痰热清注射液对产ESBLs大肠埃希菌耐药性影响的研究

【作者】 李渊；

【导师】 何明；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 大肠埃希菌是最常见的社区及医院内获得性感染的病原菌之一。其中,产超广谱β-内酰胺酶(ESBLs)大肠埃希菌引起的感染又在大肠埃希菌引起的感染中占据首位,该细菌能够水解青霉素类抗生素,以及第一、第二、第三代和部分第四代头孢菌素,并且部分产ESBLs大肠埃希菌同时对氨基糖苷类和氟喹诺酮类抗生素也耐药。于是这些产ESBLs大肠埃希菌株引起的感染,可供选择的抗生素较少,并且治疗效果往往不理想,导致患者病死率上升,给临床治疗带来了较大的困难,同时也使医疗费用增加,带来了较重的社会经济负担。许多研究报道某些中药方剂在体内具有良好的抗菌作用,尤其是在治疗或联合抗生素治疗耐药菌感染的疾病上,取得了较好的临床疗效。而且某些中药在体外对临床常见耐药菌具有较强的抗菌作用,因此深入研究中药对耐药菌耐药性的影响,有望为解决目前严峻的细菌耐药问题提供新的途径。本论文主要包括文献综述、北京中医药大学东方医院2010年主要细菌耐药性监测、实验研究3部分。1文献综述分为3部分,分别介绍了中药抗菌作用的研究进展、痰热清抗感染作用的研究进展以及产ESBLs大肠埃希菌的研究进展。2北京中医药大学东方医院2010年主要病原菌耐药性监测目的：了解2010年北京中医药大学东方医院主要细菌的检出率及对抗生素的耐药状况。方法：参照《全国临床检验操作规程》第2版获取细菌标本。参照CLSI推荐的方法,采用全自动微生物鉴定和药敏分析系统VITEK-32进行细菌鉴定以及药敏实验。结果：2010年1月至12月东方医院共分离细菌3489株,占前5位的细菌及其比例分别是：大肠埃希菌(17.71%)、铜绿假单胞菌(17.25%)、金黄色葡萄球菌(12.81%)、鲍曼复合醋酸钙不动杆菌(10.66%)、肺炎克雷伯菌(8.94%)。大肠埃希菌、肺炎克雷伯菌中ESBLs的检出率为分别为68.12%、41.03%,金黄色葡萄球菌中MRSA的检出率为91.95%。大肠埃希菌对亚胺培南的耐药率为1.62%,对美罗培南的耐药率达到了5%；对哌拉西林/他唑巴坦、头孢哌酮/舒巴坦的耐药率分别为17%、16%,对头孢他啶、头孢噻肟、头孢吡肟的耐药率均超过了75%；对头孢西丁的耐药率为26%,对阿卡米星仍保持较高的敏感性,耐药率仅为15%,对左氧氟沙星的耐药率为68%。铜绿假单胞菌对亚胺培南、美罗培南的耐药率分别为36%、46%；对哌拉西林/他唑巴坦、头孢哌酮/舒巴坦的耐药率分别为27%、44%；对头孢他啶、头孢吡肟的耐药率分别为55%、68%,对阿卡米星的耐药率为14%,对左氧氟沙星的耐药率为50%。金黄色葡萄球菌对万古霉素的耐药率为0.22%、对呋喃妥因、复方新诺明、利福平、四环素、庆大霉素的耐药率分别为25%、40%、74%、76%、89%。鲍曼复合醋酸钙不动杆菌对亚胺培南、美罗培南的耐药率分别为73%、70%,对哌拉西林/他唑巴坦、头孢哌酮/舒巴坦的耐药率分别为62%、73%,对头孢他啶、阿卡米星、左氧氟沙星的耐药率分别为84%、69%、74%。肺炎克雷伯菌对亚胺培南、美罗培南的耐药率分别为4%、11%,对哌拉西林/他唑巴坦、头孢哌酮/舒巴坦的耐药率分别为41%、31%。结论：我院2010年主要分离的细菌中,大肠埃希菌居第1位,其中ESBLs的检出率高达68.12%,对多种抗生素的耐药率均较高,并且出现了多重耐药菌株,给临床治疗带来较大的困难。目前,我院在治疗大肠埃希菌引起的感染时,抗生素的选择压力较大,成为一个亟待解决的问题。另外,铜绿假单胞菌、金黄色葡萄球菌、鲍曼复合醋酸钙不动杆菌、肺炎克雷伯菌的耐药状况同样不容乐观。3实验研究目的：通过研究痰热清注射液对产ESBLs大肠埃希菌超广谱β-内酰胺酶的影响,探讨痰热清对产ESBLs大肠埃希菌的耐药性产生影响的机制,为解决当前严峻的细菌耐药性问题提供可能的途径,也为进一步研究中药影响细菌耐药性的机制打下基础。方法：取来源于临床,经全自动微生物鉴定和药敏分析系统(VITEK-32)进行鉴定和表型筛选的产ESBLs大肠埃希菌。采用试管2倍稀释法测定痰热清注射液对产ESBLs大肠埃希菌的MIC。采用纸片扩散法测定3个不同浓度的痰热清(痰热清1组、痰热清2组、痰热清3组)作用于产ESBLs大肠埃希菌后的药敏实验抑菌圈直径,以及经舒巴坦作用后(阳性组)和不经任何药物作用(阴性组)的耐药菌药敏实验的抑菌圈直径,并用分光光度法测定以上各组耐药菌的ESBLs活性；痰热清的浓度均选择低于其MIC的浓度。结果：痰热清注射液对10株产ESBLs的大肠埃希菌的MIC为31.25μl/ml-125μl/ml。其中痰热清对菌株1、菌株2、菌株5、菌株6、菌株8、菌株9的MIC为62.5μl/ml。选取以上6株菌行药敏实验及ESBLs活性的测定实验。经痰热清作用的菌株1、6、8,其头孢他啶、头孢噻肟、头孢哌酮的抑菌圈直径与阴性组比均具有统计学差异(P<0.05),菌株1、6、8对3种抗生素的敏感性均增强；经痰热清作用的菌株5,其头孢噻肟、头孢哌酮的抑菌圈直径与阴性组比具有统计学差异(P<0.05),菌株5对头孢噻肟、头孢哌酮的敏感性增强；经痰热清作用的菌株9,其头孢他啶的抑菌圈直径与阴性组比具有统计学差异(P<0.05),菌株9对头孢他啶的敏感性增强；经痰热清作用的菌株2,其3种抗生素的抑菌圈直径与阴性组比均无统计学差异(P<0.05),菌株2对3种抗生素的敏感性均未增强。经痰热清作用的菌株1、5、6、9的ESBLs活性降低,与阴性组比均具有统计学差异(P<0.05)；经痰热清作用的菌株2、8的ESBLs活性未降低,与阴性组比均无统计学差异(P<0.05)。结论：痰热清注射液对某些产ESBLs大肠埃希菌的ESBLs活性具有抑制作用,使其对某些抗生素的敏感性增强；痰热清对某些耐药菌的ESBLs活性无抑制作用,也不能使其对抗生素的敏感性增强；痰热清可能对不同的ESBLs的活性具有不同的影响；痰热清对某些耐药菌的ESBLs活性无抑制作用,但能使其对某些抗生素的敏感性增强,那么痰热清可能存在其他使耐药菌对抗生素敏感性增强的机制。更多还原

【Abstract】 The Escherichia coli (E. coli) was the most common community acquired and hospital acquired pathogen. Among them, the infection caused by the extended-spectrum beta-lactamase (ESBLs) producing E. coli took the first place. ESBLs producing E. coli can hydrolyze penicillin, and the first, second, third generation and part of the fourth generation cephalosporin. Part of the strains can also resist aminoglycoside and fluoroquinolones antibiotics. So the infection caused by it was treated by less antibiotics, and the effect was often not ideal which increased mortality. This made the treatment difficult, made treatment fee increase, and also brought heavier social economic burden.It was reported that some formulas had certain antibacterial effect on some bacteria in vivo, especially achieved good curative effect in the therapy of some drug-resistant bacteria infection diseases. And some Chinese medicine had stronger antimicrobial effect on some drug-resistant bacteria. So it is possible to give method for the current grim drug-resistant bacteria problems by further research on antibacterial effect on Chinese medicine.This paper mainly included literature review, monitoring drug resistance of main bacteria in Dongfang hospital in 2010 and experimental research.1 Literature reviewThe literature review introduced the research progress of traditional Chinese medicine antibacterial function, Tanreqing, and E. coli producing ESBLs.2 Monitoring drug resistance of main bacteria in Dongfang hospital in 2010 Objective:To investigate the detection rate of main bacteria and the drug resistance of bacteria to antibiotics in Dongfang hospital of Beijing university of Chinese medicine in 2010.Methods:According to certain regulations, to isolate and culture bacteria. according to CLSI,to identify and do drug susceptibility test by VITEK-32.Results:From January to December of 2010,3489 strains were isolated in Dongfang hospital, the top five respectively were:E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter calcoaceticus-baumanii complex and Klebsiella pneumonia, and the detection rates were 17.71%,17.25%,12.81%, 10.66%,8.94% respectively. The detection rates of ESBLs producing E. coli and ESBLs producing Klebsiella pneumoniae respectively were 68.12% and 41.03%, and the rate of MRSA was 91.95%. The resistance rate of E.coli to imipenem was 1.62% and that of E.coli to meropenem was 5%. The resistanc rates of E. coli to piperacillin/tazobactam and cefoperazone/sulbactam respectively were 17% and 16%. To ceftazidime, cefota-xime and cefepime, the rates were more than 75%. To cefoxitin, the rate was 26%. It remained high sensitive to amikacin with only 15% of antimicrobial resistance. The rate to Levof loxacin was 68%. The resistance rates of Pseudomonas aeruginosa to imipenem and meropenem respectively were 36% and 46%. The rates to piperacillin/tazobactam and cefoperazone/sulbactam were 27%,44% respecttively. The rates to ceftazidime, cefepime were 55% and 68% respectively, to amikacin was 14% and to levof loxacin was 50%. The resistance rate of Staphylococcus aureus to vancomycin was 0.22%, to nitrofurantoin, cotrimoxazole, rifampin, tetracycline, gentamicin were 25%,40%,74%,76% and 89% respectively. The resistance rates of Acinetobacter calcoaceticus-baumanii complex to imipenem and meropenem were 73%,70% respectively, to piperacillin/tazobactam and cefoperazone/sulbactam were 62%,73% respectively, to ceftazidime was 84%, to amikacin was 69%, and to levof loxacin was 74%. The resistance rates of Klebsiel la pneumoniae to imipenem and meropenem were 4%,11% respectively, to piperacillin/tazobactam and cefoperazone/sulbactam were 41%,31% respectively.Conclusion:E. coli took the first place among the bacteria isolated from the hospital in 2010. The detection rate of ESBLs producing E.coli reached up to 68.12%, the resistance rates of E. coli to many antibiotics were higher on average and multiple drug-resistant strains appeared which brought greater difficulties in clinical treatment. The hospital had to face the problem that how to choose the antibiotics to treat the infections caused by E.coli. Furthermore, the situations in Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter calcoaceticus, and Klebsiella pneumoniae were not optimistic.3 Experiment researchObjective:To study the impact of Tanreqing injection on ESBLs, discussing the mechanism of it. It was the basic for further study to antibacterial mechanism of Chinese medicine, and giving possible method for the current grim drug-resistant bacteria problems.Methods:To take ESBLs producing E.coli from the clinical laboratory which identified by VITEK-32. To adopt two-folded dilution method to determine MIC of Tanreqing injection to E.coli producing ESBLs. To measure inhibition zone diameters of Tanreqing injection with different concentrations effecting on E. coli producing ESBLs by disc diffusion method. To use spectrophotometry to determine the ESBLs activity of E.coli affected by Tanreqing.Results:The MIC of Tanreqing to 10 ESBLs producing E.coli strains were 31.25μl/ml-125μl/ml. The MIC of Tanreqing to strains 1,2,5,6,8 and 9 were 62.5μl/ml. To select the six strains to do drug sensitive test and determine the ESBLs activity.Affected by Tanreqing, the inhibition zone diameters of ceftazidime, cefoperazone, cefotaxime of strains 1,6 and 8 were increased and their antibiotics sensitivity were enhanced, statistically significantly (P<0.05). The inhibition zone diameters of cefotaxime and cefoperazone of strain 5 were increased, and its sensitivity to cefotaxime and cefoperazone were enhanced, statistically significantly (P<0.05). The inhibition zone diameter of ceftazidime of strain 9 was increased, and its sensitivity to ceftazidime was enhanced, statistically significantly (P<0.05). The inhibition zone diameters of three kinds antibiotics of strains 2 and 8 were not increased and their sensitivity were not enhanced (P>0.05).Affected by Tanreqing, ESBLs activity of strainsl,5,6 and 9 decreased statistically significantly(P<0.05), ESBLs activity of the strains 2,8 did not reduce (P>0.05).Conclusion:Tanreqing inhibited the ESBLs activity of some E. coli strains and enhanced their sensitivity to some antibiotics; Tanreqing had no inhibition on the ESBLs activity of some E. coli strains, and also did not strengthen their sensitivity to antibiotics; Tanreqing may had different impact on different ESBLs of E. coli. Tanreqing had no impact on ESBLs activity of some strains, but increased their sensitivity to some antibiotics. There may be other mechanism for Tanreqing to strengthen antibiotics sensitivity of drug-resistant strains.更多还原