Occurrence and characterization of Escherichia coli ST410 co-harbouring blaNDM-5, blaCMY-42 and blaTEM-190 in a dog from the UK.

Background/Objectives:Carbapenemase-producing Enterobacteriaceae (CPE) are a public health threat, and have been found in humans, animals and the environment. Carbapenems are not authorized for use in EU or UK companion animals, and the prevalence of carbapenem-resistant Gram-negative bacilli (CRGNB) in this population is unknown. Methods:We investigated CRGNB isolated from animal specimens received by one diagnostic laboratory from 34 UK veterinary practices (September 2015-December 2016). Any Gram-negative isolates from clinical specimens showing reduced susceptibility to fluoroquinolones and/or aminoglycosides and/or cephalosporins were investigated phenotypically and genotypically for carbapenemases. A complete genome assembly (Illumina/Nanopore) was generated for the single isolate identified to investigate the genetic context for carbapenem resistance. Results:One ST410 Escherichia coli isolate [(CARB35); 1/191, 0.5%], cultured from a wound in a springer spaniel, harboured a known carbapenem resistance gene (blaNDM-5). The gene was located in the chromosome on an integrated 100 kb IncF plasmid, also harbouring other drug resistance genes (mrx, sul1, ant1 and dfrA). The isolate also contained blaCMY-42 and blaTEM-190 on two separate plasmids (IncI1 and IncFII, respectively) that showed homology with other publicly available plasmid sequences from Italy and Myanmar. Conclusions:Even though the use of carbapenems in companion animals is restricted, the concurrent presence of blaCMY-42 and other antimicrobial resistance genes could lead to co-selection of carbapenemase genes in this population. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst humans and companion animals are needed

Analysis of complex genetic variation using population reference graphs

In this thesis, I study the problem of genome inference from short-read DNA sequencing data, with the goal of accurate characterisation of genomic regions with high sequence diversity. I describe a set of novel algorithms based on a generalised reference genome that captures sequence variation within a species. In Chapter 3, I propose a novel data structure that extends the traditional reference genome with known variants, providing a compressed representation of genetic diversity. I present algorithms to match sequencing reads to this extended reference structure and infer a personalised reference genome within close genetic distance from the sample under analysis. Coupled with existing variant calling tools, this personalised reference confers increased power to detect complex variants in diverse regions, compared to the traditional reference genome. In Chapter 4, I evaluate the performance of the method on simulated data and show that it is viable for megabase-sized genomes such as the malaria parasite Plasmodium falciparum -- a typical sample can be analysed in 5.7h on a single CPU, using a small amount of memory. I suggest a number of future optimisations to improve computational efficiency. In Chapter 5, I apply my method to 1300 Plasmodium falciparum samples from across the world and study two hyper-diverse genes that encode surface antigens in Plasmodium falciparum. I show that the personalised references recover variants of these genes that are missed by standard techniques of mapping reads to the traditional reference genome. Next, I build the first global variation catalogue incorporating dimorphic alleles of a region of functional interest and study their frequency patterns.</p

A natural encoding of genetic variation in a Burrows-Wheeler Transform to enable mapping and genome inference

We show how positional markers can be used to encode genetic variation within a Burrows-Wheeler Transform (BWT), and use this to construct a generalisation of the traditional “reference genome”, incorporating known variation within a species. Our goal is to support the inference of the closest mosaic of previously known sequences to the genome(s) under analysis. Our scheme results in an increased alphabet size, and by using a wavelet tree encoding of the BWT we reduce the performance impact on rank operations. We give a specialised form of the backward search that allows variation-aware exact matching. We implement this, and demonstrate the cost of constructing an index of the whole human genome with 8 million genetic variants is 25GB of RAM. We also show that inferring a closer reference can close large kilobase-scale coverage gaps in P. falciparum

Occurrence and characterization of Escherichia coli ST410 co-harbouring blaNDM-5, blaCMY-42 and blaTEM-190 in a dog from the UK

Background/Objectives:Carbapenemase-producing Enterobacteriaceae (CPE) are a public health threat, and have been found in humans, animals and the environment. Carbapenems are not authorized for use in EU or UK companion animals, and the prevalence of carbapenem-resistant Gram-negative bacilli (CRGNB) in this population is unknown. Methods:We investigated CRGNB isolated from animal specimens received by one diagnostic laboratory from 34 UK veterinary practices (September 2015-December 2016). Any Gram-negative isolates from clinical specimens showing reduced susceptibility to fluoroquinolones and/or aminoglycosides and/or cephalosporins were investigated phenotypically and genotypically for carbapenemases. A complete genome assembly (Illumina/Nanopore) was generated for the single isolate identified to investigate the genetic context for carbapenem resistance. Results:One ST410 Escherichia coli isolate [(CARB35); 1/191, 0.5%], cultured from a wound in a springer spaniel, harboured a known carbapenem resistance gene (blaNDM-5). The gene was located in the chromosome on an integrated 100 kb IncF plasmid, also harbouring other drug resistance genes (mrx, sul1, ant1 and dfrA). The isolate also contained blaCMY-42 and blaTEM-190 on two separate plasmids (IncI1 and IncFII, respectively) that showed homology with other publicly available plasmid sequences from Italy and Myanmar. Conclusions:Even though the use of carbapenems in companion animals is restricted, the concurrent presence of blaCMY-42 and other antimicrobial resistance genes could lead to co-selection of carbapenemase genes in this population. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst humans and companion animals are needed

Flow instability in a vertical annulus under steady state and transient conditions

Experimental data have been obtained for the occurrence of flow instability in a vertical annulus under steady state and transient down-flow conditions. The results show that the primary factor affecting the onset of flow instability is the Q{sub ratio} parameter (a measure of the void production in a channel). In addition, it is shown that for steady state conditions good agreement exists between the single annulus data and single tube data based upon the same L/D ratio. The results also indicate that under transient operation the Q{sub ratio} can be used as a stability criterion