Scientists advancing next generation probiotic development with synergy and oxygen adaptation

A recent study introduces a novel technique for developing next-generation probiotics, achieved by enabling strictly anaerobic bacteria to tolerate oxygen exposure without compromising their potential beneficial properties.
Table of Contents

What is already known
The human gut microbiota has gained attention as an environmental element that might play a role in influencing health or disease. The advancement of next-generation probiotics stands as a promising approach to manipulate the gut microbiota and enhance human well-being. However, a number of potential next-generation probiotics are strictly anaerobic and might require synergy with other bacteria for optimal growth. Faecalibacterium prausnitzii is a highly prevalent and abundant bacterium within the human gut, associated with human health, but efforts to incorporate it into probiotic formulations have not yet been realized.

What this research adds
A recent study outlines the simultaneous isolation of F. prausnitzii and Desulfovibrio piger, a bacterium capable of sulfate reduction, and their mutual exchange of nutrients, which facilitated growth and the generation of butyrate. In the present study, F. prausnitzii was adapted to tolerate exposure to oxygen, with the aim of producing a next-generation probiotic formulation, which was then demonstrated to be well tolerated and effective in both mice and humans.

The present study introduces a novel technique for developing next-generation probiotics, achieved by enabling strictly anaerobic bacteria to tolerate oxygen exposure without compromising their potential beneficial properties. This technology can be used for future development of additional strictly anaerobic strains as next-generation probiotics.

The adult human gut microbiota is populated by as many bacterial cells as our total number of cells, with their genomes (the microbiome) containing over 500 times more genes than our human genome. A healthy microbiome is often associated with increased microbial diversity and a higher abundance of butyrate-producing bacteria, such as Faecalibacterium prausnitzii, whose abundance varies with age and lifestyle, and its presence is reduced in Western populations.

Human gut microorganisms form intricate ecological interactions that play a significant role in maintaining intestinal balance. A key function of the gut microbiota is the fermentation of carbohydrates into short-chain fatty acids (SCFAs), including butyrate, which offers various benefits to the host. Fermentation is a primary energy-generating process for gut microorganisms, and it is crucial to manage by-products like lactate and hydrogen to sustain these processes. To this end, hydrogen scavengers like sulfate-reducing bacteria play a vital role in establishing metabolic networks within the gut.

Targeting the gut microbiota presents a significant potential for enhancing human well-being, and over the last twenty years, metagenomic studies have pinpointed a wide spectrum of bacteria that could serve as potential candidates for development of next-generation probiotics. Nevertheless, as 70% of the identified bacterial species lack cultivated counterparts, a limited number of potential candidates have been assessed in human studies.

Significant obstacles in the development of human gut bacteria as next-generation probiotics encompass demanding growth conditions, such as the need for specific nutrients or environments, and susceptibility to oxygen.

Bäckhed and colleagues, have recently published a study on Nature journal, where they isolated a novel strain of F. prausnitzii by co-culturing it with a novel Desulfovibrio piger sulfate-reducing strain. They developed a novel approach to produce F. prausnitzii as a next-generation probiotic, and evaluated its safety for human consumption.

Co-isolation and cross-feeding of F. prausnitzii and D. piger in vitro

By collecting fecal material from healthy individuals and culturing it under anaerobic conditions, the authors were able to successfully isolate a strain of F. prausnitzii that grew in co-culture with a strain of D. piger, an obligate anaerobic and non-fermenting Gram-negative bacillus. D. piger is a sulfate-reducing organism and classified as gut-specific commensal, as it exclusively inhabits the intestinal environment. The two isolated strains had not been characterized before.

The authors investigated the probiotic potential of the isolated F. prausnitzii compared to different F. prausnitzii strains and found similar anti-inflammatory properties, such as reductions in the pro-inflammatory factor interleukin-1β (IL-1β)-induced IL-8 secretion, further validating the probiotic potential of this strain.

The co-isolation of F. prausnitzii and D. piger and their potential symbiotic connection was hypothesized to result from complementary metabolic needs. To test this hypothesis, the authors cultivated both strains together in a modified culture medium supplemented with glucose to support F. prausnitzii, and observed a noteworthy increase in the growth of F. prausnitzii when co-cultured, as compared to monocultures in the same medium.

Metabolite analysis of this medium following 24 hours of growth corroborated the expectations: in monocultures, D. piger solely consumed lactate and generated acetate, without utilizing glucose; in contrast, F. prausnitzii monocultures produced minimal lactate, but when co-cultured with D. piger, the fermentation of glucose and the generation of lactate and butyrate were notably induced.

As acetate is required for butyrate synthesis, it did not accumulate in the co-culture medium, suggesting that D. piger served as an electron sink within the co-culture by consuming lactate. Through this process, D. piger generated acetate, which in turn was used by F. prausnitzii for its growth and butyrate production.

Development of oxygen tolerance in F. prausnitzii by stepwise adaptation

Developing products for next-generation probiotics is challenging, particularly due to the sensitivity of human gut bacteria to oxygen. As demonstrated in prior studies, the longevity of formulations containing F. prausnitzii can be extended through the incorporation of antioxidants like cysteine. However, this approach has limited utility for large-scale production, as viability diminishes within 24 hours of exposure to ambient air. 

To enhance F. prausnitzii tolerance to oxygen, an adaptation technique use employed by using a bioreactor. The strain was exposed to successive subculture steps, with decreasing cysteine concentrations and escalating anodic potential under oxidized conditions.

During this process, distinct colony morphologies emerged and five morphotypes were selected and further characterized for oxygen tolerance. Remarkably improved oxygen tolerance was noted in two morphotypes and the one with the highest increase of oxygen tolerance was subsequently chosen for assessing its synergistic growth with D. piger.

As a result of oxygen tolerance and co-culture with D. piger, a sufficient amount of F. prausnitzii was generated for human administration. Oxygen-tolerant F. prausnitzii was successfully freeze-dried, making it suitable for capsule development with minimal loss of viability. 

As the acquired oxygen tolerance did not exert any influence on the strain immune-modulatory characteristics, cellular physiology, metabolism, and potential for host interactions at the mucosal interface, F. prausnitzii was selected for the production of an investigational product.

The safety of the product was next evaluated by administering a bacterial suspension containing F. prausnitzii and D. piger to male and female mice, and no adverse responses were observed. To further examine the tolerability of the bacteria, a group of 50 healthy individuals aged 20 to 40 years was administered with a low or a high dose of F. prausnitzii and D. piger over an 8-week period.

The investigational product was well-tolerated, regardless of the administered dose. No study participants withdrew due to adverse events, and there was no increase in the frequency of adverse events or gastrointestinal symptoms within the treatment groups. Importantly, no clinically significant or statistically notable differences between groups were observed in terms of changes of parameters such as renal function, blood cell count, liver enzymes, markers of inflammation, hemoglobin levels, glycosylated hemoglobin, or fasting blood glucose.

Abundance of D. piger and F. prausnitzii in healthy volunteers after administration of D. piger and F. prausnitzii for eight weeks

To assess potential impact of the probiotic formulation on the human gut microbiota, whole-genome sequencing was performed and no differences between the groups of individuals were observed in overall composition of microbiota at baseline or at the conclusion of the study. 

Both parental strains F. prausnitzii and D. piger were notably prevalent at baseline, and the proportions of D. piger increased in the high-dose group and particularly in individuals with initially low relative abundance. However, these levels remained within the range observed for the placebo group. 

The proportions of F. prausnitzii remained unchanged and the levels of faecal short-chain fatty acids (SCFAs) exhibited no change, as well as no alteration was observed in faecal hydrogen sulfide levels. Taken together, these findings suggest that the probiotic formulation developed in this study is not only safe for the host, as evidenced by the absence of adverse events and gastrointestinal symptoms, but also for the gut microbiota, as indicated by the absence of compositional and metabolic changes.

The overall species-level abundance of F. prausnitzii ranged from 3.4% to 25.9%, similar to the abundance found in similarly aged healthy individuals from the USA, as well as older healthy individuals from Sweden and the UK. Consequently, it is plausible that further increase of F. prausnitzii was constrained by niche saturation.

However, considering the observed increase in D. piger, this elevation could potentially influence the abundance of other butyrate producers. A significant positive correlation was found between the change in butyrate production potential and the change in D. piger levels at the end of the administration period compared with baseline in all individuals, as well as in those who received either a low or high dose. This correlation was not evident in the placebo group.

These findings suggest that the probiotic formulation might support the overall butyrate production potential within communities present in the human gut. These observations emphasize the potential significance of baseline microbiota configurations for microbiota-based therapeutic strategies.

Markers of the oxygen-tolerant F. prausnitzii variant were found in fecal samples from some participants at baseline or in the placebo group. Additional variants and/or various combinations were discovered at the end of the product administration among a subset of study participants within the low-dose and high-dose groups.

The authors hypothesized that their probiotic formulation might have the capability to enhance F. prausnitzii in patient groups with lower abundance of this bacterium (e.g., individuals with type 2 diabetes) and in those with intestinal inflammation (such as inflammatory bowel disease). This hypothesis is supported by the observation that administration of F. prausnitzii ameliorates colitis and partially restores the microbiota in mice.

In conclusion, the researchers have developed a strategy based on the symbiotic interplay between F. prausnitzii and D. piger, that leads to increased growth of F. prausnitzii and butyrate production in laboratory settings. This holds the potential to impact the production potential of butyrate within the human gut environment.

Reduced amount of F. prausnitzii has been observed in the microbiota of patients affected by hyperlipidaemia, prediabetes, type 2 diabetes, non-alcoholic fatty liver disease, and inflammatory bowel disease. Therefore the production of F. prausnitzii as next-generation probiotic is of great interest. The approach developed in this study, grounded in leveraging existing synergistic interactions between gut microorganisms and improving oxygen tolerance, outlines the potential transformation of F. prausnitzii into a next-generation probiotic suitable for human consumption. 

This technology can also be adapted to other highly oxygen-sensitive bacteria, enabling their transformation into next-generation probiotics to target patient populations exhibiting reduced levels of these bacteria.