We evaluated the incidence of Streptococcus pseudopneumoniae in clinical isolates by phenotypic methods and DNA-DNA hybridization. The pathogenic role of this organism was investigated with the mouse peritonitis/ sepsis model. Our results show a low incidence (1/120 pneumococcal isolates) and a potential pathogenic effect for S. pseudopneumoniae.Streptococcus pneumoniae, a major cause of morbidity and mortality worldwide, remains a significant health threat. Correct identification of pneumococci from clinical samples and differentiation from other oral streptococci are essential for appropriate diagnosis and treatment. A novel species of viridans group streptococci resembling S. pneumoniae was recently described as Streptococcus pseudopneumoniae (1); phenotypic identification is based on susceptibility to optochin (OPT) in ambient atmosphere and OPT resistance in a 5% CO 2 atmosphere, bile salt insolubility, and the absence of pneumococcal capsule. Genotypic identification is based on DNA-DNA hybridization, whereas sodA sequencing and 16S rRNA gene sequencing are not discriminating. While the new species tested positive for the pneumolysin gene (ply), its pathogenic role was not investigated. In the present study, we assessed the clinical significance of this new species by examining its incidence among pneumococci from clinical samples and investigating its virulence in a mouse peritonitis/sepsis model system.In total, 120 isolates presumptively identified as S. pneumoniae on the basis of Gram stain, catalase activity, and OPT susceptibility in ambient atmosphere were obtained from consecutive clinical samples of patients admitted to University Hospital, Strasbourg, France, during the period October 2004 to April 2005. Single pneumococcal isolates/patients/infections were analyzed. Eighteen isolates were collected from the lower respiratory tract, 57 were collected from the upper respiratory tract, 24 were collected from blood, 1 was collected from cerebrospinal fluid, 3 were collected from pleural fluid, and 12 were collected from other sterile body sites. Controls (S. pseudopneumoniae CCUG 49455 T and S. pneumoniae CCUG 28588 T ) were included in all assays. Additional clinical strains (S. pseudopneumoniae CCUG 48465, CCUG 50866, CCUG 50867, CCUG 50868, CCUG 50869, CCUG 50870, and CCUG 50871) described by Arbique et al. (1) were included in virulence assays.OPT susceptibility testing was performed by the disk diffusion method with a 6-mm disk (Bio-Rad) on sheep blood agar (Trypticase soy agar [bioMérieux] supplemented with 5% sheep blood); plates were incubated for 18 to 24 h at 35°C in ambient air and in a 5% CO 2 atmosphere. Solubility in bile salt (sodium deoxycholate [Merck]) was determined in tubes (4). Capsules were detected by observing a halo around pneumococci with India ink at a ϫ100 to ϫ400 magnification. Capsular agglutination tests were performed by the Pastorex test (BioRad) according to the instructions of the manufacturer.DNA-DNA hybridization was performed as described previously (8). Hybridi...