Microbiology Commons^™

Development Of A Novel, High Resolution Melting Analysis Based Genotyping Method For Cryptosporidium Parvum, Jennifer K. O'Leary, Liam Blake, Gerard D. Corcoran, Roy D. Sleator, Brigid Lucey

Department of Biological Sciences Publications

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three …

A Novel Genotyping Method For Cryptosporidium Hominis, Jennifer K. O'Leary, Liam Blake, Gerard D. Corcoran, Roy D. Sleator, Brigid Lucey

Department of Biological Sciences Publications

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominis gp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between …

Japanese Quail (Coturnix Japonica) As A Novel Model To Study The Relationship Between The Avian Microbiome And Microbial Endocrinology-Based Host-Microbe Interactions, James Keane, Joshua M. Lyte, Julia Eckenberger, Nicholas Anthony, Sandip Shrestha, Daya Marasini, Karrie M. Daniels, Valentina Caputi, Annie M. Donoghue, Mark Lyte

Articles

Background

Microbial endocrinology, which is the study of neuroendocrine-based interkingdom signaling, provides a causal mechanistic framework for understanding the bi-directional crosstalk between the host and microbiome, especially as regards the effect of stress on health and disease. The importance of the cecal microbiome in avian health is well-recognized, yet little is understood regarding the mechanisms underpinning the avian host-microbiome relationship. Neuroendocrine plasticity of avian tissues that are focal points of host-microbiome interaction, such as the gut and lung, has likewise received limited attention. Avian in vivo models that enable the study of the neuroendocrine dynamic between host and microbiome are …

Predicting, Monitoring And Controlling The Growth Of Listeria In Complex Food Matrices, Laura Mary Nyhan

Theses

Listeria monocytogenes is a foodborne pathogen which is a significant challenge in food production, particularly for ready-to-eat (RTE) products. Incidence of Listeria in RTE foods can be reduced by the application of intelligent combinations of preservative factors or hurdles, while data quantifying the efficacy of hurdle combinations can be exploited and used in the area of predictive microbiology. Traditional culture-based techniques, such as viable plate counts, are commonly used to monitor the growth/survival of microorganisms in foods, however these methods are tedious, time-consuming and due to their destructive nature, are associated with low reproducibility and high variability. Therefore, rapid, non-invasive …

Comparison Of The Effects Of Land Use / Land Cover On Water Quality And The Intertidal Marine Macroalgae Communities At The Fluvial Outflows Of The Dingle Peninsula, Cassandra Lew

Theses

The Milltown, Feohanagh, and Owenmore river basins located on the Dingle Peninsula in Ireland were studied to see how variation in land use and land cover (LU/LC) affected water quality and the macroalgal communities at each river’s respective fluvial outflow. Abundance and composition of macroalgae in river and coastal water bodies have been designated as an indicator of water quality according to the Water Framework Directive 2000. The results of this study found that the Feohanagh’s macroalgal community had the highest species richness for most months while the Milltown had the highest Shannon Diversity Index. The Owenmore’s community was consistently …

Development Of Novel Methodology For The Molecular Differentiation Of Cryptosporidium Parvum Gp60 Subtypes Via High Resolution Melting Analysis, Jennifer K. O'Leary, Liam Blake, Gerard D. Corcoran, Roy D. Sleator, Brigid Lucey

Department of Biological Sciences Publications

Cryptosporidium species subtypes are generally identified via DNA sequencing of the gp60 gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products, gp60 is subject to host selective pressures, genetic recombination and evolutionary processes that drive extensive polymorphism at this locus. The elucidation of the polymorphic nature of this gene has led to the current mainstay in Cryptosporidium subtyping nomenclature.

This study aimed to develop a real-time polymerase chain reaction based method utilising a post-PCR application, high resolution melting (HRM) analysis, in conjunction with the abovementioned gp60 nomenclature system, in order to differentiate between Cryptosporidium parvum …

Covid-19: In The Absence Of Vaccination – ‘Mask-The-Nation’, Roy D. Sleator, Steven Darby, Alan Giltinan, Niall Smith

Department of Biological Sciences Publications

“In the absence of a vaccine, or effective antiviral, one of our only remaining strategies for controlling COVID-19 is to physically block the spread of SARS-CoV-2 in the community”

Validation Of A Lysis Buffer Containing 4 M Guanidinium Thiocyanate (Gitc)/ Triton X-100 For Extraction Of Sars-Cov-2 Rna For Covid-19 Testing: Comparison Of Formulated Lysis Buffers Containing 4 To 6 M Gitc, Roche External Lysis Buffer And Qiagen Rtl Lysis Buffer, Martina F. Scallan, Catherine Dempsey, John Mcsharry, Isabelle O'Callaghan, Paula M. O'Connor, Conor P. Horgan, Edel Durack, Paul D. Cotter, Sarah Hudson, Humphrey A. Moynihan, Brigid Lucey

Dept. of Biological Sciences Preprints

The COVID-19 pandemic has resulted in increased need for diagnostic testing using reverse transcriptase real-time PCR (RT-PCR). An exponential increase in demand has resulted in a shortage of numerous reagents in particular those associated with the lysis buffer required to extract the viral RNA. Herein, we describe a rapid collective effort by hospital laboratory scientists, academic researchers and the biopharma industry to generate a validated lysis buffer. We have formulated a 4M Guanidinium thiocyanate (GITC)/ Triton X-100 Lysis buffer which provides comparable results with the recommended reagents. This buffer will ease the burden on hospital labs in their heroic efforts …

Group B Streptococcus: Molecular Epidemiology, Pathogenic Profiling And Control Strategies, Katherine Mary Hayes

Theses

Group B Streptococcus (GBS) are frequent colonisers of the gastrointestinal tract of healthy adults with worldwide rates of 18% colonisation; however, they are also opportunistic pathogens capable of causing invasive disease, particularly in neonates and adults with underlying conditions. Indeed, GBS is the leading cause of invasive neonatal disease worldwide with a global incidence of 0.49 per 1000 live births and GBS disease in adults is increasing globally, with estimated rates of 10.9 cases per 100,000 people. While GBS remain largely susceptible to penicillin, there have been reports of reduced beta-lactam susceptibility in certain countries and resistance to other antibiotic …

A Rapid Viability And Drug‑Susceptibility Assay Utilizing Mycobacteriophage As An Indicator Of Drug Susceptibilities Of Anti‑Tb Drugs Against Mycobacterium Smegmatis Mc2 155, Gillian Catherine Crowley, Jim O'Mahony, Aidan Coffey, Riona G. Sayers, Paul D. Cotter

Department of Biological Sciences Publications

Background: A rapid in-house TM4 mycobacteriophage-based assay, to identify multidrug resistance against various anti-tuberculosis drugs, using the fast-growing Mycobacterium smegmatis mc² 155 in a microtiter plate format was evaluated, based on phage viability assays. Methods: A variety of parameters were optimized before the study including the minimum incubation time for the drugs, phage and M. smegmatis mc² 155 to be in contact. An increase in phage numbers over 2 h was indicative that M. smegmatis mc² 155 is resistant to the drugs under investigation, however when phage numbers remained static, M. smegmatis mc² 155 found to …

Crystallographic Studies On Host-Interacting Proteins Of Phage K Virion, Antonio Pichel Beleiro

Theses

Phage K is a lytic bacteriophage that infects a broad range of staphylococci, including some methicillin-resistant Staphylococcus aureus strains. Phage K attaches to its host via a baseplate. The contracted conformation of the baseplate contains a fiber and two receptorbinding proteins responsible for the interaction with the host. The native conformation of the baseplate also includes a tail central spike that shears off after contraction. The aim of the thesis is the structural characterization of phage K receptor-binding proteins and tail central spike. Experimentally, the thesis relies on crystallographic methods, which encompass: production of suitable sample, crystallization, diffraction, and data …

The Occurrence Of Listeria Monocytogenes In The Mushroom Production Chain And The Use Of Bacteriophage For Its Control, Vincenzo Pennone

Theses

In Agaricus bisporus production, the occurrence of L. monocytogenes is still poorly understood. A number of studies have shown the presence of L. monocytogenes in the mushroom processing environment, suggesting its source from raw materials storage areas (Viswanath et al, 2013; Murugesan et al, 2015). In this study, 10 mushroom producers and three raw material (compost and casing) producers participated in two surveys on the occurrence of L. monocytogenes in the most high-risk areas of the production environment. In the first survey, floors, drains, platforms, crates, raw materials and mushrooms were tested for L. monocytogenes presence and the results were …

Crystallographic Structure Determination Of Bacteriophage-Encoded Enzymes That Specifically Target Pathogenic Bacteria, Marta Sanz Gaitero

Theses

Antibiotic resistance is becoming a serious public health concern. Infections that some decades ago could be treated with antibiotics now sometimes do not respond to traditional treatment, causing higher mortality and economic losses. An alternative to the use of antibiotics are bacteria's natural predators, bacteriophages (or phages), and specifically their lytic enzymes. These proteins are produced by phages to degrade bacterial peptidoglycan to inject their genetic material into the bacteria (virion-associated peptidoglycan hydrolases) or to release their progeny once the infection is finished (endolysins). They can be applied exogenously to lyse Gram-positive bacteria or be genetically engineered to lyse Gram-negative …

A Rapid And Sensitive System For Recovery Of Nucleic Acids From Mycobacteria Sp. On Archived Glass Slides, Balkis A. Talip, William J. Snelling, Roy D. Sleator, Colm Lowery, James S.G. Dooley

Department of Biological Sciences Publications

The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM …

Identification And Characterisation Of Novel Phages Of Pectobacterium And Erwinia, Colin Buttimer

PhDs

Losses in crop yields due to disease need to be reduced to meet increasing global food demands associated with growth in the human population. There is a well-recognised need to develop new environmentally-friendly control strategies to combat bacterial crop diseases. There are several crop diseases for which no effective bactericidal agents are currently available, such as potato blackleg and soft rot disease caused by Pectobacterium atrosepticum and other members of soft rot Enterobacteriaceae (Czajkowski et al., 2011). Furthermore, current control measures involving the use of traditional chemicals or antibiotics are losing their efficacy due to the natural development of bacterial …

Inhibition Of Listeria Monocytogenes By Human Gut Bacteria, Patrycja Sylvia Olesky

Masters

Listeria monocytogenes is a foodborne pathogen, which continues to be problematic to the food industry due to its ability survive within food products and persist within food processing environments. The growing trend towards natural food preservatives and antimicrobial agents increases the need for the development of novel, natural anti-Listeria agents. Evidence presented in the literature suggests that the human gut microbiota is a reservoir of novel antimicrobial agents. It was therefore hypothesized that novel anti-Listeria agents are produced by human gut-derived bacteria. The objective of this study was to isolate and characterize gut bacteria with anti-Listeria monocytogenes activity. A total …

Comparison Of Staphylococcus Phage K With Close Phage Relatives Commonly Employed In Phage Therapeutics, Jude Ajuebor, Colin Buttimer, Sara Arroyo-Moreno, Nina Chanishvili, Emma M. Gabriel, Jim O'Mahony, Olivia Mcauliffe, Horst Neve, Charles Franz, Aidan Coffey

Department of Biological Sciences Publications

The increase in antibiotic resistance in pathogenic bacteria is a public health danger requiring alternative treatment options, and this has led to renewed interest in phage therapy. In this respect, we describe the distinct host ranges of Staphylococcus phage K, and two other K-like phages against 23 isolates, including 21 methicillin-resistant S. aureus (MRSA) representative sequence types representing the Irish National MRSA Reference Laboratory collection. The two K-like phages were isolated from the Fersisi therapeutic phage mix from the Tbilisi Eliava Institute, and were designated B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1). The sequence relatedness of B1 and JA1 to phage K …

Marine Bioactives From Saccharina Latissima, Orlaith O'Connell

Masters

Due to rapid global urbanization and therefore increased adoption of a 'Westernized' lifestyle, including decreased physical activity and increased consumption of highly processed and refined foods with a high sugar, fat and salt content, prevalence of gastrointestinal disorders are on the rise. Inflammatory bowel disease (IBD) in particular is closely associated with this type of lifestyle. IBD is a term used to describe chronic inflammatory conditions which affects all or parts of the gastrointestinal tract. It includes conditions such as Crohn's Disease, which affects all the gastrointestinal tract and Ulcerative Colitis which mainly affects the colan. Due to the role …

Emergence Of The L Phenotype In Group B Streptococci In The South Of Ireland, Katherine Hayes, Lesley Cotter, L. Barry, Fiona O'Halloran

Department of Biological Sciences Publications

Group B Streptococcal isolates (n = 235) from the South of Ireland were characterised by serotyping, antimicrobial susceptibility and determination of the phenotypic and genotypic mechanisms of resistance. Resistance to erythromycin and clindamycin was observed in 21·3% and 20·4% of the total population, respectively. The c-MLSB phenotype was the most common phenotype detected (62%), with ermB being the predominant genetic determinant, present in 84% of resistant isolates. The rare L phenotype was observed in 2·9% (n = 7) of isolates, four of which harboured the lsaC gene responsible for clindamycin resistance. Serotypes Ia, III and II were the most common …

Is Tb Testing Associated With Increased Blood Interferon-Gamma Levels?, Aideen E. Kennedy, Jim O'Mahony, Noel Byrne, John Macsharry, Riona G. Sayers

Department of Biological Sciences Publications

The Republic of Ireland reports a relatively low prevalence of Johne’s disease (JD) compared to international counterparts. Postulated reasons for this include a lower average herd size and a grass-based production system. Ireland also engages in high levels of bovine tuberculosis (bTB) testing. As interferon-gamma (IFN-γ) is believed to play a key role in protecting against JD, it is our hypothesis that administration of purified protein derivative (PPD), as part of the bTB test, is associated with a systemic increase in IFN-γ production, which may potentially limit clinical progression of the disease. We studied 265 cows (202 Friesian and 63 …

A Blueprint To Evaluate One Health, Simon R. Rüegg, Barry J. Mcmahon, Barbara Häsler, Roberto Esposito, Helen O'Shea, Et Al

Department of Biological Sciences Publications

One Health (OH) positions health professionals as agents for change and provides a platform to manage determinants of health that are often not comprehensively captured in medicine or public health alone. However, due to the organization of societies and disciplines, and the sectoral allocation of resources, the development of transdisciplinary approaches requires effort and perseverance. Therefore, there is a need to provide evidence on the added value of OH for governments, researchers, funding bodies, and stakeholders. This paper outlines a conceptual framework of what OH approaches can encompass and the added values they can provide. The framework was developed during …

Microbiota, Diet And Medication In Human Gut Disorders, Adam Clooney

Theses

The microbiota, a community of micro-organisms in a particular environment, have been intensely researched in the past decade and interest appears to continue to increase. The human body is richly populated with bacterial cells (3.8x10'^) and alterations in these communities are associated with disease. The work presented in this thesis explores aspects of microbiota research methodologies with regard to optimisation and concerns over a lack of reproducibility. Additionally, we investigated the effects of medication, in particular Proton Pump Inhibitors, on the human gut microbiota, along with comparisons between the microbiota of patients with active and inactive Inflammatory Bowel Disease.

Microbiota …

A Diagnostic And Control Programme For Johne’S Disease On Irish Dairy Farms, Aideen Kennedy

Theses

Johne’s disease (JD) is an enteritis of ruminants with potential zoonotic implications. JD diagnosis and control present difficulties given JDs prolonged subclinical nature. In order to improve JD control in Ireland, PhD aims included conducting a national survey documenting high-risk husbandry practices employed on Irish farms, thereby targeting areas for improved management. Furthermore as JD has been associated with on-farm economic losses internationally, this research programme aimed to analyse production losses associated with testing JD ELISA positive on Irish dairy farms. A longitudinal study was also conducted to assess if successful JD control can be achieved using gold-standard protocols. Due …

The Development Of New Molecular Tools To Detect Mycobacterium Avium Subspecies Paratuberculosis From A Bovine Faecal Environment By Real-Time Pcr, Marcel De Kruijf

Theses

The slow-growing intracellular pathogen Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in domesticated and wild ruminants. Johne’s disease inflicts severe annual economic losses to the dairy industry worldwide. This thesis is divided into five chapters that consist of a literature review' chapter and four experimental chapters focusing on MAP strain genotyping and the development of novel molecular MAP detection tools using real-time PCR. Chapter 1 describes published information regarding MAP characteristics, Johne’s disease, MAP genomes, available MAP detection methods, MAP epidemiology and genotyping.

To efficiently control and monitor MAP infection, molecular t>ping of MAP strains provides extensive knowledge …

Mycobacterium Species Identification In An Acute Hospital: Towards Rapid Identification, And Improved Epidemiology, Using Novel Mass Spectrometry Analysis, James Anthony O'Connor

Theses

Mycobacteria are a heterogeneous group of bacteria that cause a wide spectrum of disease, including Tuberculosis (TB), the single biggest killer worldwide. The genus also incorporates over 170 nontuberculosis (NTM) species, many of which are opportunistic pathogens. The aim of this project was to ascertain the burden of mycobacterial disease in the region and to determine the efficacy of Matrix-Assisted Laser Desorption/Ionisation time-of-flight (MALDI-tof) mass spectrometry (MS) for novel uses as a rapid identification method for clinical mycobacteria and as a typing tool for Mycobacterium tuberculosis complex isolates. A five-year retrospective epidemiological study of the region was conducted, the first …

Metabolic Characteristics Of Macrophage Activation With Phenotype Microarrays, Tanya Kathryn D'Souza

Theses

Inflammation is a natural protective response and is characterised by the activation of numerous immune cells, including macrophages which can release a variety of inflammatory mediators. Macrophages play a flexible role in the initiation and effector phases of the immune response and change their behaviour to respond to environmental stimuli. The main objectives in this study were to create and characterise different macrophage behaviours and employ a novel metabolic characterisation platform the Phenotype Microarray^TM to attribute additional characteristics to the currently well recognised phenotypes. Five activated macrophage states were demonstrated and characterised. Unactivated macrophages (MO) demonstrated undetectable levels of …

Comparative Genomic Analysis Of Two Serotype 1/2b Listeria Monocytogenes Isolates From Analogous Environmental Niches Demonstrates The Influence Of Hypervariable Hotspots In Defining Pathogenesis, Aidan Casey, Kieran Jordan, Aidan Coffey, Edward M. Fox, Olivia Mcauliffe

Department of Biological Sciences Publications

The vast majority of clinical human listeriosis cases are caused by serotype 1/2a, 1/2b, 1/2c, and 4b isolates of Listeria monocytogenes. The ability of L. monocytogenes to establish a systemic listeriosis infection within a host organism relies on a combination of genes that are involved in cell recognition, internalization, evasion of host defenses, and in vitro survival and growth. Recently, whole genome sequencing and comparative genomic analysis have proven to be powerful tools for the identification of these virulence-associated genes in L. monocytogenes. In this study, two serotype 1/2b strains of L. monocytogenes with analogous isolation sources, but …

Prevalence And Distribution Of Gastrointestinal Nematode Parasites And Dung Beetles Associated With Upland And Lowland Grazing Of Irish Native Dexter Cattle, Noel Dineen

Theses

Diseases such as parasitic gastroenteritis are very common in cattle and can have detrimental economic consequences on agricultural productivity. This study was conducted to establish the prevalence and distribution of gastrointestinal nematode parasites associated with Dexter cattle under an organic lowland grazing regime and an extensive upland system. A secondary aim to the study was to investigate dung beetle assemblages in upland and lowland sites and their interactions with gastrointestinal nematode parasites as they offer an alternative method of natural control to organic farming systems in particular.

Cattle weights were recorded and faecal samples were collected at the beginning and …

Analysis Of Johne's Disease Elisa Status And Associated Performance Parameters In Irish Dairy Cows, A. E. Kennedy, N. Byrne, A. B. Garcia, Jim O'Mahony, R. G. Sayers

Department of Biological Sciences Publications

Background

Infection with Mycobacterium avium subspecies paratuberculosis (MAP) has been associated with reductions in milk production in dairy cows and sub optimal fertility. The aim of this study was to highlight the production losses associated with testing MAP ELISA positive in Irish dairy cows. Secondary objectives included investigation of risk factors associated with testing MAP ELISA positive. A survey of management practices on study farms was also conducted, with examination of associations between management practices and herd MAP status.

Blood samples were collected from 4188 breeding animals on 22 farms. Samples were ELISA tested using the ID Screen Paratuberculosis Indirect …

Comparing Apples And Oranges?: Next Generation Sequencing And Its Impact On Microbiome Analysis, Adam G. Clooney, Fiona Fouhy, Roy D. Sleator, Aisling O'Driscoll, Stanton Catherine, Paul D. Cotter, Marcus J. Claesson

Department of Biological Sciences Publications

Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to …