Metabolomics as a tool to explore the staphylococcal biofilm

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.

Using an end-of-life care pathway in acute stroke: a mixed methods study of decision-making and care experiences

Background: The evidence base on end-of-life care in acute stroke is limited, particularly with regard to recognising dying and related decision-making. There is also limited evidence to support the use of end-of-life care pathways (standardised care plans) for patients who are dying after stroke. Aim: This study aimed to explore the clinical decision-making involved in placing patients on an end-of-life care pathway, evaluate predictors of care pathway use, and investigate the role of families in decision-making. The study also aimed to examine experiences of end-of-life care pathway use for stroke patients, their relatives and the multi-disciplinary health care team. Methods: A mixed methods design was adopted. Data were collected in four Scottish acute stroke units. Case-notes were identified prospectively from 100 consecutive stroke deaths and reviewed. Multivariate analysis was performed on case-note data. Semi-structured interviews were conducted with 17 relatives of stroke decedents and 23 healthcare professionals, using a modified grounded theory approach to collect and analyse data. The VOICES survey tool was also administered to the bereaved relatives and data were analysed using descriptive statistics and thematic analysis of free-text responses. Results: Relatives often played an important role in influencing aspects of end-of-life care, including decisions to use an end-of-life care pathway. Some relatives experienced enduring distress with their perceived responsibility for care decisions. Relatives felt unprepared for and were distressed by prolonged dying processes, which were often associated with severe dysphagia. Pro-active information-giving by staff was reported as supportive by relatives. Healthcare professionals generally avoided discussing place of care with families. Decisions to use an end-of-life care pathway were not predicted by patients’ demographic characteristics; decisions were generally made in consultation with families and the extended health care team, and were made within regular working hours. Conclusion: Distressing stroke-related issues were more prominent in participants’ accounts than concerns with the end-of-life care pathway used. Relatives sometimes perceived themselves as responsible for important clinical decisions. Witnessing prolonged dying processes was difficult for healthcare professionals and families, particularly in relation to the management of persistent major swallowing difficulties.

The role of oxidative stress as an underlying mechanism in life-history trade-offs

A key aspect underpinning life-history theory is the existence of trade-offs. Trade-offs occur because resources are limited, meaning that individuals cannot invest in all traits simultaneously, leading to costs for traits such as growth and reproduction. Such costs may be the reason for the sub-maximal growth rates that are often observed in nature, though the fitness consequences of these costs would depend on the effects on lifetime reproductive success. Recently, much attention has been given to the physiological mechanism that might underlie these life-history trade-offs, with oxidative stress (OS) playing a key role. OS is characterised by a build-up of oxidative damage to tissues (e.g. protein, lipids and DNA) from attack by reactive species (RS). RS, the majority of which are by-products of metabolism, are usually neutralised by antioxidants, however OS occurs when there is an imbalance between the two. There are two main theories linking OS with growth and reproduction. The first is that traits like growth and reproduction, being metabolically demanding, lead to an increase in RS production. The second involves the diversion of resources away from self-maintenance processes (e.g. the redox system) when individuals are faced with enhanced growth or reproductive expenditure. Previous research investigating trade-offs involving growth or reproduction and self-maintenance has been equivocal. One reason for this could be that associations among redox biomarkers can vary greatly so that the biomarker selected for analysis can influence the conclusion reached about an individual’s oxidative status. Therefore the first aim of my thesis was to explore the strength and pattern of integration of five biomarkers of OS (three antioxidants, one damage and one general oxidation measure) in wild blue tit (Cyanistes caeruleus) adults and nestlings (Chapter 2). In doing so, I established that all five biomarkers should be included in future analyses, thus using this collection of biomarkers I explored my next aims; whether enhanced growth (Chapters 3 and 4) or reproductive effort (Chapter 5) can lead to increased OS levels, if these traits are traded off against self-maintenance. I accomplished these aims using both a meta-analytic and experimental approach, the latter involving manipulation of brood size in wild blue tits in order to experimentally alter growth rate of nestlings and provisioning rate (a proxy for reproductive expenditure) of adults. I also investigated the potential for redox integration to be used as an index of body condition (Chapter 2), allowing predictions about future fitness consequences of changes to oxidative state to be made. A growth – self-maintenance trade off was supported by my meta-analytic results (Chapter 4) which found OS to be a constraint on growth. However, when faced with experimentally enhanced growth, animals were typically not able to adjust this trade-off so that oxidative damage resulted. This might support the idea that energetically expensive growth causes resources to be diverted away from the redox system; however, antioxidants did not show an overall reduction in response to growth in the meta-analysis suggesting that oxidative costs of growth may result from increased RS production due to the greater metabolism needed for enhanced growth. My experimental data (Chapter 3) showed a similar pattern, with raised protein damage levels (protein carbonyls; PCs) in the fastest growing blue tit chicks in a brood, compared with their slower growing sibs. These within-brood differences in OS levels likely resulted from within-brood hierarchies and might have masked any between-brood differences, which were not observed here. Despite evidence for a growth – self-maintenance trade off, my experimental results on blue tits found no support for the hypothesis that self-maintenance is also traded off against reproduction, another energetically demanding trait. There was no link between experimentally altered reproductive expenditure and OS, nor was there a direct correlation between reproductive effort and OS (Chapter 5). However, there are various factors that likely influence whether oxidative costs are observed, including environmental conditions and whether such costs are transient. This emphasises the need for longitudinal studies following the same individuals over multiple years and across a wide range of habitats that differ in quality. This would allow investigation into how key life events interact; it might be that raised OS levels from rapid early growth have the potential to constrain reproduction or that high parental OS levels constrain offspring growth. Any oxidative costs resulting from these life-history trade-offs have the potential to impact on future fitness. Redox integration of certain biomarkers might prove to be a useful tool in making predictions about fitness, as I found in Chapter 2, as well as establishing how the redox system responds, as a whole, to changes to growth and reproduction. Finally, if the tissues measured can tolerate a given level of OS, then the level of oxidative damage might be irrelevant and not impact on future fitness at all.