| AIMMethicillin-resistant S. aureus (MRSA) has become a worldwide problem in many areas of medical care, and the number of infections caused by MRSA is increasing every year. These infections are more difficult to treat than others because of the resistance of the bacterium to the most antibiotics and the only one of the first line antibiotics is vancomycin. As vancomycin resistant staptytococous aureus comes, the impact of the infections on mortality has been stupendous. These bacteria all show a phenotype of B -lactam resistance. The resistance is due to production of either B -lactamase or an extra penicillin-binding protein, PBP2a. B -lactamase is encoded by the chromosomal gene blaZ in MRSA. Transcription of blaZ is regulated by the sensor-transducer proteins, BlaRl and its partner repressor, BlaI, which bind to DNA sequence in blaZ gene. Inactivation of the BlaI repressor through proteolytic cleavage results in derepression of the blaZ and 3 -lactamase production. DNAzyme is a catalytic active DNA molecular isolated from artifical random-sequence population using evolutionary process in vitro selection protocols. A general RNA-cleaving DNA enzyme is 10-23 consisting of an internal loop as its catalytic domain and two flankingsubstrate-recognition domains which were complementary to substrate. It could cleave almost any RNA sequence contains a purine-pyrimidine under simulated physiological conditions. DNAzyme has been applied in fields such as anti-tumor, cardiovascular diseases and antiviral infection. Therefore, in this study, DNAzyme has been used to inhibit the expression of sensor-transducer protein, BlaR1 in an attempt to the blockade of BlaR1 signal pathway and converse MRSA to be antibiotic susceptible.METHODS1. Identification of MRSA: The National Committee for Clinical Laboratory Standards (NCCLS)-recommended "Screening Test for Oxacillin-resistant S. aureus" was used to identify three strains of S. aureus (WHO-2, SA-1 and ATCC25923) and Kirby-Bauer have been applied for detecting the antibiotic resistance of S. aureus. The target gene, blaRI and blaZ were detected by PCR.2. Dz1556 to converse MRSA (WHO-2) to be antibiotic susceptible: mRNA of blaRl was chosen as a target gene and DNAzyme Dzl556 specific to it was designed and synthesized. Dzl556s (10ug mL-1, 15ug mL-1 and 20ug mL-1) were introduced into the MRSA through electroporation (voltage 900V, intitial field stength 9KV/cm; capacitance 25uF). Drug-resistant characters of MRSA were evaluated by cloning efficiency. The inhibition effects of Dz1556 on the expressions of Drug-resistant gene blaRl and blaZ were observed by RT-PCR in MRSA.3. Dz1556 to converse MRSA (SA-1) to be antibiotic susceptible: After Dz 1556s (10ng mL-1, 15ug mL-1 and 20ug mL-1) were introduced into the MRSA (SA-1), drug-resistant characters of MRSA were evaluated by cloning efficiency. The inhibition effects of Dz1556 on the expressions of Drug-resistant gene blaRI and blaZ were observed by RT-PCR in MRSA.RESULTS1. Identification of MRSA: After MRSA incubated for 24h onagar, there was a circulus of 20mm around the oxa disc (1ug/piece) in the plat of ATCC25923. However, no circulus was detected in the plat of WHO-2 or SA-1. There were a lot of Colonys in the plat of WHO-2 or SA-1 containing Oxacillin (oxa, 6ug mL-1) and no Colonys in the plat of ATCC25923. The results of PCR showed that WHO-2 and SA-1 carried blaR1 and blaZ gene, while no blaR1 or blaZ gene was detected in ATCC25923.2. CFU of MRSA (WHO-2) incubated with Dz1556 on the M-H agar added oxa (6ug mL-1') are less than that of control group (P < 0.05 or P< 0.01). Level of blaR1 mRNA of the Dzl556 group was lower than that of the control group (P < 0.05 or P < 0.01).3. CFU of MRSA (SA-1) incubated with Dzl556 on the M-H agar added oxa (6ug mL-1) are less than that of control group (P < 0.05 or P< 0.01). Level of blaRl mRNA of the Dzl556 group was lower than that of the control group (P < 0.05 or P < 0.01)... |
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