The use of a self-paced cardiopulmonary exercise test in the pre and post-operative care of patients with cardiovascular disease

The aim of this thesis was to assess the ability of a self-paced (SPV) cardiopulmonary exercise test (CPET) in assessing patient fitness prior to elective surgery, and its ability to predict postoperative outcomes. The SPV is a 10 minute test which is comprised of 5 × 2 minute stages. Each stage is fixed to a level on the ratings of perceived exertion (RPE) scale, in an incremental format (RPE: 11, 13, 15, 17 and 20). This test eliminates the need of practitioners having to choose the most appropriate work rate increments to ensure a patient reaches volitional exhaustion within the recommended time period (8-12 min). Study 1 aimed to assess the reliability of the maximal exercise test parameters obtained from the SPV. Twenty-five (12 females, 13 males) healthy participants completed three SPV tests on three separate occasions. Results demonstrated a coefficient of variation (CV) for V?O2peak (ml·kg-1·min-1) of 4.2% (95% CI: 3.4-5.6%) for trials 2-1, and 5.1% (95% CI: 4.2-6.8%) for trials 3-2. Repeated measures ANOVA analysis demonstrated no significant difference in V?O2peak across the repeated tests (p > 0.05). The limits of agreement (LOA) were ± 5.59 ml·kg-1·min-1 for trials 2-1, and ± 5.86 ml·kg-1·min-1 for trials 3-2. The mean intraclass correlation coefficient (ICC) was 0.95, which represents good reproducibility. It was concluded that the SPV is a reliable indicator of the main CPET derived variables in a healthy population, with comparable values to previous work on standard CPET protocols. Study 2 investigated the physiological responses between the SPV and a standard CPET (sCPET) protocol between a young (18-30 years) and a middle aged to older adult (50-75 years) population. This was in the attempt to gain an understanding of the response to the protocol and whether these responses differ with age. Expired gases, Q, SV, muscular deoxyhaemoglobin (deoxyHb) and electromyography (EMG) at the vastus lateralis were recorded throughout both tests. Results demonstrated a significantly higher V?O2max in the SPV (49.68 ± 10.26 ml·kg-1·min-1) vs. a sCPET (47.70 ± 9.98 ml·kg-1·min-1) in the young, but no differences in the middle aged to older adult group (> 0.05). Q and SV were significantly higher in the SPV vs. a sCPET in the young ( 0.05). No differences were seen in both age groups in the deoxyHb and EMG response (> 0.05). Findings from this study suggest that in the young group, the SPV produces higher V?O2max values as a result of an increase in oxygen delivery (enhanced Q). However, likely due to age-related differences, particularly in the cardiovascular response to exercise, the middle aged to older adult group achieved similar V?O2max values regardless of them reaching a higher physiological workload. Study 3 aimed to assess the validity and reliability of the SPV in post myocardial infarction (post-MI) patients, this was the first study to assess the use of the SPV in a clinical population. Twenty-eight post-MI patients completed one sCPET and two SPVs in a randomised, counterbalanced crossover design. Each patient completed one sCPET and two SPVs. Results demonstrated the SPV to have a coefficient of variation for V?O2peak of 8.2%. The limits of agreement were ± 4.22 ml·kg-1·min-1, with intraclass correlation coefficient of 0.89. There was a significantly higher V?O2peak achieved in the SPV (23.07 ± 4.90 ml·kg-1·min-1) against the sCPET (21.29 ± 4.93 ml·kg-1·min-1). It was concluded that the SPV is a safe and valid test of exercise capacity in post-MI patients, with acceptable levels of reliability when compared to previous work on sCPET protocols. Study 4 aimed to determine if the SPV can assess patient's preoperative risk similar to sCPET and if exercise variables obtained from the test can accurately predict post-operative outcome. Fifty patients with cardiovascular related co-morbidities completed one sCPET and one SPV, although only thirty of those patients when ahead with surgery. Post-surgery, patients were monitored for incidence of morbidity on postoperative days 3 and 5, length of hospital stay, and incidence of mortality in the 30 days after surgery. Patients achieved a significantly higher V?O2peak, HR, V?E, peak PO and TTE in the SPV compared to the sCPET (P 0.05). Although when combining postoperative morbidity at days 3 and 5, logistic regression analysis showed that oxygen pulse at AT obtained from the SPV was significantly related to postoperative complications (P < 0.05). ROC curve analysis demonstrated oxygen pulse at AT to provide an AUC of 0.72 a.u. (95%CI 0.51 to 0.92), with an optimal cut-off point of 8.5 ml/beat-1 which provided 72.7% sensitivity and 71.4% specificity. It was concluded that the SPV was able to assess preoperative fitness comparable to the sCPET. Although none of the CPET variable from either test were associated with postoperative morbidity, which is likely a result of the small sample size. The conclusion for this thesis is that a self-paced CPET test is able to reliably assess cardiovascular patient's fitness comparable to traditional methods. This type of test may be seen as advantageous, this is because the SPV takes away the need of clinicians having to choose the most appropriate work rate increments, it allows patients to have full control over the test, and it ensures that regardless of fitness all patients will be exercise for the recommended test time. The fixed test duration of 10 minutes may also help to improve the efficiency of running busy CPET clinics. There are clear benefits to using the SPV, although further research is required first to assess its ability of predicting postoperative outcome in a much larger sample, and to determine if it can be used to the same advantages sCPET protocols have previously demonstrated

Evolution of a zoonotic pathogen:investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing

Enterohaemorrhagic Escherichia coli (EHEC) O157 is a zoonotic pathogen for which colonization of cattle and virulence in humans is associated with multiple horizontally acquired genes, the majority present in active or cryptic prophages. Our understanding of the evolution and phylogeny of EHEC O157 continues to develop primarily based on core genome analyses; however, such short-read sequences have limited value for the analysis of prophage content and its chromosomal location. In this study, we applied Single Molecule Real Time (SMRT) sequencing, using the Pacific Biosciences long-read sequencing platform, to isolates selected from the main sub-clusters of this clonal group. Prophage regions were extracted from these sequences and from published reference strains. Genome position and prophage diversity were analysed along with genetic content. Prophages could be assigned to clusters, with smaller prophages generally exhibiting less diversity and preferential loss of structural genes. Prophages encoding Shiga toxin (Stx) 2a and Stx1a were the most diverse, and more variable compared to prophages encoding Stx2c, further supporting the hypothesis that Stx2c-prophage integration was ancestral to acquisition of other Stx types. The concept that phage type (PT) 21/28 (Stx2a+, Stx2c+) strains evolved from PT32 (Stx2c+) was supported by analysis of strains with excised Stx-encoding prophages. Insertion sequence elements were over-represented in prophage sequences compared to the rest of the genome, showing integration in key genes such as stx and an excisionase, the latter potentially acting to capture the bacteriophage into the genome. Prophage profiling should allow more accurate prediction of the pathogenic potential of isolates

Acquisition and loss of CTX-M plasmids in Shigella species associated with MSM transmission in the UK

Shigellosis in men who have sex with men (MSM) is caused by multidrug resistant Shigellae, exhibiting resistance to antimicrobials including azithromycin, ciprofloxacin and more recently the third-generation cephalosporins. We sequenced four bla (CTX-M-27)-positive MSM Shigella isolates (2018–20) using Oxford Nanopore Technologies; three S. sonnei (identified as two MSM clade 2, one MSM clade 5) and one S. flexneri 3a, to explore AMR context. All S. sonnei isolates harboured Tn7/Int2 chromosomal integrons, whereas S. flexneri 3a contained the Shigella Resistance Locus. All strains harboured IncFII pKSR100-like plasmids (67-83kbp); where present bla (CTX-M-27) was located on these plasmids flanked by IS26 and IS903B, however bla (CTX-M-27) was lost in S. flexneri 3a during storage between Illumina and Nanopore sequencing. IncFII AMR regions were mosaic and likely reorganised by IS26; three of the four plasmids contained azithromycin-resistance genes erm(B) and mph(A) and one harboured the pKSR100 integron. Additionally, all S. sonnei isolates possessed a large IncB/O/K/Z plasmid, two of which carried aph(3’)-Ib/aph(6)-Id/sul2 and tet(A). Monitoring the transmission of mobile genetic elements with co-located AMR determinants is necessary to inform empirical treatment guidance and clinical management of MSM-associated shigellosis

Whole-genome sequencing for national surveillance of Shiga toxin–producing Escherichia coli O157

Background. National surveillance of gastrointestinal pathogens, such as Shiga toxin–producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. Methods. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. Results. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. Conclusions. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations

Optimisation of a lozenge-based sensor for detecting impending blockage of urinary catheters

Catheter-associated urinary tract infections resulting from urease-positive microorganisms are more likely to cause a urinary catheter blockage owing to the urease activity of the microbes. Catheter blockage can be dangerous and increases the risk of severe infections, such as sepsis. Ureases, a virulence factor in Proteus mirabilis, cause an increase in urine pH - leading to blockage. An optimised biosensor "lozenge" is presented here, which is able to detect impending catheter blockage. This lozenge has been optimised to allow easy manufacture and commercialisation. It functions as a sensor in a physiologically representative model of a catheterised urinary tract, providing 6.7 h warning prior to catheter blockage. The lozenge is stable in healthy human urine and can be sterilized for clinical use by ethylene oxide. Clinically, the lozenge will provide a visible indication of impending catheter blockage, enabling quicker clinical intervention and thus reducing the morbidity and mortality associated with blockage.</p

Enhanced Colorimetric Differentiation between Staphylococcus aureus and Pseudomonas aeruginosa Using a Shape-Encoded Sensor Hydrogel

Herein, we demonstrate a combined fluorescent probe/shape-encoded hydrogel strategy for the fast, sensitive, and selective detection of bacterial species via their characteristic enzymes. A poly(vinyl alcohol) (PVA) hydrogel loaded with the fluorescent probe N,N′-(3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-3′,6′-diyl)bis(2,2,3,3,3-pentafluoropropanamide) (ACS-HNE) was designed for the detection of elastase, an enzyme produced by Pseudomonas aeruginosa. Likewise, a chitosan-derived hydrogel was loaded with the fluorescent probe 4-methylumbelliferyl-α-d-glucopyranoside (MUD) by entrapment for the selective detection of α-glucosidase, an enzyme produced by Staphylococcus aureus. For an observation time of 60 min, limits of detection (LODs) of ≤20 nM for elastase and ≤30 pM for α-glucosidase were obtained, which in the latter case is 3 orders of magnitude better than related chitosan systems with covalently coupled substrate. To illustrate the potential utility of these highly sensitive sensor hydrogels as a simple point-of-care test system, shaped hydrogel slabs representing the letters P and S were manufactured to detect P. aeruginosa and S. aureus, respectively. These shapes were shown to provide an additional unique color code under UV illumination corresponding to the characteristic enzyme produced by the corresponding bacteria. This study shows potential for the future development of an effective and simple point-of-care test for the rapid identification of bacterial species that can be operated by nonspecialists

Boronate ester cross-linked PVA hydrogels for the capture and H2O2-mediated release of active fluorophores

A new set of PVA hydrogels were formed using the boronate ester fluorescent probe, PF1 and the novel boronate fluorescent probe PT1 as the covalent crosslinkers. Treatment with aqueous H2O2 allowed triggered release of the fluorescent dye accompanied by complete dissolution of the hydroge

Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in Escherichia coli O157.

Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens.IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.This work was supported by the Wellcome Trust (grant number WT098051). A.K.C. and C.J.B. were supported by the Medical Research Council (grant number G1100100/1). D.L.G. and A.S.L. were supported by BBSRC (UKRI) funding (programme number P013740)

Rapid geographical source attribution of Salmonella enterica serovar Enteritidis genomes using hierarchical machine learning.

Salmonella enterica serovar Enteritidis is one of the most frequent causes of Salmonellosis globally and is commonly transmitted from animals to humans by the consumption of contaminated foodstuffs. In the UK and many other countries in the Global North, a significant proportion of cases are caused by consumption of imported food products or contracted during foreign travel, therefore making the rapid identification of the geographical source of new infections a requirement for robust public health outbreak investigations. Herein, we detail the development and application of a hierarchical machine learning model to rapidly identify and trace the geographical source of S. Enteritidis infections from whole genome sequencing data. 2,313 S. Enteritidis genomes, collected by the UKHSA between 2014-2019, were used to train a 'local classifier per node' hierarchical classifier to attribute isolates to 4 continents, 11 sub-regions and 38 countries (53 classes). The highest classification accuracy was achieved at the continental level followed by the sub-regional and country levels (macro F1: 0.954, 0.718, 0.661 respectively). A number of countries commonly visited by UK travellers were predicted with high accuracy (hF1: >0.9). Longitudinal analysis and validation with publicly accessible international samples indicated that predictions were robust to prospective external datasets. The hierarchical machine learning framework provided granular geographical source prediction directly from sequencing reads in <4 minutes per sample, facilitating rapid outbreak resolution and real-time genomic epidemiology. The results suggest additional application to a broader range of pathogens and other geographically structured problems, such as antimicrobial resistance prediction, is warranted