Investigation of the molecular pathogenesis of the multi-host bacterial pathogen Staphylococcus aureus

Staphylococcus aureus is a major human and animal bacterial pathogen that causes an array of diseases. The main aim of this thesis is to investigate the key host-pathogen interactions that underpin S. aureus infection. S. aureus abundantly secretes two isoforms of the enzyme lipase into the extracellular milieu, where they scavenge upon polymeric triglycerides. It has previously been suggested that these lipases may interfere with the function of innate immune cells, such as macrophages and neutrophils, but the impact of lipases on phagocytic killing mechanisms remains unknown. We showed that there were no differences in the survival of S. aureus USA300 LAC wild type and its lipase-deficient isogenic mutant after incubation with human whole blood or neutrophils. Furthermore, there was no detectable lipase-dependent effect on phagocytosis, intracellular survival, or escape from both human primary and immortalised cell line macrophages, even upon supplementation with exogenous recombinant lipases. Therefore, we showed that S. aureus lipases do not inhibit bacterial killing mechanisms of human macrophages, neutrophils, or whole blood. Furthermore, the capacity of S. aureus to adapt to distinct host-species ecologies is a major public health and economic concern. Approximately 60 years ago, a human-to-poultry host jump and adaptation of S. aureus belonging to the widespread CC5 clade, led to the avian-adaptation and global expansion of S. aureus in broiler poultry. Our research aims to combine transposon (Tn) mutagenesis of S. aureus with experimental models of infections to identify the immune cell repertoire and bacterial genes involved in avian host-adaptation. To determine the avian immune cell tropism for S. aureus, mCherry-integrated clones from common avian (CC385, CC5) and human (CC8) S. aureus clonal lineages were screened in blood extracted from the transgenic chicken line Runx1-eGFP. We demonstrate that monocytes and heterophils generate the first-line response to S. aureus infection, with avian strains exhibiting differential uptake by heterophils compared to a human strain. Furthermore, our analysis demonstrated that avian S. aureus strains may have adapted to the avian host through the inhibition of degranulation of heterophils as a novel survival mechanism. TraDIS analysis of genes involved in the fitness of S. aureus during infection of peripheral blood leukocytes demonstrated that regulators of the Type VII secretion system and Spl-proteases were required for survival of S. aureus in the face of the innate immune response of PBLs. Taken together, these studies provide new insights into the evolution of S. aureus and the key host-pathogen interactions underpinning S. aureus infections

Investigating hill sheep farmers and crofters’ experiences of blackloss in the Highlands and Islands of Scotland

Hill sheep farming is an important component of Scottish agriculture and comprises a significant land use in much of the Highlands and Islands. However it faces significant challenges due to the natural constraints of the landscape. Hill sheep farming uses hardy traditional breeds, such as the Scottish blackface and North Country Cheviot to graze extensive areas, where the sheep are not housed and tend to lamb on the open hill. Flocks are gathered several times a year for stock checks, husbandry, and health treatments. Between these handling events, stock will disappear and be unaccounted for. These unexplained losses are known as blackloss in the Highlands and Islands. Previously reported figures for annual lamb blackloss give an average of 18.6%. These losses are in addition to the known losses of lambs and represent a significant welfare and sustainability issue. High parasite burdens, predation, a photosensitisation disease known as plochteach or yellowses, and poor nutrition are often given as presumed reasons for blackloss. A questionnaire was developed to assess the experiences, impacts and understanding flock managers have of blackloss. Typology analysis using partitioning around medoids was used to cluster respondents into three distinct groups: 1- very large extensive farms and Sheep Stock Clubs, 2- medium sized farms, and 3- small-scale crofts. The responses of these groups were subsequently analysed to see if their experiences and perceptions of blackloss differed with relation to lamb health challenges and predation impacts. The groups reported similar health challenges, apart from Group 1 which had a significantly higher plochteach challenge. In terms of predators, Group 1 also perceived white-tailed eagles (Haliaeetus albicilla) as a much higher threat to their lambs than the other groups. It was observed that many of the respondents believed blackloss is inevitable and that predators pose a large threat to lambs. However, most agreed that reducing these losses is important and that understanding the causes would enable them to do so.</p

Characterisation of autophagy disruption in the ileum of pigs infected with Lawsonia intracellularis

Lawsonia intracellularis is the aetiological agent of proliferative enteropathy, an enteric disease endemic in swine. Survival in its intracellular niche of the ileum epithelial lining requires the capacity to subvert, repress or exploit the host immune response to create an environment conducive to bacterial propagation. To better understand how L. intracellularis survives in its intracellular niche, we have performed an investigation into the dynamic relationship between infection and the host autophagy response by immunohistochemistry in experimentally infected porcine ileum samples. Beclin1, a protein required early in the autophagy pathway was observed to be distributed with a basal to apical concentration gradient in the crypts of healthy piglets, whilst infected piglets were observed to have no gradient of distribution and an increase in the presence of Beclin1 in crypts with histological characteristics of L. intracellularis residence. Detecting microtubule-associated protein light chain 3 (LC3) is used as a method for monitoring autophagy progression as it associates with mature autophagosomes. For LC3 there was no notable change in signal intensity between crypts with characteristic L. intracellularis infection and healthy crypts of uninfected pigs. Finally, as p62 is degraded with the internal substrate of an autophagosome it was used to measure autophagic flux. There was no observed reduction or redistribution of p62. These preliminary results of the autophagy response in the ileum suggest that L. intracellularis affects autophagy. This disruption to host ileum homeostasis may provide a mechanism that assists in bacterial propagation and contributes to pathogenesis

Investigating the perceived versus actual gastrointestinal nematode challenge on extensive sheep farms

Extensive farming systems form an integral part of sheep production systems across Europe. However, with innate production handicaps, declining sheep numbers and narrow economic margins, production is becoming increasingly challenging threatening the future sustainability of the industry. Gastrointestinal nematodes (GINs) are a significant cause of production losses to the global sheep industry, with well-established resistance to the major anthelmintic groups. Traditionally, extensive farming systems are not thought to have a significant parasite challenge compared with intensive farms, but there is a need to identify the scale and importance of GINs on extensive farms to inform the need for sustainable control strategies. In this study, a questionnaire of extensive farmers (n=34) was conducted and parasitological data were collected from nine study farms to investigate the perceived versus actual GIN and anthelmintic resistance challenge faced by extensive farms. The results showed a production-limiting challenge on most farms, with a higher GIN challenge observed on improved pastures. Furthermore, over half of the extensive farmers perceived anthelmintic resistance to be a greater problem for intensive farmers, with only 20% of respondents reporting known anthelmintic resistance. However, all study farms had evidence of resistance to at least one group of anthelmintics. Consequently, this study has demonstrated that despite the traditional perception of parasitism on extensive farms, there is a need to increasingly consider its impact and take a proactive approach to sustainable control, with solutions tailored to their unique management

A case report: Insights into Reducing Plastic Waste in a Microbiology Laboratory

Single-use plastics have often replaced more sustainable materials in microbiology laboratories. Keeping in mind that one of the objectives of the United Nations Sustainable Development Goals is responsible consumption and production, we wanted to document how many single-use plastic items could be saved by taking reduction and reuse approaches in a microbiology laboratory. After taking 4 weeks to document the baseline levels of single-use plastic waste being generated in our laboratory and identifying ways to reduce our reliance on them, we implemented various reduction and reuse approaches and then documented our plastic use over a 7-week period. Reduction approaches included moving to sustainable materials, such as reusable wooden sticks for patch plating and metal loops for inoculation. Reuse approaches focused on reusing plastic tubes via a chemical decontamination station and autoclaving, facilitating the reduction of single-use plastics and a decrease in the amount of waste generated. By utilizing reduction and reuse strategies, which could be implemented in other microbiology laboratories, substantial single-use plastic savings were achieved. These savings had an impact on the amount of biohazard waste being autoclaved and incinerated, as well as generating substantial cost savings for the research institute. The reductions in waste documented in this study could act as a benchmark for others wanting to implement the changes described

Time-lapse mesoscopy of Candida albicans and Staphylococcus aureus dual-species biofilms reveals a structural role for the hyphae of C. albicans in biofilm formation

Polymicrobial infection with Candida albicans and Staphylococcus aureus may result in a concomitant increase in virulence and resistance to antimicrobial drugs. This enhanced pathogenicity phenotype is mediated by numerous factors including metabolic processes and direct interaction of S. aureus with C. albicans hyphae. The overall structure of biofilms is known to contribute to their recalcitrance to treatment, however the dynamics of direct interaction between species and how it contributes to pathogenicity is poorly understood. To address this, a novel time-lapse mesoscopic optical imaging method was developed to enable the formation of C. albicans/S. aureus whole dual-species biofilms to be followed. It was found that yeast-form or hyphal-form C. albicans in the biofilm founder-population profoundly affects the structure of the biofilm as it matures. Different sub-populations of C. albicans and S. aureus arise within each biofilm as a result of the different C. albicans morphotypes, resulting in distinct sub-regions. These data reveal that C. albicans cell morphology is pivotal in the development of global biofilm architecture and the emergence of colony macrostructures and may temporally influence synergy in infection

Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing

Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires' disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing. The bacterium Legionella pneumophila can cause severe respiratory infection, but is typically a symbiont of free-living amoeba. Here, the authors analyse the genomes of 902 clinical and environmental isolates, and identify a bacterial gene that is strongly associated with human infection and confers resistance to complement-mediated killing.Peer reviewe

Introgression of Ivermectin Resistance Genes into a Susceptible Haemonchus contortus Strain by Multiple Backcrossing

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci