Mechanism Of Synergy Between SIPI-8294 And β-Lactam Antibiotics Against MRSA

Staphylococcus aureus strains with methicillin resistance(MRSA) are one of the most frequent aetiology of community and nosocomially bacterial acquired infections currently. Infections caused by MRSA are associated with increased mortality and morbidity, aggressive course, multiple drug resistance and hospital outbreaks, which has become a major public health concern. Conventional anti-MRSA antibiotics like vancomycin, teicoplanin, linezolid and daptomycin are currently in clinical use. However, development of MRSA resistant to many of these drugs has been identified worldwide recently. Therefore, the development of new agents remains a priority to ensure the availability of efficacious therapies. Recent study has revealed that SIPI-8294, as a new derivative of erythromycin, has remarkable synergistic effect with β-lactam antibiotics against MRSA. However, the mechanism for the synergistic effect of SIPI-8294 and β-lactams is still unclear. The aim of this study was to find out the detailed synergistic mechanism of SIPI-8294 and β-lactams against MRSA.The synergistic effects between SIPI-8294 and β-lactam antibiotics against MRSA were comprehensively and systematically studied in our research.The results showed that SIPI-8294 has remarkable synergistic effect with most β-lactam antibiotics against MRSA. However, none of synergistic effect between SIPI-8294 and β-lactams was found against MSSA. Moreover, SIPI-8294 was not able to synergize 10 non-β-lactam antibiotics tested in our research against MRSA and MSSA. A murine systemic MRSA infection model was established.The synergy between SIPI-8294 and β-lactams against MRSA in vivo were also detected, suggesting the possibility that SIPI-8294 together with β-lactam antibiotics to treat MRSA-infected patients.ATCC 43300 is used as a type strain of MRSA in our research.The regulatory effects of SIPI-8294 on mec A, the key gene for MRSA resistance against β-lactams, were investigated with RT-PCR and Western blot. With gene knock out technologies, the role of mec A in the synergism of SIPI-8294 and β-lactams against MRSA were studied. Mec A gene knock out and complemented strains were constructed. The synergistic effects of SIPI-8294 and β-lactams against the mutants of ATCC 43300 were detected. The results demenstrated that mec A played a key and decisive role in the synergsim of SIPI-8294 and β-lactams against MRSA.MecR1 and mecI were he pivotal regulatory genes of mec A.With gene knock out technologies, the role of mecR1 and mecI in the synergism of SIPI-8294 and β-lactams against MRSA were studied. mecR1 gene knock out and complemented strains as well as mecI gene knock out strain of ATCC 43300 were constructed.The synergistic effects of SIPI-8294 and β-lactams against thesd mutants were detected.The regulatory effects of SIPI-8294 on mec A in these mutants were also detected. The results reveal that mecR1 and mecI don‘tparticipate in the synergism of SIPI-8294 and β-lactam antibiotics. Morever, SIPI-8294 does not suppress mec A expression in MRSA through regulating mecR1 and mecI.The impacts of SIPI-8294 on bacterial cell wall were examined. The results demonstranted that the damage effect of SIPI-8294 to cell wall was very strong. Meanwhile, SIPI-8294 displayed an inhibitory effect on cell wall biosynthesis. The impacts of SIPI-8294 on protein synthesis, nuclear acid synthesis and peptidoglycan synthesis were also examined. We found that SIPI-8294 may not have inhibitory effect on protein synthesis, nuclear acid synthesis and peptidoglycan synthesis.In summary, our resech demontrates that SIPI-8294 inhibits cell wall biosynthesis through suppressing mec A expression, resulting in direct damage of cell wall, thus leading SIPI-8294 to synergize β-lactams against MRSA. The research is of important significance for SIPI-8294 development as a new drug and lay a solid foundation for its clinical application.