Phenotypic and Molecular Evaluation for Accurate Differentiation of Staphylococcus Aureus and Staphylococcus Xylosus Under Standardized Laboratory Conditions

Abstract

The genus Staphylococcus comprises a diverse group of Gram-positive bacteria that include both major human pathogens and commensal species. Among them, Staphylococcus aureus is a clinically significant pathogen, whereas Staphylococcus xylosus is a coagulase-negative species that is generally non-pathogenic. Due to shared phenotypic traits and occasional ambiguity in molecular assays, accurate discrimination between these species remains challenging in routine microbiological diagnostics. This study aimed to comparatively evaluate phenotypic and molecular methods for reliable differentiation of S. aureus and S. xylosus under standardized laboratory conditions. Under strictly controlled laboratory conditions, eleven S. aureus reference strains and three Staphylococcus xylosus isolates were examined using a tiered diagnostic approach. Phenotypic characterization was performed on Columbia blood agar, assessing colony morphology, pigmentation, and hemolysis. Species confirmation was evaluated using latex agglutination targeting clumping factor and Protein A, followed by molecular confirmation via endpoint PCR targeting the femB gene. PCR results were interpreted based on the presence of a clear ~120 bp amplicon reproducible across duplicate runs. Diagnostic performance of phenotypic methods was assessed using femB PCR as the reference criterion, calculating sensitivity, specificity, predictive values, accuracy, exact 95% confidence intervals, and Cohen’s kappa for inter-method agreement. All S. aureus strains produced uniformly golden-pigmented, β-hemolytic colonies, whereas all S. xylosus isolates were non-pigmented and non-hemolytic. Latex agglutination yielded strong positive reactions in all S. aureus strains (11/11) and remained negative for all S. xylosus isolates (0/3). femB PCR generated strong, reproducible ~120 bp amplicons in all S. aureus strains, while S. xylosus isolates exhibited only very faint, non-reproducible bands near the expected size, interpreted as non-specific amplification or partial sequence homology with related CoNS genes. Both colony morphology and latex agglutination demonstrated 100% sensitivity and 100% specificity relative to PCR classification, with perfect agreement (κ = 1.00). When applied under standardized laboratory conditions, colony morphology and latex agglutination provide highly reliable phenotypic discrimination between S. aureus and S. xylosus, while femB PCR serves as a robust confirmatory tool. The observation of faint, non-reproducible femB-like signals in S. xylosus highlights the importance of cautious molecular interpretation and supports an integrated phenotypic-molecular diagnostic strategy for accurate species-level identification.