Microbiome research – from the tip of the iceberg to the full glacier

Although often used interchangeably, differences exist between the terms “microbiota” and “microbiome.” “Microbiota” describes the microorganisms that are found in a defined environment, while the term “microbiome” is broader and refers to the collection of genomes from all the microorganisms in the environment, including the microorganisms themselves as well as microbial structural elements, metabolites, and the environmental conditions (Berg et al., Reference Berg, Rybakova, Fischer, Cernava, Vergès, Charles, Chen, Cocolin, Eversole, Corral, Kazou, Kinkel, Lange, Lima, Loy, Macklin, Maguin, Mauchline, McClure, Mitter and Schloter2020).

As many microbes do not grow on standard media, traditional agar or culture-based approaches in microbiology provide scientists with only a fraction, or the “tip of the iceberg,” of the potential picture of the microbiome present in a particular environment. The emergence of culture-independent techniques, such as DNA sequencing, allows the microorganisms present to be more fully investigated based on the DNA found in any sample, giving information about microbes that are difficult to grow or currently cannot be grown in the laboratory. Notably, the sample being prepared for analysis must be of good quality and collected in a manner to ensure that it is representative of the DNA from the environment under investigation.

Two main types of DNA sequencing are used to study the microbiome – amplicon sequencing and shotgun metagenomics. Amplicon sequencing techniques mostly focuses on the 16S rRNA sequences present in all bacteria. In the case of fungi, Internal Transcribed Spacer sequences are used. The 16S rRNA gene contains conserved DNA regions easily found by a universal PCR primer. Between these conserved regions, bacteria have highly variable sequences, which are then amplified and can be used to identify the taxonomic status of bacteria using reference databases. This technique facilitates identification of the microorganisms down to the genus level (e.g., Lactobacillus or Bifidobacterium) but rarely at the species level. This might be important as functional benefits have been shown to be species-specific or even strain-specific.

For this reason, shotgun metagenomics, allowing identification at species and strain levels, has become more popular. Shotgun metagenomic-based approaches sequence all the genes present in a community, including genes from bacteria, yeasts, fungi, viruses, phages, and small eukaryotes. In addition to identifying the members present in the microbial communities, shotgun metagenomics also provides information about the pathways encoded within those microorganisms. This yields valuable information about the functional potential of the ecosystem; for example, it can also provide an assessment of the number of genes associated with health-promoting attributes or undesirable traits (e.g., virulence factor, antibiotic resistance genes). The identification at the strain level is key for probiotic research because the majority of probiotic traits are strain-specific. Shotgun sequencing allows researchers to assemble the entire genome of a microorganism by creating the so-called metagenome assembled genomes, even if the microorganism has never been grown in a laboratory. Based on the genome sequences, it is possible to predict metabolic pathways and whether the organism is likely to be health promoting or not. DNA sequencers have evolved considerably in the last 4–5 years. Overall, the quality of the sequencing has improved, as well as the length of the sequence fragments, leading to more reliable assemblies of more complete genomes.

Why does microbial diversity matter?

DNA sequence analysis also allows an assessment of the microbial diversity of a sample. Microbial diversity appears to be critical in maintaining resilience, which can be defined as the speed or extent to which the microbial ecosystem will recover its initial function or composition following perturbations (Fassarella et al., Reference Fassarella, Blaak, Penders, Nauta, Smidt and Zoetendal2021). Using an ecological analogy, the large number of species present in a rainforest makes it more resilient to the loss of a few species, for example, due to predators or severe weather events. The reason is that the functions of the lost species may more likely be replaced by other species in that environment, allowing the complete ecosystem to continue to thrive. Conversely, in a desert environment with less species diversity, such an event has more serious implications, as there is less capability to replace the functional activities of the lost species. Similarly, a gut microbiota that is more diverse before exposure to stressors such as infection, antibiotic use, stress, or illness may resist better against major changes that can impact host health. Diet may play a key role in developing a diverse and resilient gut microbiota (Fassarella et al., Reference Fassarella, Blaak, Penders, Nauta, Smidt and Zoetendal2021).

Alpha and beta diversity refer to the species richness (number) or distribution (evenness) within a community and the difference in the number of species between different ecosystems, respectively (Andermann et al., Reference Andermann, Antonelli, Barrett and Silvestro2022). For example, a study of microbial diversity in a selection of fermented foods detected 301 different microbial species in brine, 242 in sugar, and 70 in dairy foods (Leech et al., Reference Leech, Cabrera-Rubio, Walsh, Macori, Walsh, Barton, Finnegan, Crispie, O’Sullivan, Claesson and Cotter2020). Beta diversity analysis highlighted that these microbiomes clustered distinctly reflecting the substrates (i.e., brine, sugar and dairy) used to produce these foods. It is important to note that species numbers should not be the only measure of diversity in microbiome research but should preferably also include gene diversity, functional potential, and metabolic capability of the microbiota.

Prebiotics to enrich next generation health-promoting microbes – a personalised approach?

Human milk oligosaccharides: There is continued interest in more personalised approaches in relation to interventions that may improve health through microbiome-targeted activities. A prebiotic group that has gained a lot of interest in this area is the human milk oligosaccharides (HMOs). These are complex multifunctional glycans are present in human breast milk at different levels and compositions, depending on the mother’s genetic background and diet. They are resistant to digestion in the small intestine and reach the infant’s colon in an intact form where they are metabolised by some gut microorganisms. Over 150 HMO structures have been identified with different functionality, sparking increased interest in their use beyond infant formula. Particular HMOs have the potential to dictate microbiota composition that may find application for personalised nutrition whereby a particular HMO may be used to target an increase in a specific Bifidobacterium that may be deficient in an individual submitted but more research is required (Lordan et al., Reference Lordan, Roche, Delsing, Nauta, Groeneveld, MacSharry, Cotter and van Sinderen2024).

Beyond Lactobacillus and Bifidobacterium: There are many additional desirable microorganisms beyond species from the genera Lactobacillus and Bifidobacterium that are associated with health benefits, such as Akkermansia muciniphila, Ruminococcus bromii, Faecalibacterium prausnitzii, and Roseburia intestinalis. A. muciniphila is associated with a lean phenotype, whereas F. prausnitzii has been found to be present at high levels in individuals without inflammatory bowel disease (IBD). By extension, the absence of this microorganism in IBD suggests that encouraging its growth might contribute to alleviating some of the associated symptoms (Lordan et al., Reference Lordan, Thapa, Ross and Cotter2020). However, challenges can exist when incorporating these microorganisms into foods as they are extremely sensitive to oxygen, difficult to grow in sufficient amounts, or have not (yet) been sufficiently characterised for safety and functionality to allow them to be used in a dietary supplement. A strategy to maintain high levels of these microorganisms is to target them through dedicated dietary interventions that encourage their growth as summarised in a recent publication (Lordan et al., Reference Lordan, Thapa, Ross and Cotter2020).

New opportunities for personalised nutrition may also come from metabolic modelling tools that can be used to predict which nutrients will support the growth of microorganisms that have never previously been cultured (Machado et al., Reference Machado, Andrejev, Tramontano and Patil2018). This “cocktail of ingredients” could contain a particular prebiotic, vitamin, mineral, or carbohydrate source that confers a selective advantage on that microorganism, providing the basis for a more personalised or targeted intervention. An alternative route to personalised nutrition is to identify a specific goal for an individual, such as an increase in the levels of desirable metabolites, such as short-chain fatty acids (SCFAs; acetate, butyrate and propionate), vitamins, or antioxidants, and target interventions that address this goal (Lordan et al., Reference Lordan, Roche, Delsing, Nauta, Groeneveld, MacSharry, Cotter and van Sinderen2024).

Focus on fermented foods

Fermented foods have been defined as “foods that are made through the desired microbial growth and enzymatic conversion of food components” (Marco et al., Reference Marco, Sanders, Gänzle, Arrieta, Cotter, De Vuyst, Hill, Holzapfel, Lebeer, Merenstein, Reid, Wolfe and Hutkins2021). These foods have generally been consumed for thousands of years (e.g., beer, wine, cheese, fermented meat, kefir, kimchi, etc.) and the microorganisms present dictate the outcome of the fermentation process. The original purpose of fermentation was to preserve food, but over time, knowledge has grown around their potential health benefits, as well as their ability to enhance texture and flavour of the final product. Arguments have been made to suggest that the move away from fermented foods in Western societies over the last century, coupled with many other factors such as increased hygiene and sterility of our food and environments, reduced rates of breastfeeding, and overuse of antibiotics, has contributed to the increase in non-communicable diseases (Sonnenburg et al., Reference Sonnenburg, Smits, Tikhonov, Higginbottom, Wingreen and Sonnenburg2016).

Consumption of fermented foods has been shown to positively influence the gut microbiota and could exert benefits through a number of potential pathways, including the delivery of live microorganisms to the gut, modulating the gut microbiota (Marco et al., Reference Marco, Sanders, Gänzle, Arrieta, Cotter, De Vuyst, Hill, Holzapfel, Lebeer, Merenstein, Reid, Wolfe and Hutkins2021; Mukherjee et al., Reference Mukherjee, Breselge, Dimidi, Marco and Cotter2024). A large-scale meta-analysis comparing the overlap of lactic acid bacteria (LAB) from food sources and human body sites, demonstrated a close relation between the two, providing increasing evidence that fermented foods can be a possible source of LAB for the gut microbiota (Pasolli et al., Reference Pasolli, De Filippis, Mauriello, Cumbo, Walsh, Leech, Cotter, Segata and Ercolini2020).

Depending on the strains present, fermented foods can alter the nutritive qualities of the food through activities such as pre-digestion (e.g., lactose), elimination of toxins (e.g., phytic acid) or production of vitamins. Microbial-derived products made during fermentation, for example, peptides, bacteriocins, and amino acids, may also exert benefits by changing intestinal or systemic function. In foods that have been pasteurised or baked, thus killing any live microbes (e.g., sourdough bread), beneficial attributes may still exist depending on the presence of microbial metabolites and the mechanism of action associated with it. While Professor Cotter cautions against heeding advice from some influencers citing fermented foods as a “cure-all,” he believes that encouraging daily consumption of fermented foods is a useful way to introduce more microbes in our diet.

Not all fermented foods are created equally

It is important to clearly differentiate between fermented foods and probiotics. The term “probiotic” is retained for a strain-specific microorganism that has been studied in-depth and for which a particular health effect has been established through well conducted clinical trials (see the following presentation by Professor Pot). For many fermented foods, evidence is lacking regarding the specific health benefits of the microbes present, and the exact quantities of microbes remain often undefined. A greater understanding of the health effects of fermented foods based on high-quality data available from population-based diet studies and randomised controlled trials (RCTs) are needed. Until such evidence is available, and depending on the regulatory environment also, such products should not be labelled as “containing probiotics” unless a specific probiotic strain has been added.

Kefir, a fermented milk beverage, has been consumed for thousands of years. Significant differences can be found in the microbial composition of individual samples of kefir. Many commercial examples of kefir do not typically contain acetic acid bacteria, which are often present in traditional kefirs, or contain Lactobacillus species that are different from those found in traditional kefir or kefir grains. Traditional kefirs also tend to have more complex yeast communities compared to commercial products (Bourrie et al., Reference Bourrie, Ju, Fouhse, Forgie, Sergi, Cotter and Willing2021).

These differences can impact not only on the final flavour of the fermented product but also on the potential health benefits as demonstrated in a recent RCT in 21 adult males fed a commercial versus a traditional kefir (350 g twice daily for a period of 4 weeks). Compared to baseline, traditional kefir consumption reduced the levels of both LDL cholesterol (LDL-C) and the intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (VCAM-1), while commercial kefir consumption increased tumour necrosis factor-α (TNF-α). The traditional kefir resulted in greater reductions in Interleukin-8 (IL-8), C-reactive protein, VCAM-1, and TNF-α when compared to commercial kefir consumption (Bourrie et al., Reference Bourrie, Forgie, Makarowski, Cotter, Richard and Willing2023). This work illustrates the importance of identifying the specific microbial composition when linking fermented foods to health benefits.

How do “good” bacteria influence the immune response?

As we consume approximately 65 tonnes of non-sterile food and drink over our lifetime, it is not unexpected that a great proportion (70%) of our immune system is situated in the gut – where it functions to filter out viruses, toxins and allergens. Pathogenic organisms have the ability to penetrate through the epithelium into the inner part of the body, where they are detected by dendritic cells and are destroyed by the macrophages of the immune system. In this process, immune cells send out signals which, in the case of an inflammatory response to a pathogen, will be characterised by the cytokine IL-12 (Preidis and Versalovic, Reference Preidis and Versalovic2009). IL-12 activates other immune cells (e.g., T-cells) to launch a cascade of inflammatory responses to fight the pathogen. This includes the activation of B lymphocytes which will secrete immunoglobulin A into the lumen of the gut where the pathogen is destroyed and excreted from the body.

Unlike pathogens, commensals (generally beneficial organisms that help maintain a healthy host environment) do not translocate through the gut epithelium, and when detected in the lumen by dendritic cells, they trigger production of the anti-inflammatory Interleuking-10 (IL-10) (Preidis and Versalovic, Reference Preidis and Versalovic2009). The balance of pro- and anti-inflammatory cytokines is important not only in health but also in the control of allergies or auto-immune diseases when the immune system is overactive and inflammation results in damage. Thus, supporting the growth of beneficial bacteria through prebiotics or introducing certain probiotics may provide a means to influence the inflammatory response.

Multiple mechanisms, multiple responses, and multiple strains – a challenge to research

To understand the challenges posed in probiotic research, it is important to look at some of the complexities faced. First, it is likely that a probiotic may exert its effects through several mechanisms that may be employed simultaneously and can be either direct effects or mediated indirectly (see Figure 1). The mechanisms include direct competition with pathogens for nutrients and place on the epithelium, production of useful metabolites such as vitamins, SCFAs, or bacteriocins, barrier effects (e.g., fortifying tight junctions), as well as immunological effects and has been summarised more extensively in Chen et al. (Reference Chen, Heyndrickx, Meynier, Ouwehand, Pot, Stahl, Theis, Vaughan and Miani2023). For example, a probiotic that produces SCFAs may at the same time also produce growth factors which will indirectly stimulate the growth of other beneficial organisms. For this reason, a careful definition of the endpoints and whether they are immunological or metabolic in nature is needed in probiotic clinical trials. With this endpoint in mind, specific strains can then be selected in the laboratory that most likely will have the best performance in a targeted activity or to obtain a particular outcome (Sánchez et al., Reference Sánchez, Delgado, Blanco-Míguez, Lourenço, Gueimonde and Margolles2017).

Figure 1. Examples of potential mechanisms observed for probiotics. The application range of probiotic organisms is linked to a wide variety of possible mechanisms. At any given time, more than one mechanism may be active. Several mechanisms may be required simultaneously to achieve a specific beneficial health effect.

Second, research shows that it is not sufficient to characterise probiotic effects based on similarities at the genus or species level. In vitro research investigating the potential of various species and strains of lactobacilli to induce different levels of the anti-inflammatory cytokine IL-10 or the pro-inflammatory cytokine IL-12 on human peripheral blood mononuclear cells was presented (Foligne et al., Reference Foligne, Nutten, Grangette, Dennin, Goudercourt, Poiret, Dewulf, Brassart, Mercenier and Pot2007). It showed that it was possible to clearly discriminate between strains that have overall anti-inflammatory potential versus those which do not, even within the same species of bacteria, such as Lacticaseibacillus (Lc.) casei (Alard et al., Reference Alard, Peucelle, Boutillier, Breton, Kuylle, Pot, Holowacz and Grangette2018). Owing to this strain specificity, probiotic products should be labelled with the exact same strain number as used for the clinical studies and healthcare professionals are advised, when recommending a particular probiotic product, to confirm that the strain used in the product indeed matches the strain used in the supporting clinical research.

Third, variation in individual response is observed in many clinical drug trials, and the same potential exists in the host responses to probiotics. Using the same strain, individuals demonstrate variable capacity to induce the key cytokines IL-10 and IL-12. People with a high potential to produce IL-10, however, will generally show a low capacity to induce IL-12 (Pot et al., Reference Pot, Foligné, Daniel and Grangette2013; Alard et al., Reference Alard, Peucelle, Boutillier, Breton, Kuylle, Pot, Holowacz and Grangette2018). This observation highlights the need for clinical trials performed on a sufficiently diverse population to accurately represent these variants and ensure that the probiotic illustrates an overall efficacy.

Systematic reviews and meta-analysis in probiotic research – proceed with caution?

Although systematic reviews and meta-analyses of RCTs are considered the “gold standard” for providing evidence of an in vivo effect, they should be approached with caution in the field of probiotic research, due to the heterogeneity of the data in terms of factors such as strain identity, dose and mode of administration used, as well as target population, endpoint variation, and season of investigation.

When closely inspecting meta-analyses in this area, it is clear that the performance of a strain significantly depends on the overall product characteristics, including the food matrix, and whether it is presented as a single strain or in a combination of strains. For example, a meta-analysis of studies investigating probiotics for antibiotic-associated diarrhoea showed a difference in efficacy when a particular probiotic strain was combined with other strains (Agamennone et al., Reference Agamennone, Krul, Rijkers and Kort2018). When Lacticaseibacillus rhamnosus GG was given as a single strain, the study results favoured the probiotic. When combined in another study with Lactobacillus acidophilus and Bifidobacterium (B.) animalis subsp. lactis strains, the performance of the multi-strain probiotic was improved. However, in a third study using Lc. rhamnosus GG but with a different combination of other strains (including another strain of Lc. rhamnosus), the effect favoured the placebo. In addition, results differed when the probiotic was given as a dairy product or as a food supplement (Agamennone et al., Reference Agamennone, Krul, Rijkers and Kort2018).

The quality of the study should also be considered. In the same meta-analysis, two studies showed a neutral effect of a probiotic on antibiotic associated diarrhoea. In one study, the conclusion is based on one diarrhoea case in 49 subjects following probiotic or placebo intervention, while the other was based on 159 cases of antibiotic associated diarrhoea in 14170 subjects receiving the probiotic and 153 cases of diarrhoea in 14171 participants taking the placebo.

Meta-analysis of probiotics should therefore be handled with care and at present well conducted RCT’s may offer the best route for direct proof of efficacy of a particular strain (Agamennone et al., Reference Agamennone, Krul, Rijkers and Kort2018).

Can we define a healthy gut microbiota?

Microbial numbers gradually increase along the intestinal tract from the relatively sterile environment of the stomach (<10³ CFU/ml) to the large intestine, where the combination of slower transit time (48–72 hours in an adult), along with a favourable pH and a constant supply of nutrients from the diet, result in a dramatic increase in microbial numbers (10¹¹–10¹² CFU/ml) (Sender et al., Reference Sender, Fuchs and Milo2016). The interrelationship between the gut microbiota and the host is usually mutually beneficial, with the host supplying nutrients via dietary intake and the microbiota helping the process of homeostasis by providing resistance to infections and reducing inflammation. Various dietary interventions, including prebiotics, are being investigated as modulators of the gut microbiota in order to improve resilience to these stressors.

As each individual has an entirely unique gut microbiota profile owing to numerous modifiable factors, including diet and physical activity as well as non-modifiable factors, such as age, ethnicity, environment, and genetics (Redondo-Useros et al., Reference Redondo-Useros, Nova, González-Zancada, Díaz, Gómez-Martínez and Marcos2020), which interrelate and react with each other (Qin et al., Reference Qin, Havulinna, Liu, Jousilahti, Ritchie, Tokolyi, Sanders, Valsta, Brożyńska, Zhu, Tripathi, Vázquez-Baeza, Loomba, Cheng, Jain, Niiranen, Lahti, Knight, Salomaa, Inouye and Méric2022), it is not possible to define an optimal gut microbiota composition. Nevertheless, a number of initial concepts important for health can be suggested to characterise a “healthy” gut microbiota. Activities that could be considered of positive value to the host include improvements in barrier function, the production of vitamins by bacteria (vitamin B₁₂ and K), stimulation of anti-inflammatory cytokines, inhibitory activities towards gastrointestinal (GI) pathogens, and the production of organic acids (e.g., acetate, propionate, butyrate, succinate and lactate). There are also activities that at present appear neither positive nor negative, such as the formation of gases such as H₂, CO₂, and CH₄ which are relatively inert. Finally, there are certain activities which do not appear to be beneficial to the host, including toxic gas formation (H₂S), certain products of proteolytic metabolism (phenol, indole, skatole, ammonia, and branched chain fatty acids), and known bacterial toxins from Shigella spp., enterotoxigenic Escherichia coli, Cl. difficile, and Clostridium perfringens. Better understanding of the exact relationship between these various activities may assist in defining and quantifying a “healthy” gut microbiota.

The prebiotic research landscape – mechanisms and impact on health and disease

Prebiotics are defined as “substrates that are selectively utilised by host microorganisms conferring a health benefit” (Gibson et al., Reference Gibson, Hutkins, Sanders, Prescott, Reimer, Salminen, Scott, Stanton, Swanson, Cani, Verbeke and Reid2017). They work by selective proliferation of beneficial bacteria, which in turn also inhibit pathogenic organisms. Both in vitro and in vivo methods are used to investigate the mechanisms of action of prebiotics, which share some common features with probiotics (Chen et al., Reference Chen, Heyndrickx, Meynier, Ouwehand, Pot, Stahl, Theis, Vaughan and Miani2023).

Prebiotics include, besides HMOs, readily fermentable dietary fibres, conjugated linoleic acids, polyunsaturated fatty acids, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), mannan-oligosaccharides, xylo-oligosaccharides, and certain phenolic compounds and phytochemicals. However, the levels of evidence for health benefits of these substances vary considerably with the highest weight of evidence and mechanistic understanding for inulin, GOS, and FOS (Gibson et al., Reference Gibson, Hutkins, Sanders, Prescott, Reimer, Salminen, Scott, Stanton, Swanson, Cani, Verbeke and Reid2017).

In vitro methods at the University of Reading apply batch culture fermenters for the initial screening of candidate prebiotics using a single pH, transit time, and substrate concentration. Once a candidate prebiotic has been identified, it is studied in a more complex, multi-chambered continuous flow system that replicates the conditions of the proximal, transverse, and distil areas of the colon. This permits prediction of the part of the colon where the prebiotic may exert its influence. A desirable trait for a prebiotic is to target the more distal areas of the gut where the majority of gut diseases are known to originate, for example, colitis, colon cancer. While these models serve a purpose in providing initial information on potential mechanisms of action, the definitive test of a prebiotic is by in vivo human intervention studies. The newer molecular microbiology methods for characterising the microbiota metabolism will also help to provide the necessary mechanistic evidence that support effects of prebiotics. A selection of RCTs of prebiotic interventions in humans across a range of health conditions were presented.

In vivo prebiotic trials in adults: The early days of prebiotic studies were characterised by feeding prebiotics and measuring the effects on gut microbiota, as exemplified in a trial of healthy participants assigned to consume a novel or commercially available GOS versus placebo (Depeint et al., Reference Depeint, Tzortzis, Vulevic, I’anson and Gibson2008). Those consuming the GOS showed significant dose-dependent increases in bifidobacteria. Such studies were followed by RCTs that attempted to additionally characterise both the health impact along with changes in the microbiota.

An RCT supplementing overweight adults at risk of metabolic syndrome (n = 45) with a GOS mixture (5.5 g/d) for 12 weeks, resulted in an increase in Bifidobacterium and a significant beneficial change in the immunomarkers, plasma C-reactive protein, and faecal calprotectin, compared to placebo. Insulin, total cholesterol, and triglyceride concentrations were also significantly lower at 12 weeks, but weight and blood pressure remained stable (Vulevic et al., Reference Vulevic, Juric, Tzortzis and Gibson2013). For outcomes such as weight management, it is likely that longer term trials are necessary.

In ageing, there is a marked decrease in the numbers of beneficial bifidobacteria in the gut and a decline in immune function. Supplementing healthy elderly subjects (n = 44) with GOS (5.5 g/d) for 10 weeks resulted in a significant increase in bifidobacteria and phagocytosis, natural killer cell activity, and production of the anti-inflammatory cytokine IL-10 compared with placebo. This demonstrates the potential of prebiotics to attenuate age-related decline in immune function (Vulevic et al., Reference Vulevic, Drakoularakou, Yaqoob, Tzortzis and Gibson2008).

Use of the prebiotic FOS has been associated with reduced recurrence of Cl. difficile-associated diarrhoea and reduced recovery time from the consequences of antibiotic treatment, accompanied by an increase in faecal Bifidobacterium (Lewis et al., Reference Lewis, Burmeister and Brazier2005). Similarly, GOS has been shown to significantly reduce the incidence and duration of travellers’ diarrhoea compared to placebo (Drakoularakou et al., Reference Drakoularakou, Tzortzis, Rastall and Gibson2010). These data led to further research into athletes and military personnel who travel frequently and where diarrhoea can have a significant impact on performance.

Prebiotics interventions have also been tested in patients suffering from irritable bowel syndrome (IBS), where a GOS supplement resulted in improvements in stool consistency, flatulence, bloating, and composite score of symptoms, which tallied with an increase in Bifidobacterium as compared to placebo (Silk et al., Reference Silk, Davis, Vulevic, Tzortzis and Gibson2009).

In vivo prebiotic trials in infants: Infants fed a formula including a combination of short-chain GOS (scGOS) and long-chain FOS (lcFOS) had significantly less cumulative gut and respiratory infections at 18 months versus those fed a standard formula (Ivakhnenko and Nyankovskyy, Reference Ivakhnenko and Nyankovskyy2013). Additionally, the prebiotics conferred a similar level of protection to that observed in breastfed infants. In a prospective RCT trial in healthy term infants at risk of atopy, a prebiotic-supplemented formula offered in the first 6 months of life significantly reduced the 2-year cumulative incidences of any allergic manifestation compared to the placebo group (Arslanoglu et al., Reference Arslanoglu, Moro, Schmitt, Tandoi, Rizzardi and Boehm2008). Furthermore, the 5-year cumulative incidences of any allergic manifestation and atopic dermatitis (AD) were significantly lower in the scGOS/lcFOS group compared to the placebo group (Arslanoglu et al., Reference Arslanoglu, Moro, Boehm, Wienz, Stahl and Bertino2012).

Future horizons for prebiotics

Moving forward, it is likely that different prebiotics will be required to promote the growth of the more recently identified probiotic organisms in genera such as Akkermansia, Roseburia, and Eubacteria. Unlike the well-established targets such as Lactobacillus and Bifidobacterium species, various newly discovered bacteria produce additional SCFAs such as propionic acid and butyric acid, which are also of interest in supporting the gut microbiota. This may lead to more work on synbiotics, which can be either complementary (pre and probiotics acting independently but administered together) or synergistic (pre and probiotics acting together). In addition, different prebiotics will also be needed to target the range of commensals found in areas beyond the GI tract, including the skin, urogenital tract, placenta, vagina, and oral cavity.

Future work should focus on generating data on mechanisms of action to support evidence, while the inclusion of human cells and dialysis techniques in artificial gut models will help improve the replication of real-life gut conditions. Increasing our understanding of the human metabolome (the complete set of metabolites found in a biological sample, e.g., blood, urine) and any relationships with microbial profiles during intervention trials will also assist in the understanding of mechanisms. It is likely that the greatest potential for prebiotics is as a prophylactic treatment in healthy populations or as an adjunct clinical therapy rather than fully replacing pharmaceutical interventions. Greater consideration should be given to recommending prebiotic interventions following the administration of antibiotics to help restore the gut microbiota.

Evidence for a role of diet in mood and depression

Beyond temporary changes in mood arising from the enjoyment of palatable food, there is growing evidence for the role of diet in maintaining positive mood and preventing or decreasing the severity of mood disorders. Observational studies suggest a stepwise increase between happiness and diet quality (Mujcic and J Oswald, Reference Mujcic and J Oswald2016). In an RCT, providing participants (n = 75) with their recommended amount of vegetables for 8 weeks improved their diet quality and ratings on the subjective happiness scale versus a placebo group who received no additional vegetables (De Leon et al., Reference De Leon, Jahns, Roemmich, Duke and Casperson2022).

Depression is a devastating mood disorder and the biggest contributor to disability worldwide. There is a scarcity of data on the pathophysiology of depression, and there is little consistent evidence that depression is caused by a chemical imbalance in the brain, particularly serotonin (Moncrieff et al., Reference Moncrieff, Cooper, Stockmann, Amendola, Hengartner and Horowitz2023). At present, available treatments induce remission in only half of the time (Casacalenda et al., Reference Casacalenda, Perry and Looper2002), and adjunct therapy based on modifiable risk factors are being explored.

The role of seven lifestyle factors (alcohol consumption, diet, physical activity, sleep, smoking, sedentary behaviour, and social connection) in depression were investigated in almost 300,000 participants followed up over 9 years (Zhao et al., Reference Zhao, Yang and Sahakian2023). The results demonstrated the independent protective roles of certain factors on clinically diagnosed depression, including a healthy diet, which decreased the risk by 6% and was assessed based on adherence to U.S. dietary guidelines.

A meta-analysis of observational studies has shown that a healthy diet and, in particular, a Mediterranean-style diet, was strongly linked to lower rates of depression (Lassale et al., Reference Lassale, Batty, Baghdadli, Jacka, Sánchez-Villegas, Kivimäki and Akbaraly2019) while diets high in ultra-processed foods were reported to be linked to a 23% higher risk of depression (Lane et al., Reference Lane, Lotfaliany, Hodge, O’Neil, Travica, Jacka, Rocks, Machado, Forbes, Ashtree and Marx2023). However, one of the issues with observational studies is reverse causality; low mood and stress can potentially change eat patterns towards more high-calorie, nutrient-poor “comfort foods” (Firth et al., Reference Firth, Gangwisch, Borisini, Wootton and Mayer2020) whilst feelings of tiredness arising from low mood could contribute to a reduced desire to cook healthy meals. For this reason, RCTs offer more concrete evidence for a link between diet and mood.

A 12-week landmark RCT in Australia (The SMILES Trial) investigated dietary changes using a modified Mediterranean diet in participants (n = 67) suffering from moderate to severe depression whose diets were of poor quality (Jacka et al., Reference Jacka, O’Neil, Opie, Itsiopoulos, Cotton, Mohebbi, Castle, Dash, Mihalopoulos, Chatterton, Brazionis, Dean, Hodge and Berk2017). The dietary intervention comprised personalised dietary advice and nutritional counselling from a clinical dietician to support optimal adherence to the recommended diet. The placebo group did not receive any dietary advice but was provided with the same schedule and length of social visits as the intervention group. The diet group demonstrated significantly greater improvement in ratings of depression using the Montgomery–Åsberg Depression Rating Scale versus the placebo group. Remission from depression was achieved for 32.3% of the diet group and only 8.0% of the social support group. Similar findings were replicated in another 12-week RCT in young men suffering from depression who followed a Mediterranean diet intervention. The diet led to a significant reduction in depression (as measured by the Beck Depression Inventory) versus the control group who only received social support (Bayes et al., Reference Bayes, Schloss and Sibbritt2022).

In summary, a combination of observational and RCTs have demonstrated the potential impact of diet on mood and mental health, which may be mediated by many factors, including the effects of nutrients on function and brain structure and also potentially through modulation of the gut microbiome.

The gut as a “chatty” neighbour to the brain

The brain is an organ that is still in discovery, as shown by the recent publication of the “largest atlas of human brain cells,” revealing more than 3,000 types of cells, many of which were newly discovered (Maroso, Reference Maroso2023). The brain is regarded as the “control centre” and communicates with many organs, including the gut, via the vagus nerve. However, evidence of a bidirectional relationship between the brain and gut has existed for some time, through knowledge around hunger and satiety signalling from the gut to the brain and conversely the impact of mental stress (triggering the flight or fight response) on gut symptoms. What is not often appreciated, however, is that 80% of the neurons in the vagus nerve are sensory afferent neurons conveying information “up” from the intestinal wall to the brain, informing the brain around the status of the gut and dietary status (Breit et al., Reference Breit, Kupferberg, Rogler and Hasler2018).

The reference to the gut being the “second brain” refers to the enteric nervous system (ENS); a network of around 500 million neurons embedded along the GI tract that connects to the vagus nerve (Fleming et al., Reference Fleming, Ehsan, Moore and Levin2020). The ENS autonomously controls digestion and can communicate with the brain via several neuromodulators and neurotransmitters, including epinephrine, norepinephrine, serotonin, dopamine, γ-aminobutyric acid, and acetylcholine as well as via microbial metabolites, such as SCFAs (Montagnani et al., Reference Montagnani, Bottalico, Potenza, Charitos, Topi, Colella and Santacroce2023). It is therefore possible that any disturbance or dysregulation of either gut or brain pathways can potentially affect the functionality of the other.

Microbiota–gut–brain axis: emerging evidence

Although it has been recognised that there is a bidirectional relationship between the gut and brain, there has been heightened interest in the role of the gut microbiota as part of this axis. At present most of the evidence for a role of the gut microbiota in brain function, behaviour or mental health originates from animal research or observational studies in humans and is therefore in the very early stages of understanding, though there is gathering evidence of the significant interaction the gut microbiota has in human behaviour, mood, and cognition.

Animal models: Animal models for investigating the gut–microbiome–brain axis include germ-free (GF) rodents (bred without a microbiota) and rodents with antibiotic-induced dysbiosis. A meta-analysis of 48 rodent studies investigating the impact of antibiotic-induced dysbiosis reported a 32.7% increase in anxiety and a 40.7% increase in depression-like behaviour in the study populations, respectively (Hayer et al., Reference Hayer, Hwang and Clayton2023). There was a significant association between symptoms of negative valence system (including anxiety and depression) and cognitive system (decreased spatial cognition) with antibiotic intake (p < 0.05). While the area is promising, the level of heterogeneity between the studies and publication bias makes it difficult to generalise results.

Human studies: Observational studies show associations between mood disorders and gut dysbiosis. In a cohort of elderly adults in China, there was a correlation between high concentrations of antibiotics in urine and the presence of depression (Liu et al., Reference Liu, Zhang, Sang, Liu, Zhu, Yang, Wang, Sheng, Wang, Zhang, Cao and Tao2021b). Using nested case–control studies from a large population‐based UK database, a single antibiotic treatment was associated with a higher risk of depression in all antibiotic groups (Lurie et al., Reference Lurie, Yang, Haynes, Mamtani and Boursi2015). However, observational studies lack the ability to prove causation. Illness and infection itself (leading to antibiotic use) may promote greater levels of mood disorders.

Faecal microbiota transplant studies: Evidence from faecal microbiota transplant (FMT) is emerging to support a role for the microbiota in mood. Transferring faecal samples from the microbiota of humans with depression into GF rats resulted in increased anxiety and reduced interest in pleasurable activities. There was also an association with reduced tryptophan metabolism, the precursor of serotonin (Kelly et al., Reference Kelly, Borre, O’Brien, Patterson, El Aidy, Deane, Kennedy, Beers, Scott, Moloney, Hoban, Scott, Fitzgerald, Ross, Stanton, Clarke, Cryan and Dinan2016).

Irritable bowel syndrome: IBS is a GI disorder characterised by chronic abdominal pain and altered bowel habits and is one of the most common GI disorders seen by clinicians. People with IBS have a threefold higher increase of anxiety and depression than healthy controls (Zamani et al., Reference Zamani, Alizadeh-Tabari and Zamani2019), and IBS is described as a disorder of gut–brain interaction (Mearin et al., Reference Mearin, Lacy, Chang, Chey, Lembo, Simren and Spiller2016) with acknowledgement that the gut microbiota likely plays a role.

In summary, the evidence for a role of the gut microbiome in the effects on mood is still in its infancy, but growing as a result of animal and FMT studies, human observational studies, and RCTs in humans investigating the effects of probiotics and prebiotics.

How might the microbiota signal to the brain?

The routes of communication between gut microbes and the brain are varied and complex, and their understanding is still evolving (Butler et al., Reference Butler, Cryan and Dinan2019). There are a number of pathways and mechanisms between the gut microbiota and the brain, including the vagus nerve and the immune system, besides metabolites and neurotransmitters (Margolis et al., Reference Margolis, Cryan and Mayer2021).

Severing the vagus nerve in mice (vagotomy) has helped identify multiple roles for this cranial nerve, including evidence that gut microorganisms can activate the vagus nerve and play a role in mediating the effects on the brain and behaviour (Forsythe et al., Reference Forsythe, Bienenstock, Kunze, Lyte and Cryan2014). Studies on mice have demonstrated how certain probiotic action desists after a vagotomy. For example, Lc. rhamnosus JB-1 has been demonstrated to have an anxiolytic effect in mice. However, mice subjected to vagotomy did not benefit from the reduction in anxiety-like behaviour or reduced corticosterone response to stress, as observed in the control mice also offered the probiotic; demonstrating that signalling is taking place via the vagus nerve (Liu et al., Reference Liu, Zhang, Sang, Liu, Zhu, Yang, Wang, Sheng, Wang, Zhang, Cao and Tao2021b).

The immune system is beginning to be appreciated as an important factor in mental health. The immune systems in both the gut and brain are connected and can be influenced by the microbiome. As previously discussed by Professor Pot, immune cells possess toll-like receptors that, when activated by the presence of bacteria, initiate a cascade of events resulting in either pro- or anti-inflammatory cytokine production, depending on the microorganisms detected. The presence of pathogens results in the release of pro-inflammatory cytokines and other activated immune cells into the peripheral circulation, which cross the blood barrier and activate microglia (CNS immune cells), potentially causing CNS inflammation (Kasarello et al., Reference Kasarello, Cudnoch-Jedrzejewska and Czarzasta2023). The role of the microbiota in influencing the immune system is supported by research in GF mice that display defects in the maturation and function of microglia, resulting in deficient innate immune responses. The key signalling pathway mediating this influence in mice was found to be bacterial SCFAs (Erny et al., Reference Erny, Hrabě de Angelis, Jaitin, Wieghofer, Staszewski, David, Keren-Shaul, Mahlakoiv, Jakobshagen, Buch, Schwierzeck, Utermöhlen, Chun, Garrett, McCoy, Diefenbach, Staeheli, Stecher, Amit and Prinz2015).

SCFA produced by gut microorganisms can communicate both directly and indirectly with the brain and have been reviewed by Berding et al. (Reference Berding, Vlckova, Marx, Schellekens, Stanton, Clarke, Jacka, Dinan and Cryan2021). SCFA bind onto receptors in the blood–brain barrier indicating the potential for direct communication, with the potential to stimulate neurotransmitter synthesis and influence neuronal behaviour, gene expression, and immune activation (Lan et al., Reference Lan, Tang, Lu, Hu and Tang2024). Butyrate, in particular, has also been shown to enhance the integrity of the blood–brain barrier, protecting it from potential neurotoxic factors which could cause inflammation.

The gut microbiota can also encourage the production of neurotransmitters and their precursors. Tryptophan is the sole precursor of the neurotransmitter serotonin (5-hydroxytryptophan [5-HT]), which is involved in mood regulation. While it is true that 95% of serotonin is produced in the gut (Terry and Margolis, Reference Terry and Margolis2017; Appleton, Reference Appleton2018), it is unlikely that this molecule can cross the blood–brain barrier; serotonin produced locally in the gut primarily contributes to gut motility (Berding et al., Reference Berding, Vlckova, Marx, Schellekens, Stanton, Clarke, Jacka, Dinan and Cryan2021). However, both precursor molecules tryptophan and 5-HTP can cross the blood–brain barrier. The gut microbiota have been suggested to be able to produce small quantities of tryptophan, the precursor of serotonin, while others metabolise tryptophan into metabolites that can signal directly or indirectly transmitted to the brain (Agus et al., Reference Agus, Planchais and Sokol2018). The gut microbiota also shifts the accessibility of tryptophan to the kynurenine pathway, an alternative metabolic route for tryptophan (Waclawiková and El Aidy, Reference Waclawiková and El Aidy2018). Recent research in a depressed mouse model has shown reduced levels of tryptophan and 5-HTP in faeces and reduced levels of 5-HT in brain tissue, which are strongly correlated with significantly decreased levels of members of the Lactobacillus group in the microbiota (Xie et al., Reference Xie, Wu, Chen, Zhong, Wu, Niu, Wang, Zeng and Wang2023). The mechanisms by which tryptophan metabolism in the gut can affect the brain remains an active area of research.

The era of psychobiotics, mood, and depression

RCTs investigating the effects of the various “biotics” on the modification of the microbiome and mood are further helping to elucidate this relationship. Psychobiotics is a term commonly used to refer to a live micro-organism that, when ingested in appropriate quantities, has a positive mental health benefit (Butler et al., Reference Butler, Cryan and Dinan2019). It has been suggested that prebiotics should also fall into this class (Yang et al., Reference Yang, Zhou, Zhang, Si, Liu and Li2023).

Trials in healthy subjects: In a 12-week double-blind RCT of 63 healthy older adults (≥65 years), a probiotic supplement containing Bifidobacterium species promoted mental flexibility (ability to switch between different thoughts and actions), improved stress scores versus placebo but did not impact on other self-reported measures of depression (Kim et al., Reference Kim, Cha, Sim, Jung, Chun, Baik and Shin2021). Brain-derived neurotrophic factor known to be crucial for learning, memory function, and stress was also significantly elevated in the probiotic group after 12 weeks. In a triple-blind RCT including 20 healthy participants, a 4-week probiotic supplement (Bifidobacterium and Lactobacillus strains) significantly reduced reactivity to sad mood, which was largely accounted for by reduced rumination and less aggressive thoughts as compared to a placebo control group (Steenbergen et al., Reference Steenbergen, Sellaro, van Hemert, Bosch and Colzato2015).

In terms of prebiotics, an RCT using a GOS supplement (5.5 g/day) offered to healthy individuals (n = 45, 18–45 years) for 3 weeks significantly reduced the waking cortisol response and shifted the focus from a negative to a positive imagery in participants versus placebo. There was no effect on self-reported mood, possibly owed to the short duration of the study (Schmidt et al., Reference Schmidt, Cowen, Harmer, Tzortzis, Errington and Burnet2015). A more recent RCT observed the effects of 4-week GOS supplementation (5.5 g/day) in healthy young women (n = 64, 16–24 years), comparing the results between those with high or low anxiety traits. Supplementation with GOS significantly reduced self-reported anxiety scores in women who were highly anxious but not in those with low-level anxiety, versus placebo (Johnstone et al., Reference Johnstone, Milesi, Burn, van den Bogert, Nauta, Hart, Sowden, Burnet and Cohen Kadosh2021). A RCT in older adults found that a 7.5-g prebiotic supplement of inulin and FOS improved cognition in older adults over a span of 12 weeks, performing better than the placebo on memory tests typically used to identify early stages of Alzheimer’s disease (Ni Lochlainn et al., Reference Ni Lochlainn, Bowyer, Moll, García, Wadge, Baleanu and Steves2024).

Probiotics for depressive symptoms: The use of psychobiotics is becoming increasingly studied for use in clinical disorders, particularly as an adjunct to current therapies. A meta-analysis demonstrated that probiotics were effective in reducing depressive symptoms when administered in addition to antidepressants but not when used alone (Nikolova et al., Reference Nikolova, Cleare, Young and Stone2021). Guidelines issued by the World Federation of Societies of Biological Psychiatry and the Canadian Network for Mood and Anxiety Disorders provisionally recommend the use of probiotic strains (e.g., Lactobacillus and Bifidobacterium spp.) at doses of between 1 and 10 billion CFUs per day for adjunctive use and are weakly recommended for monotherapy use in major depressive disorder (MDD) (Sarris et al., Reference Sarris, Ravindran, Yatham, Marx, Rucklidge, McIntyre, Akhondzadeh, Benedetti, Caneo, Cramer, Cribb, de Manincor, Dean, Deslandes, Freeman, Gangadhar, Harvey, Kasper, Lake, Lopresti and Berk2022).

In summary, psychobiotics are an emerging area of research that appears to show a tendency for greater effects in those with mood disorders and with some organisations provisionally recommending probiotics for adjunctive use in MDD. However, additional RCTs are necessary to evaluate their therapeutic benefit in patient populations with psychiatric conditions.

Practical dietary advice

While there are still gaps in our understanding of the links between specific diets and mood disorders, Dr Leeming indicated that dietary changes to support the gut microbiota include a focus on nutrients, which are key components of a Mediterranean-style diet including fibre, polyphenols, and omega-3 fatty acids.

It is generally recognised that a diet rich in fibre promotes an increase in bacterial diversity and beneficial microorganisms in particular (Berding et al., Reference Berding, Vlckova, Marx, Schellekens, Stanton, Clarke, Jacka, Dinan and Cryan2021). In a recent meta-analysis, a higher fibre intake was linked to lower odds of depression (Fatahi et al., Reference Fatahi, Matin, Sohouli, Găman, Raee, Olang, Kathirgamathamby, Santos, Guimarães and Shidfar2021), and a separate analysis showed that every 5 g increase in fibre was associated with a 5% lower risk of depression (Saghafian et al., Reference Saghafian, Hajishafiee, Rouhani and Saneei2023). This is of particular concern as many countries fall short of daily fibre recommendations. In the United Kingdom, the average fibre intake among adults is 19.2 g per day (Public Health England (PHE), 2020) compared to the recommended 30 g per day (Scientific Advisory Committee on Nutrition (SACN), 2015), with similar gaps found in other European countries (Rippin et al., Reference Rippin, Hutchinson, Jewell, Breda and Cade2017).

Polyphenols are plant metabolites found typically in colourful fruit and vegetables as well as in coffee, chocolate, tea, and red wine. Most polyphenols are not absorbed in the small intestine but are metabolised further down the digestive tract by colonic microflora and emerging data support their effects as prebiotics on the microbiota (Zhou et al., Reference Zhou, Gu, Zhuang, Xu, Yang and Zhou2020). The inclusion of fermented foods such as kefir, sauerkraut, kimchi, and yoghurt potentially provides beneficial bacteria (Marco et al., Reference Marco, Sanders, Gänzle, Arrieta, Cotter, De Vuyst, Hill, Holzapfel, Lebeer, Merenstein, Reid, Wolfe and Hutkins2021). Omega-3 fatty acids contained in foods such as oily fish may also have a prebiotic-like effect (Vijay et al., Reference Vijay, Astbury, Le Roy, Spector and Valdes2021).

As a note of caution, while diet and lifestyle measures can be used to help those who have sub-clinical depression, the emerging research on food and mood should not delay or desist access to mental healthcare practitioners or prescribed mental health medication, particularly in severe depression.

Hormonal changes during menopause

Menopause is the culmination of hypothalamic and ovarian ageing, both of which are influenced by genetic factors, environment, lifestyle, and systemic disease, for example, autoimmune disorders, diabetes, and hypertension. Specific stages of menopausal transition have been defined using the Stages of Reproductive Aging Workshop criteria (Davis et al., Reference Davis, Lambrinoudaki, Lumsden, Mishra, Pal, Rees, Santoro and Simoncini2015). These are based on menstrual, endocrine, and ovarian markers. The menopausal transition is divided into an early stage characterised by fluctuating length of menstrual cycles and variable levels of follicle-stimulating hormone (FSH) and a later stage, defined by amenorrhoea (>60 days) and elevated FSH. Progressing through the stages of menopause, there is a decline in ovarian follicles accompanied by a decline in ovarian hormones (oestradiol, inhibin B, and anti mullerian hormone) and an increase in the gonadotropin-luteinising hormone and FSH. Post menopause, ovarian hormone levels remain low for the remainder of life, and adipose tissue mostly becomes the primary source of remaining oestrogen (Davis et al., Reference Davis, Lambrinoudaki, Lumsden, Mishra, Pal, Rees, Santoro and Simoncini2015).

Sex- and age-related changes in the microbiota

Menopause-related changes to the gut microbiome need to be considered against the context of age- and sex-related changes in the microbiota. The largest shift in microbiota diversity occurs in early weaning with progression from breast milk to mixed solid foods, and it increases drastically within the first year of life for the infant. Up to approximately 3 years of age is a critical window of opportunity to modulate the gut microbiota composition. After this age, the microbiota is more stable and mature (40%–60% similarity with the adult microbiota) (Koenig et al., Reference Koenig, Spor, Scalfone, Fricker, Stombaugh, Knight, Angenent and Ley2011; Yatsunenko et al., Reference Yatsunenko, Rey, Manary, Trehan, Dominguez-Bello, Contreras, Magris, Hidalgo, Baldassano, Anokhin, Heath, Warner, Reeder, Kuczynski, Caporaso, Lozupone, Lauber, Clemente, Knights, Knight and Gordon2012; Bäckhed et al., Reference Bäckhed, Roswall, Peng, Feng, Jia, Kovatcheva-Datchary, Li, Xia, Xie, Zhong, Khan, Zhang, Li, Xiao, Al-Aama, Zhang, Lee, Kotowska, Colding, Tremaroli and Wang2015). During puberty, changes are observed in the gut microbiota, highlighting the influential role of sex hormones. Research on adolescent mono- and dizygotic twins shows significant differences between the microbiota of male and female twins, not observed in infancy (Yatsunenko et al., Reference Yatsunenko, Rey, Manary, Trehan, Dominguez-Bello, Contreras, Magris, Hidalgo, Baldassano, Anokhin, Heath, Warner, Reeder, Kuczynski, Caporaso, Lozupone, Lauber, Clemente, Knights, Knight and Gordon2012). Microbiota diversity continues to increase gradually during adulthood and appears to reach a plateau around 40 years of age. Interestingly, women also have slightly higher microbiota diversity than men in younger adulthood, but not in older adulthood, which may suggest sex differences in microbiota ageing (de la Cuesta-Zuluaga et al., Reference de la Cuesta-Zuluaga, Kelley, Chen, Escobar, Mueller, Ley, McDonald, Huang, Swafford, Knight and Thackray2019). With increasing age, there is a shift towards a state of uniqueness within each person with the highest uniqueness in long lived individuals which may be related to healthy ageing (Wilmanski et al., Reference Wilmanski, Diener, Rappaport, Patwardhan, Wiedrick, Lapidus, Earls, Zimmer, Glusman, Robinson, Yurkovich, Kado, Cauley, Zmuda, Lane, Magis, Lovejoy, Hood, Gibbons, Orwoll and Price2021).

Sex hormones and the gut microbiome – a bidirectional relationship?

A bidirectional relationship has been proposed between the gut microbiota and sex hormones. Sex hormones can influence the microbiota and therefore impact microbiota-related mechanisms in health and disease. Similarly, gut microbiota deconjugation of sex hormones can contribute to hormone retention which may, in turn, have an impact on hormone-related mechanisms in health and disease.

Sex hormones originate in various tissues. The ovaries and adipose tissue are the main source of oestrogen in premenopausal and postmenopausal women, respectively. Oestrogens enter the systemic circulation to reach target tissues as well as the liver, where they undergo hepatic conjugation with glucuronide or sulphate groups. Free oestrone and oestradiol are conjugated with glucuronides to form oestrone-3-glucuronide and estrodiol-17-glucuronide. This facilitates their excretion in the bile into the gut, where microbial-derived β-glucuronidase deconjugate and restore oestrogen for reabsorption into the enterohepatic circulation. Similarly, gut microbial sulphatases return oestrone-sulphate and dehydroepiandrosterone-sulphate into the free unconjugated forms oestrone and DHEA; the latter is a precursor hormone which can be converted to oestrogens and androgens. The aggregate of bacterial genes capable of deconjugating and recycling oestrogens is termed the “estrobolome” and may help in the retention of oestrogen in the body, with potential benefits on hormonal symptoms in menopause (Peters et al., Reference Peters, Lin, Qi, Usyk, Isasi, Mossavar-Rahmani, Derby, Santoro, Perreira, Daviglus, Kominiarek, Cai, Knight, Burk and Kaplan2022). A recent review, however, also discusses the potential carcinogenic role of oestrogen in the light of hormone-susceptible cancers, such as breast, prostate, endometrial, and lung cancers, while also highlighting other factors associated with, for example, oestrogen-driven breast cancer (Al-Shami et al., Reference Al-Shami, Awadi, Alsheikh, Al-Sharif, Ala’Bereshy, Al-Eitan and Al Zoubi2023).

“Estrobolome” enzymes are expressed across a diverse range of microbes and across multiple bacterial phyla, especially species in Firmicutes (now Bacillota) and Bacteroidetes (now Bacteroidota) which express β-glucuronidase enzymes (Pollet et al., Reference Pollet, D’Agostino, Walton, Xu, Little, Biernat, Pellock, Patterson, Creekmore, Isenberg, Bahethi, Bhatt, Liu, Gharaibeh and Redinbo2017). An in vitro study showed that different gut bacterial β-glucuronidases had the capacity to deconjugate oestradiol and oestrone glucuronides, to varying degrees, providing a proof-of-principle that human gut microbiota can reactivate oestrogens (Ervin et al., Reference Ervin, Li, Lim, Roberts, Liang, Mani and Redinbo2019). Similarly, human gut bacterial sulphatases are expressed mostly in the Bacteroidota, followed by the Bacillota phyla. A small cross-sectional study (n = 25 men, n = 7 postmenopausal women, n = 19 premenopausal women) found a significant positive correlation between gut microbial diversity and total urinary oestrogens in postmenopausal women and men, indicating a role of the microbiota in systemic non-ovarian oestrogen levels (Flores et al., Reference Flores, Shi, Fuhrman, Xu, Veenstra, Gail, Gajer, Ravel and Goedert2012). This correlation was not observed in premenopausal women whose levels of oestrogen can vary significantly and where the additional impact of the gut microbiota may be minimal or obscured.

What happens to the microbiome in menopause?

To determine the effect of oestrogen and menopause status on gut microbiome diversity, premenopausal and postmenopausal women were compared to each other and to men of similar ages in a large prospective cohort study (n = 2300; Peters et al., Reference Peters, Lin, Qi, Usyk, Isasi, Mossavar-Rahmani, Derby, Santoro, Perreira, Daviglus, Kominiarek, Cai, Knight, Burk and Kaplan2022). Shotgun sequencing of stool samples was carried out to determine gut microbiota taxonomic and functional composition. In the alpha-diversity analysis, premenopausal women had a trend towards a higher gut microbial diversity than postmenopausal women (p = 0.06) and significantly higher diversity compared to both young and old men (p = 0.03) while postmenopausal women did not differ from men. In beta-diversity analysis, postmenopausal women differed significantly in gut microbiome composition from premenopausal women. Furthermore, gut microbiome composition in premenopausal women differed more from younger men than postmenopausal women did from older men. Together, these various comparisons support an effect of female sex hormones on the gut microbiome and that menopause is associated with a gut microbiome that is more similar to men. β-Glucuronidase abundance was significantly higher in premenopausal versus postmenopausal women while sulphatase abundance only differed significantly between premenopausal women compared to men (both young and old). This may suggest that the estrobolome potential, that is, deconjugation activity towards sex steroid hormones, is diminished after menopause due to oestrogen depletion.

The association of sex hormones with the gut microbiome was studied in a sample of 200 postmenopausal women in the USA (Peters et al., Reference Peters, Hanna, Wang, Weber, Topper, Appleton, Sharma, Hodis, Santoro, Guillemette, Caron, Knight, Burk, Kaplan and Qi2023a). In terms of alpha diversity, higher levels of serum oestrogens were associated with greater gut microbiome diversity, but this was not consistently observed for other hormones, including androgens and adrenal precursors. In the beta-diversity analysis, serum levels of oestradiol and oestrone were significantly associated with the overall microbiota composition. In this study, a microbiota score (the sum of all gut microbiota species associated with each hormone) was calculated for each of the 15 hormones measured, and a significant positive correlation was found between oestrogen-related microbiota scores and β-glucuronidase abundance. Oestrogen-related species, such as Alistipes, were most strongly correlated with β-glucuronidase abundance, which is consistent with prior literature that shows this species to harbour β-glucuronidase genes (Peters et al., Reference Peters, Hanna, Wang, Weber, Topper, Appleton, Sharma, Hodis, Santoro, Guillemette, Caron, Knight, Burk, Kaplan and Qi2023a). Thus, in postmenopausal women, higher serum oestrogens are associated with higher gut microbiota diversity, an altered gut microbiome composition, and greater estrobolome potential, suggesting metabolic interaction of the gut microbiota with oestrogens (i.e., “recycling” of oestrogens).

Given that the estrobolome may play a key role in determining oestrogen levels in postmenopausal women, further research is required to determine its impact on health, menopause symptoms, and the influence of diet and lifestyle interventions on its function. For now, a focus on following general healthy eating recommendations including fibre rich foods, can help maintain the diversity of the microbiota while also bringing benefits for other areas related to menopause (e.g., body composition, bone and heart health). Women suffering from symptoms associated with menopause should speak with their healthcare practitioner about options, including hormone replacement therapy (HRT) as well as lifestyle changes, including a healthy diet.

It was highlighted that dietary patterns rich in minimally processed plant foods (i.e, wholegrains, fruits and vegetables, nuts and seeds) and low in refined grains, red or processed meats, and saturated fats are associated with beneficial gut microbiota and also with better cardiometabolic health – which is particularly important in menopause when body composition and cardiometabolic health are affected (Peters et al., Reference Peters, Xing, Chen, Usyk, Wang, McClain and Qi2023b).

The vaginal microbiota in menopause and the gut–vagina axis

The vaginal microbiome is also of interest during menopause when lower levels of oestrogen cause glycogen levels in vaginal cells to reduce, depleting Lactobacillus of their food source and elevating the pH with potential colonisation by pathogens as a consequence (Laniewski and Herbst-Kralovets, Reference Laniewski and Herbst-Kralovetz2022). A healthy vaginal microbiota is characterised by high Lactobacillus dominance and low diversity. Lactobacillus uses glycogen as an energy source and produces a lower pH. The gut microbiome with its “estrobolome” activity could promote oestrogen retention in the circulation, reaching the vagina, and promoting glycogen retention in vaginal cells. This hypothesised connection is referred to as the gut–vagina axis. There are small RCTs which have investigated the effects of probiotics, administered either locally or orally, in both bacterial vaginosis and vulvovaginal candidiasis. These studies have been reviewed by Han and Ren (Reference Han and Ren2021); larger RCTs are needed in this area.

Comfortable in your own skin microbiome

The skin is the largest organ in the body, and it functions to prevent infection, retain moisture, and regulate body temperature. It is rich in immune cells and heavily colonised by microbial cells. As with the gut, the skin microbiome refers to the community of microorganisms colonising the skin, including structural elements, metabolites, signalling molecules, and the surrounding environmental conditions (Berg et al., Reference Berg, Rybakova, Fischer, Cernava, Vergès, Charles, Chen, Cocolin, Eversole, Corral, Kazou, Kinkel, Lange, Lima, Loy, Macklin, Maguin, Mauchline, McClure, Mitter and Schloter2020). The skin is a harsh environment containing a unique set of microorganisms that can tolerate the salty, dry, nutrient-poor, and acidic conditions, along with fluctuating temperatures and exposure to ultraviolet (UV) radiation (Byrd et al., Reference Byrd, Belkaid and Segre2018). Microbial communities on the skin vary between individuals and between their sites on the body. A diverse range of bacterial species can be found, including the genera Corynebacterium, Staphylococcus, Cutibacterium (representing some former species of the genus Propionibacterium), Brevibacterium, Micrococcus, and Malassezia as the main fungal genus (Mahmud et al., Reference Mahmud, Akter, Tamanna, Mazumder, Esti, Banerjee, Akter, Hasan, Acharjee, Hossain and Pirttilä2022). The specific functions of the microbiota living on the skin can be strain-specific; therefore, need careful consideration if therapeutic modulation of the microbiome is to be explored. For example, Cutibacterium acnes is a major commensal on skin and plays an important role in skin health, but strains have also been associated with acne (Johnson et al., Reference Johnson, Kang, Barnard and Li2016). Novel species have recently been identified including phages although there is little evidence on how viruses impact skin health (Saheb Kashaf et al., Reference Saheb Kashaf, Proctor, Deming, Saary, Hölzer, Taylor, Kong, Segre, Almeida and Finn2022).

The skin microbiota provides protection both directly by competing with pathogens for resources, the release of various chemicals, including antimicrobial factors, and indirectly by interacting with the immune system of the skin, which can differentiate between the commensal bacteria and harmful strains. Thus, the skin microbiota determines the production of pro- or anti-inflammatory cytokines and is also involved in wound healing and strengthening of epithelial tight junctions (De Pessemier et al., Reference De Pessemier, Grine, Debaere, Maes, Paetzold and Callewaert2021); all of which are of relevance to inflammatory skin conditions such as AD. Increasingly, observational studies have shown that the abundance of some pathogenic microbes is associated with skin conditions, but it is difficult to ascertain whether the altered skin microbiome is a cause or consequence of skin disorders (De Pessemier et al., Reference De Pessemier, Grine, Debaere, Maes, Paetzold and Callewaert2021).

The gut–skin axis in dermatological conditions

The skin and gut share many structural similarities; both are highly innervated and vascularised and have important immune and neuroendocrine functions (De Pessemier et al., Reference De Pessemier, Grine, Debaere, Maes, Paetzold and Callewaert2021). The gut–skin axis describes a bidirectional relationship whereby the gut can influence the condition of the skin and vice versa. Evidence of this interaction is seen in food allergies manifesting in skin symptoms such as urticaria and AD or, in the case of coeliac disease, dermatitis herpetiformis (De Pessemier et al., Reference De Pessemier, Grine, Debaere, Maes, Paetzold and Callewaert2021). Conversely, UV-dependent cutaneous synthesis of vitamin D can influence gut microbial diversity (Bosman et al., Reference Bosman, Albert, Lui, Dutz and Vallance2019), which may affect GI tract integrity. The role of oral prebiotics and probiotics in the amelioration of some skin conditions, such as AD and acne also provide emerging evidence of a gut–skin connection (Mahmud et al., Reference Mahmud, Akter, Tamanna, Mazumder, Esti, Banerjee, Akter, Hasan, Acharjee, Hossain and Pirttilä2022).

Dysbiosis in skin conditions

While the term dysbiosis is not well defined, it is commonly used to indicate an imbalance of the microbiota correlated with either reduced beneficial strains, over-proliferation of pathogenic strains, and/or reduced overall microbiota variety. Dysbiosis of the gut has been associated with many dermatologic conditions, including acne vulgaris, AD, psoriasis, and rosacea (De Pessemier et al., Reference De Pessemier, Grine, Debaere, Maes, Paetzold and Callewaert2021). What often remains unclear is whether gut dysbiosis influences the dermatological disorder or if the dermatological disorder influences the gut dysbiosis. This cause or consequence question is difficult to solve as many patients with dermatological conditions are frequently prescribed antibiotics that can destroy and/or inhibit the growth of specific microbial groups and/or change the molecular patterns associated with the (skin) microbiome. In these cases, an additional drug factor should be considered. In severe burn patients, an acute systemic inflammatory response spreads throughout the body, and the need to implement antibiotic therapy can quickly result in microbiome dysregulation within the skin, lungs, and gut, which can influence the inflammatory response (Corcione et al., Reference Corcione, Lupia and De Rosa2020). Disruption of the intestinal barrier in burn patients leads to increased intestinal permeability and the translocation of bacteria and/or endotoxins, causing systemic inflammatory response syndrome. Research is investigating how rehabilitating the gut microbiota may be an efficient adjunct therapeutic strategy in burn patients.

Acquired skin conditions – AD

AD, often referred to as eczema, is a chronic, relapsing inflammatory skin disease characterised by barrier dysfunction, chronic inflammation, and microbial dysbiosis on the skin (De Pessemier et al., Reference De Pessemier, Grine, Debaere, Maes, Paetzold and Callewaert2021). A range of genetic and environmental factors can increase the risk of developing AD. Specifically, genetic filaggrin gene mutations can predispose individuals to a disrupted skin barrier and dry skin; however, environmental factors such as water hardness, allergens, and irritants can also play a role in the pathogenesis. The severity of AD symptoms varies; therefore, treatment ranges from mild cases necessitating topical treatments only to severe cases requiring hospital admission for wet wrapping or systemic treatments, such as biologics. Treatment often involves antibiotics, which also has implications for the gut microbiome.

Probiotic research in AD and guidelines for use: Evidence suggests that individuals displaying gut dysbiosis as infants are much more likely to develop AD (Cheung et al., Reference Cheung, Leung, Tam, Leung, Chan, Ng, Yau, Yuen, Tong, Ho, Yeung, Chen and Chan2023). Consequently, research has investigated the potential therapeutic use of oral probiotics to modulate GM and its subsequent effects on AD. Lacticaseibacillus rhamnosus GG in early infancy has been shown to reduce the incidence of AD in at-risk infants through to the age of 7 years (Wickens et al., Reference Wickens, Stanley, Mitchell, Barthow, Fitzharris, Purdie, Siebers, Black and Crane2013), but meta-analyses present a mixed picture. A significant reduction in AD severity scores was observed in those consuming probiotics compared to the control group (25 RCTs; n = 1,599). This effect was observed in children and adults with moderate-to-severe AD but not in infants. Treatment effect was improved with combinations of bacterial species or Lactobacillus alone versus Bifidobacterium (Kim et al., Reference Kim, Ah, Yu, Choi, Shin and Lee2014). In a meta-analysis of eight RCTs conducted on the use of probiotics in infants with AD (n = 741), subgroup analysis revealed that probiotics were protective in moderate-to-severe patients and in preparations containing Lactobacillus (Zhao et al., Reference Zhao, Shen and Ma2018). However, substantial heterogeneity exists with studies differing in duration, type, dosing, and timing of supplementation, diagnostic criteria, and duration of follow-up (Halken et al., Reference Halken, Muraro, de Silva, Khaleva, Angier, Arasi, Arshad, Bahnson, Beyer, Boyle, du Toit, Ebisawa, Eigenmann, Grimshaw, Hoest, Jones, Lack, Nadeau, O’Mahony and Szajewska2021), making conclusions difficult. Lc. rhamnosus, Bifidobacterium animalis subsp. lactis, and L. acidophilus probiotics in single- or multi-strain formulations appear to currently hold the most promise for prevention and treatment of AD. The difference between single- and multi-strain products is further discussed by D’Elios et al. (Reference D’Elios, Trambusti, Verduci, Ferrante, Rosati, Marseglia, Drago and Peroni2020).

Guidelines from the American Academy of Paediatrics (Thomas et al., Reference Thomas and Greer2010) and the European Academy of Allergy and Clinical Immunology (Halken et al., Reference Halken, Muraro, de Silva, Khaleva, Angier, Arasi, Arshad, Bahnson, Beyer, Boyle, du Toit, Ebisawa, Eigenmann, Grimshaw, Hoest, Jones, Lack, Nadeau, O’Mahony and Szajewska2021) do not currently contain recommendations for probiotics to prevent the development of allergic diseases due to the lack of conclusive evidence. However, the World Allergy Organization guidelines conditionally recommend the use of probiotics in pregnant women and breastfeeding women with infants at high risk of allergies and in infants at high risk of developing allergies, with the evidence cited as very low quality (Fiocchi et al., Reference Fiocchi, Pawankar, Cuello-Garcia, Ahn, Al-Hammadi, Agarwal, Beyer, Burks, Canonica, Ebisawa, Gandhi, Kamenwa, Lee, Li, Prescott, Riva, Rosenwasser, Sampson, Spigler, Terracciano and Schünemann2015). Safety of probiotics is important, and their use in patients with compromised immune function or serious underlying disease should be considered carefully and restricted to strains with proven safety and efficacy for these specific population groups (World Gastronenterology Organisation (WGO), 2023).

Practical dietary measures for AD: Dietary strategies that may offer benefits in high-risk infants include early introduction of allergens during weaning and allergen avoidance only in cases of proven allergic reactions (BDA and BSACI, 2018). Vitamin D levels have been reported to be lower in AD patients of all ages, with symptoms reporting to improve following vitamin D supplementation (Kim et al., Reference Kim, Kim, Lee, Choe and Ahn2016). For this reason, vitamin D supplementation as per guidelines for the general population (e.g., 10 μg/day for >4 years age) is recommended (National Health Service (NHS), 2023). While more specific evidence is needed, a varied diet high in fibre and omega-3 fatty acids and containing fermented foods is also thought to be beneficial in ensuring a diverse gut microbiota (Berding et al., Reference Berding, Vlckova, Marx, Schellekens, Stanton, Clarke, Jacka, Dinan and Cryan2021; Marco et al., Reference Marco, Sanders, Gänzle, Arrieta, Cotter, De Vuyst, Hill, Holzapfel, Lebeer, Merenstein, Reid, Wolfe and Hutkins2021; Vijay et al., Reference Vijay, Astbury, Le Roy, Spector and Valdes2021), and show promise in helping either prevent or alleviate allergic inflammatory conditions.

Congenital skin conditions – EB

EB refers to a group of rare inherited skin disorders that cause the skin to become very fragile, leading to painful blistering. In normal healthy skin, the three layers (dermis, basement layer, and epidermis) are held together by “velcro-like” collagen proteins. However, in EB patients, expression of these proteins is absent or patchy, depending on the particular EB type (Yerlett et al., Reference Yerlett, Petrof, Bageta, Balboa and Martinez2022). With friction or tension, skin comes away, leaving large areas of acute dissuasion, similar to burns, alongside regular episodes of inflammation, infection, itchiness, and pain. EB patients often present with high losses of fluid, plasma, and electrolytes from skin exudate, have high nutritional requirements, and often present with significant chronic micronutrient deficiencies.

Elucidation of the role of the skin and gut microbiomes in EB, particularly in reducing inflammation or the need for antibiotics, may pave the way for adjunct therapies intended to improve both skin- and gut-related symptoms as well as quality of life. However, more direct data are clearly needed. One study (n = 28) showed that patients with recessive dystrophic EB (RDEB-S; a faulty gene is inherited from both parents) have significantly reduced alpha diversity in their skin microbiome compared to healthy controls (Reimer-Taschenbrecker et al., Reference Reimer-Taschenbrecker, Künstner, Hirose, Hübner, Gewert, Ibrahim, Busch and Has2022). In particular, a predominance of Staphylococcus aureus was observed in both wounded and unwounded skin of RDEB-S patients, with higher numbers seen in those with more severe disease.

Dysbiosis has also been associated with EB patients due to frequent antibiotic use and reduced dietary intake. This may consequently result in numerous GI symptoms, including mucosal damage, constipation, protein-losing enteropathy, and bloating (Yerlett et al., Reference Yerlett, Petrof, Bageta, Balboa and Martinez2022). A small study in three children with RDEB-S supplemented with a multi-strain probiotic for 12 weeks found improved stool consistency, reducing the need for chronic macrogol gel use. All three patients reported significant improvements in abdominal pain, bloating, flatulence, and an increase in appetite (Yerlett et al., Reference Yerlett, Petrof, Bageta, Balboa and Martinez2022). As EB is a rare condition, cohort numbers are a challenge, but further research is planned to confirm the benefits of probiotics in this condition. However, improving the quality of life for these patients through alleviation of GI symptoms should not be underestimated.

How is FMT administered in practice?

Water and excess fibre is extracted from donated faecal samples, resulting in a dry mass which is composed of approximately 55% microbes (bacteria, fungi, archaea, viruses) and 24% soluble components (mucus, protein, lipids, SCFAs, colon cells). The FMT sample is homogenised and blended with saline and administered via nasogastric tube, enema, or provided in supplement form. The treatment is regulated in the United Kingdom under the guidance of the Medicines and Healthcare products Regulatory Authority for human medicine regulation. In the United States, the FDA recently approved two procedures for the treatment of recurrent Cl. difficile associated diarrhoea (Mullard, Reference Mullard2023).

Notably, as the majority of species in the microbiota are anaerobes, there can be a loss of these important microbes during FMT processing; therefore, adapted, anaerobic techniques are being developed to ensure the preservation of the natural diversity present. The human gut also contains a vast array of viruses, mostly bacteriophages (virus that infect bacteria and Archaea), which are increasingly being recognised as an important part of the gut microbiome and, as present in the transplant, warrant further investigation (Shkoporov et al., Reference Shkoporov, Clooney, Sutton, Ryan, Daly, Nolan, McDonnell, Khokhlova, Draper, Forde, Guerin, Velayudhan, Ross and Hill2019).

Donors are generally voluntary (in non-commercial organisations) and recommended to be within a certain age range and absent of chronic or metabolic diseases, as confirmed by mandatory screening of donor blood and stool samples (Cammarota et al., Reference Cammarota, Ianiro, Kelly, Mullish, Allegretti, Kassam, Putignani, Fischer, Keller, Costello, Sokol, Kump, Satokari, Kahn, Kao, Arkkila, Kuijper, Vehreschild, Pintus, Lopetuso, Masucci, Scaldaferri, Terveer, Nieuwdorp, López-Sanromán, Kupcinskas, Hart, Tilg and Gasbarrini2019). Provision of donor blood and stool samples is strongly recommended during screening for a number of criteria, including the presence of pathogens and multi-drug-resistance bacteria (Mullish et al., Reference Mullish, Quraishi, Segal, McCune, Baxter, Marsden, Moore, Colville, Bhala, Iqbal, Settle, Kontkowski, Hart, Hawkey, Williams and Goldenberg2018). One of the biggest challenges in FMT is accessing and producing sufficient quantities of donor faecal samples. Industrialising the process as a high-throughput intervention could lead to the identification of key strains and their respective effects and greater standardisation of the FMT. However, at present, commercialisation of this process is limited.

FMT is generally considered safe when rigorous screening procedures are in place but there is limited data on the long-term impact and potential risks (Merrick et al., Reference Merrick, Allen, Masirah, Zain, Forbes, Shawcross and Goldenberg2020), and for this reason, its therapeutic indications are currently limited. There have been some reported rare side effects of pathogenic organisms being transferred from donor to recipient (Food and Drink Administration (FDA), 2020) as well as reported complications regarding certain routes of administration, for example, risk of regurgitation/aspiration associated with upper GI administration of FMT (Baxter et al., Reference Baxter, Ahmad, Colville and Sheridan2015). Professional protocols are becoming increasingly more stringent to manage and prevent such issues (Mullish et al., Reference Mullish, Quraishi, Segal, McCune, Baxter, Marsden, Moore, Colville, Bhala, Iqbal, Settle, Kontkowski, Hart, Hawkey, Williams and Goldenberg2018).

Hair raising questions about mechanisms

The precise way in which FMT works remains largely unknown; however, multiple mechanisms are likely to be involved. At its simplest, it is an ecological intervention to address the loss of diversity (e.g., after antibiotic use) or to outcompete the presence of a pathogen (e.g., Cl. difficile). Besides ecological remodelling, potential mechanisms of action may include bioremediation (removal of toxins), re-establishment of gut barrier function, enteroendocrine cell function, hormone bioavailability, immune modulation, and interactions within the ENS (Hanssen et al., Reference Hanssen, de Vos and Nieuwdorp2021). FMT also contains highly biologically active substances such as SCFAs and bile acids, which may also contribute to restoring gut function and barrier health. Mullish et al. (Reference Mullish, McDonald, Pechlivanis, Allegretti, Kao, Barker, Kapila, Petrof, Joyce, Gahan, Glegola-Madejska, Williams, Holmes, Clarke, Thursz and Marchesi2019) alluded to this multiplicity of mechanisms by hypothesising that in the treatment of Cl. difficile infection, the loss of microbiota-derived bile salt hydrolases (BSHs) affects bile metabolism in the gut and that restoring BSHs could be a key mediator of FMTs efficacy in treating Cl. difficile infection. In this study, faecal samples from recovered patients demonstrated restored BSH activity and sustained changes in deoxycholic acid excretion. Whilst limited in their scientific rigour, case studies have also reported hair regrowth following FMT in two patients with alopecia universalis treated for recurrent Cl. difficile infection, which may be mediated through changes in immune function (Rebello et al., Reference Rebello, Wang, Yen, Lio and Kelly2017).

Super donors, super recipients, or best of both?

The phenomenon of “super-donors” has been proposed following the observation that the FMT of some donors results in significantly more successful outcomes than the stool of other donors (El-Salhy et al., Reference El-Salhy, Hatlebakk, Gilja, Bråthen Kristoffersen and Hausken2020). However, without a complete understanding of the mechanisms of FMT, this may not be a true phenomenon, and it has been questioned in at least some indications (Olesen and Gerardin, Reference Olesen and Gerardin2021). FMT efficacy outcomes are complicated and difficult to predict, and the recipient’s existing microbiota as well as the interaction between donor and recipients strains as well as the conditions of the FMT procedure (dose/frequency/route of administration) may also be important. Some research studies suggest that understanding the recipient’s gut microbiota and selecting a donor with a complementary microbiota or metabolic profile could be crucial (Schmidt et al., Reference Schmidt, Li, Maistrenko, Akanni, Coelho, Dolai, Fullam, Glazek, Hercog, Herrema, Jung, Kandels, Orakov, Thielemann, von Stetten, Van Rossum, Benes, Borody, de Vos, Ponsioen and Bork2022). Therefore, beyond standard donor screening, matching donors and recipients for microbiota complementarity based on differences in community, species, strains, or even function may improve colonisation success (Schmidt et al., Reference Schmidt, Li, Maistrenko, Akanni, Coelho, Dolai, Fullam, Glazek, Hercog, Herrema, Jung, Kandels, Orakov, Thielemann, von Stetten, Van Rossum, Benes, Borody, de Vos, Ponsioen and Bork2022), paving the way for more personalised selection strategies with better prediction of clinical outcomes.

FMT in clinical practice

In the United Kingdom, current clinical guidelines only recommend FMT for recurrent Cl. difficile infection that has not responded to standard therapy, but it is also actively being explored for treating other clinical conditions or as adjunct therapy. The majority of research to date has focused on the use of FMT in gut disorders and was reviewed as part of guidelines from the British Gastroenterology Society and Healthcare Infection Society (Mullish et al., Reference Mullish, Quraishi, Segal, McCune, Baxter, Marsden, Moore, Colville, Bhala, Iqbal, Settle, Kontkowski, Hart, Hawkey, Williams and Goldenberg2018). Meta-analyses have been published on the efficacy of FMT for various GI conditions (some of which are discussed later on), but there is frequently substantial heterogeneity in studies based on the type of processing (aerobic or anaerobic FMT), stool donors (single or pooled), route of administration, and use of antibiotics prior to FMT or bowel lavage. These factors, combined with significant variations in the composition of healthy faecal microbiomes make interpretation of the meta-analysis in relation to the FMT complex.

Cl-difficile infection: The National Institute of Clinical Excellence (NICE) in the United Kingdom currently recommends FMT as an option for adults who have been treated more than twice for recurrent Cl. difficile infection without success (National Institute of for Health and Care Excellence (NICE), 2022). Clinical trial evidence has shown that FMT treatment is significantly more effective than antibiotics at resolving a Cl. difficile infection and is cheaper than almost all other treatment options with antibiotics (National Institute of for Health and Care Excellence (NICE), 2022).

Inflammatory bowel disease: FMT has also been considered for the treatment of ulcerative colitis (UC), which can result in changes to the gut microbiota composition, with a loss of Clostridium cluster XIVa, butyrate producers, and an overabundance of Bacteroides, which may be corrected by FMT (Fuentes et al., Reference Fuentes, Rossen, van der Spek, Hartman, Huuskonen, Korpela, Salojärvi, Aalvink, de Vos, D’Haens, Zoetendal and Ponsioen2017). Despite significant sources of heterogeneity in the data as mentioned previously, a meta-analysis appears to favour FMT for UC, based on a reduction in inflammation (El Hage Chehade et al., Reference El Hage Chehade, Ghoneim, Shah, Chahine, Mourad, Francis, Binion, Farraye and Hashash2023). In this same meta-analysis, short-term clinical and endoscopic remission were significantly higher in patients who received FMT compared with placebo groups. In terms of Crohn’s disease, a recent meta-analysis (n = 228) showed that 57% of patients (95% CI = 49–64%) experienced some form of clinical remission following FMT, with less heterogeneity in the datasets compared to UC (Zhou et al., Reference Zhou, Cui, Zhang, Zhao and Cong2023).

Irritable bowel syndrome: Meta-analyses investigating the impact of FMT on IBS have proved more challenging to interpret, with no clear benefit or mixed results observed. However, many of these studies have not properly phenotyped the IBS subtype and there is significant heterogeneity in the same factors as discussed for other conditions, including the route of administration, which may be of particular significance in IBS. Recent work has shown that administering FMT to the upper GI (via duodenoscopy or nasojejunal tube) may be more effective than colonoscopy in IBS patients (Rokkas and Hold, Reference Rokkas and Hold2023) but more research is needed.

Overcoming drug resistance: FMT is also being explored as an adjunct to medical therapy and in exciting ways to radically change the efficacy of drugs. A relatively new class of drugs, called “checkpoint inhibitors” is used as treatment of advanced metastatic melanoma; however, drug resistance is observed in some patients. The use of FMT has been shown to change the gut microbiota and help overcome drug resistance in some patients (Davar et al., Reference Davar, Dzutsev, McCulloch, Rodrigues, Chauvin, Morrison, Deblasio, Menna, Ding, Pagliano, Zidi, Zhang, Badger, Vetizou, Cole, Fernandes, Prescott, Costa, Balaji, Morgun and Zarour2021). Thus, the potential exists to use the gut microbiota as a tool to improve the efficacy for certain drugs and to identify potential negative effects of the microbiota on drug metabolism. In parallel, the ability of probiotics to impact drug function, both positively and negatively, is a growing area of research (Merenstein et al., Reference Merenstein, Pot, Leyer, Ouwehand, Preidis, Elkins, Hill, Lewis, Shane, Zmora, Petrova, Collado, Morelli, Montoya, Szajewska, Tancredi and Sanders2023).

Feeding the FMT

Ensuring a nutritionally adequate dietary intake post-FMT treatment may play a role in sustaining the engraftment, thereby encouraging a successful and effective response following treatment. However, the optimal diet for donors and recipients in FMT still remains to be elucidated, and currently donor screening guidelines do not require a specific diet. In practice, when dietary advice is provided, promotion of government healthy eating guidelines that inherently promote higher fibre diets through emphasis on wholegrains, vegetables, and fruit appear to be most commonly applied (Clancy et al., Reference Clancy, Gunaratne and Borody2021).

Dietary fibre has been shown to be an important modulator of the gut microbiota and there have been some indications that different types of dietary fibre may be useful. An RCT in severely obese patients with metabolic syndrome receiving oral FMT combined with either high-fermentable or low-fermentable (LF) fibre supplements or placebo supplements only found that patients in the FMT-LF group alone had significant improvements in insulin resistance (HOMA2-IR) at 6 weeks compared to baseline, with no improvement in the other groups (Mocanu et al., Reference Mocanu, Zhang, Deehan, Kao, Hotte, Karmali, Birch, Samarasinghe, Walter and Madsen2021).

Beyond FMT and the gut microbiome – exploring new frontiers

Both viruses and fungi have remained unmeasured in any great detail to date, but they are important components of the gut microbiota. There are a small number of studies investigating faecal virome transplants. Patients with Cl. difficile infection who were infused with a donor FMT richer in Caudovirales were all cured by FMT, although the mechanisms are still to be defined (Zuo et al., Reference Zuo, Wong, Lam, Lui, Cheung, Tang, Ching, Chan, Chan, Wu, Chan, Yu, Sung and Ng2018). Specific fungi appear to be important in regulating mucosal and intestinal barrier health and can also be transplanted (Zhang et al., Reference Zhang, Zuo, Yeoh, Cheng, Liu, Tang, Cheung, Yang, Cheung, Mo, Hui, Chan, Li, Chan and Ng2021). However, research is in its infancy, and the significance and implications of these alternations in fungal compositions on disease outcomes warrant further investigations.

Microbiota transplants from non-GI area are also being researched. Vaginal microbiota transplants (VMTs) are currently being studied in women suffering from infertility. A single case report in a 30-year-old woman with a history of miscarriages and stillbirths who had a Lactobacillus-depleted microbiota had a VMT resulting in an increase in L. crispatus and Lactobacillus jensenii. This concurred with resolution of vaginal symptoms and a pregnancy 5 months post VMT with consequent live birth (Wrønding et al., Reference Wrønding, Vomstein, Bosma, Mortensen, Westh, Heintz, Mollerup, Petersen, Ensign, DeLong, van Hylckama Vlieg, Thomsen and Nielsen2023).