Co-blockade of mecR1/blaR1 signal pathway to restore antibiotic susceptibility in clinical isolates of methicillin-resistant Staphylococcus aureus.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is caused by the production of low-affinity penicillin-binding protein 2a and ß-lactamases, which are encoded by mecA and blaZ, respectively. Expressions of the two key genes are mutually regulated by MecI and BlaI. The aim of this study was to design specific anti-mecR1 and anti-blaR1 deoxyribozymes and identify the restoration of susceptibility in MRSA isolates with mecI or blaI or no deletions by interfering with the mutual regulation of mecA and blaZ. MATERIAL AND METHODS: Specific deoxyribozymes were designed by using the program RNA structure 4.6. RNA substrates were obtained by transcription in vitro and used to assess the target cleavage of DNAzymes. Transcription of mecR1-mecA and blaR1-blaZ was analysed by real time RT-PCR. The susceptibility of MRSA was tested. RESULTS: Specific deoxyribozymes showed efficient catalytic activity to each own substrate mecR1 or blaR1 in vitro and caused the reduction of mecR1 and blaR1 transcription in vivo. Furthermore, simultaneous administration of two DNAzymes to knockdown mecR1 and blaR1 resulted in increased susceptibility of all MRSA strains tested in this study. CONCLUSIONS: These results demonstrated that combined use of the two specific phosphorothioate deoxyribozymes could be a viable and promising strategy to restore the susceptibility of almost all MRSA clinical isolates.