Genome-Based Reclassification of Fusobacterium nucleatum Subspecies at the Species Level

Abstract

Fusobacterium nucleatum is classified as four subspecies, subsp. nucleatum, polymorphum, vincentii, and animalis, based on DNA-DNA hybridization (DDH) patterns, phenotypic characteristics, and/or multilocus sequence analysis (MLSA). The gold standards for classification of bacterial species are DDH and 16S ribosomal RNA gene (16S rDNA) sequence homology. The thresholds of DDH and 16S rDNA similarity for delineation of bacterial species have been suggested to be >70 and 98.65%, respectively. Average nucleotide identity (ANI) and genome-to-genome distance (GGD) analysis based on genome sequences were recently introduced as a replacement for DDH to delineate bacterial species with ANI (95-96%) and GGD (70%) threshold values. In a previous study, F. hwasookii was classified as a new species based on MLSA and DDH results. 16S rDNA similarity between F. hwasookii type strain and F. nucleatum subspecies type strains was higher than that between F. nucleatum subspecies type strains. Therefore, it is possible that the four F. nucleatum subspecies can be classified as Fusobacterium species. In this study, we performed ANI and GGD analyses using the genome sequences of 36 F. nucleatum, five F. hwasookii, and one Fusobacterium periodonticum strain to determine whether the four F. nucleatum subspecies could be classified as species using OrthoANI and ANI web-based softwares provided by ChunLab and Kostas lab, respectively, and GGD calculator offered by German Collection of Microorganisms and Cell Cultures. ANI values calculated from OrthoANI and ANI calculators between the type strains of F. nucleatum subspecies ranged from 89.80 to 92.97 and from 90.40 to 91.90%, respectively. GGD values between the type strains of F. nucleatum subspecies ranged from 42.3 to 46.0%. ANI and GGD values among strains belonging to the same F. nucleatum subspecies, subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis were >96 and >68.2%, respectively. These results strongly suggest that F. nucleatum subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis should be classified as F. nucleatum, F. polymorphum, F. vincentii, and F. animalis, respectively.

Strong oral plaque microbiome signatures for dental implant diseases identified by strain-resolution metagenomics

“…F. nucleatum is a key bacterium in the plaque microbiome associated with periodontal diseases 74 , 75 , and we showed here also that it is strongly associated with peri-implant diseases and especially with mucositis. Given the diversity of the strains present in this species 76 , we further investigated the subspecies structure of F. nucleatum in relation to peri-implantitis and mucositis (see “Methods”). By using a pangenome-based strain profiling tool 77 , we characterized the dominant F. nucleatum strains in the 113 samples considered in this study as well as in 135 additional plaque samples from an American healthy metagenomic cohort 63 and 48 plaque samples from patients with periodontitis 62 .…”

Analysis of 16S rRNA genes reveals reduced Fusobacterial community diversity when translocating from saliva to GI sites

“… 32 There is considerable genomic variability among F. nucleatum , with the average nucleotide identity (ANI) between subspecies of less than 93%. 33 Thus, it is important to not only distinguish between subspecies, but also identify relevant strains within the subspecies.…”

Mash Screen: High-throughput sequence containment estimation for genome discovery

“…Also notable are points closer to the diagonal, but still in distinct clusters, highlighted in magenta and orange in Figure 3 (left). These represent two distinct clades of F. nucleatum, a gram-negative commensal of the human oral cavity that has welldelineated subspecies [18]. The expected reference strain, ATCC 25586, was noted by Awad et al to be present in low abundance, which is likely what is causing the Mash containment to be lower than expected for this and similar strains (below the diagonal, circled in magenta).…”