Bacterial extracellular vesicles: future postbiotics?

Why is it so challenging to clarify what is and what is not a postbiotic?

In 2021, a large panel of experts in the field of pro- and prebiotics, the International Scientific Association for Probiotics and Prebiotics (ISAPP), joined forces and re-defined postbiotics. Welcoming them officially into the biotics family, the definition described postbiotics as a “preparation of inanimate microorganisms and/or their components that confer a health benefit on the host”. Opposition was expressed shortly after the ISAPP consensus paper was published, allowing for further discussion and scientific engagement on the topic. Among the caveats discussed was the challenge of ensuring the stability and efficacy of postbiotics during product shelf life, as the intrinsic components (inorganic, organic or enzymatic) can inevitably participate in processes that profoundly affect stability.

The GMFH editing team connected with Dr. Gabriel Vinderola, Associate Professor at Universidad Nacional del Litoral, Argentina, Principal Researcher from CONICET, and senior co-author of the ISAPP definition paper, to further discuss the matter. “Science is an evolving field and the first definition of any given concept is not necessarily the one that best represents science and follows scientific evolution,” explained Dr. Vinderola. “This happened with the definition of probiotics, first introduced in 1965, modified in 1989 and the latest one from 2002 still survives. For postbiotics, 6 definitions were proposed by different scientists from 2013 until 2021 when ISAPP proposed a new definition. The limitations of the previous definitions were discussed and clarified here.”

Separating the contribution to host health benefits made by postbiotics already present in probiotic products is not straightforward

ISAPP’s discussion around the definition focused especially on the starting material and the question: must a postbiotic be produced from a probiotic? As such a requirement would solely place capricious restrictions on a postbiotic, it was removed from the ISAPP definition. However, such a move does not necessarily exclude the presence of postbiotics in a probiotic formula sold over the counter. For instance, overfilling practices—loading a probiotic product with multiple times the expected number of live cells to ensure the amount reported on the package dosage is delivered by the end of the product’s shelf life—are barely reported or studied. Nevertheless, they are a strong indication of the presence of non-viable microorganisms alongside live cells in the initial probiotic formula.

Of all the cell fragments and components fragments derived from inanimate bacterial cells, membrane vesicles have increasingly attracted scientific interest in the field of postbiotics. And while membrane vesicles may already be present and active in current probiotic products, what are bacterial vesicles and why do they offer potential as postbiotics?

“The contribution of non-viable microorganisms present in probiotic products may be really significant,” explains Dr. Vinderola, “but it is difficult to manage and to know what percentage of the benefit is due to non-viable microbes.” He further discusses the issue in this blog post.

Bacterial vesicles as a novel strategy to enhance the efficacy of probiotics

Extracellular vesicles (EVs) are non-viable elements, yet they are carriers of biological information produced in all domains of life. According to Dr. Vinderola, “EVs have a lot of potential and they fit the ISAPP definition of postbiotics as they are generated from the bacterial membrane. In fact, they will fit the definition when they are proven to have a health benefit.”

Bacterial EVs, in particular, contain various biomolecules derived from their progenitor bacteria, including proteins, lipids, nucleic acids, metabolites, and numerous surface molecules. And all that biological information represents the inanimate structures of their progenitor bacteria (i.e., Lactobacillus, Bifidobacterium, Akkermansia). In the case of probiotic bacteria, EVs may mediate interactions with immune cells and enterocytes, potentially conferring a health benefit on the host and therefore enlisting them as postbiotics. A representative example is the contribution of extracellular membrane vesicles in immune-dependent properties of Lactobacillus rhamnosus GG and L. reuteri DSM 17938.

Various research areas have put bacterial EVs increasingly in the spotlight, yet the underlying mechanisms and corresponding molecules driving their health effects remain poorly understood. In vitro findings show the involvement of Lactobacillus-derived extracellular vesicles in reducing pro-inflammatory responses mediated by monocytes. Other in vitro findings reveal that EVs produced from the probiotic Propionibacterium freudenreichii CIRM-BIA 129 mitigate inflammation by modulating the NF-kB pathway. Studies in mice indicate that Bifidobacterium longum-derived EVs alleviate food allergy through mast cell suppression, while Akkermansia muciniphila-derived EVs ameliorate obesity by reducing intestinal inflammation and permeability.

Most information so far comes from laboratory and mice experiments, and ongoing clinical trials have only recently been investigating the role of postbiotics for obesity, type 2 diabetes and IBS. To our knowledge, there are no clinical trials to date examining probiotic-derived EVs’ health benefits against infectious or auto-inflammatory diseases. Therefore, it is essential we use the current information cautiously. Findings in mice have been promising, yet there is an unmet need for clinical proof before we move to microbial EV technology and its potential in innovative diagnostic and therapeutic solutions in precision medicine.

Moreover, the safety of EVs will be assessed and confirmed before administration is suggested in a clinical setting. “I do not foresee advantages compared to other postbiotics,” explains Dr. Vinderola. “EVs will have their own specific uses. I foresee challenges instead. By using vesicles, you are leaving out an important part of the cell that may also deliver a health benefit.”

Vesicles’ very small size means they can penetrate the gut barrier, reach host tissues and interact with distinct cell types that the original whole bacterial cell could not otherwise reach, and that may have a potentially negative systemic effect on the host. In other words, there is a long way to go before that possibility can be excluded and the move to clinical application of EVs can be made.

“EVs may be another tool that medical doctors may use to modulate the gut immune system and the microbiome,” proposes Dr. Vinderola, “maybe, with the possible plus that, as they are not living microbes, the safety issue concerning translocation in immunocompromised patients may be lower. In any case, safety cannot be presumed, but properly demonstrated.”

References:

Aguilar-Toalá J. E., Arioli S., Behareet P. et al. Postbiotics — when simplification fails to clarify. Nat. Rev. Gastroenterol. Hepatol. 2021. doi: 10.1038/s41575-021-00521-6

Salminen S., Collado M. C., Endo A. et al. Reply to: Postbiotics — when simplification fails to clarify.  . Nat. Rev. Gastroenterol. Hepatol. 2021. doi: 10.1038/s41575-021-00522-5

Salminen S., Collado M. C., Endo A. et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. 2021. doi: 10.1038/s41575-021-00440-6

Kim E., Kim D., Yang S. M. et al. Validation of probiotic species or subspecies identity in commercial probiotic products using high-resolution PCR method based on large-scale genomic analysis Int. Food Res. J. 2022. doi: 10.1016/j.foodres.2022.111011

Fiore W., Arioli S., Guglielmetti S. The neglected microbial components of commercial probiotic formulations Microorganisms 2020. doi: 10.3390/microorganisms8081177

Vinderola G., Senders M. E, Salminen S. The Concept of Postbiotics. Foods. 2022. doi: 10.3390/foods11081077

Domínguez Rubio A. P., Martínez J. H., Martínez Casilla D. C. et al. Lactobacillus casei BL23 Produces Microvesicles Carrying Proteins That Have Been Associated with Its Probiotic Effect. Front. Microbiol. 2017. doi: 10.3389/fmicb.2017.01783

de Rezende Rodovalho V., da Luz B. S., Rabah H. et al. Extracellular vesicles produced by the probiotic Propionibacterium freudenreichii CIRM-BIA 129 mitigate inflammation by modulating the NF-κB pathway. Front. Microbiol. 2020. doi: 10.3389/fmicb.2020.01544

Kim J. H., Jeun E. J., Hong C. P. et al. Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression. J Allergy Clin Immunol. 2016. doi: 10.1016/j.jaci.2015.08.016

Ashrafian F., Shahriary A., Behrouzi A. et al. Akkermansia muciniphila-derived extracellular vesicles as a mucosal delivery vector for amelioration of obesity in mice. Front. Microbiol. 2019. doi: 10.3389/fmicb.2019.02155