Deciphering the secondary resistome of Methicillin-Resistant Staphylococcus aureus (MRSA) to Beta-lactams - a Multi-Omics Approach

Introduction: Antimicrobial resistance (AMR) is a significant global health issue, impacting both human and livestock populations. Methicillin-resistant Staphylococcus aureus (MRSA), a common multidrug-resistant bacterium, has been identified as a high-priority pathogen for the development of new antibiotics. The approach of restoring susceptibility to existing antimicrobials presents a promising strategy in the battle against AMR, as the full expression of resistance relies on auxiliary enzymes.

Goal: This study aims to identify the auxiliary genes and proteins involved in the resistance of MRSA to oxacillin, which could potentially serve as targets for re-establishing susceptibility.

Materials and Methods: Using Transposon-Directed Insertion site Sequencing (TraDIS), we screened a large MRSA transposon mutant library post-exposure to subinhibitory concentrations of oxacillin and cefazolin. The output reads were analyzed via the BioTradis pipeline. Subsequently, label-free quantitative proteomics was performed on the antibiotic-exposed cultures, the mass spectrometer data was processed using Proteome Discoverer, and a comprehensive statistical analysis was conducted with Perseus v1.6.

Results: Our high-throughput transposon mutagenesis assay identified a set of conditionally essential genes potentially contributing to beta-lactam resistance. Subsequently, Post-antimicrobial exposure proteomics analysis highlighted significant alterations in protein expression, notably within the methionine and D-alanylation pathways of wall teichoic acid, which are linked to peptidoglycan integrity. Our findings underscore a limited overlap between TraDIS and proteomics results.

Conclusion: Our investigation sheds light on the identification of auxiliary genes and proteins linked to beta-lactam resistance in MRSA. By merging large-scale transposon mutagenesis and proteomics, we provide valuable data about the secondary resistome and potential targets for tackling antimicrobial resistance. These insights set the stage for devising novel strategies to reinstate MRSA"s susceptibility to existing antimicrobials, addressing the pressing global health issue of AMR.