Studies On The Effects Of Chinese Medicine Resistance Inhibitor On Serum Pharmacology And Sequence Mutation Of Related Gene

Staphylococcus aureus that is clear in drug resistance mechanism was used as the test organisms in this study to investigate the drug resistance inhibitor of medicinal preparation and the traditional Chinese medicine. Research compound prescription Chinese medicine, goldthread rhizome injections, bears the drug inhibitory action in vitro, and the blood serum's pharmacological function and bacteriostasis ability in vivo. To futher understood the molecular mechanism of the drug resistance by compound prescription Chinese goldthread rhizome injection, the influence of the compound prescription Chinese goldthread rhizome injection on the induction of Staphylococcus aureus gyrA gene was also investigated at the molecular level. These data provide some scientific foundation that the compound prescription Chinese goldthread rhizome injection and the traditional Chinese medicine as potent drug resistance inhibitors.The first part of this study is 1) to determine the resistance spectruma and drug resistance of Staphylococcus aureus from milk; 2) to establish the bank of strains possing drug resistance for investigating the inhibition of drug resistance by medicinal preparation and the traditional Chinese medicine; 3) to understand the molecular basis of drug resistance of Chinese goldthread rhizome injection by plasmids eliminating test. The results indicated that 6 Staphylococcus aureus from milk had durg resistance to Ofloxacin, Norfloxacin, Amoxicillin, Cefoxitin and Ciprofloxacin as multidrug resistance. The bank of strains possing drug resistance was established by using different concentration gradient of Norfloxacin, Ciprofloxacin, Amoxicillin and Cefoxitin. The results from borehole method and tpaper disk method indicated that the compound prescription of Chinese goldthread rhizome injection may strengthen sensitivity to the Fluoroquinolones. The plasmids eliminating test revealed that the drug resistance against Staphylococcus aureus had no relationship with plasmid. In other words, there drug resistant stains did not contain the drug resistance plasmid. The second part of this study is 1) to determine the RP-HPLC method for me asuring the content of hydrochloric acid berberine in compound Chinese goldthr ead rhizome injection and serum with Chinese drug; The RP-HPLC method wa s established to determine the content of hydrochloric acid berberine in compo und Chinese goldthreadrhizome injection and serum with Chinese drug through the examination of mobile phase composition, column temperature, retention time, ultraviol et detection wave.2) to study serum pharmacology and in in vivo effects of compound Chinese goldthread rhizome injection by measuring the blood level of drug. Serum pharmacological analysis by using the compound prescription Chinese goldthre ad rhizome injection showed that the compound prescription Chinese goldthread rhizome injection has the obvious inhibitory action to the drug resistance of S taphylococcus aureus, MIC for S1-S5 is 0.125g/ml, MIC for Sa6 is 0.25g/ml. The inhibition zone diameter is between 14-17 mm. These results suggested th at hydrochloric acid berberine is principal constituen possing antibacterial functi on in the compound prescription Chinese goldthread.The 3rd part of this study is to explorate relationship of the drug resistance strength of Staphylococcus aureus to Fluoroquinolones with sequence mutation of gyrA gene, and the relationship of the compound prescription Chinese goldthread rhizome injection to the sequence mutation of Staphylococcus aureu gyrA gene and the amino acids. The results revealed that higher drug resistance of bacterium accompanied with an increase rate of sequence and the amino acid mutation. The sequence nutation of gyrA gene at 32,43 and 125 base caused drug resistance of Staphylococcus aureus to Norfloxacin. The sequence nutation of gyrA gene at 251 and 298 base caused higher level of drug resistance. The retrieval sequence mutations of gyrA gene at base 251 (Câ†'T) and 259 (Câ†'T) were observed in staphylococcus aureu treated with compound prescription Chinese goldthread rhizome injection. In the same style, the retrieval sequence mutations of amino acids were also observed at amino acid 88 (Valâ†'Ser) and 99 (Gluâ†'Lys). That is the reason that Staphylococcus aureus gyrA caused inhibitory effects to drug resistance.