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High-throughput genomic analysis of virulence and resistance mechanisms among microbial pathogens

  • Author(s): Tewhey, Ryan Stephen
  • et al.
Abstract

The rapid development of high-throughput sequencing technologies poses to transform many aspects of human health. It is without question that within the next decade the ability to rapidly sequence any infection to determine the genetic structure of the etiologic agent will be feasible. To effectively utilize such an approach we must first develop a greater understanding of the genetic variation of clonal microbial pathogens as well as the functional significance of the variation found. To this extent I have focused on three projects. i.) By sequencing 36 community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) from within a defined geographical area we are able to understand how the genome is evolving overtime. Sequence analysis of the strains suggests the population is under diversifying selection for genes related in virulence. In addition, mutational burden of individual isolates correlates with the severity of infection suggesting the CA-MRSA is evolving away from it's hyper-virulent ancestor to become less pathogenic. ii.) Acquiring resistance to the gram-positive antimicrobial daptomycin has previously been shown to be a complicated process that can be achieved through multiple routes. I describe the non-linear multi-allelic acquisition of resistance in a laboratory evolved strain of Bacillus subtilis. Through the use of allelic replacement and transcriptomic analysis three loci (dctP/ ybdI, yvrG, yqeG) are shown to be involved in the resistance phenotype through the alteration of cell wall and cell membrane metabolism pathways. iii.) Finally, I present methods to acquire rare sequences from complex pools of DNA. I demonstrate two such methods, microdroplet PCR and RNA/DNA hybridization to accomplish this. I apply these methods to the capturing of Plasmodium vivax from dried whole blood spots thus alleviating the need to leukocyte filtration. I discuss future application for the integrating all three studies towards the use of clinical sequencing for the detection and monitoring of infectious diseases

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