大肠杆菌对恩诺沙星耐药性研究
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摘要
细菌对抗菌药物产生抗药性是兽医临床的常见现象,检测临床分离细菌的抗药性可及时掌握细菌的抗药趋势,研究细菌抗药机制可以为新药物的开发及合理用药提供理论支持。
本文检测了山东地区大肠杆菌药敏情况并改进了药敏试验方法,建立了测定MPC的坡面平板法,研究了大肠杆菌对恩诺沙星的耐药机制。
利用96点阵药敏检测盒检测山东地区禽源和猪源大肠杆菌的抗药性结果显示：大肠杆菌呈现严重的多重抗药性,多抗耐药率超过90%,禽源性大肠杆菌抗药性明显高于猪源性大肠杆菌。
建立了坡面平板法,结果显示坡面平板上的药物浓度呈良好的梯度分布,可以把对药物敏感性不同的细菌区分开。研究坡面平板上大肠杆菌的性质,发现高浓度药物下生长大肠杆菌发生了基因突变,而低浓度药物下生长的没有发生基因突变。
利用基因芯片和实时荧光定量PCR研究恩诺沙星对大肠杆菌全基因表达谱的影响,结果显示：(1)恩诺沙星作用5h后,大肠杆菌519个基因出现表达差异,外排泵基因表达水平提高,外膜蛋白基因表达量急剧减少,恩诺沙星的靶酶parC基因表达量增加。说明细菌生理机制发生了广泛而复杂的变化。(2)处理菌株的恩诺沙星靶酶没有基因突变,提示低水平的细菌耐药不是由基因突变造成的。
本研究结果表明山东地区大肠杆菌对抗生素的耐受性极高；大肠杆菌可以通过自身的生理变化和相关基因表达量的变化对低浓度的抗生素产生耐药性,通过基因突变对高浓度抗生素产生抗药性。
Resistance to antibiotic is inevitable for bacterium, we can know the resistance tendency of bacterium in time by testing drug sensitivity of pathogenic from clinical, it can supply theory support to product new antibiotic by researching drug resistance mechanism.
This study tested the drug sensitivity of pathogenic from Shandong Province, researched the resistance mechanism of E.coli to enrofloxacin, created slope plate which can measuermen MPC quickly, reaserched the peresistance mechanism of E.coli to enrofloxacin.
We tested drug sensitivity of E. coli from pigs and poultries in Shandong Province by large flux drug susceptibility test kit. The results indicated:Bacterium present high multi-resistance in Shandong Province, the ratio of multi-resistance is more than 90%, the drug resistance of E.coli from poultries is higher than pigs obviously.
Researched the effect of enrofloxacin on gene expression of E.coli by gene array and RT-PCR. The results showed:(1)519 genes of E.coli appeared difference affected by enrofloxacin compared with the control group. Efflux pump gene expressed higher; outer membrane protein gene expressed decrease sharp; the target enzyme of enrofloxacin gene parC expressed higher. It illustrated the physiology of E.coli changed extensive and complicated. (2)The target enzyme of test group did not mutant, it illustrated low persistence was not caused of mutation.
Experiment results indicated the drug resistance is high of bacterium in Shandong Province, E.coli can adapt low antibiotic pressure by physiologic change and genes expression change, called persistence, can adapt high antibiotic pressure by gene mutantion, called resistance.
本文检测了山东地区大肠杆菌药敏情况并改进了药敏试验方法,建立了测定MPC的坡面平板法,研究了大肠杆菌对恩诺沙星的耐药机制。
利用96点阵药敏检测盒检测山东地区禽源和猪源大肠杆菌的抗药性结果显示：大肠杆菌呈现严重的多重抗药性,多抗耐药率超过90%,禽源性大肠杆菌抗药性明显高于猪源性大肠杆菌。
建立了坡面平板法,结果显示坡面平板上的药物浓度呈良好的梯度分布,可以把对药物敏感性不同的细菌区分开。研究坡面平板上大肠杆菌的性质,发现高浓度药物下生长大肠杆菌发生了基因突变,而低浓度药物下生长的没有发生基因突变。
利用基因芯片和实时荧光定量PCR研究恩诺沙星对大肠杆菌全基因表达谱的影响,结果显示：(1)恩诺沙星作用5h后,大肠杆菌519个基因出现表达差异,外排泵基因表达水平提高,外膜蛋白基因表达量急剧减少,恩诺沙星的靶酶parC基因表达量增加。说明细菌生理机制发生了广泛而复杂的变化。(2)处理菌株的恩诺沙星靶酶没有基因突变,提示低水平的细菌耐药不是由基因突变造成的。
本研究结果表明山东地区大肠杆菌对抗生素的耐受性极高；大肠杆菌可以通过自身的生理变化和相关基因表达量的变化对低浓度的抗生素产生耐药性,通过基因突变对高浓度抗生素产生抗药性。
Resistance to antibiotic is inevitable for bacterium, we can know the resistance tendency of bacterium in time by testing drug sensitivity of pathogenic from clinical, it can supply theory support to product new antibiotic by researching drug resistance mechanism.
This study tested the drug sensitivity of pathogenic from Shandong Province, researched the resistance mechanism of E.coli to enrofloxacin, created slope plate which can measuermen MPC quickly, reaserched the peresistance mechanism of E.coli to enrofloxacin.
We tested drug sensitivity of E. coli from pigs and poultries in Shandong Province by large flux drug susceptibility test kit. The results indicated:Bacterium present high multi-resistance in Shandong Province, the ratio of multi-resistance is more than 90%, the drug resistance of E.coli from poultries is higher than pigs obviously.
Researched the effect of enrofloxacin on gene expression of E.coli by gene array and RT-PCR. The results showed:(1)519 genes of E.coli appeared difference affected by enrofloxacin compared with the control group. Efflux pump gene expressed higher; outer membrane protein gene expressed decrease sharp; the target enzyme of enrofloxacin gene parC expressed higher. It illustrated the physiology of E.coli changed extensive and complicated. (2)The target enzyme of test group did not mutant, it illustrated low persistence was not caused of mutation.
Experiment results indicated the drug resistance is high of bacterium in Shandong Province, E.coli can adapt low antibiotic pressure by physiologic change and genes expression change, called persistence, can adapt high antibiotic pressure by gene mutantion, called resistance.
引文
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