| AIM: Methicillin-resistant Staphylococcus aureus (MRSA), a important human pathogen causing serious hospital infections and community infections, is multi-resistant to virtually all of the p-lactams and a wide variety of antibiotics. It thereby threatens the most potent antibiotics we have. This situation limits treatment options to a very few agents, such as vancomycin and teicoplanin. However, in 1997 an MRSA clinical isolate that responded poorly to vancomycin therapy was reported in Japan. Five years later, isolation of vancomycin-resistant S. aureus was documented in the United States. It is evident that the need to seek a new defending strategy against MRSA is now urgent. One apparently feasible approach to the problem is to restore the antibacterial activity of the current antibiotics. The resistance is duo to acquiring drug-resistant gene blaZ and mecA, which encode P-lactamase and PBP2a. Transcription of mecA is regulated by the sensor-transducer proteins, MecRl and its partner repressor, Mecl. Similarily, transcription of blaZ is regulated by BlaRl and Blal. In our experiment, to restore the susceptibility of MRSA to oxacillin, we block resistant signalpathway in MRSA by antisense oligonucleotides, which target mecRl mRNA or blaRl mRNA. METHODS:1. Reversal of resistance of MRSA by PS-ODNs6087 and PS-ODNs6088 anti-mecR1 mRNA: Different concentrations (5 μg/ml 10μg/ml and 15 μg/ml) of PS-ODNs were introduced into competent MRSA by electroporation. After electroporation, the total colony forming unit (CFU) per sample was determined by correcting the colony count for the dilution. Drug-resistant characters of MRSA were evaluated by measuring minimal inhibitory concentration (MIC) of oxacillin. To semiquantify the expression of mecRl and mecA in MRSA, RT-PCR was used. The amplified products were analyzed by electrophoresis on 1% agarose gel and visualized with ethidium bromide. The relative expression level of mecRl and mecA in comparison with 16srRNA was calculated and used to determine whether the expression of mecRl and mecA was inhibited after anti-mecRl PS-ODNs treatment.WHO-2 strains are divided into control group, random chain group(PS-ODNs1810,15 jig/ml) and PS-ODNs6087 or PS-ODNs6088 treated groups.2. Reversal of resistance of MRSA by PS-ODNs8016 and PS-ODNs8017 anti-blaRl mRNA: PS-ODNs8016 and PS-ODNs8017 were desinned and synethesized, targeting blaRl mRNA. Different concentrations ( 5μg/ml 10 ug/ml and 15 μg/ml) of PS-ODNs were introduced into competent MRSA by electroporation. After electroporation, CFU per sample was determined by correcting the colony count for the dilution. Drug-resistant characters of MRSA were evaluated by measuring MIC of oxacillin. To semiquantify the expression of blaRl and blaZ in MRSA, RT-PCR was used. The amplified products were analyzed by electrophoresis on 1% agarose gel and visualized with ethidium bromide. The relative expression level of blaRl and blaZ in comparison with \6srRNA was calculated and used to determine whether the expression of blaRl and blaZ was inhibited after anti-blaRl PS-ODNstreatment. WHO-2 strains are divided into control group, random chain group(PS-ODNs3414, 15 μg/ml) and PS-ODNs8016 or PS-ODNs8017 treated groups.RESULTS:1. Reversal of resistance of MRSA by PS-ODNs6087 and PS-ODNs6088 anti-mecRl mRNA: The number of MRSA colonies on the Mueller-Hinton agar containing oxicillin (6 ug/ml) was significantly decreased in all anti-mecRl PS-ODNs6087 and PS-ODNs6088 treated groups, in concentration dependent manner with 5,10,15 μg/ml of PS-ODNs6088 respectively(P<0.05), while CFU of MRSA was not influenced in random chain PS-ODNsl810 treated group(P>0.05). MICs of all PS-ODNs6087 and PS-ODNs6088 treated groups were 16 μg/ml, which were lower than control group and PS-ODNsl810 treated group(MIC=32 μg/ml). PS-ODNs6087 and PS-ODNs6088 inhibited mRNA expression of mecRl and mecA concentration-dependently. The PS-ODNsl810 showed no effects on the expression of both genes.2. Reversal of resistance of MRSA by PS-ODNs8016 and PS-ODNs8017 anti-blaRl mRNA: The number of MRSA colonies on the Mueller-Hinton agar containing oxicillin (6 ug/ml) was significantly decreased in 10,15 μg/ml of PS-ODNs8016 and all PS-ODNs8017 treated groups(P<0.05), while CFU of MRSA was not influenced in 5 ug/ml of PS-ODNs8016 and PS-ODNs3414 treated group(P>0.05). 15 ug/ml of PS-ODNs8016 and all PS-ODNs8017 treated groups decreased MIC of WHO-2, while 5,10 μg/ml of PS-ODNs8016. and PS-ODNs3414 treated group showed no effects on MIC of WHO-2. PS-ODNs8017 inhibited mRNA expression of blaRl and blaZ in concentration dependent manner. The PS-ODNs3414 showed no effects on the expression of both genes. CONCLUSION: 1. Anti-mecRl mRNA PS-ODNs6087 and PS-ODNs6088 selectively...
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