Exploiting the bioactive potential of marine sponge microbiota

Endospore-forming bacteria are often isolated from different marine sponges, but their abundance varies, and they are frequently missed by culture-independent studies. Within endospore-formers, Bacillus are renowned for the production of antimicrobials and other compounds of medical and industrial importance. Although this group has been well studied in many different environments, very little is known about the actual diversity and properties of sporeformers associated with marine sponges. Identification of the endospore-forming bacteria associated with the marine sponges; Haliclona simulans, Amphilectus fucorum and Cliona celata, has uncovered an abundant and diverse microbial population composed of Bacillus, Paenibacillus, Solibacillus, Halobacillus and Viridibacillus species. This diversity appears to be overlooked by other non-targeted approaches where spore-formers are masked by more dominant species within the ecosystem. In addition to the identification of two antibiotic resistant plasmids, this bank of sporeformers produce a range of bioactive compounds. New antimicrobial compounds are urgently needed to combat the spread of multidrug resistant pathogens, as few new options are entering the drug discovery pipelines for clinical trials. Based on the results of this project, endospore-formers associated with marine sponges may hold the answer. The power of coupling functional based assays with genomic approaches has enabled us to identify a novel class 1 lantibiotic, subtilomycin, which is active against several clinically relevant pathogens. Subtilomycin is encoded in the genomes of all the marine sponge B. subtilis isolates analysed. They cluster together phylogenetically and form a distinct group from other sequenced B. subtilis strains. Regardless of its potential clinical relevance, subtilomycin may be providing these strains with a specific competitive advantage(s) within the stringent confines of the marine sponge environment. This work has outlined the industrial and biotechnological potential of marine sponge endospore-formers which appear to produce a cocktail of bioactive compounds. Genome sequencing of specific marine sponge isolates highlighted the importance of mining extreme environments and habitats for new lead compounds with potential therapeutic applications.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms