Microbiology - Research by Masters Theses

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Development of a synergistic synbiotic containing arabinoxylan and Bifidobacterium longum using in vivo selection
(University College Cork, 2024) Jones, Evan; Walter, Jens; van Sinderen, Douwe; University College Cork; Synbiotic Health
Colonisation and metabolic activity of orally ingested bacteria in the colon rely on competitive ecological and niche-based factors that often limit the functionality of commonly used probiotics. Synergistic synbiotics, which involve the parallel administration of a microorganism with its cognate substrate, have the potential to improve persistence and ecological performance of putative probiotic microbes. However, real synergism has not yet been established for synbiotics in human trials, and most synbiotic combinations have not been designed using an approach that accounts for the ecological constraints of the GI tract. Here we use in vivo selection (IVS) to identify strains of Bifidobacterium longum that are adapted toward the utilization of arabinoxylan (AX) in the human gut. To achieve this, bifidobacteria were quantitatively cultured from fecal samples collected during a human trial which showed that a high dose of corn bran AX leads to a significant but highly individualised increase of B. longum. Isolates were randomly picked and genotyped by a rapid, high throughput gyrB sequencing method that was developed for this project. Bacterial counts and strain composition were compared between baseline and week 6, and B. longum strains enriched in vivo were then tested through in vitro fermentations to investigate their growth on AX and its constituents. These monoculture experiments confirmed the ability of representative isolates to use free arabinose, xylo-oligosaccharides (XOS) and the complete corn AX fibre, which suggests that these B. longum strains are primary AX degraders. Viable cell counts revealed a high level of consistency in growth patterns among the fecal isolates compared to reference strains on AX. Whole genome sequencing (WGS) of selected strains followed by comparative genomic analysis revealed an enrichment of relevant glycoside hydrolase family 43 (GH43) genes and the presence of three specific carbohydrate utilisation clusters associated with xylan and AX metabolism in a number of in vivo-selected isolates which was not observed in reference strains. Finally, gas production experiments helped to further characterise the fermentation profiles of the AX-degrading isolates and highlighted their capacity to facilitate cross-feeding with other members of the microbiota. This study demonstrates the value of an ecologically relevant process for selecting improved synbiotic combinations, with the B. longum strains identified here representing promising candidates based on their predicted ecological performance in vivo.
The impact of bile and anaerobic stress on Listeria monocytogenes isolates
(University College Cork, 2023) Lynch, Mary Jane; Gahan, Cormac G.; Hill, Colin
Listeria monocytogenes is regarded as an important food pathogen that can cause serious illness. All individuals are susceptible to L. monocytogenes infection with pregnant women, immunocompromised people and the elderly particularly at high risk. L. monocytogenes infection can cause febrile gastroenteritis and a more serious invasive form where the bacteria cross the blood/brain barrier or the blood/foetal barrier. In this study the effect of bile and anaerobic stress on L. monocytogenes was assessed. Bile has antimicrobial properties and it is one of the stressors that L. monocytogenes must overcome in the human gastrointestinal tract to establish infection. L. monocytogenes uses an enzyme, bile salt hydrolase (BSH), to detoxify bile. The bile resistance response may be triggered by anaerobic conditions. In total 154 food/environmental isolates and 74 clinical isolates were screened. A bile tolerance assay and a BSH enzymatic agar plate assay were developed. Experiments were carried out at a lowered pH and/or anaerobic conditions to simulate conditions in the human gastrointestinal tract where L. monocytogenes is exposed to bile. Results indicate that there are bile tolerance variations in L. monocytogenes strains from food, environmental and clinical sources. In this large-scale study, cultures grown anaerobically were less tolerant to bile than cultures grown aerobically and generated more varied resistance patterns. The effect of anaerobiosis is not as well understood as aerobic and results provide an insight into the influence of environmental factors. Interestingly, the bile resistance assay revealed that the clinical isolates were significantly more tolerant to bile than the food and environmental isolates (P < 0.001). This important finding demonstrates the evolutionary pressure on these strains to survive in the gut. Correlation of results from the BSH agar plate assay with the bile tolerance assay demonstrated that the food and environmental isolates had slightly higher levels of BSH activity than the clinical isolates. However, the difference between the groups was not as significant as the bile resistance results. Results were also analysed on the level of lineage and clonal complex to identify possible links to the bile response. The results generated from this study provide insight into important associations between bile tolerance of individual strains/lineages and efficacy of L. monocytogenes infection.
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.

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