Phenotypic and genomic characterization of pneumococcus-like streptococci isolated from HIV-seropositive patients

1 Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Oslo, Norway 2 Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 3 Department of Oral Biology, University of Oslo, Oslo, Norway 4 Department of Microbiology, Ullevål University Hospital Oslo, Norway 5 Institute of Medical Microbiology, Rikshospitalet University Hospital, Oslo, Norway Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae , as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci. Correspondence Truls M. Leegaard truls.leegaard{at}rikshospitalet.no Abbreviations: CGH, comparative genomic hybridization; EPP, estimated probability of presence; MLSA, multilocus sequence analysis; MLST