The metabolomic characterisation of probiotic microbial communities

Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder, with an increasing prevalence in the western world. Despite this, there is no cure for the condition, with both diagnosis and treatment being based on the symptoms. In attrition, treatments typically include lifestyle changes and medication, which may be difficult for the patient to adjust to and may possess side effects. Probiotic supplements have been postulated as promising treatment, which may combat some of the symptoms and also treat some of the potential causes (including dysbiosis and inflammation. A formulation combining probiotic strains PD01 and PD12 was developed by an industrial partner, MRM Health. Bacillus cibi strain PD01, isolated during the Colorspore project, was a modulator of the Short Chain Fatty Acid (SCFA) butyrate. SCFA are organic acids produced solely by gastrointestinal bacteria. They fulfil a range of roles in the gut, including an energy source for intestinal epithelial cells, reduction of inflammation and maintenance of the gut barrier. In addition, PD01 produced 4,4’-diapocarotenoid derivatives which are novel compounds potentially possessing potent antioxidant activity which may aid in gut health, such as reducing inflammation. Strain PD12 was supplied by MRM Health, and was a high butyrate producer, hence the probiotic activity, and PD01 positively modulates PD12 probiotic activity.
Bacterial co-cultures and IBS intervention studies in animal models were used to study the interaction between these strains and the host microbiota at metabolite level, focusing on the SCFA production in the caecum. Targeted profiling (via GC-MS and SPME-GC-MS) and untargeted metabolomics (via LC-MS) was undertaken to determine differences between the PD01/PD12 and two other treatments on non-stressed and stressed animal models, via sampling of both the caecal content and the serum. These other treatments consisted of a placebo as well as a second probiotic combination from MH002, consisting of six different strains intended for use against Ulcerative Colitis (UC) which may have analogous against IBS within these animal models. The direct effects of PD01 on other probiotic strains (including PD12 and Lactiparacasei bacillus parapacasei Shirota strain) were determined, including via the metabolomics platforms described previously as well as with Optical Density (OD) growth curves. Due to the novel nature of the 4,4’-diapocarotenoids, chemical mutagenesis was attempted to produce mutants possessing altered 4,4’-diapocarotenoid profiles.
Targeted profiling showed overall significant increases in the production of butyrate in the PD01/PD12 supplementation vs the two other treatments, with differing effects on both the serum and caecum. The key finding was that the PD01/PD12 supplement was acting to help stabilise the gut metabolome in response to stress, which can exacerbate IBS.
Co-cultivation analysis of PD01 with both PD12 and Lactiparacasei bacillus paraparacasei Shirota strain have indicated a range of significant differences when compared to monoculture samples, as well as significant changes with the profiles of butyrate, propionate and acetate, with a greater effect on PD12 than with L. paracasei Shirota. In addition, PD01 showed a positive influence on the metabolism and growth of the co-cultivated strains, with the specific combination of PD01 and PD12 having the greatest synergistic effect. This was likely due to the specific mechanism utilised by PD12 for the production of SCFAs compared to L. paracasei Shirota.
Chemical mutagenesis yielded three viable mutants, two of which produced the methyl 4-glycosyl-diapo-4,4’-lycopenoate esters, and the third which produced 4,4’-diapophytoene, a squalene analogue which may be a sustainable alternative for this compound, in products such as vaccines and cosmetics.
Key future work will include further analysis on animal models and on PD01 via co-cultivation, with other probiotic and pathogenic strains to further determine whether PD01 has a synergistic effect on other beneficial strains as well as reducing the prevalence on pathogenic species. These experiments will also include flux analysis, which will determine any changes in the rates of consumption and production key compounds, such as SCFAs and 4,4’-diapocarotenoids. Further mutagenesis will be attempted to create additional strains with desirable characteristics.
Nonetheless, these results indicate that PD01 may be a viable treatment to help mitigate some IBS symptoms in combination with strains such as PD12, as well as a source of novel compounds possessing comparable or superior antioxidant activity compared to potent carotenoid equivalents, as well as potentially exhibiting immunoregulatory function.