臨床微生物樣本中持留菌的篩選及對肺炎克雷伯桿菌持留株持留機制的研究
發(fā)布時間：2018-08-05 20:01
【摘要】：抗生素在殺菌抗感染方面為人類帶來了巨大貢獻,但隨之而來的耐藥問題,給全球抗感染治療帶來極大挑戰(zhàn)。目前,隨著細菌耐藥機制研究的越發(fā)深入,研究者越來越關(guān)注一種特殊的耐藥情況,即病原體表型耐藥,其耐藥機制不涉及細菌基因組突變,但在高濃度抗生素作用條件下,仍有一小部分細菌存活,抗生素壓力撤銷后,這部分細菌又恢復(fù)增殖生長。其實,這一現(xiàn)象在抗生素應(yīng)用早期即已發(fā)現(xiàn),1944年,Joseph Bigger在研究盤尼西林藥效動力學(xué)時,首先描述了持留菌(persister)和細菌持留(Bacterial persistence)現(xiàn)象,其發(fā)現(xiàn),向處于生長期的金黃色葡萄球菌菌液中加入盤尼西林,有一小部分細菌仍能存活,但這些細菌并不是耐抗生素的突變株。Bigger推測,持留菌能躲避抗生素殺傷,是由于這些細菌處于休眠狀態(tài),細胞整體代謝水平下調(diào),且不進行分裂生長,因此能躲避抗生素殺傷。由于持留菌在細菌種群里比例極低,關(guān)于它的研究停滯了相當(dāng)長的時間,直到上世紀(jì)80年代,對持留菌的分離培養(yǎng)才取得些許進展,目前各國科學(xué)家已成功分離培養(yǎng)出了大腸桿菌、結(jié)核桿菌、金黃色葡萄球菌、納豆芽孢桿菌等細菌的持留變異株,在真菌方面,也有研究報道了白色念珠菌的持留現(xiàn)象,這些發(fā)現(xiàn)大大推動了對該領(lǐng)域的研究。特別是在慢性感染、抗生素失效、臨床耐藥現(xiàn)象越來越突出的情況下,迫切需要重新面對這個老問題,開展新的研究。本研究通過分析臨床微生物數(shù)據(jù),篩選出大腸埃希菌、肺炎克雷伯桿菌、銅綠假單胞菌、金黃色葡萄球菌慢性感染樣本,并通過持留菌在抗生素條件下的特異生長曲線,從中篩選持留株,最終共獲得4株大腸桿菌持留株,23株肺炎克雷伯桿菌持留株,8株銅綠假單胞菌持留株,6株金黃色葡萄球菌持留株。分析慢性感染患者持留菌樣本后發(fā)現(xiàn),持留菌有以下特性:持留菌具有多重耐藥性,但不同菌種對不同抗生素的持留水平不同,其中大腸埃細菌對頭孢噻肟的持留水平最低,肺炎克雷伯桿菌對環(huán)丙沙星的持留水平最低,銅綠假單胞菌對左氧氟沙星的持留水平最低,金黃色葡萄球菌對亞胺培南的持留水平最低;持留菌能耐受營養(yǎng)不足的生長條件;分離純化的單個持留菌菌落,在普通培養(yǎng)基中可恢復(fù)生長,加入抗生素后,其生長曲線與親代一致。其中,持留菌的多藥耐受表型與文獻中結(jié)果一致,但各菌種在不同抗生素中的持留水平與文獻報道不同,該差異可能是由持留菌所處環(huán)境及持留形成過程中各種刺激因素不同所導(dǎo)致。由持留菌特性可證實,持留是一種菌群整體的自我保護機制,保證菌群在各種殺傷因素作用下,仍有部分細菌存活下來,并把整個菌落的基因組信息保留傳遞下去,且持留菌的持留水平存在地域差異。用轉(zhuǎn)座子誘導(dǎo)突變法對肺炎克雷伯桿菌持留株的持留相關(guān)基因進行定位。綜合文獻結(jié)果,本課題組發(fā)現(xiàn),尚未見肺炎克雷伯桿菌持留機制的報道,因此本研究特選取1株肺炎克雷伯桿菌持留株,用轉(zhuǎn)座子誘導(dǎo)突變法構(gòu)建肺炎克雷伯桿菌持留株突變體庫,共獲得3282個突變株,并從中篩選到1株失去持留特性的突變株,通過反向PCR等方法確定持留基因為NC_016845.1基因,該基因編碼磷酯酶D,參與細菌磷脂代謝。由此推斷,細菌通過調(diào)節(jié)代謝水平來改變細胞膜通透性及表面電荷,從而使自身轉(zhuǎn)化為持留狀態(tài),以抵御抗生素、酸、堿等因素的殺傷。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5
本文編號：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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5
【共引文獻】
相關(guān)期刊論文 前1條
1 姜旭;王麗敏;張桂敏;于波;曾慶韜;;基因工程菌發(fā)酵生產(chǎn)L-乳酸研究進展[J];生物工程學(xué)報;2013年10期
，本文編號：2166864
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