Integrating Phenotypic Testing, Targeted PCR and Long-Read Genomics in Staphylococcus aureus: Defining the Scope of Genotype–Phenotype Concordance

Phenotypic antimicrobial susceptibility testing (AST), targeted PCR and whole-genome sequencing (WGS) provide complementary information on Staphylococcus aureus, but their results are not directly interchangeable. We integrated these approaches in a diagnostic collection of 450 nasal and throat S. aureus isolates from outpatients (2016–2020). AST was performed for 17 agents using EUCAST 2019 breakpoints. Ten deliberately selected isolates (four MRSA, six MSSA) underwent long-read WGS and screening against MEGARes and VFDB; targeted PCR was compared with their genomes and applied to 40 randomly selected isolates. Benzylpenicillin and cefoxitin resistance occurred in 86.0% and 38.9% of isolates; resistance within three or more categories (30.4%) was confined to MRSA. MEGARes returned 31 resistance-associated entries, 13 in all ten genomes. Concordance across 100 directly evaluable isolate–agent pairs was 92.0% (bootstrap 95% CI 85.0–98.0%), with discordances confined to four isolates; fluoroquinolone resistance lay outside the evaluable set, without canonical target-site substitutions. VFDB identified 75 virulence-associated genes; lukF-PV was returned in four isolates, but in three, it was lukD and only one carried the PVL locus. PCR matched the genomic loci in 179 of 180 paired observations, although ten of 18 targets were invariant. Genotype–phenotype concordance should be assessed within a clearly defined analytical scope.