Conversion Of Antibiotic Resistance To Antibiotic Sensitivity In MRSA By Antisnese Phosphorothioate Oligonucleotides

Staphylocicus aureus (S. aureus) is an aggressive pathogen that is a common cause of infection both in the community and in hospitals. A growing prevalence of S aureus displaying methecillin resistance currently has become a serious problem because these bacteria show a phenotype of b-lactam resistance. The resistance is due to production of either b-lactamase or an extra penicillin-binding protein, PBP2a, which has low affinity to P-lactams and is encoded by the chromosomal gene mecA in methicillin resistant Staphylocicus aureus (MRSA), confers resistance not only to penicillin, but also to all p lactam antibiotics. Transcription of mecA is regulated by the sensor-transducer proteins, MecRl and its partner represser, Mecl, which bind to DNA sequence in mecA gene. Inactivation of the Mecl represser through proteolytic cleavage results in de-repression of the mecA and PBP2a production. We propose that disruption of PBP2a regulatory signal pathway may lead to repression of PBP2a gene and restore the activities of P-lactams against MRSA. Therefore, in this study, the antisense phosphorothiate oligonucleotides (PS-ODN) have been used toinhibit the expression of sensor-transducer protein, MecRl in an attempt to converse MRSA to be antibiotic susceptible.1. Identification of MRSAMicroscan WalkAway-40 microbiol identification system was used to identify two strains of S. aureus and Kirby-Bauer have been applied for detecting the antibiotic resistance of S. aureus. The target gene, mecR1, was detected by PCR. The results showed that both bacteria strain of WHO-2 and ATCC25923 were S aureus and WHO-2 was identified as an MRSA which carried mecRl gene, while ATCC25923 was methicillin sensitive and no mecRl was detected in this strain.2. Design and synthesis of antisense PS-ODNmRNA of mecRl was chosen as a target gene and the appropriate target sites of it for antisense PS-ODNs were carefully selected by analysis of free energy and thermodynamic parameters of DNA and RNA interaction through RNA STRUCTURE software on computer. Three antisense PS-ODNs were selected and sequences of them were displayed in Tab 1.Antisense PS-ODNs(500ng) were introduced into the MRSA (5+1010 cells) through electroporation.(voltage 900V, intitial field stength 9KV/cm; capacitance 25F). The growth curve of MRSA incubated with oligl and olig2 in the broth containing Oxacillin(oxa, 6g/ml, 18g/ml) is lower than the curve of control(n=6, P<0.05 vs control, the curve of MRSA incubated with olig3 is similar with that of control (n=6, P>0.05 vs control). Colony forming unit (CPU) of MRSA incubated with oligl and olig2 on the M-H agar added oxa (6g/ml) are less than that of control(n=9, P<0.05 vs control), CPU of MRSA incubated with olig3 are similar with that of control(n=9, P>0.05 vs control). After MRSA incubated with oligl and olig2 for 20h on agar , There was a circulus around the oxa disc (1 g/piece) .However, no circulus was detected under the condition of incubating with olig3. These preliminary data demonstrated that antibiotic sensitivity on MRSA could be partially restored by exogenously delivered antisense oligonucleotides targeted to mRNA of mecR1 in blocking PBP2a regulatory pathway.