Taxonomy of bacteria in the genomic era

• Author / Creator

  Orata, Fabini D

• Identification of bacterial isolates is important for taxonomic purposes, as well as to predict behaviour and properties of organisms in evolutionary, ecological, industrial, or medical contexts. This process embraces a polyphasic approach (phenotypic, genotypic, and phylogenetic), but has been ultimately based on comparisons of 16S rRNA gene sequences and genomic DNA–DNA hybridization (DDH). It was only in the past decade that modern taxonomy has begun the process of incorporating whole-genome sequencing (WGS) data, thanks to significant advancements in next-generation sequencing (NGS) technologies, to perform taxonomic assignments of bacteria. As such, genome-based pairwise similarity indexes were introduced as part of the taxonomic polyphasic approach. This includes multilocus sequence analysis of the core genome, in silico (digital) DDH (dDDH) and average nucleotide identity (ANI) for species delineations, and average amino acid identity (AAI) and the percentage of conserved proteins (POCP) for delineations within higher taxonomic ranks. For this thesis, WGS was employed in characterizing a novel species of Vibrio, Vibrio cidicii, which was only distinguishable from its closest known relative, Vibrio navarrensis, in one phenotypic test. The former, but not the latter, was able to utilize L-rhamnose as a sole carbon source. Pairwise ANI was calculated from the genomes of four isolates of each species, which ranged from 95.4–95.8% (same-species cutoff for ANI of 95–96%). Although ANI showed borderline results, pairwise dDDH results were more conclusive and ranged from 61.9–64.3% (same-species cutoff for DDH is 70%). This was further supported by phylogenetic analysis of the core genome, which placed the V. cidicii isolates into their own monophyletic clade, distinct from V. navarrensis. Whereas dDDH and ANI are becoming more commonly used in species characterizations and specifically demonstrated here to be useful in the characterization of a novel species, higher taxonomic ranks are still mostly circumscribed based on 16S rRNA gene sequence phylogeny. The use of 16S rRNA gene sequences was predominant in identifying organisms within the family Rhodobacteraceae, with the first isolates discovered in the early 1990s, the heyday of sequencing for this marker gene. Within this family is a monophyletic group of organisms called the roseobacter clade, plagued with inconsistencies in nomenclature. In another study described in this thesis, pairwise AAI and POCP were calculated from 290 high-quality whole-genome sequences with the aim of properly delineating genera within this family. Whereas the POCP metric did not show distinct delineations for genus, a cutoff for genus (70%) is proposed based on AAI. Using this cutoff, strains were reclassified to remove polyphyly and paraphyly in some genera. Additionally, we were able to determine 60% AAI as the newly proposed cutoff for family, reclassifying some members into a new family with the proposed name Stappiaceae fam. nov. WGS is useful beyond identification purposes. In a third study described in this thesis, the extent of interspecies horizontal gene transfer (HGT) was determined between two very closely related organisms, Vibrio cholerae and Vibrio metoecus, to demonstrate the role HGT plays in diversification and speciation. Vibrio metoecus is a recently described species that showed high 16S rRNA gene sequence identity with V. cholerae (98%), the causative agent of the potent diarrheal disease cholera. It was previously thought to be an atypical variant of V. cholerae. However, genome comparisons resulted in only 86–87% ANI between species, indicating that V. metoecus is a different species. Both species were co-isolated in a brackish coastal pond in the United States East Coast, which suggests that they are likely in constant interaction with each other. This presented an opportunity to study interspecies gene exchange in natural populations. Comparative genomic analysis showed a bias in interspecies recombination, where V. metoecus was a recipient of up to three times more genes from V. cholerae as it was the donor. Interestingly, it was determined by qPCR that V. cholerae is three times more abundant in the environment than V. metoecus, and the former was present throughout the summer but the latter was only detectable at the end of the season. This difference in abundance and seasonality could be major contributors in the HGT bias from V. cholerae to V. metoecus. WGS has also shown promise in epidemiological investigations and was proven to be a powerful tool in investigating the origin of the cholera outbreak in Haiti. After the devastating 2010 earthquake in Haiti, cholera spread quickly around the country. Initial epidemiological reports linked the outbreak to the United Nations peacekeeping troops deployed to Haiti from Nepal after news reports showed improper sewage treatment in their camp. Traditional epidemiological investigations were enhanced by WGS and phylogenetic analysis and allowed the determination of the exact source of the outbreak. The clinical V. cholerae isolates from Haiti showed very high genomic resemblance to clinical isolates from Nepal, with only one to two nucleotide differences in their core genome, supporting the hypothesis that pathogenic V. cholerae was imported to Haiti from an external source through human transmission. The various studies presented herein demonstrate the usefulness of WGS in taxonomy, population genetics, and microbial epidemiology. This is made possible by significant advances in NGS technologies as well as sequence analysis methods.

• Subjects / Keywords

  • Taxonomy
  • Genomics
  • DNA-DNA hybridization
  • Whole-genome sequencing
  • Average amino acid identity
  • Average nucleotide identity
  • Next-generation sequencing

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R38S4K53H

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.