Exploring the susceptome of methicillin-resistant Staphylococcus aureus to oxacillin and cefazolin using integrated transposon-directed insertion site sequencing and proteomics

Objective: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge to public health due to its evolving resistance to antibiotics. This study aimed to identfy the determinants that modulate MRSA susceptibility to β-lactam antibiotics. Methods: An integrated approach combining transposon-directed insertion site sequencing (TraDIS) and proteomics was applied. TraDIS was used to identify genetic determinants influencing susceptibility to oxacillin and cefazolin while proteomics was applied to characterise the adaptive response under sub-inhibitory antibiotic concentrations. Results: Proteomic analysis revealed significant shifts in the protein spectrum under sub-inhibitory concentrations of oxacillin and cefazolin, characterised by stringent response. This included upregulation of amino acid metabolism and oligopeptide transport pathways, and downregulation of arginine biosynthesis. Concurrently, TraDIS screening identified 50 genes whose inactivation conferred a fitness advantage in the presence of both β-lactams, implicating processes such as nucleotide second messenger signalling, DNA repair, and histidine transport. Validation with selected mutants (relA::Tn, mutY::Tn, nsaR::Tn, aroC::Tn, SAUSA300_0432::Tn, SAUSA300_0846::Tn, SAUSA300_0595::Tn) confirmed the improved fitness in the presence of β-lactams. Integrative analysis of proteomic and TraDIS datasets highlighted the implication of the stringent response in mediating MRSA adaptation to oxacillin and cefazolin. Conclusions: These findings establish the 'susceptome' concept, a collection of genes that maintain or potentiate the vulnerability of MRSA strains to β-lactams, providing new targets for therapeutic intervention and strategies to counteract resistance evolution.