Reverse Effects Of APS From Ampelopsis Grossedentata (Hand-mazz) W.T.Wang In Guangxi On Resistance Of Methicilin-Resistant Staphylococcus. Aureus

[Objective] MRS A has been recognized as one of the most important pathogens,which is endemic in hospitals worldwide. It was resistant to most antibiotics including methicillin. Since the methicillin resistant emerged severely, the limited antibiotics, such as glycopetide vancomycin which has been the best choice to treat MRSA infections, and the emergence of vacomycin-nonsusceptible S.aureus reported in recent years had made therapy and drugs of MRSA infections even more challenging for clinicians and researchers.Great amount of reports have demonstrated that the traditional Chinese medicine has distinctive activity to the therapy of MRSA. We have improved that APS which has been extracted from Ampelopsis grossedentata (Hand-Mazz)W.T.Wang from Guangxi had good antibacterial activity against MSSA and MRSA in vitro and in vivo. The cell wall and membrane of MSSA and MRSA were observed the morphology changes in various degrees with electron microscopy.The bacterium biosynthesis of TdR and UR were inhibited obviously.The purpose of this dissertation is to investigate the effects of APS alone and in combination with (3-lactam antibiotics on MRSA, to present the mechanisms of antibacterial, to provide the evidence for APS regaining the susceptible or decreasing the resistant against MRSA.[Methods]1. Kirby-Bauer and mecA gene test were used to identify 15 MRSA strains isolated from hospital. The MICs of 4 MRSA strains including MSAR-6, MRSA-8, MRSA-9, MRSA-12 and the standand MSSA were tested by agar dilution method. The synergistic effects of APS with 6 ?-lactam antibiotics against MRSA-6 were measured by checkerboard method with FICI.2. To evaluate the protective effect of APS alone and in combination with ?-lactam antibiotics in vivo, MRSA abdominal infection and lung infection mice models were performed with intraperitoneal injection and intranasal inhalation of MRSA-6 respectivly.3. Scanning and transmission electron microscopy were used to observe the ultrastructure of bacterial morphology treating with different concentrate APS alone and in combination with ?-lactam antibiotics against MSSA and MRSA.4. Cephalosporin nitrocefin hydrolysis method was applied to analyze the inhibition effects of APS against (3-lactamase which extracted from MRSA-6 by ultrasonic fragmentation method.5. To evaluate the suppressive effects of PBP2a protein of MRS A on APS,spectrophotometric scanning was used to detecte the contents of PBP2a protein which have been treated by APS alone and in combination with tienam and cefoxitin.6. The total RNA of MRSA treated with APS alone and in combination with tienam and cefoxitin were extracted with column extraction purification kit for total bacteria RNA. RT-PCR was used to detecte the expression of mRNA of mecA,femA and femB.7. The total RNA of MRSA treated with APS alone and in combination with tienam and cefoxitin were extracted with column extraction purification kit for total bacteria RNA. RT-PCR was used to detecte the expression of mRNA of blaZ.[Results] 4 strain MRSAs were identified with Kirby-Bauer and mecA gene test.The MICs of APS alone were 125?g/mL. Synergistic effect of APS and 5 ?-lactam antibiotics such as penicillin, oxacillin, cefazolin, tienam, cefoxitin were detected with checkerboard-method to MRSA in vitro except cefepime which showed the additive effect with APS.APS in combination with tienam and cefoxitin can increase the survival rates of abdominal infected mice by MRSA, decrease the bacterial counting of lung of infected mice, improved the inflammation of lung.The changes of morphology of MRSA were observed with scanning and transmission electron microscopy. Most cells wall of MRS A and MSS A were destroyed, the permeability of plasma membrane were changed and cytoplasmic contents of the cells leaking out.Tazobactam showed the good inhibition for ?-lactamase, while APS which alone or combined with tienam or cefoxitin had no effects on ?-lactamase.The contents of PBP2a were reduced when MRSA was treated with APS alone or tienam alone instead of cefoxitin. It showed the significant inhibition of expression of PBP2a when APS combinated with tienam and cefoxitin.Expression of mRNA of mecA, femA and femB gene was reduced after the MRSA treated with APS combinated with tienam and cefoxitin. It had no effect on the expression of mRNA of blaZ gene whether APS alone or combinated with tienam or cefoxitin.[Conclusions] APS combined with ?-lactams can increase the antibacterial effect against MRSA in intro or in vivo. The mechanism of reverse effects of it may due to the breakage of bacteria cell wall, the reducing expression of PBP2a, the down-regulation of mecA, femA and femB gene.