Optimisation of a 1,000 year-old antimicrobial recipe

Leechdoms is a term given to medieval era Anglo-saxon remedies which the historian ML Cameron believed to be a meticulous collection of medical texts and knowledge from several cultures. His hypothesis was that these recipes, for years dismissed as useless and ineffective, had merit as medical treatments. In 2015, Harrison et al. published a paper in which they recreated a recipe from a 10th century book known as Bald’s Leechbook. This recipe was that of an eyesalve designed to treat a stye in the eye. The authors showed that their preparation of Bald’s eyesalve proved to be a powerful antimicrobial, effective against antibiotic resistant S. aureus strains. This thesis builds on this work in two ways. First to simplify and optimise the recipe without sacrificing efficacy. Second to refine and isolate active components from the eyesalve to narrow down the list of candidate active components. Attempts to simplify the recipe showed that the wine required in the recipe is key to activity, but is independent of the type of wine used for the preparation. It was demonstrated that the number of days it takes to prepare an active batch of eyesalve can be reduced from nine days to one. The eyesalve remained stable when incorporated into molten agar which was demonstrated to be useful in screening some species for sensitivity to the eyesalves antimicrobial effects. Four different species, S. aureus, K. pneumoniae, A. baumanii and E. coli were screened for growth on eyesalve treated plates and only S. aureus was found to grow, demonstrating that the salve may specifically target certain species of bacteria. Fractionation experiments generated fractions with antimicrobial activity, however the specific method used was not very sensitive and as such narrowed the potential active components to a relatively broad spectrum of compounds. In summary, the data presented here provides significant advancements into understanding how old recipes may have functioned against ancient bacterial infections and provides a unique novel avenue for the discovery of antibiotics

Tuberculosis in badgers where the bovine tuberculosis epidemic is expanding in cattle in England.

Bovine tuberculosis (bTB) is an important animal health and economic problem for the cattle industry and a potential zoonotic threat. Wild badgers (Meles meles) play a role on its epidemiology in some areas of high prevalence in cattle, particularly in the UK and Republic of Ireland and increasingly in parts of mainland Europe. However, little is known about the involvement of badgers in areas on the spatial edge of the cattle epidemic, where increasing prevalence in cattle is seen. Here we report the findings of a study of found-dead (mainly road-killed) badgers in six counties on the edge of the English epidemic of bTB in cattle. The overall prevalence of Mycobacterium tuberculosis complex (MTC) infection detected in the study area was 51/610 (8.3%, 95% CI 6.4-11%) with the county-level prevalence ranging from 15 to 4-5%. The MTC spoligotypes of recovered from badgers and cattle varied: in the northern part of the study area spoligotype SB0129 predominated in both cattle and badgers, but elsewhere there was a much wider range of spoligotypes found in badgers than in cattle, in which infection was mostly with the regional cattle spoligotype. The low prevalence of MTC in badgers in much of the study area, and, relative to in cattle, the lower density of sampling, make firm conclusions difficult to draw. However, with the exception of Cheshire (north-west of the study area), little evidence was found to link the expansion of the bTB epidemic in cattle in England to widespread badger infection

Optimisation of a 1,000 year-old antimicrobial recipe

Leechdoms is a term given to medieval era Anglo-saxon remedies which the historian ML Cameron believed to be a meticulous collection of medical texts and knowledge from several cultures. His hypothesis was that these recipes, for years dismissed as useless and ineffective, had merit as medical treatments. In 2015, Harrison et al. published a paper in which they recreated a recipe from a 10th century book known as Bald’s Leechbook. This recipe was that of an eyesalve designed to treat a stye in the eye. The authors showed that their preparation of Bald’s eyesalve proved to be a powerful antimicrobial, effective against antibiotic resistant S. aureus strains. This thesis builds on this work in two ways. First to simplify and optimise the recipe without sacrificing efficacy. Second to refine and isolate active components from the eyesalve to narrow down the list of candidate active components. Attempts to simplify the recipe showed that the wine required in the recipe is key to activity, but is independent of the type of wine used for the preparation. It was demonstrated that the number of days it takes to prepare an active batch of eyesalve can be reduced from nine days to one. The eyesalve remained stable when incorporated into molten agar which was demonstrated to be useful in screening some species for sensitivity to the eyesalves antimicrobial effects. Four different species, S. aureus, K. pneumoniae, A. baumanii and E. coli were screened for growth on eyesalve treated plates and only S. aureus was found to grow, demonstrating that the salve may specifically target certain species of bacteria. Fractionation experiments generated fractions with antimicrobial activity, however the specific method used was not very sensitive and as such narrowed the potential active components to a relatively broad spectrum of compounds. In summary, the data presented here provides significant advancements into understanding how old recipes may have functioned against ancient bacterial infections and provides a unique novel avenue for the discovery of antibiotics

Improving bacterial genomic characterisation in M. bovis and E. coli

This thesis explores the use of genomics to understand two organisms, Mycobacterium bovis and Escherichia coli. M. bovis, the causative agent of tuberculosis in cattle, was isolated from a known wildlife host, badgers. The badgers in this study were collected as roadkill by a network of stakeholders and the study investigators. Over 700 badgers were collected, and approximately 640 useable carcasses were obtained. From these carcasses, ~12% were culture positive for M. bovis colonies, as confirmed through IS6110 PCR. Spoligotyping was performed on these isolates using a microarray. M. bovis colonies were sequenced and analysed in a variety of ways. Molecular spoligotyping was found to conform to In silico spoligotyping, but In silico typing was better able to resolve ambiguous types. Comparative analysis of the genomes found gaps in the genomes across several isolates. A control set of M. bovis reads from a badger were assembled using the same process to confirm that these regions were not an error in the method. Only one genomic gap was found not to be an artefact of the methods used. SNP analysis of the badger M. bovis sequences, combined with their geographical metadata, shows that there might be evidence of an additional transmission driver between badgers - due to the distances separating the identified clades. The small numbers of M. bovis isolates obtained in this study would likely not be enough to perform quantitative transmission dynamics analysis but highlights the difficulty in obtaining useful sequence from M. bovis in badgers, which is key to understanding multi-host transmission dynamics. One isolate in this study M. bovis 867, was selected for sequencing using long-read sequencing technologies. Comparative analysis of the hybrid assembled M. bovis genome to the existing reference genomes found several SNPs distinguishing it from the reference. These SNPs were explored based on their predicted effects on protein translation and how this may phenotypically alter these isolates. However, phenotypic studies must be carried out to confirm these predictions. Overall, a method is presented for getting HMW, long-fragment DNA from M. bovis, from which long-read data can be generated. The data show the improvements to assemblies using a hybrid approach. An additional study involving E. coli is presented as the final chapter of this thesis. This chapter investigates the improvements that can be made using hybrid assembly methods in microbial genomics. Twelve putative E. coli isolated from the feet of sheep on a farm were selected for hybrid assembly. From these 12 E. coli, whole contiguous plasmid sequences were resolved, including a 120kb megaplasmid carrying several AMR genes. The use of hybrid assembly methods also elucidated a Tn21-like transposon, a powerful driver of AMR not often seen in farm environments in the UK

Improving bacterial genomic characterisation in M. bovis and E. coli

This thesis explores the use of genomics to understand two organisms, Mycobacterium bovis and Escherichia coli. M. bovis, the causative agent of tuberculosis in cattle, was isolated from a known wildlife host, badgers. The badgers in this study were collected as roadkill by a network of stakeholders and the study investigators. Over 700 badgers were collected, and approximately 640 useable carcasses were obtained. From these carcasses, ~12% were culture positive for M. bovis colonies, as confirmed through IS6110 PCR. Spoligotyping was performed on these isolates using a microarray. M. bovis colonies were sequenced and analysed in a variety of ways. Molecular spoligotyping was found to conform to In silico spoligotyping, but In silico typing was better able to resolve ambiguous types. Comparative analysis of the genomes found gaps in the genomes across several isolates. A control set of M. bovis reads from a badger were assembled using the same process to confirm that these regions were not an error in the method. Only one genomic gap was found not to be an artefact of the methods used. SNP analysis of the badger M. bovis sequences, combined with their geographical metadata, shows that there might be evidence of an additional transmission driver between badgers - due to the distances separating the identified clades. The small numbers of M. bovis isolates obtained in this study would likely not be enough to perform quantitative transmission dynamics analysis but highlights the difficulty in obtaining useful sequence from M. bovis in badgers, which is key to understanding multi-host transmission dynamics. One isolate in this study M. bovis 867, was selected for sequencing using long-read sequencing technologies. Comparative analysis of the hybrid assembled M. bovis genome to the existing reference genomes found several SNPs distinguishing it from the reference. These SNPs were explored based on their predicted effects on protein translation and how this may phenotypically alter these isolates. However, phenotypic studies must be carried out to confirm these predictions. Overall, a method is presented for getting HMW, long-fragment DNA from M. bovis, from which long-read data can be generated. The data show the improvements to assemblies using a hybrid approach. An additional study involving E. coli is presented as the final chapter of this thesis. This chapter investigates the improvements that can be made using hybrid assembly methods in microbial genomics. Twelve putative E. coli isolated from the feet of sheep on a farm were selected for hybrid assembly. From these 12 E. coli, whole contiguous plasmid sequences were resolved, including a 120kb megaplasmid carrying several AMR genes. The use of hybrid assembly methods also elucidated a Tn21-like transposon, a powerful driver of AMR not often seen in farm environments in the UK

Mobilisation and analyses of publicly available SARS-CoV-2 data for pandemic responses

The COVID-19 pandemic has seen large-scale pathogen genomic sequencing efforts, becoming part of the toolbox for surveillance and epidemic research. This resulted in an unprecedented level of data sharing to open repositories, which has actively supported the identification of SARS-CoV-2 structure, molecular interactions, mutations and vari-ants, and facilitated vaccine development and drug reuse studies and design. The European COVID-19 Data Platform was launched to support this data sharing, and has resulted in the deposition of several million SARS-CoV-2 raw reads. In this paper we describe (1) open data sharing, (2) tools for submission, analysis, visualisation and data claiming (e.g. ORCiD), (3) the systematic analysis of these datasets, at scale via the SARS-CoV-2 Data Hubs as well as (4) lessons learnt. This paper describes a component of the Platform, the SARS-CoV-2 Data Hubs, which enable the extension and set up of infrastructure that we intend to use more widely in the future for pathogen surveillance and pandemic preparedness.</p

Mobilisation and analyses of publicly available SARS-CoV-2 data for pandemic responses

The COVID-19 pandemic has seen large-scale pathogen genomic sequencing efforts, becoming part of the toolbox for surveillance and epidemic research. This resulted in an unprecedented level of data sharing to open repositories, which has actively supported the identification of SARS-CoV-2 structure, molecular interactions, mutations and vari-ants, and facilitated vaccine development and drug reuse studies and design. The European COVID-19 Data Platform was launched to support this data sharing, and has resulted in the deposition of several million SARS-CoV-2 raw reads. In this paper we describe (1) open data sharing, (2) tools for submission, analysis, visualisation and data claiming (e.g. ORCiD), (3) the systematic analysis of these datasets, at scale via the SARS-CoV-2 Data Hubs as well as (4) lessons learnt. This paper describes a component of the Platform, the SARS-CoV-2 Data Hubs, which enable the extension and set up of infrastructure that we intend to use more widely in the future for pathogen surveillance and pandemic preparedness.</p