Whole Genome Analyses Accurately Identify Neisseria spp. and Limit Taxonomic Ambiguity

Abstract

Genome sequencing facilitates the study of bacterial taxonomy and allows the re-evaluation of the taxonomic relationships between species. Here, we aimed to analyze the draft genomes of four commensal Neisseria clinical isolates from the semen of infertile Lebanese men. To determine the phylogenetic relationships among these strains and other Neisseria spp. and to confirm their identity at the genomic level, we compared the genomes of these four isolates with the complete genome sequences of Neisseria gonorrhoeae and Neisseria meningitidis and the draft genomes of Neisseria flavescens, Neisseria perflava, Neisseria mucosa, and Neisseria macacae that are available in the NCBI Genbank database. Our findings revealed that the WGS analysis accurately identified and corroborated the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) species identities of the Neisseria isolates. The combination of three well-established genome-based taxonomic tools (in silico DNA-DNA Hybridization, Ortho Average Nucleotide identity, and pangenomic studies) proved to be relatively the best identification approach. Notably, we also discovered that some Neisseria strains that are deposited in databases contain many taxonomical errors. The latter is very important and must be addressed to prevent misdiagnosis and missing emerging etiologies. We also highlight the need for robust cut-offs to delineate the species using genomic tools.

Phylogenomic analysis of the understudiedNeisseriaceaespecies reveals a poly- and paraphyleticKingellagenus

“…Core genes were clustered at the 75%, 80%, 85%, 90%, and 95% sequence similarity level and were limited to only those clusters of orthologous groups (COGs) found in all genomes (-cd 100), unless otherwise noted. Additionally, other recent evolutionary study of these taxa has used less stringent similarity and gene presence cutoff to define the core genome for species in the Neisseriaceae for subsequent phylogenetic analyses (8, 34). We replicated this analysis by reanalyzing the data using a sequence similarity cut off of 50% for genes present in 99% or 80% of analyzed genomes.…”

“…As whole genome sequences became readily available, a newer version of this system, taxonomogenomics, seeks to incorporate this rich genomic data into taxonomical descriptions (3)(4)(5)(6). This approach also has the benefit of being explicitly phylogenetic, providing a non-arbitrary approach to classification (5,7,8). While there is still controversy about how best to integrate phenotypic and phylogenomic data in bacterial taxonomy, there is a general consensus that taxonomy should be reflective of the evolutionary relationships and that monophyly is a desirable characteristic of taxonomic groups.…”

“…The majority of prior work devoted to understanding the taxonomic relationships between these genera has focused primarily on the pathogenic Neisseria species, N. meningitidis and N. gonorrhoeae (2,8,(12)(13)(14)(15)(16)(17). Early work using 16S rRNA failed to properly resolve species boundaries, resulting in several polyphyletic genera (18).…”

“…The Neisseriaceae family is a group of Gram-negative, β-proteobacteria that currently includes 20 genera: Neisseria, Wielerella, Vitreoscilla, Uruburuella, Stenoxybacter, Snodgrassela, Simonsiella, Rivicola, Prolinoborus, Paralysiella, Morococcus, Kingella, Eikenella, Crenobacter, Craterilacuibacter, Conchiformibius, Bergeriella, Aquella, Amantichitinum , and Alysiella (9–11). The majority of prior work devoted to understanding the taxonomic relationships between these genera has focused primarily on the pathogenic Neisseria species, N. meningitidis and N. gonorrhoeae (2, 8, 12–17). Early work using 16S rRNA failed to properly resolve species boundaries, resulting in several polyphyletic genera (18).…”

“…HGT results in the formation of ill-defined species with ambiguous species boundaries (20). To help resolve this ambiguity, recent studies of the commensal Neisseria species employed core genome MLST (cgMLST) and proposed genus specific cut-offs for defining species using genome relatedness indices (8, 19, 20). However, these studies did not comprehensively address other polyphyletic genera within the Neisseriaceae .…”