The Secondary Resistome of Methicillin-Resistant to β-Lactam Antibiotics.

: Therapeutic strategies for methicillin-resistant (MRSA) are increasingly limited due to the ability of the pathogen to evade conventional treatments such as vancomycin and daptomycin. This challenge has shifted the focus towards novel strategies, including the resensitization of β-lactams, which are still used as first-line treatments for methicillin-susceptible (MSSA). To achieve this, it is essential to identify the secondary resistome associated with the clinically relevant β-lactam antibiotics. : Transposon-Directed Insertion Site Sequencing (TraDIS) was employed to assess conditional essentiality by analyzing the depletion of mutants from a highly saturated transposon library of MRSA USA300 JE2 exposed to ½ minimal inhibitory concentration (MIC) of oxacillin or cefazolin. : TraDIS analysis led to the identification of 52 shared fitness genes involved in β-lactam resistance that are primarily linked to cell wall metabolism and regulatory systems. Among these, both known resistance factors and novel conditionally essential genes were highlighted. As proof of concept, transposon mutants corresponding to nine genes (, , , , , , , , and ) were grown in the presence of β-lactam antibiotics and their MICs were determined. All mutants showed significantly reduced resistance to β-lactam antibiotics. : This comprehensive genome-wide investigation provides novel insights into the resistance mechanisms of β-lactam antibiotics, and suggests potential therapeutic targets for combination therapies with helper drugs.