Project Summary: Currently, identification of microbial species in clinical, food, environmental and industrial samples relies mainly on traditional microbiological methods. Such methods are slow and identification to species level requires a battery of additional biochemical, serological or immunoassay based tests to be performed once the bacterial species under investigation is isolated from the sample. However, nucleic acid diagnostic tests have been adapted in clinical microbiology for viral pathogen identification, for diagnosis of microbial causes of human sepsis and sexually transmitted diseases. These nucleic acid diagnostic tests are a replacement technology that are of higher sensitivity, specificity and give faster turn-around time to results. The proposed project aims to utilize and develop RiboTech technology, a novel genomic target platform diagnostics technology invented by the Molecular Diagnostics Research Group at NUI, Galway. This technology will be applied in Nucleic Acid Diagnostic (NAD) tests for the identification of microorganisms associated with sepsis (24 microorganisms inclusive of Gram- and Gram+ bacteria, yeast and fungi).

Objectives: 1. Generation of a nucleotide sequence database of information for the RiboTech target in a relevant microbial species.
2: In-silico analysis of RiboTech nucleotide sequence database to assess its suitability for molecular microbial identification. Design of Real-time PCR in-vitro amplification primers and probes for a selected relevant microbial species to experimentally demonstrate the value of RiboTech technology for microbial detection and identification.
3: RiboTech target nucleic acid based assay design and development for microorganism detection and identification using Real-time PCR in-vitro amplification technology.

Project Relevance: The proposed project will contribute to with the research currently being conducted in Dr. Barry’s laboratory, the main objectives of which are to develop nucleic acid-based diagnostic tests for the diagnosis of human disease.

Benefits to the student: The student will have the opportunity to undertake an in-depth research experience and develop skills in identifying novel targets and platforms for nucleic acid diagnostic tests. The longer-term objective is to make this training experience a launching pad for their research career.

The Pathogenic Mechanisms group utilises molecular and cellular approaches to investigate the relationships between bacteria and the cells of humans and animals. The main research goal of the Pathogenic Mechanisms group is to understand the pathogenic mechanisms of Vibrio parahaemolyticus. This bacterium causes diarrhoea and inflammatory gastroenteritis in humans following consumption of contaminated raw or improperly prepared seafood. This is a significant problem in Asia, where up to 80% of all cases of foodborne gastroenteritis are due to V. parahaemolyticus and the occurrence of V. parahaemolyticus infections is increasing in Europe as the temperature of our marine waters increases due to global warming. The virulence mechanisms of this emerging pathogen need to be better understood in order to develop more effective measures to prevent and treat infection. We are currently investigating how V. parahaemolyticus alters the normal regulation of eukaryotic cell signalling proteins, thereby leading to changes in host cell behaviours that favour bacterial survival.

The aim of this project is to characterise how alteration of eukaryotic cell signalling events by V. parahaemolyticus affects epithelial cell behaviour. The student will use V. parahaemolyticus strains which have been genetically modified to inactivate specific virulence proteins. These bacteria will be used in studies to compare the ability of wild-type V. parahaemolyticus and bacteria with defective genes to influence cell signalling events and/or the activities of human intestinal cells. Techniques to be used for this project include aseptic procedures, prokaryotic culture, eukaryotic cell culture, bacteria+host cell co-incubation studies, cell signalling analysis and/or analysis of host cell behaviours.

The results of this project will be integrated with the ongoing research in the Pathogenic Mechanisms Group investigating the effect of V. parahaemolyticus on host cell behaviours. As well as increasing understanding of Vibrio pathogenesis, this research would promote a broader knowledge of the control of eukaryotic cell function by pathogenic organisms.

The Pathogenic Mechanisms group utilises molecular and cellular approaches to investigate the relationships between bacteria and the cells of humans and animals. The main research aim of the Pathogenic Mechanisms group is to understand the pathogenic mechanisms of Vibrio parahaemolyticus. This bacterium causes diarrhoea and inflammatory gastroenteritis in humans following consumption of contaminated raw or improperly prepared seafood. This is a significant problem in Asia, where up to 80% of all cases of foodborne gastroenteritis are due to V. parahaemolyticus and the occurrence of V. parahaemolyticus infections is increasing in Europe as the temperature of our marine waters increases due to global warming. The virulence mechanisms of this emerging pathogen need to be better understood in order to develop more effective measures to prevent and treat infection. We have found that V. parahaemolyticus can enter human epithelial cells. This may provide a protective niche for the bacteria during infection.

Currently we are identifying the V. parahaemolyticus proteins involved in the ability of the bacteria to adhere to and invade intestinal epithelial cells. The aim of this project is to study these proteins and confirm their role in bacterial pathogenesis. The student will co-incubate eukaryotic cells with various bacterial strains which possess or lack these proteins in order to compare their adherent and invasive capabilities. In experiments to assess adherence the cells will be washed after the co-incubation to remove non-adherent bacteria and the remaining adherent bacteria will be enumerated by plating on selective agar. In experiments to measure invasion, extracellular bacteria will be killed after the co-incubation by incubation with gentamicin. Enumeration of intracellular bacteria will then be performed by plating out lysates of the eukaryotic cells. In further experiments microscopy will be performed to visualise the location of the bacteria. Techniques to be used for this project include aseptic procedures, prokaryotic and eukaryotic cell culture, bacteria+host cell co-incubation studies, gentamicin protection assays, microscopy.

The results of this project will be integrated with the ongoing research in the Pathogenic Mechanisms Group to increase understanding of Vibrio pathogenesis.

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are recognized as a major cause of human gastroenteritis worldwide. In Ireland, campylobacteriosis is the commonest bacterial cause of human gastrointestinal illness. Campylobacteriosis is mainly a food-borne infection, in which foods of animal origin, and in particular raw or undercooked poultry meat, play an important role. Good hygiene practice routinely employs the use of disinfectants to decontaminate surfaces and clear them of pathogenic bacteria. The misuse of disinfectants could be counterproductive however, as an increase in disinfectant tolerance may in some cases co-select for drug-resistant strains. Studies at Dr Carroll’s lab have been investigating the effect of sub-inhibitory concentrations of different disinfectants on the dynamics of selection of disinfectant tolerance in C. jejuni. We have shown that increased tolerance to Benzalkonium Chloride (BKC), the active biocide ingredient in a number of commonly used disinfectants, is accompanied by an overall increase in the level of multiple drug resistance.

This study proposes to investigate the effect of sub-inhibitory concentrations of Benzalkonium chloride (BKC) on the dynamics of selection for resistance. C. jejuni strains have been challenged to different sub-inhibitory levels of disinfectant and antibiotic using a bio-processor-controlled chemostat and the adapted populations to the agents have been isolated. In this study, colonies isolated at different levels of biocide/antibiotic stress will be assayed for tolerance to commercial disinfectants. This project seeks to determine the nature and extent of the ATR to disinfectant stress, and to characterise the genomic and proteomic stress response in Campylobacter.

Key words: Campylobacter, disinfectants, selective pressure, resistance, Gene expression.

Campylobacter jejuni infections are the leading cause of bacterial gastroenteritis in Ireland. There is significant evidence to suggest that poultry meat is not only a primary source of Campylobacter infection but also that the conditions prevalent during meat processing may modulate, and enhance, virulence of the bacteria.

Although unable to grow at the chill temperatures used for storage of most fresh poultry and meat products, C. jejuni can survive the conditions prevalent during meat processing to subsequently cause illness. There is increasing molecular and physiological evidence from studies with other pathogens that stresses such as acidity and cold-shock may modulate, and enhance, virulence. Campylobacter strains have the ability to induce an Adaptive Tolerance Response, which enables them to survive hostile environments. The use of disinfectants in food processing has consequences for bacterial pathogenicity and virulence. Treatment with sub-inhibitory concentrations of certain disinfectants can lead to increased survival of Multiple Antibiotic Resistant (MAR) strains. Presently, little is known about the physiological basis of disinfectant tolerance in Campylobacters. The information gained will allow this phenomenon to be taken into account in the design of treatment regimes aimed at the reduction in the numbers of Campylobacters in foods and a concomitant decrease in the rate of associated illness.

This study proposes to investigate the effect of sub-inhibitory concentrations of a food processing disinfectant such as citric acid on the dynamics of selection for resistance. C. jejuni strains will be challenged to different sub-inhibitory levels of disinfectant and antibiotic using a bio-processor-controlled chemostat and adapted populations will be isolated. Colonies isolated at different levels of disinfectant will be assayed for to determine the nature and extent of the disinfectant stress at the genomic and/or proteomic level.

Key words: Campylobacter, citric acid, selective pressure, Adaptation, Gene expression.

Beta-lactamases are enzymes that hydrolyse and inactivate one or more of the beta-lactam antibiotics (penicillins and cephalosporins). The TEM-1 beta-lactamase (encoded on blaTEM-1) is one of the most widely disseminated plasmid encoded beta-lactamases. This enzyme has a relatively narrow substrate range including ampicillin but not newer cephalosporins such as cefotaxime. More recently there has been rapid dissemination of extended-spectrum beta-lactamase (ESBL) enzymes and other enzymes with a border substrate range (including both ampicillin and cefotaxime). These enzymes are encoded by blaCTX-M, blaTEM variants and blaOXA genes amongst others. A number of strains that possess extended spectrum beta-lactamases also retain genes for enzymes with a narrower substrate range. The hypothesis is that these narrow substrate range enzymes are retained because under certain circumstances where beta-lactams are present in the environment they confer a survival advantage.

The aims of this project are to identify and characterize plasmids from antimicrobial resistant Enterobacteriaceae which encode for both narrow substrate range and broad substrate range beta-lactams. Time permitting gene knock-out techniques will be used to knock out (1) the narrow substrate range enzyme and (2) the broad substrate range enzyme from representative plasmids. The minimum inhibitory concentrations (MIC) of a broad range of beta-lactam antimicrobial agents and beta-lactam/beta-lactamase combinations will the be determined for E. coli transfected with the native plasmid and with the knock-out plasmids to assess the impact of the genes and the individually and in combination on the MIC.

Salmonella enterica is an important human pathogen. Some serovars are associated with enteric fever while others are associated predominantly with gastroenteritis. Subclassification (typing) within specific serovars of S. enterica, such as S. Enteritidis, is important in managing investigations of outbreaks of infection. Molecular sub-typing may include pulsed-field gel electrophoresis, plasmid profiles and multi-locus variable number tandem repeat typing. Plasmid profiles are performed by comparing the number and size of plasmids in a collection of isolates of interest. Plasmids can be classified into Inc (inclusion) groups. Recently a series of multiplex PCR technique has been reported that allows the characterization of the Inc group of most common plasmids in Enterobacteriaceae. This project aims to assess if the multiplex PCR to identify Inc group of plasmids in Salmonella can be used on whole genomic DNA as a typing technique.

Anti retroviral therapy (ART) became available in the Karonga District, Malawi in 2005. With more than 10% of the adult population of Malawi possibly taking ART within 5-7 years, the effectiveness and long-term consequences for the control of the HIV epidemic remain uncertain due to the very large numbers of adults receiving long-term therapy for HIV in a resource-limited setting. HIV is less infectious in persons receiving ART, however in terms of transmission this can be counterbalanced by improved survival, prolonging the time available for transmission and increasing the probability of new partnership formation. If drug resistance develops in treated individuals there will be a rise in viral load and infectiousness, sometimes leading to transmission of drug resistant virus and hence new infections that are not amenable to ART. The proposed project will monitor the emergence and transmission of drug resistant virus in this primarily subtype C infected population, by recording the extent of drug-resistant HIV among new infections in Karonga.

Campylobacter jejuni is a common and leading cause of bacterial gastroenteritis and food-poisoning in humans. Infection by certain strains of C. jejuni is also associated with development of the autoimmune paralytic disorder, Guillain-Barré syndrome. Previously, we have documented the significant role played by the C. jejuni lipooligosaccharide (LOS) in triggering this autoimmune disease. Poultry represent an important reservoir of C. jejuni for human infection, and consumption of poultry has been identified as an important risk factor in developing C. jejuni enteritis. Unlike in humans, however, C. jejuni colonization of poultry does not generally cause overt disease.

The cell-surface LOS of C. jejuni is an important component of the outer membrane of this bacterium required for survival and adaptation to its environment. Our preliminary studies indicate that the structure and properties of C. jejuni LOS can vary when the bacterium is grown at 37^oC or 42^oC. Importantly, the body core temperature of humans is 37^oC and that of birds is 42^oC, and thus the observed variation in LOS properties may play an important role in the adaptation and/or response of the bacterium to these hosts. Understanding this phenomenon could have central importance in controlling the infection in poultry, and hence spread to humans, but also in part explain the difference in outcome of C. jejuni colonization of poultry and humans. Thus, the central aim of the present project will be to examine and characterize LOS produced by a model strain of C. jejuni when grown at 37^oC and 42^oC.

Preparations of C. jejuni LOS will be examined using electrophoretic and staining procedures to assess macromolecular changes in LOS structure. Additionally, these LOS preparations will be probed with antibodies and other reactive ligands, including lectins (carbohydrate-recognizing proteins), using Western blotting and thin-layer chromatography immuno-overlay techniques to give further insights into the structural and antigenic variation of the LOS. Finally, a variety of biochemical assays and chemical analytical techniques will be used to assess further the structural changes occurring within the LOS molecule. The project will thus give multidisciplinary training in the emerging field of microbial glycobiology using a range of microbiological, serological, biochemical and chemical techniques. The results of the study will provide important insights for our ongoing aim of understanding the pathogenesis of this important human pathogen and the role played by LOS.

Escherichia coli is found as a commensal in the gastrointestinal tract of all humans. These strains coexist with the host throughout life without causing any problems. However there a several strains of E. coli that are pathogenic for humans, most notably the enterohemorrhagic strains such as H7:O157, which has claimed the lives of many people following food borne infections. In addition E. coli is one of the leading causes of hospital acquired infections, where it is often associated with urinary tract infections.

In my lab we are investigating the possibility that a bacterial metabolite called gamma aminobutyrate (GABA) might play a role in pathogenesis. Many bacteria including E. coli can produce this compound and they can secrete large amounts of it under certain conditions. GABA also happens to be important neurotransmitter in the human nervous system. It is possible that pathogenic bacteria might exploit this during pathogenesis in order to manipulate the host.

In this project we will survey a range of pathogenic and non pathogenic strains of E. coli with the aim of determining whether GABA production correlates with the ability to establish infections in humans. The polymerase chain reaction will be used to determine whether individual strains carry the genes required for GABA production. Then the GABA levels produced by each strain will be measured using an assay that we have recently developed in the lab. The results of these experiments will reveal any strain to strain differences that exist in the ability to produce GABA and will also reveal whether GABA production correlates in any way with pathogenicity.

The success of Listeria monocytogenes as a pathogen can be partly attributed to its tolerance of a range of environmental stresses, including high salt, low pH, and cold temperatures. These attributes enable the organism to persist in diverse environments, including foods destined for human consumption, and also enable the pathogen to establish infections within the host, as the harsh conditions in the stomach and gastrointestinal tract must be endured prior to colonization. My lab investigates how L. monocytogenes responds to environmental stresses. Understanding how this pathogen can thrive in stressful environments is important as it may help us prevent this bacterium from growing in foods that are preserved by the addition of salt. Our work to date indicates that the sigma factor (a subunit of RNA polymerase required for promoter recognition) SigmaB (σB) plays a central role in coordinating the expression of stress resistance genes in L. monocytogenes.

This project will involve the construction of transcriptional reporter to investigate the regulation of σB activity. The promoter region of a known σB–dependent gene (lmo2230) will be cloned upstream from the lacZ reporter gene in plasmid that can replicate in L. monocytogenes (pTVC-lac). This plasmid will then be transformed into L. monocytogenes wild type and a ΔsigB mutant, and LacZ (β-galactosidase) assays will be performed to confirm that cloned promoter can drive lacZ transcription. Next the recombinant L. monocytogenes strain will be subjected to a variety of physical (salt and acidic pH) and chemical (hydrogen peroxide and bile) stresses and the response of the reporter fusion will be measured to each. These experiments will establish which conditions trigger the activation of σB. Overall this approach will give new insights into how L. monocytogenes protects itself against stressful environments, which will have important implications for our understanding of how this pathogen causes disease in humans.

Degeneration of the intervertebral disc is the main cause of neck and low back pain. The cause of low back pain is unclear but imaging studies have identified degeneration of the intervertebral disc as a leading factor. Current research in this area in our group is focussed on the development of a cell-specific gene delivery system for targeted disc regeneration using phage display technology and nanotechnology. Phage display is a technique in which large, highly diverse libraries of antibody fragments are screened in vitro to isolate antibodies that bind a particular target of interest. These are then linked to hollow, biodegradable nanoshells containing a therapeutic gene for improved targeting to specific cell types in vivo.

This project will involve the production of a lead antibody fragment in an Escherichia coli expression system. The protein will be purified and attached to pre-prepared nanoshells containing a fluorescent marker for ease of imaging. The efficacy of gene delivery will be measured in a cell culture-based assay in vitro using fluorescence microscopy and, as appropriate, RTPCR and microarray analysis. The experimental techniques learned will be broadly applicable in the production, engineering and immobilisation of antibodies for the detection and therapy of a wide variety of infectious diseases.

The project will provide specific experience in PCR, cloning and various DNA manipulation techniques; recombinant protein production, purification and characterisation; and, depending on progress, fluorescence microscopy and RTPCR techniques.