Microbiology - Research by Masters Theses

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An investigation of factors influencing early life gut microbiome in the piglet model, and potential functional food ingredients
(University College Cork, 2023) Skillington, Orla; Stanton, Catherine; Ross, R. Paul; Vistamilk; Science Foundation Ireland
Increasingly regarded as an “invisible organ”, the microbiological supersystem that is the gut microbiome, has become a central field of research in recent years. Thanks to multi-omics approaches and modern day bioinformatic techniques, characterization of the gut microbiome has advanced hugely in the last three decades, and continues to evolve. Characterization of the gut microbiome to-date, has concerned infanthood through to old-age, across a variety of species, from humans to aquatic life. Due to ethical considerations involving human-targeted therapeutics and interventions for the gut microbiome, animal models must be considered. The porcine pig model is an attractive model for putative human interventions and their associated pre-clinical trials, due to certain physiological analogies. Herein, a porcine model was developed, to mimic the early life gut microbiota and to investigate the effects of prenatal stress exposure on the seeding of this microbial consortia. Piglets and neonatal infants share an early life gut microbiota rich in Bacteroides, Escherichia, Lactobacillus and Clostridium¸ however, whilst Lactobacillus dominate the early life porcine gut, the nascent infant gut may be characterized by high abundances of bifidobacterial species. Analysis by 16s rRNA sequencing revealed that, at two weeks of age, piglets exposed to low stress levels in utero harboured higher levels of Prevotella, Fusobacterium and Bacteroides, whilst high stress exposure influenced increased levels of Lactobacillus. The effects of stress on the gut microbiome projected into later life, following weaning, whereby piglets of the low stress group were seen to have significantly higher levels of Prevotella NK3B31 and Helicobacter and piglets of the high stress group harboured higher levels of Faecalbacterium and Megasphaera.. Taxa driving microbial clustering as the piglet aged were seen to be stress-related, with high stress piglets becoming enriched in Deferribacterota, Treponema and Pyramidobacter at the post-weaning stage. Conversely, increased populations of Ruminococcus were observed in the low stress groups at two weeks of age, and remained higher than that of their counterpart post- weaning. Commonalities between the stress groups existed at two weeks, with phyla such as Campilobacterota, Bacteroidota, Actinobacteria and Firmicutes being enriched and later declining with age. At post-weaning, both groups were found to have clusterings of Cyanobacterota, Fibrobacterota, Spirochaetota and Patescibacteria. Alpha and beta diversity metrics were unaffected by prenatal stress exposure, however differed significantly with respect to age, at two weeks of age versus four weeks (post-weaning). We also investigated the putative probiotic potential of a range of indigenous African fermented food that may serve to provide human health benefits. It is postulated that the succession of the development of probiotic strains from these foods, may be suited for further investigations within infant formula matrices, to help to equilibrate the microbial consortia of a dysbiotic infant gut which had been exposed to prenatal stresses. The bacterial isolates, obtained from seventeen different food sources, on a variety of growth media, were initially subjected to a preliminary subtractive process in order to reduce the 218 isolates to a smaller number. Following exopolysaccharide production screening, bile salt hydrolase production screening, and antimicrobial activity screening, seven isolates were chosen for further analyses. These were found to be dominated by lactic acid bacteria such as Enterococcus and Lactobacillus as identified by 16s rRNA Sanger Sequencing, with all isolates displaying average growth kinetics of between 8 and 10 log cfu mL-1. Analysis by whole genome sequencing revealed Enterococcus populations to be dominated by E. faecium and E. casselflavus, whilst Lactobacillus delbrueckii and Pediococcus pentosaceus were also identified in the isolated cultures. In silico mining and comparative genomic analysis revealed the presence of antimicrobial substances such as enterolysins, enterocins and helveticins in the samples, whilst predicted secondary biosynthetic metabolites revealed the presence of polyketide synthases and antimicrobial ribosomally synthesized and post-translationally modified peptides (RiPPs) such as lanthipeptides. Subjection of the seven isolates to simulated gastric environments revealed the ability of two strains, L. delbrueckii and P. pentosaceus, to survive gastric pH conditions after 4 hours. Furthermore, both of these strains were found to be capable of growing in simulated bile conditions. Haemolytic assays revealed all strains were non-hazardous to red blood cells, and produce no signs of lysis when grown on blood agar plates. African fermented foods present a largely untapped area of unidentified strains with potentially valuable technical properties, however may also serve as vehicles for antimicrobial resistant and pathogenic strains. There lies a substantial gap in knowledge surrounding these food products and their applications, which warrant investigation to benefit both the native consumers, and the market potential of these foods.
Optimisation of growth and storage conditions of lactic streptococci
(University College Cork, 1987) Lyne, John Gerard; Daly, Charlie
The application of defined strain culture systems in the Irish Cheese Industry required detailed knowledge of culture preparation and preservation. Concentrated starters were prepared for strains of Streptococcus lactis and Streptococcus cremoris in a low lactose medium (2.5% milk solids) with external pH control. Initial studies indicated that doubling the level of yeast extract in the medium did not have a significant effect on specific growth rate or on final cell numbers. For most strains, final cell numbers at 22°C were greater than obtained in 10%.milk solids medium. Individual strains showed variable ability to maintain activity once the culture entered the stationary phase. When optimum conditions for cell harvesting had been established, individual strains were stored at refrigeration and sub-zero temperatures - both with and without cryoprotectants. Samples were assayed at various intervals for cell viability and culture activity. At -80°C most strains maintained activity and viability whether cryoprotectants were added or not. Strains which showed a decline in viability were best protected by glycerol with lactose being slightly less effective. At -20°C, the spent ferementation medium was not able to protect the strains from freezing or from frozen storage. Some strains lost activity without showing a decline in viability. With others, activity decreased at a rate corresponding to the loss in viability. For the former strains, subculturing at 21°C did not restore full activity but the latter cultures were able to regain full activity on sub-culture. The addition of glycerol and lactose prior to freezing aided in cell protection as did freezing at low temperature. However, the degree of protection was strain dependent. Some strains retained activity at refrigeration temperatures for up to 73 days, with all strains remaining active at 4°C for at least 14 days. One S. cremoris strain did not grow well under pHcontrol. A bacteriophage was detected in the culture, initially at low levels. The phage propagated and eventually lysed the culture. This occurred in milk based media which were pH controlled between pH values 6.0 and 7.3. Growth under pH controlled conditions at pH 5.5 did not cause phage release even though in separate experiments the phage was able to multiply at this pH. Inducing agents, such as U.V. and mitomycin-C failed to release lytic phage, nor were phage detected in broth or in phage-inhibitory media. Single strain isolates also exhibited phage release, even though these isolates differed from the parent strain in phenotype and phage typing. With the exception of one S. cremoris strain, conditions of culture growth, harvesting and storage were established. These have proved useful for the growth of lactic streptococci for commercial cheese making.
Potential of bacteriocin producing bacteria to control VRE infection
(University College Cork, 2021-01-08) Wiggins Savage, Kelsie; Ross, R. Paul; Hill, Colin
Antimicrobial resistance has been a growing and concerning problem over the last decade. It is estimated that antibiotic resistant pathogens cause up to 700,000 deaths each year. The Centre for Disease Control ranks vancomycin resistant enterococci as a serious threat. They estimate that in 2017 alone there were at least 54,500 cases in hospitalized patients. The rise of antibiotic resistance globally has led to a pressing need to develop therapeutic alternatives. Bacteriocins, small ribosomally synthesised peptides, have been proposed as one such alternative. Bacteriocins are non-toxic to producer strains and have been shown to exhibit antimicrobial ability against multi-drug resistant species including VRE and MRSA. This makes them excellent candidates as a substitute to antibiotics. In Chapter 1 the mechanism behind vancomycin resistance in Enterococcus faecium and Enterococcus faecalis is discussed. Also discussed are the chemotherapeutic treatments currently in use in treatment of VRE infections. Finally, a number of novel alternatives to antibiotics such as bacteriocins and bacteriophage and their efficiency are evaluated. In Chapter 2, the ability of Avicin, a Class II bacteriocin to inhibit growth of vancomycin resistant enterococci is investigated. Avicin was found to inhibit all six VRE strains in this experiment. One of these strains is a multi-drug resistant E. faecium APC1031 which is highly resistant to vancomycin (MIC 62μg/mL). MIC (Minimum inhibitory concentration the lowest concentration of an antibiotic that will prevent visible growth of bacteria. E. faecium APC1031 is sensitive to Avicin (MIC 0.9μg/mL). It was found that Avicin initially has a dramatic impact on cfu/mL of an established culture of APC1031. Addition of concentrations as low as 2μg/mL to a culture resulted in a 9 log decrease in cfu/mL after a two hour period but a number of cells did survive. The phenotype of these cells was assessed, and they exhibited a high level of resistance to Avicin (MIC 45-90μg/mL). More importantly these colonies exhibited decreased resistance to vancomycin (from 62μg/mL to 0.9μg/mL). This phenotypic change post-treatment with Avicin remained consistent when varying concentrations of Avicin were used. A combination of both Avicin and vancomycin resulted in an 8 log reduction of cfu of 108 cfu/mL to less than 3 cells surviving after 48 hours of incubation. Finally, whole genome sequencing of the Avicin resistant “mutants” revealed SNP’s in both the vanR region and the manZ region. VanR is a part of the two component regulatory system (vanS-vanR) which confers resistance to the glycopeptide, vancomycin. The results in this thesis highlight the potential of Avicin to be used in a combination with vancomycin for the treatment of VRE infections – VRE are killed by Avicin and where Avicin resistance occurs the resulting cells would revert to vancomycin sensitivity and consequently be killed by vancomycin.
Laboratory scale aerobic bioreactor conditioning of dairy processing wastewater as feedstock for Lemna minor production
(University College Cork, 2019) Broughton, Róisín; O'Leary, Niall
Dairy processing wastewater is a nutrient-rich resource, containing chemical oxygen demand (COD) ranging from 1,150 – 68,814 mg/L, nitrogen (N) from 14 – 1,462 mg/L and phosphorus (P) from 7.2 – 650 mg/L, depending on the product stream. This resource has potential to be utilised for cultivation of Lemna minor as a value-added product, in line with EU circular economy principles. The purpose of this project was to condition dairy processing wastewater for downstream application as growth media for Lemna minor. Effluent from both systems were tested for nutrient removal, and IASBR effluent was more suitable for the coupled system, with removal efficiencies of >90% COD, 4 – 94% NH4+ and 30 – 80% P respectively. The coupled system yielded poor nutrient removal and little to no plant growth, with plants reaching senescence by day 28 of the trial. It can be concluded that Lemna minor cultivation failed as a result of both A/O and IASBR operational issues (including starting pH) and insufficient seeding of cultivation tanks. It is therefore recommended to focus future studies on the suitability of anaerobic treatment technologies for conditioning of dairy processing wastewater for Lemna minor cultivation. Future work with coupled wastewater treatment/duckweed cultivation systems should also involve investigation into plant-symbiont interactions, as well as profiling of wastewater microbial communities and their potential impacts on duckweed growth.
Natural transformation in lactic acid bacteria
(University College Cork, 2020-10-16) O'Connell, Laura Michelle; van Sinderen, Douwe; Mahony, Jennifer; Science Foundation Ireland
Fermentation has been commercially exploited to produce food products for millennia. The most intensely exploited microorganisms used in modern industry to commercially produce fermented products are yeasts and lactic acid bacteria (LAB). LAB are most widely employed in dairy processing, where they contribute to the biochemical changes that convert milk to cheese and yoghurt. To ensure consistency, large-scale, industrial fermentations use starter cultures of LAB, meaning a known strain or a sometimes-undefined combination of multiple strains with well-defined characteristics. Hypothetically, through natural genetic modification, which exploits DNA transfer systems occurring in nature, desirable traits could be introduced to strains to improve the efficacy of the starter culture. The focus of this thesis is natural competence. Competence refers to the specialised physiological state in which bacteria are able to undergo transformation through the internalisation of exogenous DNA. Recently an early com system was described for the industrially relevant LAB, Streptococcus thermophilus and a late com system homologous to that of S. pneumoniae was described in another LAB widely employed in cheese production, Lactococcus lactis. This thesis aimed to explore the functionality of the proven and predicted natural com systems encoded these LAB. In the case of S. thermophilus, this was achieved by defining the nutritional requirements of competence development, which involved defining a minimal cultivation medium. This medium was used in competence assays involving a model strain, S. thermophilus LMD-9, to confirm the functionality of the competence system in the minimal medium. The optimised medium and assay were then applied to a panel of industrial strains to determine the transferability of the transformation protocol. While it was observed that the minimal medium could support the growth of the industrially relevant strains, no transformation was detected in any industrial strains. A different approach was taken to study competence in L. lactis, as the system is far less defined compared to that of S. thermophilus. For this reason, an investigation of the gene activity of the lactococcal competence components was conducted, as opposed to competence assays. Specifically, the activity of the com-promoters was assessed via blue/white selection, using a promoter-probe vector constructed in this study. Several conditions were trialled to identify those optimal for competence development. This included the nisin-induction of the master competence regulator, ComX, which was the only condition that proved capable of activating the com-promoters. Chemical mutagenesis was employed in an attempt to generate random mutants that did not require comX-induction; however, this method did not prove effective, and no such mutants were isolated and the mechanism of lactococcal competence remains elusive.

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