were small.

WHAT IS ONE THING YOU HAVE CHANGED IN YOUR DAY TO DAY BEHAVIOUR AS A RESULT OF YOUR WORK.

I try to consider my microbiome in how I live my life. I feed them plenty of dietary fibre so that we can co-exist in harmony. And until there is further solid research available, as a healthy individual, I don’t think I would take probiotics after antibiotics.

IF YOU WERE A MICROBE, WHICH ONE WOULD YOU BE?

If I were not restricted to bacteria, I would pick the parasite Giardia lamblia, which, in one of its life stages, puts on a happy face when you look at it though the microscope.

Links to the studies themselves:

https://www.cell.com/cell/pdf/S0092-8674(18)31102-4.pdf

https://www.cell.com/cell/pdf/S0092-8674(18)31108-5.pdf