Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles

Articular cartilage defects are common after joint injuries. When left untreated, the biomechanical protective function of cartilage is gradually lost, making the joint more susceptible to further damage, causing progressive loss of joint function and eventually osteoarthritis (OA). In the process of translating promising tissue-engineering cartilage repair approaches from bench to bedside, pre-clinical animal models including mice, rabbits, goats, and horses, are widely used. The equine species is becoming an increasingly popular model for the in vivo evaluation of regenerative orthopaedic approaches. As there is also an increasing body of evidence suggesting that successful lasting tissue reconstruction requires an implant that mimics natural tissue organization, it is imperative that depth-dependent characteristics of equine osteochondral tissue are known, to assess to what extent they resemble those in humans. Therefore, osteochondral cores (4-8 mm) were obtained from the medial and lateral femoral condyles of equine and human donors. Cores were processed for histology and for biochemical quantification of DNA, glycosaminoglycan (GAG) and collagen content. Equine and human osteochondral tissues possess similar geometrical (thickness) and organizational (GAG, collagen and DNA distribution with depth) features. These comparable trends further underscore the validity of the equine model for the evaluation of regenerative approaches for articular cartilage.

Nurse-administered procedural sedation and analgesia in the cardiac catheter laboratory : an integrative review

Objectives: To identify and appraise the literature concerning nurse-administered procedural sedation and analgesia in the cardiac catheter laboratory. Design and data sources: An integrative review method was chosen for this study. MEDLINE and CINAHL databases as well as The Cochrane Database of Systematic Reviews and the Joanna Briggs Institute were searched. Nineteen research articles and three clinical guidelines were identified. Results: The authors of each study reported nurse-administered sedation in the CCL is safe due to the low incidence of complications. However, a higher percentage of deeply sedated patients were reported to experience complications than moderately sedated patients. To confound this issue, one clinical guideline permits deep sedation without an anaesthetist present, while others recommend against it. All clinical guidelines recommend nurses are educated about sedation concepts. Other findings focus on pain and discomfort and the cost-savings of nurse-administered sedation, which are associated with forgoing anaesthetic services. Conclusions: Practice is varied due to limitations in the evidence and inconsistent clinical practice guidelines. Therefore, recommendations for research and practice have been made. Research topics include determining how and in which circumstances capnography can be used in the CCL, discerning the economic impact of sedation-related complications and developing a set of objectives for nursing education about sedation. For practice, if deep sedation is administered without an anaesthetist present, it is essential nurses are adequately trained and have access to vital equipment such as capnography to monitor ventilation because deeply sedated patients are more likely to experience complications related to sedation. These initiatives will go some way to ensuring patients receiving nurse-administered procedural sedation and analgesia for a procedure in the cardiac catheter laboratory are cared for using consistent, safe and evidence-based practices.

Trends in nurse-administered procedural sedation and analgesia across cardiac catheterisation laboratories in Australia and New Zealand : results of an electronic survey

Background Knowledge of current trends in nurse-administered procedural sedation and analgesia (PSA) in the cardiac catheterisation laboratory (CCL) may provide important insights into how to improve safety and effectiveness of this practice. Objective To characterise current practice as well as education and competency standards regarding nurse-administered PSA in Australian and New Zealand CCLs. Design A quantitative, cross-sectional, descriptive survey design was used. Methods Data were collected using a web-based questionnaire on practice, educational standards and protocols related to nurse-administered PSA. Descriptive statistics were used to analyse data. Results A sample of 62 nurses, each from a different CCL, completed a questionnaire that focused on PSA practice. Over half of the estimated total number of CCLs in Australia and New Zealand was represented. Nurse-administered PSA was used in 94% (n = 58) of respondents CCLs. All respondents indicated that benzodiazepines, opioids or a combination of both is used for PSA (n = 58). One respondent indicated that propofol was also used. 20% (n = 12) indicated that deep sedation is purposefully induced for defibrillation threshold testing and cardioversion without a second medical practitioner present. Sedation monitoring practices vary considerably between institutions. 31% (n = 18) indicated that comprehensive education about PSA is provided. 45% (n = 26) indicated that nurses who administer PSA should undergo competency assessment. Conclusion By characterising nurse-administered PSA in Australian and New Zealand CCLs, a baseline for future studies has been established. Areas of particular importance to improve include protocols for patient monitoring and comprehensive PSA education for CCL nurses in Australia and New Zealand.

Issues and challenges associated with nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory : a qualitative study

Aims and objectives To explore issues and challenges associated with nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory from the perspectives of senior nurses. Background Nurses play an important part in managing sedation because the prescription is usually given verbally directly from the cardiologist who is performing the procedure and typically, an anaesthetist is not present. Design A qualitative exploratory design was employed. Methods Semi-structured interviews with 23 nurses from 16 cardiac catheterisation laboratories across four states in Australia and also New Zealand were conducted. Data analysis followed the guide developed by Braun and Clark to identify the main themes. Results Major themes emerged from analysis regarding the lack of access to anaesthetists, the limitations of sedative medications, the barriers to effective patient monitoring and the impact that the increasing complexity of procedures has on patients' sedation requirements. Conclusions The most critical issue identified in this study is that current guidelines, which are meant to apply regardless of the clinical setting, are not practical for the cardiac catheterisation laboratory due to a lack of access to anaesthetists. Furthermore, this study has demonstrated that nurses hold concerns about the legitimacy of their practice in situations when they are required to perform tasks outside of clinical practice guidelines. To address nurses' concerns, it is proposed that new guidelines could be developed, which address the unique circumstances in which sedation is used in the cardiac catheterisation laboratory. Relevance to clinical practice Nurses need to possess advanced knowledge and skills in monitoring for the adverse effects of sedation. Several challenges impact on nurses' ability to monitor patients during procedural sedation and analgesia. Preprocedural patient education about what to expect from sedation is essential.

Risk factors for impaired respiratory function during nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory : a matched case-control study

Background: Side effects of the medications used for procedural sedation and analgesia in the cardiac catheterisation laboratory are known to cause impaired respiratory function. Impaired respiratory function poses considerable risk to patient safety as it can lead to inadequate oxygenation. Having knowledge about the conditions that predict impaired respiratory function prior to the procedure would enable nurses to identify at-risk patients and selectively implement intensive respiratory monitoring. This would reduce the possibility of inadequate oxygenation occurring. Aim: To identify pre-procedure risk factors for impaired respiratory function during nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory. Design: Retrospective matched case–control. Methods: 21 cases of impaired respiratory function were identified and matched to 113 controls from a consecutive cohort of patients over 18 years of age. Conditional logistic regression was used to identify risk factors for impaired respiratory function. Results: With each additional indicator of acute illness, case patients were nearly two times more likely than their controls to experience impaired respiratory function (OR 1.78; 95% CI 1.19–2.67; p = 0.005). Indicators of acute illness included emergency admission, being transferred from a critical care unit for the procedure or requiring respiratory or haemodynamic support in the lead up to the procedure. Conclusion: Several factors that predict the likelihood of impaired respiratory function were identified. The results from this study could be used to inform prospective studies investigating the effectiveness of interventions for impaired respiratory function during nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory.

Australian and New Zealand nurses’ perceptions of procedural sedation and analgesia in the cardiac catheterisation laboratory : a qualitative study

Background: Australian and New Zealand College of Anaesthetists’ guidelines for procedural sedation and analgesia (PSA) are intended to apply across all clinical settings. As nurses are frequently responsible for patient care during PSA in the cardiac catheterisation laboratory (CCL), their perspectives can provide insight into the effectiveness of these guidelines within this particular setting. Methods: A cross-sectional sampling design was used to recruit nurses from urban, regional, public and private CCLs across Australia and New Zealand. Semi-structured interviews were conducted, digitally recorded and transcribed. Data were analysed using thematic analysis. Findings: Twenty-three nurses from 16 CCLs across four states in Australia and New Zealand participated. Most held senior positions (managers=14; educators=5) and CCL experience ranged from 4 to 26 years (mean 11). Participants were concerned about the legitimacy of their practice as they administered PSA outside of guideline recommendations and deemed present education and training as deficient. Participants noted also that guideline recommendations were sometimes not adhered to as it was difficult to balance the increasingly complex PSA requirements of their case-mix with limited access to anaesthetists while trying not to delay procedures. Conclusion: Findings suggest that application of current PSA guidelines may be impractical for CCL nurses and, as a consequence, they are often not followed. Participants were concerned about risks to patient safety as they felt education and training was not commensurable with practice requirements. The findings suggest existing guidelines should be reviewed or new guidelines developed which address nursing practice, education and competency standards for PSA in the CCL

Respiratory monitoring practices during procedural sedation and analgesia in the cardiac catheterisation laboratory could be improved by using capnography to assess ventilation

Background: Procedural sedation and analgesia (PSA) administered by nurses in the cardiac catheterisation laboratory (CCL) is unlikely to yield serious complications. However, the safety of this practice is dependent on timely identification and treatment of depressed respiratory function. Aim: Describe respiratory monitoring in the CCL. Methods: Retrospective medical record audit of adult patients who underwent a procedure in the CCLs of one private hospital in Brisbane during May and June 2010. An electronic database was used to identify subjects and an audit tool ensured data collection was standardised. Results: Nurses administered PSA during 172/473 (37%) procedures including coronary angiographies, percutaneous coronary interventions, electrophysiology studies, radiofrequency ablations, cardiac pacemakers, implantable cardioverter defibrillators, temporary pacing leads and peripheral vascular interventions. Oxygen saturations were recorded during 160/172 (23%) procedures, respiration rate was recorded during 17/172 (10%) procedures, use of oxygen supplementation was recorded during 40/172 (23%) procedures and 13/172 (7.5%; 95% CI=3.59–11.41%) patients experienced oxygen desaturation. Conclusion: Although oxygen saturation was routinely documented, nurses did not regularly record respiration observations. It is likely that surgical draping and the requirement to minimise radiation exposure interfered with nurses’ ability to observe respiration. Capnography could overcome these barriers to respiration assessment as its accurate measurement of exhaled carbon dioxide coupled with the easily interpretable waveform output it produces, which displays a breath-by-breath account of ventilation, enables identification of respiratory depression in real-time. Results of this audit emphasise the need to ascertain the clinical benefits associated with using capnography to assess ventilation during PSA in the CCL.

Nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory : a mixed methods study

The cardiac catheterisation laboratory (CCL) is a specialised medical radiology facility where both chronic-stable and life-threatening cardiovascular illness is evaluated and treated. Although there are many potential sources of discomfort and distress associated with procedures performed in the CCL, a general anaesthetic is not usually required. For this reason, an anaesthetist is not routinely assigned to the CCL. Instead, to manage pain, discomfort and anxiety during the procedure, nurses administer a combination of sedative and analgesic medications according to direction from the cardiologist performing the procedure. This practice is referred to as nurse-administered procedural sedation and analgesia (PSA). While anecdotal evidence suggested that nurse-administered PSA was commonly used in the CCL, it was clear from the limited information available that current nurse-led PSA administration and monitoring practices varied and that there was contention around some aspects of practice including the type of medications that were suitable to be used and the depth of sedation that could be safely induced without an anaesthetist present. The overall aim of the program of research presented in this thesis was to establish an evidence base for nurse-led sedation practices in the CCL context. A sequential mixed methods design was used over three phases. The objective of the first phase was to appraise the existing evidence for nurse-administered PSA in the CCL. Two studies were conducted. The first study was an integrative review of empirical research studies and clinical practice guidelines focused on nurse-administered PSA in the CCL as well as in other similar procedural settings. This was the first review to systematically appraise the available evidence supporting the use of nurse-administered PSA in the CCL. A major finding was that, overall, nurse-administered PSA in the CCL was generally deemed to be safe. However, it was concluded from the analysis of the studies and the guidelines that were included in the review, that the management of sedation in the CCL was impacted by a variety of contextual factors including local hospital policy, workforce constraints and cardiologists’ preferences for the type of sedation used. The second study in the first phase was conducted to identify a sedation scale that could be used to monitor level of sedation during nurse-administered PSA in the CCL. It involved a structured literature review and psychometric analysis of scale properties. However, only one scale was found that was developed specifically for the CCL, which had not undergone psychometric testing. Several weaknesses were identified in its item structure. Other sedation scales that were identified were developed for the ICU. Although these scales have demonstrated validity and reliability in the ICU, weaknesses in their item structure precluded their use in the CCL. As findings indicated that no existing sedation scale should be applied to practice in the CCL, recommendations for the development and psychometric testing of a new sedation scale were developed. The objective of the second phase of the program of research was to explore current practice. Three studies were conducted in this phase using both quantitative and qualitative research methods. The first was a qualitative explorative study of nurses’ perceptions of the issues and challenges associated with nurse-administered PSA in the CCL. Major themes emerged from analysis of the qualitative data regarding the lack of access to anaesthetists, the limitations of sedative medications, the barriers to effective patient monitoring and the impact that the increasing complexity of procedures has on patients' sedation requirements. The second study in Phase Two was a cross-sectional survey of nurse-administered PSA practice in Australian and New Zealand CCLs. This was the first study to quantify the frequency that nurse-administered PSA was used in the CCL setting and to characterise associated nursing practices. It was found that nearly all CCLs utilise nurse-administered PSA (94%). Of note, by characterising nurse-administered PSA in Australian and New Zealand CCLs, several strategies to improve practice, such as setting up protocols for patient monitoring and establishing comprehensive PSA education for CCL nurses, were identified. The third study in Phase Two was a matched case-control study of risk factors for impaired respiratory function during nurse-administered PSA in the CCL setting. Patients with acute illness were found to be nearly twice as likely to experience impaired respiratory function during nurse-administered PSA (OR=1.78; 95%CI=1.19-2.67; p=0.005). These significant findings can now be used to inform prospective studies investigating the effectiveness of interventions for impaired respiratory function during nurse-administered PSA in the CCL. The objective of the third and final phase of the program of research was to develop recommendations for practice. To achieve this objective, a synthesis of findings from the previous phases of the program of research informed a modified Delphi study, which was conducted to develop a set of clinical practice guidelines for nurse-administered PSA in the CCL. The clinical practice guidelines that were developed set current best practice standards for pre-procedural patient assessment and risk screening practices as well as the intra and post-procedural patient monitoring practices that nurses who administer PSA in the CCL should undertake in order to deliver safe, evidence-based and consistent care to the many patients who undergo procedures in this setting. In summary, the mixed methods approach that was used clearly enabled the research objectives to be comprehensively addressed in an informed sequential manner, and, as a consequence, this thesis has generated a substantial amount of new knowledge to inform and support nurse-led sedation practice in the CCL context. However, a limitation of the research to note is that the comprehensive appraisal of the evidence conducted, combined with the guideline development process, highlighted that there were numerous deficiencies in the evidence base. As such, rather than being based on high-level evidence, many of the recommendations for practice were produced by consensus. For this reason, further research is required in order to ascertain which specific practices result in the most optimal patient and health service outcomes. Therefore, along with necessary guideline implementation and evaluation projects, post-doctoral research is planned to follow up on the research gaps identified, which are planned to form part of a continuing program of research in this field.

Acute illness is a risk factor for impaired respiratory function during nurse-administered procedural sedation and analgesia in the cardiac catheterization laboratory

Impaired respiratory function (IRF) during procedural sedation and analgesia (PSA) poses considerable risk to patient safety as it can lead to inadequate oxygenation and ventilation. Risk factors that can be screened prior to the procedure have not been identified for the cardiac catheterization laboratory (CCL).

Clinical practice guidelines for nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory : A modified Delphi study

Aim To develop clinical practice guidelines for nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory. Background Numerous studies have reported that nurse-administered procedural sedation and analgesia is safe. However, the broad scope of existing guidelines for the administration and monitoring of patients who receive sedation during medical procedures without an anaesthetist presents means there is a lack of specific guidance regarding optimal nursing practices for the unique circumstances in which nurse-administered procedural sedation and analgesia is used in the cardiac catheterisation laboratory. Methods A sequential mixed methods design was utilised. Initial recommendations were produced from three studies conducted by the authors: an integrative review; a qualitative study; and a cross-sectional survey. The recommendations were revised in accordance with responses from a modified Delphi study. The first Delphi round was completed by nine senior cardiac catheterisation laboratory nurses. All but one of the draft recommendations met the pre-determined cut-off point for inclusion. There were a total of 59 responses to the second round. Consensus was reached on all recommendations. Implications for nursing The guidelines that were derived from the Delphi study offer twenty four recommendations within six domains of nursing practice: Pre-procedural assessment; Pre-procedural patient and family education; Pre-procedural patient comfort; Intra-procedural patient comfort; Intra-procedural patient assessment and monitoring; and Post-procedural patient assessment and monitoring. Conclusion These guidelines provide an important foundation towards the delivery of safe, consistent and evidence-based nursing care for the many patients who receive sedation in the cardiac catheterisation laboratory setting.

Depth of anaesthesia monitoring during procedural sedation and analgesia: A systematic review protocol

Background Procedural sedation and analgesia (PSA) is used to attenuate the pain and distress that may otherwise be experienced during diagnostic and interventional medical or dental procedures. As the risk of adverse events increases with the depth of sedation induced, frequent monitoring of level of consciousness is recommended. Level of consciousness is usually monitored during PSA with clinical observation. Processed electroencephalogram-based depth of anaesthesia (DoA) monitoring devices provide an alternative method to monitor level of consciousness that can be used in addition to clinical observation. However, there is uncertainty as to whether their routine use in PSA would be justified. Rigorous evaluation of the clinical benefits of DoA monitors during PSA, including comprehensive syntheses of the available evidence, is therefore required. One potential clinical benefit of using DoA monitoring during PSA is that the technology could improve patient safety by reducing sedation-related adverse events, such as death or permanent neurological disability. We hypothesise that earlier identification of lapses into deeper than intended levels of sedation using DoA monitoring leads to more effective titration of sedative and analgesic medications, and results in a reduction in the risk of adverse events caused by the consequences of over-sedation, such as hypoxaemia. The primary objective of this review is to determine whether using DoA monitoring during PSA in the hospital setting improves patient safety by reducing the risk of hypoxaemia (defined as an arterial partial pressure of oxygen below 60 mmHg or percentage of haemoglobin that is saturated with oxygen [SpO2] less than 90 %). Other potential clinical benefits of using DoA monitoring devices during sedation will be assessed as secondary outcomes. Methods/design Electronic databases will be systematically searched for randomized controlled trials comparing the use of depth of anaesthesia monitoring devices with clinical observation of level of consciousness during PSA. Language restrictions will not be imposed. Screening, study selection and data extraction will be performed by two independent reviewers. Disagreements will be resolved by discussion. Meta-analyses will be performed if suitable. Discussion This review will synthesise the evidence on an important potential clinical benefit of DoA monitoring during PSA within hospital settings.

Inadvertent hypothermia after procedural sedation and analgesia in a cardiac catheterisation laboratory: A prospective observational study

Objective To identify the prevalence of and risk factors for inadvertent hypothermia after procedures performed with procedural sedation and analgesia in a cardiac catheterisation laboratory. Design Single-centre, prospective observational study. Setting Tertiary care private hospital in Australia. Participants A convenience sample of 399 patients undergoing elective procedures with procedural sedation and analgesia were included. Propofol infusions were used when an anaesthetist was present. Otherwise, bolus doses of either midazolam or fentanyl or a combination of these medications was used. Interventions None Measurements and main results Hypothermia was defined as a temperature <36.0° Celsius. Multivariate logistic regression was used to identify risk factors. Hypothermia was present after 23.3% (n=93; 95% confidence interval [CI] 19.2%-27.4%) of 399 procedures. Sedative regimens with the highest prevalence of hypothermia were any regimen that included propofol (n=35; 40.2%; 95% CI 29.9%-50.5%) and the use of fentanyl combined with midazolam (n=23; 20.3%; 95% CI 12.9%-27.7%). Difference in mean temperature from pre to post-procedure was -0.27°C (Standard deviation [SD] 0.45). Receiving propofol (odds ratio [OR] OR 4.6 95% CI 2.5-8.6), percutaneous coronary intervention (OR 3.2 95% CI 1.7-5.9), body mass index <25 (OR 2.5 95% CI 1.4-4.4) and being hypothermic prior to the procedure (OR 4.9; 95% CI 2.3-10.8) were independent predictors of post-procedural hypothermia. Conclusions A moderate prevalence of hypothermia was observed. The small absolute change in temperature observed may not be a clinically important amount. More research is needed to increase confidence in our estimates of hypothermia in sedated patients and its impact on clinical outcomes.

Capnography monitoring during procedural sedation and analgesia: A systematic review protocol

Background An important potential clinical benefit of using capnography monitoring during procedural sedation and analgesia (PSA) is that this technology could improve patient safety by reducing serious sedation-related adverse events, such as death or permanent neurological disability, which are caused by inadequate oxygenation. The hypothesis is that earlier identification of respiratory depression using capnography leads to a change in clinical management that prevents hypoxaemia. As inadequate oxygenation/ventilation is the most common reason for injury associated with PSA, reducing episodes of hypoxaemia would indicate that using capnography would be safer than relying on standard monitoring alone. Methods/design The primary objective of this review is to determine whether using capnography during PSA in the hospital setting improves patient safety by reducing the risk of hypoxaemia (defined as an arterial partial pressure of oxygen below 60 mmHg or percentage of haemoglobin that is saturated with oxygen [SpO2] less than 90 %). A secondary objective of this review is to determine whether changes in the clinical management of sedated patients are the mediating factor for any observed impact of capnography monitoring on the rate of hypoxaemia. The potential adverse effect of capnography monitoring that will be examined in this review is the rate of inadequate sedation. Electronic databases will be searched for parallel, crossover and cluster randomised controlled trials comparing the use of capnography with standard monitoring alone during PSA that is administered in the hospital setting. Studies that included patients who received general or regional anaesthesia will be excluded from the review. Non-randomised studies will be excluded. Screening, study selection and data extraction will be performed by two reviewers. The Cochrane risk of bias tool will be used to assign a judgment about the degree of risk. Meta-analyses will be performed if suitable. Discussion This review will synthesise the evidence on an important potential clinical benefit of capnography monitoring during PSA within hospital settings. Systematic review registration: PROSPERO CRD42015023740

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

Chlamydia pneumoniae is genetically diverse in animals and appears to have crossed the host barrier to humans on (at least) two occasions

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non- Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials.