Management of pathogenic bacteria with Bacillus probiotics

Infections caused by resistant microorganisms impose a significant threat to human health. Developments in our society, resulting from a growing and migrating population of humans and animals, antibiotics, which are overused and misused, and preventive measures that are insufficient, cause microorganisms to change, why they become resistant. Antibiotic resistant microorganisms are responsible for a wide range of health complications in the community and healthcare settings, but infections caused by bacterial pathogens, such as Staphylococcus aureus (S. aureus) and Clostridioides difficile (C. difficile) are associated with an increased risk of hospitalisation and death. Antimicrobial resistance (AMR) imposes a noticeable challenge since standard of care and surgical actions can fail, which will increase the morbidity and mortality for patients and the financial burden for healthcare facilities. If this problem is not addressed either by regulating the use of antibiotics or implementing alternative solutions, AMR will cause the annual loss of 10 million human in 2050. The use of probiotic bacteria, such as the members of the Bacillus subtilis (B. subtilis) group (BSG) can contribute to limit the problem by controlling the growth of pathogens through various mechanisms, where they reduce available nutrients, produce inhibiting compounds, or active host-defences.

This thesis aimed to improve the understanding of how the members of the BSG could be used to limit and
control resistant pathogens in humans. Firstly, selected methods that were used to study the probiotic
properties of the BSG members were presented before addressing their biological and technical challenges
(Chapter I). Hereafter, we investigated how the diversity of 115 unique BSG members, which was reflected by their phylogenetic relationship and potential to produce secondary metabolites influenced the ability of the strains to inhibit human pathogens. Initially, the growth-limiting potential of the strains was established for a panel of pathogenic microorganisms as was their metabolome with liquid chromatography and mass spectrometry (LC-MS). Additionally, complete genomes were generated to facilitate bioinformatic analysis. To identify the bioactive metabolites that were predictive for the control of S. aureus, a random forest (RF) model was applied to the inhibition profiles and LC-MS data, which found that growth-limiting compounds, quorum-sensing molecules, and uncharacterised metabolites were among the most important features (Chapter II, Manuscript I). Next, we investigated the protective properties of the BSG members in the context of the chronic, inflammatory, skin condition atopic dermatitis (AD), where S. aureus colonisation contribute to exacerbation of clinical manifestations. Our findings showed that Bacillus strains were able to protect an in vitro skin layer against damage included by staphylococcal protease. Attempts to understand the underlying mechanisms revealed that the probiotic bacteria increased the rate of wound healing and activated an immune response (Chapter III, Manuscript II). Thereafter, we explored the ability of Bacillus velezensis (B. velezensis) DSM 33864 to selective manage C. difficile in the context of antibiotic-induced dysbiosis of the gut microbiota in both in vitro and in vivo settings. Analysis revealed that supplementation with B. velezensis DSM 33864s controlled the growth of C. difficile without changing the composition of the microbiota, delaying the reestablishment of the gut community, or causing adverse effects (Chapter IV, Manuscript III). Lastly, I presented my conclusions on the findings of this thesis and proposed, where efforts could be made to build on these results and further advance the field of probiotic research (Chapter V). The findings presented here show that the members of the BSG hold great potential for application to limit and control infections and provide an alternative solution that could be used to address the challenges imposed by AMR.