An investigation into antimicrobial production in the Lactobacillus genus and the fish microbiome

This thesis outlines a study of the identification and characterisation of antimicrobials from two primary sources; the Lactobacillus genus and the microbiome of fish. Through the incorporation of a wide variety of techniques, this study successfully demonstrates how a variety of methods can be used for the identification and production of novel antimicrobials. Chapter 1 gives an overview of the variety of different antimicrobials which can be produced by the lactic acid bacteria (LAB) which can play an important role in a number of processes, such as the preservation of fermented foods. These antimicrobials can vary from organic acids, such as lactic and acetic acid, to antimicrobial peptides known as ‘bacteriocins’. Chapter 2 outlines the identification of a novel bacteriocin known as ‘formicin’ through traditional colony isolation and screening methods. Formicin was identified from Bacillus paralicheniformis APC1576, an antimicrobial producing strain isolated from the intestine of a fish. Using a combination of mass spectrometry and genomic screening, formicin was found to be a two-peptide lantibiotic, displaying antimicrobial activity against a broad range of Gram positive microbes. In Chapter 3, through a combination of in silico and lab-based screening of the Lactobacillus pangenome, it was possible to determine the extent and diversity of bacteriocins encoded and produced by the genus. This study shows that bacteriocin production may not be as prevalent as previously believed, however of the bacteriocins which were identified from within the genomes, many were found to be novel. By screening the strains identified as harbouring bacteriocin-related genes, five novel active bacteriocins were identified. Many strains of lactobacilli were found to encode bacteriocins, however upon analysis, these failed to display in vitro antimicrobial activity. Often the regulation of bacteriocin operons, or the loss of key bacteriocin-associated genes, results in the failure of these strains to produce bacteriocins when tested in vitro. Chapter 4 outlines a method for the heterologous expression of a particular class of bacteriocins, the Class IIa ‘pediocin-like’ bacteriocins. Here, using an expression designed for Class IIa bacteriocins, it was possible to produce eight novel bacteriocins identified from genomic data. In Chapter 5 shotgun metagenomic sequencing is used to characterise the compositional and functional properties of the intestinal microbiome of deep sea fish. Here it can be seen how bacteria have adapted to live in this environment by encoding systems to relieve the stress associated with life at higher pressures. The study also outlines the diversity of the potential antimicrobials which may be produced within the microbiome of such fish whilst also highlighting an apparent lack of genes associated with known mechanisms of antibiotic resistance. Overall, the results of this work demonstrate the effectiveness of a variety of methods for identifying novel antimicrobials from a range of bacterial sources

Influence of Anode Functional Layers on Electrochemical Performance and Mechanical Strength in Microtubular Solid Oxide Fuel Cells Fabricated by Gel-Casting

Anode-supported microtubular solid oxide fuel cells (mT-SOFCs) using samaria-doped ceria (SDC) as electrolyte were fabricated, varying the composition and number of anode functional layers (AFLs), by combining the aqueous gel-casting and spray-coating techniques. Suitable aqueous slurry formulation of NiO-SDC was prepared using agarose as a gelling agent for gel casting of tubular supports. Afterward, 40:60 and 50:50 NiO:SDC (wt %) as AFLs and SDC electrolyte were deposited by spray-coating and subsequently co-sintered. Finally, mT-SOFCs with 2.5 mm outer diameter and thicknesses of 380 pm support; 0, 12, and 24 mu m AFLs; 15 mu m electrolyte; and 30 mu m cathode were obtained. The influence of AFLs on the performance and mechanical integrity was investigated for the three cells. For this purpose, electrochemical and mechanical tests at both macroscopic and micro-/nanometric scales (at the AFLs region) were determined by flexural strength and nanoindentation techniques, respectively. The results evidence that the use of AFLs with an adequate composition and microstructure in the mT-SOFCs is required to improve the performance and mechanical strength of the cell. The cell with a single-layer AFL of 50:50 NiO:SDC (wt %) and 12 pm thickness exhibited the best performance (0.52 W.cm(-2)) at 650 degrees C using hydrogen as fuel and air as oxidant

Synchronous genitourinary lichen sclerosus signals a distinct urinary microbiome profile in men with urethral stricture disease.

PurposeAlterations in the urinary microbiome have been associated with urological diseases. The microbiome of patients with urethral stricture disease (USD) remains unknown. Our objective is to examine the microbiome of USD with a focus on inflammatory USD caused by lichen sclerosus (LS).MethodsWe collected mid-stream urine samples from men with LS-USD (cases; n = 22) and non-LS USD (controls; n = 76). DNA extraction, PCR amplification of the V4 hypervariable region of the 16S rRNA gene, and sequencing was done on the samples. Operational taxonomic units (OTUs) were defined using a > 97% sequence similarity threshold. Alpha diversity measurements of diversity, including microbiome richness (number of different OTUs) and evenness (distribution of OTUs) were calculated and compared. Microbiome beta diversity (difference between microbial communities) relationships with cases and controls were also assessed.ResultsFifty specimens (13 cases and 37 controls) produced a 16S rRNA amplicon. Mean sample richness was 25.9 vs. 16.8 (p = 0.076) for LS-USD vs. non-LS USD, respectively. LS-USD had a unique profile of bacteria by taxonomic order including Bacillales, Bacteroidales and Pasteurellales enriched urine. The beta variation of observed bacterial communities was best explained by the richness.ConclusionsMen with LS-USD may have a unique microbiologic richness, specifically inclusive of Bacillales, Bacteroidales and Pasteurellales enriched urine compared to those with non-LS USD. Further work will be required to elucidate the clinical relevance of these variations in the urinary microbiome

HMOX1 genetic polymorphisms and outcomes in infectious disease:a systematic review

INTRODUCTION: Heme-oxygenase 1 (HMOX1) is a critical stress response gene that catalyzes the multistep oxidation of heme. A GT(n) repeat of variable length in the promoter in has been associated with a wide range of human diseases, including infections. This paper aims to summarise and systematically review associations between the length of the HMOX1 GT(n) promoter and infectious disease in humans. METHODS: A search using relevant terms was performed in PubMED and EMBASE through to 15/01/21 identifying all research that studied an association between the HMOX1 GT(n) repeat polymorphism and the incidence and/or outcome of any human infectious disease. Citations were screened for additional studies. Potential studies were screened for inclusion by two authors. Data was extracted on allele frequency, genotype, strength of association, mechanism of genotyping, and potential biases. A narrative review was performed across each type of infection. RESULTS: 1,533 studies were identified in the search, and one via citation screening. Sixteen studies were ultimately included, seven in malaria, three in HIV, three in sepsis, and one each in pneumonia, hepatitis C, and acute respiratory distress syndrome (ARDS). Sample sizes for nearly all studies were small (biggest study, n = 1,646). Allelic definition was different across all included studies. All studies were at some risk of bias. In malaria, three studies suggested that longer alleles were associated with reduced risk of severe malaria, particularly malaria-induced renal dysfunction, with four studies identifying a null association. In sepsis, two studies suggested an association with longer alleles and better outcomes. CONCLUSIONS: Despite the importance of HMOX1 in survival from infection, and the association between repeat length and gene expression, the clinical data supporting an association between repeat length and incidence and/or outcome of infection remain inconclusive

Adolescent ambivalence about diabetes technology—The Janus faces of automated care

The Janus face metaphor approach highlights that a technology may simultaneously have two opposite faces or properties with unforeseen paradoxes within human‐technology interaction. Suboptimal acceptance and clinical outcomes are sometimes seen in adolescents who use diabetes‐related technologies. A traditional linear techno‐determinist model of technology use would ascribe these unintended outcomes to suboptimal technology, suboptimal patient behavior, or suboptimal outcome measures. This paradigm has demonstratively not been successful at universally improving clinical outcomes over the last two decades. Alternatively, the Janus face metaphor moves away from a linear techno‐determinist model and focuses on the dynamic interaction of the human condition and technology. Specifically, it can be used to understand variance in adoption or successful use of diabetes‐related technology and to retrospectively understand suboptimal outcomes. The Janus face metaphor also allows for a prospective exploration of potential impacts of diabetes‐related technology by patients, families, and their doctors so as to anticipate and minimize potential subsequent tensions

Reincarnation of Bacteriocins From the Lactobacillus Pangenomic Graveyard

peer-reviewedBacteria commonly produce narrow spectrum bacteriocins as a means of inhibiting closely related species competing for similar resources in an environment. The increasing availability of genomic data means that it is becoming easier to identify bacteriocins encoded within genomes. Often, however, the presence of bacteriocin genes in a strain does not always translate into biological antimicrobial activity. For example, when analysing the Lactobacillus pangenome we identified strains encoding ten pediocin-like bacteriocin structural genes which failed to display inhibitory activity. Nine of these bacteriocins were novel whilst one was identified as the previously characterized bacteriocin “penocin A.” The composition of these bacteriocin operons varied between strains, often with key components missing which are required for bacteriocin production, such as dedicated bacteriocin transporters and accessory proteins. In an effort to functionally express these bacteriocins, the structural genes for the ten pediocin homologs were cloned alongside the dedicated pediocin PA-1 transporter in both Escherichia coli and Lactobacillus paracasei heterologous hosts. Each bacteriocin was cloned with its native leader sequence and as a fusion protein with the pediocin PA-1 leader sequence. Several of these bacteriocins displayed a broader spectrum of inhibition than the original pediocin PA-1. We show how potentially valuable bacteriocins can easily be “reincarnated” from in silico data and produced in vitro despite often lacking the necessary accompanying machinery. Moreover, the study demonstrates how genomic datasets such as the Lactobacilus pangenome harbor a potential “arsenal” of antimicrobial activity with the possibility of being activated when expressed in more genetically amenable hosts

Accurate crop yield predictions from modelling tree-crop interactions in gliricidia-maize agroforestry

Agroforestry systems, containing mixtures of trees and crops, are often promoted because the net effect of interactions between woody and herbaceous components is thought to be positive if evaluated over the long term. From a modelling perspective, agroforestry has received much less attention than monocultures. However, for the potential of agroforestry to impact food security in Africa to be fully evaluated, models are required that accurately predict crop yields in the presence of trees. The positive effects of the fertiliser tree gliricidia (Gliricidia sepium) on maize (Zea mays) are well documented and use of this tree-crop combination to increase crop production is expanding in several African countries. Simulation of gliricidia-maize interactions can complement field trials by predicting crop response across a broader range of contexts than can be achieved by experimentation alone. We tested a model developed within the APSIM framework. APSIM models are widely used for one dimensional (1D), process-based simulation of crops such as maize and wheat in monoculture. The Next Generation version of APSIM was used here to test a 2D agroforestry model where maize growth and yield varied spatially in response to interactions with gliricidia. The simulations were done using data for gliricidia-maize interactions over two years (short-term) in Kenya and 11 years (long-term) in Malawi, with differing proportions of trees and crops and contrasting management. Predictions were compared with observations for maize grain yield, and soil water content. Simulations in Kenya were in agreement with observed yields reflecting lower observed maize germination in rows close to gliricidia. Soil water content was also adequately simulated, except for a tendency for slower simulated drying of the soil profile each season. Simulated maize yields in Malawi were also in agreement with observations. Trends in soil carbon over a decade were similar to those measured, but could not be statistically evaluated. These results show that the agroforestry model in APSIM Next Generation adequately represented tree-crop interactions in these two contrasting agro-ecological conditions and agroforestry practices. Further testing of the model is warranted to explore tree-crop interactions under a wider range of environmental conditions

Retention of brivaracetam in adults with drug-resistant epilepsy at a single tertiary care center

INTRODUCTION: Brivaracetam (BRV) is licensed as an adjunctive treatment for focal epilepsy. We describe our clinical experience with BRV at a large UK tertiary center. METHODS: Adults initiated on BRV between July 2015 and July 2020 were followed up until they discontinued BRV or September 2021. Data on epilepsy syndrome, duration, seizure types, concomitant and previous antiseizure medication (ASM) use, BRV dosing, efficacy, and side effects were recorded. Efficacy was categorized as temporary (minimum three months) or ongoing (at last follow-up) seizure freedom, ≥50% seizure reduction, or other benefits (e.g., no convulsions or daytime seizures). Brivaracetam retention was estimated using Kaplan-Meier survival analysis. RESULTS: Two-hundred people were treated with BRV, of whom 81% had focal epilepsy. The mean (interquartile range [IQR]) follow-up time was 707 (688) days, and the dose range was 50-600 mg daily. The mean (IQR) of the previous number of used ASMs was 6.9 (6.0), and concomitant use was 2.2 (1.0). One-hundred and eighty-eight people (94%) had previously discontinued levetiracetam (LEV), mainly due to side effects. 13/200 (6.5%) were seizure free for a minimum of six months during treatment, and 46/200 (23%) had a ≥50% reduction in seizure frequency for six months or more. Retention rates were 83% at six months, 71% at 12 months, and 57% at 36 months. Brivaracetam was mostly discontinued due to side effects (38/75, 51%) or lack of efficacy (28/75, 37%). Concomitant use of carbamazepine significantly increased the hazard ratio of discontinuing BRV due to side effects (p = 0.006). The most commonly reported side effects were low mood (20.5%), fatigue (18%) and aggressive behavior (8.5%). These side effects were less prevalent than when the same individuals took LEV (low mood, 59%; aggressive behavior, 43%). Intellectual disability was a risk factor for behavioral side effects (p = 0.004), and a pre-existing mood disorder significantly increased the likelihood of further episodes of low mood (p = 0.019). CONCLUSIONS: Brivaracetam was effective at a broad range of doses in managing drug-resistant epilepsy across various phenotypes, but less effective than LEV in those who switched due to poor tolerability on LEV. There were no new tolerability issues, but 77% of the individuals experiencing side effects on BRV also experienced similar side effects on LEV