Comprehensive in silico genomic surveillance of β-lactam and methicillin resistance in Staphylococcus aureus: Machine...

According to the World Health Organization, methicillin-resistant Staphylococcus aureus (MRSA) is classified as a "High" priority pathogen on its Global Priority Pathogens List. The aim of this study is to perform a comprehensive genome-based in silico analysis of S. aureus to elucidate the evolutionary dynamics of β-lactam and methicillin resistance, focusing on resistance genes and minimum inhibitory concentration (MIC) phenotypes. This study analyzes all publicly available S. aureus genomes in NCBI to investigate β-lactam and methicillin resistance, including resistance genes and MIC phenotypes. The dataset covers isolates from multiple countries worldwide, representing global diversity.

A total of 111,350 S. aureus genomes (1880s-2020s) were retrieved from GenBank and rigorously quality-filtered. Antimicrobial resistance genes were identified with AMRFinderPlus, and phenotypic MIC data for key β-lactams were integrated via genome-BioSample linkage from the National Center for Biotechnology Information AST Browser. Multi-locus sequence typing was used to resolve sequence types (STs), while machine learning models (Random Forest, eXtreme Gradient Boosting, Elastic Net regularized regression, and Partial Least Squares) were trained on gene/mutation profiles to predict log₂ MICs under repeated 5-fold cross-validation. Temporal and lineage-specific trends were assessed with correlation and generalized linear models, and genotype-phenotype associations tested using chi-square or Fisher's exact test with Bonferroni correction. All genomic, clustering, and phylogenetic analyses were performed in R using fully reproducible pipelines.

The dataset included 111,350 S. aureus genomes, 78 % clinical and 10 % environmental, spanning 1884-2025 from 137 countries. Core genome size remained stable (mean 2.87 Mb; ∼2874 genes), with modest increases in total genes (r = 0.15, p < 0.001) and pseudogenes (r = 0.09, p < 0.001), reflecting subtle genomic plasticity. MLST analysis identified >2000 STs, but ST8 (16.6 %), ST5 (13.8 %), and ST22 (6.9 %) together accounted for ∼37 % of isolates, illustrating epidemic clone dominance and temporal turnover from pre-1980 hospital lineages to modern polyclonal populations. β-lactam resistance showed a marked upward trend: the bla operon (blaZ-blaI-blaR1) increased from ∼60 % in the early 2000s to >95 % by 2024, while blaPC1 declined from near ubiquity in the 1990s to <10 %. Methicillin resistance, driven by mecA (62.7 %), was accompanied by progressive loss of regulators mecR1 (52.0 %) and mecI (13.8 %), reflecting evolutionary streamlining for constitutive PBP2a expression. Clinical isolates carried higher frequencies of blaZ (+17.4 %) and mecA (+15.5 %) than environmental strains (p < 0.01). MIC data revealed rising resistance to ticarcillin and cefoxitin, while carbapenems and ceftaroline remained active. Machine-learning models accurately predicted ceftaroline and penicillin MICs but poorly predicted oxacillin. Clustering of β-lactam/methicillin loci identified 146 gene-presence patterns, with the five-gene MRSA cassette (blaI-blaR1-blaZ-mecA-mecR1) dominating ∼30 % of genomes, illustrating the evolutionary consolidation of resistance modules over time.

S. aureus has maintained a stable core genome while consolidating β-lactam and methicillin resistance around the bla and mecA cassettes, with epidemic lineages driving global spread. Despite widespread resistance to older β-lactams, carbapenems, and ceftaroline remain effective, underscoring the value of WGS-based surveillance and stewardship.