Antimicrobial activities and diversity of sponge derived microbes

In this study, marine sponges collected in Irish waters were analysed for their associated microbiota. Of the approximately 240 bacterial isolates obtained from two sponges several showed antimicrobial activity; among them members of genera which have rarely been shown to produce antimicrobial compounds. Differences observed from the sponge-derived groups of isolates in terms of bioactivity suggests that S. carnosus isolates may be a better source of antibacterial compounds, while Leucosolenia sp. isolates appear to be a better source of antifungal compounds. More than 60% of fungal isolates obtained from 12 sponge samples proved to be bioactive. One of the isolates, which was closely related to Fusarium oxysporum and showed activity against bacteria and fungi, was investigated for its secondary metabolite genes. At least 5 different NRPS genes, with a sequence similarity as low as 50 % to known genes, were identified highlighting the likelihood that this isolate may be capable of producing novel secondary metabolites. A Micromonospora sp. was isolated from a Haliclona simulans sample collected in Irish waters. The isolate inhibited the growth of Gram positive bacterial test strains in three different antimicrobial assays. Employing preparative layer chromatography the compound responsible for the bioactivity could be isolated. According to LC-MS andNMR data the bioactive compound could indeed be novel. Finally, two deep water sponges were shown to host a remarkably different bacterial and archaeal diversity by application of 454 Pyrosequencing. The L. diversichela –proteobacterial community was dominated by a single ƴ-proteobacterial bacterium whereas the S. normani sample hosted a largely sponge specific microbial community, even more diverse than has been previously reported for shallow water sponges. Organisms potentially involved in nitrification, sulphate reduction and secondary metabolite production were found to be spatially distributed in the sponge. Furthermore, a deep sea specific population was implied.

Nutraceutical and functional food bioactive peptides in beef

In recent years, the potential to positively modulate human health through dietary approaches has received considerable attention. Bioactive peptides which are released during the hydrolysis or fermentation of food proteins or following digestion may exert beneficial physiological effects in vivo. The aim of this work was to isolate, characterise and evaluate Angiotensin-І-converting enzyme (ACE-І) inhibitory, antimicrobial and antioxidant peptides from the bovine myofibrillar proteins actin and myosin. In order to generate these peptides, the myofibrillar proteins actin and myosin were hydrolysed with digestive enzymes pepsin, trypsin and α-chymotrypsin, or with the industrial thermolysin-like enzyme “Thermoase”, Amano Inc. It was found that each hydrolysate generated contained peptides which possessed ACE inhibitory, antioxidant and antimicrobial activity. The peptides responsible in part for the observed ACE inhibitory, antioxidant and antimicrobial activity of a number of hydrolysates were isolated using the method of RP-HPLC and the bioactive peptides contained within each active fraction was determined using either MALDI-TOF MS/MS or N-terminal peptide sequencing. During the course of this thesis six ACE inhibitory and five antimicrobial peptides were identified. It was determined that the reported antioxidant activity was a direct result of a number of peptides working in synergy with each other. The IC50 values of the six ACE inhibitory peptides ranged in values of 6.85 to 75.7 µM which compare favourably to values previously reported for other food derived ACE inhibitory peptides, particularly the well known milk peptides IPP and VPP, IC50 values of 5 and 9 µM respectively. All five antimicrobial peptides identified in this thesis displayed activity against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Listeria innocua with MIC values ranging from 0.625 to10 mM. The activity of each antimicrobial peptide was strain specific. Furthermore the role and importance of charged amino acids to the activity of antimicrobial peptides was also determined. Generally the removal of charged amino acids from the sequence of antimicrobial peptides resulted in a loss of antimicrobial activity. In conclusion, this thesis revealed that a range of bioactive peptides exhibiting ACE inhibitory, antioxidant and antimicrobial activities were encrypted in bovine myofibrillar proteins that could be released using digestive and industrial enzymes. Finally enzymatic hydrolysates of muscle proteins could potentially be incorporated into functional foods; however, the potential health benefits would need to be proven in human clinical studies.

Characterisation of the microbiota of traditional fermented beverages and screening these and other populations for novel antimicrobial producers and gene clusters

To screen for novel ribosomally synthesised antimicrobials, in-silico genome mining was performed on all publically available fully sequenced bacterial genomes. 49 novel type 1 lantibiotic clusters were identified from a number of species, genera and phyla not usually associated with lantibiotic production, and indicates high prevalence. A crucial step towards the commercialisation of fermented beverages is the characterisation of the microbial content. To achieve this goal, we applied next-generation sequencing techniques to analyse the bacterial and yeast populations of the organic, symbiotically-fermented beverages kefir, water kefir and kombucha. A number of minor components were revealed, many of which had not previously been associated with these beverages. The dominant microorganism in each of the water kefir grains and fermentates was Zymomonas, an ethanol-producing bacterium that had not previously been detected on such a scale. These studies represent the most accurate description of these populations to date, and should aid in future starter design and in determining which species are responsible for specific attributes of the beverages. Finally, high-throughput robotics was applied to screen for the presence of antimicrobial producers associated with these beverages. This revealed a low frequency of bacteriocin production amongst the bacterial isolates, with only lactococcins A, B and LcnN of lactococcin M being identified. However, a proteinaceous antimicrobial produced by the yeast Dekkera bruxellensis, isolated from kombucha, was found to be active against Lactobacillus bulgaricus. This peptide was patially purified.

Fundamental studies on the application of enzymes when brewing with unmalted oats and sorghum

The use of unmalted oats or sorghum in brewing has great potential for creating new beer types/flavors and saving costs. However, the substitution of barley malt with oat or sorghum adjunct is not only innovative but also challenging due to their specific grain characteristics. The overall objectives of this Ph.D. project were: 1) to investigate the impact of various types and levels of oats or sorghum on the quality/processability of mashes, worts, and beers; 2) to provide solutions as regards the application of industrial enzymes to overcome potential brewing problems. For these purposes, a highly precise rheological method using a controlled stress rheometer was developed and successfully applied as a tool for optimizing enzyme additions and process parameters. Further, eight different oat cultivars were compared in terms of their suitability as brewing adjuncts and two very promising types identified. In another study, the limitations of barley malt enzymes and the benefits of the application of industrial enzymes in high-gravity brewing with oats were determined. It is recommended to add enzymes to high-gravity mashes when substituting 30% or more barley malt with oats in order to prevent filtration and fermentation problems. Pilot-scale brewing trials using 10–40% unmalted oats revealed that the sensory quality of oat beers improved with increasing adjunct level. In addition, commercially available oat and sorghum flours were implemented into brewing. The use of up to 70% oat flour and 50% sorghum flour, respectively, is not only technically feasible but also economically beneficial. In a further study on sorghum was demonstrated that the optimization of industrial mashing enzymes has great potential for reducing beer production costs. A comparison of the brewing performance of red Italian and white Nigerian sorghum clearly showed that European grown sorghum is suitable for brewing purposes; 40% red sorghum beers were even found to be very low in gluten.

Antimicrobial stewardship in Ireland, with a focus on long term care facilities

Background: Antimicrobial resistance is a major public health concern, and its increasing incidence in the Long Term Care Facility (LTCF) setting warrants attention (1). The prescribing of antimicrobials in this setting is often inappropriate and higher in Ireland than the European average (2). The aim of the study was to generate an evidence base for the factors influencing antimicrobial prescribing in LTCFs and to investigate Antimicrobial Stewardship (AMS) strategies for LTCFs. Methods: An initial qualitative study was conducted to determine the factors influencing antimicrobial prescribing in Irish LTCFs. This allowed for the informed implementation of an AMS feasibility study in LTCFs in the greater Cork region. Hospital AMS was also investigated by means of a national survey. A study of LTCF urine sample antimicrobial resistance rates was conducted in order to collate information for incorporation into future LTCF AMS initiatives. Results: The qualitative interviews determined that there are a multitude of factors, unique to the LTCF setting, which influence antimicrobial prescribing. There was a positive response from the doctors and nurses involved in the feasibility study as they welcomed the opportunity to engage with AMS and audit and feedback activities. While the results did not indicate a significant change in antimicrobial prescribing over the study period, important trends and patterns of use were detected. The antimicrobial susceptibility of LTCF urine samples compared to GPs samples found that there was a higher level of antimicrobial resistance in LTCFs. Conclusion: This study has made an important contribution to the development of AMS in LTCFs. The complexity of care and healthcare organisation, and the factors unique to LTCFs must be borne in mind when developing quality improvement strategies.