Discrimination between Streptococcus pneumoniae and Streptococcus mitis based on sorting of their MALDI mass spectra.

Accurate species-level identification of alpha-hemolytic (viridans) streptococci (VGS) is very important for understanding their pathogenicity and virulence. However, an extremely high level of similarity between VGS within the mitis group (S. pneumoniae, S. mitis, S. oralis and S. pseudopneumoniae) often results in misidentification of these organisms. Earlier, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a tool for the rapid identification of S. pneumoniae. However, by using Biotyper 3.0 (Bruker) or Vitek MS (bioMérieux) databases, Streptococcus mitis/oralis species can be erroneously identified as S. pneumoniae. ClinProTools 2.1 software was used for the discrimination of MALDI-TOF mass spectra of 25 S. pneumoniae isolates, 34 S. mitis and three S. oralis. Phenotypical tests and multilocus gene typing schemes for the S. pneumoniae (http://spneumoniae.mlst.net/) and viridans streptococci (http://viridans.emlsa.net/) were used for the identification of isolates included in the study. The classifying model was generated based on different algorithms (Genetic Algorithm, Supervised Neural Network and QuickClassifier). In all cases, values of sensitivity and specificity were found to be equal or close to 100%, allowing discrimination of mass spectra of different species. Three peaks (6949, 9876 and 9975 m/z) were determined conferring the maximal statistical weight onto each model built. We find this approach to be promising for viridans streptococci discrimination.

Misidentification of alpha-hemolytic streptococci by routine tests in clinical practice.

Accurate species-level identification of viridans group streptococci (VGS) is very important for understanding of their pathogenicity and virulence. However, an extremely high level of the similarity between VGS, especially Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis and Streptococcus pseudopneumoniae, often results in misidentification of these organisms, so there is an urgent need of novel approaches to species identification. A set of 50 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (alpha-hemolysis, colony morphology, Gram stain and optochin susceptibility). Modern proteomic and genetic approaches - the direct bacterial profiling (DBP) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique and multilocus sequence analysis (MLSA) scheme (http://viridans.emlsa.net/) - were applied for the accurate species identification. After that all isolates were stored at -70°C. Later they were re-inoculated, and a number of additional tests (bile solubility, latex agglutination by commercial "Slidex® pneumo-kit" and repeated optochin test) were performed. A considerable discrepancy was discovered in the results of the different approaches. Looking in the future, one could say that MLSA-like schemes based on the analysis of the nucleotide sequences of seven or more loci of the bacterial genome, appeared to be the most useful instrument in the VGS discrimination, in contrast to the numerous one-target identification schemes, which have been introduced into practice by now.