Genome characterization and taxonomy of Actinomyces acetigenes sp. nov., and Actinomyces stomatis sp. nov., previously isolated from the human oral cavity

Background: Actinomyces strains are commonly found as part of the normal microflora on human tissue surfaces, including the oropharynx, gastrointestinal tract, and female genital tract. Understanding the diversity and characterization of Actinomyces species is crucial for human health, as they play an important role in dental plaque formation and biofilm-related infections. Two Actinomyces strains ATCC 49340 ^T and ATCC 51655 ^T have been utilized in various studies, but their accurate species classification and description remain unresolved. 

Results: To investigate the genomic properties and taxonomic status of these strains, we employed both 16S rRNA Sanger sequencing and whole-genome sequencing using the Illumina HiSeq X Ten platform with PE151 (paired-end) sequencing. Our analyses revealed that the draft genome of Actinomyces acetigenes ATCC 49340 ^T was 3.27 Mbp with a 68.0% GC content, and Actinomyces stomatis ATCC 51655 ^T has a genome size of 3.08 Mbp with a 68.1% GC content. Multi-locus (atpA , rpoB, pgi , metG , gltA , gyrA, and core genome SNPs) sequence analysis supported the phylogenetic placement of strains ATCC 51655 ^T and ATCC 49340 ^T as independent lineages. Digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) analyses indicated that both strains represented novel Actinomyces species, with values below the threshold for species demarcation (70% dDDH, 95% ANI and AAI). Pangenome analysis identified 5,731 gene clusters with strains ATCC 49340 ^T and ATCC 51655 ^T possessing 1,515 and 1,518 unique gene clusters, respectively. Additionally, genomic islands (GIs) prediction uncovered 24 putative GIs in strain ATCC 49340 ^T and 16 in strain ATCC 51655 ^T, contributing to their genetic diversity and potential adaptive capabilities. Pathogenicity analysis highlighted the potential human pathogenicity risk associated with both strains, with several virulence-associated factors identified. CRISPR-Cas analysis exposed the presence of CRISPR and Cas genes in both strains, indicating these strains might evolve a robust defense mechanism against them. Conclusion: This study supports the classification of strains ATCC 49340 ^T and ATCC 51655 ^T as novel species within the Actinomyces, in which the name Actinomyces acetigenes sp. nov. (type strain ATCC 49340 ^T = VPI D163E-3 ^T = CCUG 34286 ^T = CCUG 35339 ^T) and Actinomyces stomatis sp. nov. (type strain ATCC 51655 ^T = PK606^T = CCUG 33930 ^T) are proposed.