临床微生物样本中持留菌的筛选及对肺炎克雷伯杆菌持留株持留机制的研究
发布时间：2018-08-05 20:01
【摘要】：抗生素在杀菌抗感染方面为人类带来了巨大贡献,但随之而来的耐药问题,给全球抗感染治疗带来极大挑战。目前,随着细菌耐药机制研究的越发深入,研究者越来越关注一种特殊的耐药情况,即病原体表型耐药,其耐药机制不涉及细菌基因组突变,但在高浓度抗生素作用条件下,仍有一小部分细菌存活,抗生素压力撤销后,这部分细菌又恢复增殖生长。其实,这一现象在抗生素应用早期即已发现,1944年,Joseph Bigger在研究盘尼西林药效动力学时,首先描述了持留菌(persister)和细菌持留(Bacterial persistence)现象,其发现,向处于生长期的金黄色葡萄球菌菌液中加入盘尼西林,有一小部分细菌仍能存活,但这些细菌并不是耐抗生素的突变株。Bigger推测,持留菌能躲避抗生素杀伤,是由于这些细菌处于休眠状态,细胞整体代谢水平下调,且不进行分裂生长,因此能躲避抗生素杀伤。由于持留菌在细菌种群里比例极低,关于它的研究停滞了相当长的时间,直到上世纪80年代,对持留菌的分离培养才取得些许进展,目前各国科学家已成功分离培养出了大肠杆菌、结核杆菌、金黄色葡萄球菌、纳豆芽孢杆菌等细菌的持留变异株,在真菌方面,也有研究报道了白色念珠菌的持留现象,这些发现大大推动了对该领域的研究。特别是在慢性感染、抗生素失效、临床耐药现象越来越突出的情况下,迫切需要重新面对这个老问题,开展新的研究。本研究通过分析临床微生物数据,筛选出大肠埃希菌、肺炎克雷伯杆菌、铜绿假单胞菌、金黄色葡萄球菌慢性感染样本,并通过持留菌在抗生素条件下的特异生长曲线,从中筛选持留株,最终共获得4株大肠杆菌持留株,23株肺炎克雷伯杆菌持留株,8株铜绿假单胞菌持留株,6株金黄色葡萄球菌持留株。分析慢性感染患者持留菌样本后发现,持留菌有以下特性:持留菌具有多重耐药性,但不同菌种对不同抗生素的持留水平不同,其中大肠埃细菌对头孢噻肟的持留水平最低,肺炎克雷伯杆菌对环丙沙星的持留水平最低,铜绿假单胞菌对左氧氟沙星的持留水平最低,金黄色葡萄球菌对亚胺培南的持留水平最低;持留菌能耐受营养不足的生长条件;分离纯化的单个持留菌菌落,在普通培养基中可恢复生长,加入抗生素后,其生长曲线与亲代一致。其中,持留菌的多药耐受表型与文献中结果一致,但各菌种在不同抗生素中的持留水平与文献报道不同,该差异可能是由持留菌所处环境及持留形成过程中各种刺激因素不同所导致。由持留菌特性可证实,持留是一种菌群整体的自我保护机制,保证菌群在各种杀伤因素作用下,仍有部分细菌存活下来,并把整个菌落的基因组信息保留传递下去,且持留菌的持留水平存在地域差异。用转座子诱导突变法对肺炎克雷伯杆菌持留株的持留相关基因进行定位。综合文献结果,本课题组发现,尚未见肺炎克雷伯杆菌持留机制的报道,因此本研究特选取1株肺炎克雷伯杆菌持留株,用转座子诱导突变法构建肺炎克雷伯杆菌持留株突变体库,共获得3282个突变株,并从中筛选到1株失去持留特性的突变株,通过反向PCR等方法确定持留基因为NC_016845.1基因,该基因编码磷酯酶D,参与细菌磷脂代谢。由此推断,细菌通过调节代谢水平来改变细胞膜通透性及表面电荷,从而使自身转化为持留状态,以抵御抗生素、酸、碱等因素的杀伤。
[Abstract]:Antibiotics have made great contributions to human beings in the field of bactericidal and anti infection, but the attendant problem of drug resistance brings great challenges to the global anti infection treatment. At present, with the further research on the mechanism of bacterial resistance, researchers pay more and more attention to a special drug resistance, that is, the phenotypic resistance of pathogens, and the mechanism of drug resistance does not involve bacteria. Genomic mutation, but a small number of bacteria still survive under the condition of high concentration of antibiotics, and this part of the bacteria restores proliferation after the antibiotic pressure is revoked. In fact, this phenomenon was discovered early in the application of antibiotics. In 1944, Joseph Bigger described the retention bacteria (persiste) in the study of the pharmacodynamics of penicillin. R) and the bacterial retention (Bacterial persistence) phenomenon, it was found that penicillin was added to the growth phase of Staphylococcus aureus, and a small number of bacteria still survived, but these bacteria were not antibiotic resistant mutants.Bigger speculates that the retained bacteria can avoid antibiotic killing because these bacteria are in a dormant state. The overall metabolic level of the cells is down and does not divide growth, so it can avoid the killing of antibiotics. Because the proportion of the retained bacteria is very low in the bacterial population, the research on it has been stagnant for quite a long time. Until the 80s of last century, some progress was made to the isolation and cultivation of the retained bacteria, and scientists in various countries have successfully separated and cultivated. The retention of Escherichia coli, Mycobacterium tuberculosis, Staphylococcus aureus and Bacillus natto, and fungi, also reported on the retention of Candida albicans, which greatly promoted the research in this field, especially in the case of chronic infection, antibiotic failure, and clinical drug resistance. Under the analysis of clinical microbiological data, this study screened Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, chronic infection samples of Staphylococcus aureus, and screened the remaining strains by the specific growth curve of the retained bacteria under the condition of antibiotics. 4 strains of Escherichia coli, 23 strains of Klebsiella pneumoniae, 8 strains of Pseudomonas aeruginosa and 6 strains of Staphylococcus aureus were found. After the analysis of chronic infected patients, the retention bacteria had the following characteristics: the retention bacteria had multiple resistance, but the retention levels of different strains to different antibiotics were not. In the same way, the retention level of Escherichia coli to cefotaxime is the lowest, Klebsiella pneumoniae has the lowest retention level to ciprofloxacin, the retention level of Pseudomonas aeruginosa to levofloxacin is the lowest, and the retention level of Staphylococcus aureus to imipenem is the lowest; the retention bacteria can tolerate the growth conditions of undernourishment; isolation and purification The growth curve of single retention bacteria can be recovered in ordinary medium. After adding antibiotics, the growth curve is consistent with the parent. Among them, the tolerance phenotype of the retained bacteria is consistent with the results in the literature, but the retention level of various strains in different antibiotics is different from that in the literature, which may be caused by the environment and retention of the retained bacteria. The retention is a self-protection mechanism of the whole bacteria group, which ensures that some bacteria survive under the action of all kinds of killing factors, and the whole bacterial genome information is preserved and transferred, and the retention level of the retained bacteria exists regional difference. The retention related genes of Klebsiella pneumoniae were located with the method of transposon induction. The results of the comprehensive literature showed that there was no report on the retention mechanism of Klebsiella pneumoniae. Therefore, 1 Klebsiella pneumoniae were selected to establish the retention strain of Klebsiella pneumoniae, and Klebsiella pneumoniae was constructed by transposon induction mutation. A total of 3282 mutant strains were obtained, and 1 mutant strains were screened from the mutant. The retained group was identified by reverse PCR as the NC_016845.1 gene, which encodes the phospholipase D and participates in the metabolism of bacterial phospholipid. Thus, the bacteria can be used to regulate the membrane permeability and surface charge by regulating the metabolic level. So as to transform itself into a state of retention, in order to resist the killing of antibiotics, acids, alkaloids and other factors.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
本文编号：2166864
[Abstract]:Antibiotics have made great contributions to human beings in the field of bactericidal and anti infection, but the attendant problem of drug resistance brings great challenges to the global anti infection treatment. At present, with the further research on the mechanism of bacterial resistance, researchers pay more and more attention to a special drug resistance, that is, the phenotypic resistance of pathogens, and the mechanism of drug resistance does not involve bacteria. Genomic mutation, but a small number of bacteria still survive under the condition of high concentration of antibiotics, and this part of the bacteria restores proliferation after the antibiotic pressure is revoked. In fact, this phenomenon was discovered early in the application of antibiotics. In 1944, Joseph Bigger described the retention bacteria (persiste) in the study of the pharmacodynamics of penicillin. R) and the bacterial retention (Bacterial persistence) phenomenon, it was found that penicillin was added to the growth phase of Staphylococcus aureus, and a small number of bacteria still survived, but these bacteria were not antibiotic resistant mutants.Bigger speculates that the retained bacteria can avoid antibiotic killing because these bacteria are in a dormant state. The overall metabolic level of the cells is down and does not divide growth, so it can avoid the killing of antibiotics. Because the proportion of the retained bacteria is very low in the bacterial population, the research on it has been stagnant for quite a long time. Until the 80s of last century, some progress was made to the isolation and cultivation of the retained bacteria, and scientists in various countries have successfully separated and cultivated. The retention of Escherichia coli, Mycobacterium tuberculosis, Staphylococcus aureus and Bacillus natto, and fungi, also reported on the retention of Candida albicans, which greatly promoted the research in this field, especially in the case of chronic infection, antibiotic failure, and clinical drug resistance. Under the analysis of clinical microbiological data, this study screened Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, chronic infection samples of Staphylococcus aureus, and screened the remaining strains by the specific growth curve of the retained bacteria under the condition of antibiotics. 4 strains of Escherichia coli, 23 strains of Klebsiella pneumoniae, 8 strains of Pseudomonas aeruginosa and 6 strains of Staphylococcus aureus were found. After the analysis of chronic infected patients, the retention bacteria had the following characteristics: the retention bacteria had multiple resistance, but the retention levels of different strains to different antibiotics were not. In the same way, the retention level of Escherichia coli to cefotaxime is the lowest, Klebsiella pneumoniae has the lowest retention level to ciprofloxacin, the retention level of Pseudomonas aeruginosa to levofloxacin is the lowest, and the retention level of Staphylococcus aureus to imipenem is the lowest; the retention bacteria can tolerate the growth conditions of undernourishment; isolation and purification The growth curve of single retention bacteria can be recovered in ordinary medium. After adding antibiotics, the growth curve is consistent with the parent. Among them, the tolerance phenotype of the retained bacteria is consistent with the results in the literature, but the retention level of various strains in different antibiotics is different from that in the literature, which may be caused by the environment and retention of the retained bacteria. The retention is a self-protection mechanism of the whole bacteria group, which ensures that some bacteria survive under the action of all kinds of killing factors, and the whole bacterial genome information is preserved and transferred, and the retention level of the retained bacteria exists regional difference. The retention related genes of Klebsiella pneumoniae were located with the method of transposon induction. The results of the comprehensive literature showed that there was no report on the retention mechanism of Klebsiella pneumoniae. Therefore, 1 Klebsiella pneumoniae were selected to establish the retention strain of Klebsiella pneumoniae, and Klebsiella pneumoniae was constructed by transposon induction mutation. A total of 3282 mutant strains were obtained, and 1 mutant strains were screened from the mutant. The retained group was identified by reverse PCR as the NC_016845.1 gene, which encodes the phospholipase D and participates in the metabolism of bacterial phospholipid. Thus, the bacteria can be used to regulate the membrane permeability and surface charge by regulating the metabolic level. So as to transform itself into a state of retention, in order to resist the killing of antibiotics, acids, alkaloids and other factors.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
【共引文献】
相关期刊论文 前1条
1 姜旭;王丽敏;张桂敏;于波;曾庆韬;;基因工程菌发酵生产L-乳酸研究进展[J];生物工程学报;2013年10期
，本文编号：2166864
本文链接：https://www.wllwen.com/huliyixuelunwen/2166864.html
最近更新
教材专著
