Towards a cure for MRSA: Novel Drug Combination and Novel Factors Critical for β-Lactam Resistance

Abstract

Methicillin-resistant Staphylococcus aureus remains a global health threat as it increases the risk of infections in both community and hospital settings. Additionally, the pathogen has become increasingly resistant to current antibiotics including β-lactams, resulting in an elevation of mortality and morbidity rate, particularly in intensive care units. The class of β-lactam antibiotics are known for their safety, selectivity, broad range of activity and being Bng the most wAly prescribed antibiotics. Hence, restoring their efficacy against MRSA is of great clinical importance. The resistance to β-lactams has been attributed to the presence of the drug insensitive transpeptidase, penicillin-binding protein-2a (PBP2a), which is encoded by the mecA gene. However, numerous auxiliary factors are required for the full expression of the resistance phenotype. In a previous screening of 1200 FDA-approved drug library, we Antified A which augments Bxicillin against MRSA USA300 strain. In this study, we demonstrated that A effectively synergised Bxicillin against a panel of representative prevalent sequence types of MRSA strains. Subsequent checkerboard test showed that A is able to synergise other β-lactams and even other antibiotics from different classes. We also showed that A/Bxicillin combination can eradicate the biofilm mass of the MRSA USA300 significantly, with a p-value of <0.05. Using Galleria mellonella infection model, A/Bxicillin combination improved survival rate of larvae up to 80%, with an efficacy comparable to the standard therapy vancomycin. The synergy of A with Bxicillin occurred independently of the mecA gene, as RT-qPCR and PBP2a expression assay showed no significant differences between treated and untreated samples. RNA-Seq data showed an upregulation of genes involved in oxidative stress pathway, as well as a disruption of components of the electron transport chain, manifested by an 11 upregulation of genes at the beginning of the chain with a downregulation of genes encoding terminal components. Moreover, downregulation of genes involved in toxins and virulence factors was observed. Using fluorescent dye 3′-(p-hydroxyphenyl) fluorescein (HPF), a significant induction ~16 fold elevation in hydroxyl radical production was observed with A/Bxicillin combination. This study also Antified two novel auxiliary factors GA (GA) and DA (DA), with increased susceptibility to β-lactams in the MRSA strain, JE2, without affecting mecA gene transcription or PBP2a expression. The complementation of both mutants restored β-lactam resistance, suggesting that both factors could play crucial role in β lactam resistance in MRSA. In addition, the transduction of GA and DA transposon mutations by phage ϕ11 into community-acquired (MW2) and hospital-acquired (COL) MRSA strains, resulted in increased susceptibility to β-lactams, including oxaciliin, cefoxitin and meropenem, confirming that the mutations also led to an increased β lactam susceptibility in different MRSA backgrounds. In a Galleria mellonella infection model, the survival rate of larvae inoculated with either GA or DA was significantly improved after treatment with Bxicillin compared to wild-type JE2 infected larvae. Collectively, this study presents a novel adjunctive compound, A, along with two novel auxiliary factors, GA and DA, critical for β-lactam resistance where targeting these factors can re-sensitise MRSA strains to β-lactam antibiotics and aids in tackling MRSA infections.