Whole-Genome Sequencing Re-Identifies Clinical “Peptoniphilus harei” Isolates as “Peptoniphilus genitalis”

Accurate species-level identification within the genus Peptoniphilus is challenging, yet routine methods, including biochemical identification, MALDI-TOF MS, and 16S rRNA gene sequencing, have not been systematically evaluated. We re-identified seven clinical isolates recovered between 2019 and 2025 from blood, ovarian abscess, diabetic foot, and skin and soft tissue, using VITEK-2 ANC, two MALDI-TOF MS platforms (Autof ms1000 and Bruker Biotyper), 16S rRNA gene sequencing, and whole-genome sequencing (WGS) combined with average nucleotide identity (ANI) analysis. VITEK-2 reported all seven isolates as P. asaccharolyticus; both MALDI-TOF platforms reported all as P. harei with high confidence (Autof ms1000 scores 9.299–9.638; Bruker Biotyper scores 2.008–2.227); and 16S rRNA sequencing could not reliably distinguish between P. vaginalis (98.60–99.23%) and P. harei (98.03–98.31%), as both identity ranges were close to the 98.65% species-level threshold and neither consistently exceeded it. WGS/ANI identified all seven isolates as P. genitalis (ANI 96.44–97.05% against the reference genome). All clinical isolates remained susceptible to β-lactams and vancomycin. Clindamycin resistance was common (6/7), and erythromycin showed a concordant pattern; the fluoroquinolones showed small inhibition zones in 5/7 isolates. However, erythromycin and the fluoroquinolones lacked a method-appropriate breakpoint and could not be formally categorized. The clinical distribution of P. genitalis is not limited by its name; conventional methods underrepresent this recently described species, and WGS/ANI should serve as the confirmatory method when identification conflicts with phenotypic observations.