Genomic characterization of novel Neisseria species

Abstract

Of the ten human-restricted Neisseria species two, Neisseria meningitidis, and Neisseria gonorrhoeae, cause invasive disease: the other eight are carried asymptomatically in the pharynx, possibly modulating meningococcal and gonococcal infections. Consequently, characterizing their diversity is important for understanding the microbiome in health and disease. Whole genome sequences from 181 Neisseria isolates were examined, including those of three well-defined species (N. meningitidis; N. gonorrhoeae; and Neisseria polysaccharea) and genomes of isolates unassigned to any species (Nspp). Sequence analysis of ribosomal genes, and a set of core (cgMLST) genes were used to infer phylogenetic relationships. Average Nucleotide Identity (ANI) and phenotypic data were used to define species clusters, and morphological and metabolic differences among them. Phylogenetic analyses identified two polyphyletic clusters (N. polysaccharea and Nspp.), while, cgMLST data grouped Nspp isolates into nine clusters and identified at least three N. polysaccharea clusters. ANI results classified Nspp into seven putative species, and also indicated at least three putative N. polysaccharea species. Electron microscopy identified morphological differences among these species. This genomic approach provided a consistent methodology for species characterization using distinct phylogenetic clusters. Seven putative novel Neisseria species were identified, confirming the importance of genomic studies in the characterization of the genus Neisseria.

Figure 1

Hierarchical gene by gene analysis. Maximum-likelihood trees generated from the aligned sequences of f_rplF (A); the concatenated sequences of 51 rMLST loci (B) and the concatenated sequences of the 1114 complete cgMLST loci out of the 1441 (C). The orange stars correspond to the isolates selected in the first ANI analysis (Table 2) and the purple stars to those added in the ANI analysis done only among the N. polysaccharea clusters. The purple circles group Np isolates from specific geographical location. Countries of isolation of Nspp clusters are indicated. Np: N. polysaccharea and Nspp: Neisseria spp.

Figure 2

Heatmap of pairwise comparison of the 1114 core genes with complete sequences among the different clusters. Two-way Average Nucleotide Identity using concatenated cgMLST loci sequences among all the clusters (A) and only among the N. polysaccharea (B); ANI measured in percentage are presented with a colour gradient, yellow for values <94 and red for values >95%, which is the threshold above which genomes are considered to be from the species. Pairwise allelic comparison of the cgMLST loci (C), Number of loci with at least one allele shared are displayed with a colour gradient, pink for values <50 and purple for values >500. Nm: N. meningitidis; Ng: N. gonorrhoeae; Np: N. polysaccharea and Nspp: Neisseria spp.

Figure 3

Maximum-likelihood tree of the putative novel Neisseria species defined following 95% cgMLST ANI.

Figure 4

Scanning Electron Microscopy images of the different clusters identified by genomic analysis. A-N. meningitidis, B-N. polysaccharea cluster 2, C-N. polysaccharea cluster 3, D-Nspp 1 (N. bergeri), E-Nspp 2 (N. bergeri), F-Nspp 3 (N. uirgultaei), G-Nspp 4 (N. blantyrii), H-Nspp 5 (N. viridiae), I-Nspp 6 (N. benedictiae), J-Nspp 7 (N. bassei), K- Nspp 8 (N. maigaei) and L-Nspp 9 (N. bergeri). The scale bar represents 0.25 µm.