| on the basis of determination of major antibiotic ingredients in 4 extracts of traditional chinese medicine, in this dissertation, 20 ESBLs-producing E.coli isolates were selected, the antibacterial activities of several kinds of antibacterial agents and extracts to producing ESBLs E.coli were measured, biochemistry characteristics of ESBLs, including TEM-7V, TEM-8V, TEM-9V and CTX-M-1V, were studied furtherly, and the inhibitory action of sulbactam and extracts were surveyed in order to effectively treat the infection of Fowl caused by E.coli, to select inhibitor toβ-Lactamases from traditional chinese medicines, and provide proof for the development of compound preparation of chinese and western medicine.The results of experiment indicated that ESBLs-producing E.coli possess severe resistance to amoxicillin and ceftiofur, their MIC50 and MIC90 were 10 times higher than non-ESBLs-producing ones. However the antimicrobial activity ofβ-lactam antibiotics to ESBLs positive strains could be raised significantly by enzyme inhibitor(EI)-sulbactam, which can lower the MIC50 of amoxicillin from 146.17μg/ml to 29.66μg/ml, and that of ceftiofur was reduced from 106.22μg/ml to 21.31μg/ml, which are both near the sensitive range. In the mean time, the multi-resistance to antibiotics, such as aminoglycosides, chloramphenicol and FQNS, were presented by producing ESBLs E.coli.Baicalin, baicalin, forsythin, and paeoniflorin are essential antibacterial component in their extracts. The content of berberine in extract of Rhizoma Coptidis is 99.32%, baicalin in that of Radix Scutellariae is 93.65%, forsythin in that of Fructus Forsythiae is 34.37%, and paeoniflorin in that of Raidix Paeoniae Alba is 12.03% with high performance liquid chromatography method. Four estracts had same antibacterial activities against ESBLs-producing and non- producing E.coli, of which the most efficient one is paeoniflorin and its MIC50 against producing ESBLs and non- producing ESBLs E.coli were 0.29 and 0.22 respectively, subsequent ones were forsythin and baicalin , and the worst one is berberine, its MIC50 against ESBLs-producing and non- producing E.coli were 10.37 and 9.56 respectively. Also, there existed the most powerful synergistic effect when ceftiofur combined with paeoniflorin, and there were 15 strains presenting FIC≤0.5 of 20 ESBLs-producing E.coli. Antagonistic effect hadn't been found among the combination of ceftiofur with 3 others.The kinetic experiment ofβ-Lactamases was carried out after purification. The outcome showed us that the hydrolysis of TEM-1 to penicillins were more powerful than to cephalosporin, nevertheless except penicillins and the first genertation cephalosporin- cefalexin, the secondary generation one-cefamandole and the third generation ones- cefotaxime and ceftiofur cannot be hydrolysed. In addition, the affinity and relative hydrolytic efficiency of TEM-1 to penicillins is the highest in the 5 enzymes. TEM-7V, TEM-8V, TEM-9V and CTX-M-1V all can hydrolyze cefotaxime and ceftiofur partly, their affinity to cephalosporins are all greater than penicillins except TEM-8V, but their hydrolytic abilities to the two kinds ofβ-lactam antibiotics are on the contrary to the affinity due to their lower Vmax to cephalosporins. CTX-M-1V possessed stronger affinity to third generation cephalosporins containing hydroxyimino -cefotaxime and ceftiofur. Sulbactam showed inhibitory action to ESBLs to different extent, the range of its Ki is 0.12～1.88, that of IC50 is 1.03～2.26, among which the highest Ki is performed when come to TEM-7V which give us the suggestion that there is certain inhibitor resistant characteristics within TEM-7V. From the Ki values performed by 4 kinds of traditional chinese medicines to ESBLs, we can get the conclusion that the inhibitory action to ESBLs of Raidix Paeoniae Alba and Fructus Forsythiae were better than Rhizoma Coptidis and Radix Scutellariae, Raidix Paeoniae Alba owned the best inhibitory action and Rhizoma Coptidis exhibited the worst , but the inhibitory action of traditional chinese medicine were remarkably lower than sulbactam.In conclusion, different ESBLs performed different affinity and hydrolytic ability, so properβ-lactam antibiotics should be chosen to the clinical treatment accordding to actual situation. At the same time, Raidix Paeoniae Alba can be associated withβ- lactam antibiotics to validly treat the clinical infection caused by producing ESBLs E.coli. |
