南京地區(qū)臨床分離大腸桿菌耐藥性基因多樣性及相關(guān)特性的研究
發(fā)布時(shí)間：2018-08-27 20:15
【摘要】： 細(xì)菌感染曾嚴(yán)重威脅著人類的生命和健康。自Fleiming發(fā)現(xiàn)青霉素后60余年來,人們不斷地從微生物次級(jí)代謝產(chǎn)物中提取出眾多的抗生素,并開發(fā)出半合成抗生素。近年來,抗生素藥物的廣泛和不合理應(yīng)用所形成的強(qiáng)選擇壓力使細(xì)菌耐藥性逐年上升,特別是多重耐藥菌株的出現(xiàn)和耐藥性的快速傳播已經(jīng)成為臨床感染性疾病治療的難題。 細(xì)菌的耐藥性可由染色體或質(zhì)粒上相關(guān)耐藥性基因介導(dǎo),耐藥性質(zhì)�？赏ㄟ^接合方式在不同菌種之間傳遞,使受體細(xì)菌成為耐藥性菌株,這是細(xì)菌獲得耐藥性的主要方式。本實(shí)驗(yàn)將從接合基因水平轉(zhuǎn)移角度研究南京臨床大腸桿菌耐藥性產(chǎn)生機(jī)制及相關(guān)基因的多樣性。 選取2008年3月到8月南京軍區(qū)總院臨床分離培養(yǎng)的200株大腸桿菌,采用平板涂布方法測(cè)定對(duì)8種不同抗生素耐藥情況,結(jié)果顯示94%的分離菌株具有超過4種抗生素耐藥性,未得到不耐受或僅耐受1種抗生素菌株。其中超過90%的菌株對(duì)Amp, Nal耐藥,Chi, Spe耐藥率相對(duì)較低,但也分別超過了30%和50%。 接合實(shí)驗(yàn)來研究耐藥基因的水平情況,篩選其中KmS或StrS的菌株75株,通過與SM10λpir (KmR)或Bw20676 (StrR)接合實(shí)驗(yàn)發(fā)現(xiàn),大部分菌株耐藥基因可通過接合方式發(fā)生水平轉(zhuǎn)移,其中Amp, Tet, Nal, Str耐藥基因發(fā)生水平轉(zhuǎn)移概率較Chl, Spe,Gm高,分別占可接合菌株的91.69%,54.70%,53.08%,33.77%。并且多種不同的耐藥基因可轉(zhuǎn)移至同一受體菌中。 對(duì)可發(fā)生水平轉(zhuǎn)移菌株,其耐藥基因水平轉(zhuǎn)移方式通過電擊轉(zhuǎn)化實(shí)驗(yàn)來確定,得到的接合子提質(zhì)粒后電擊轉(zhuǎn)化實(shí)驗(yàn)發(fā)現(xiàn),90%以上Amp耐藥基因通過質(zhì)粒方式轉(zhuǎn)移,其它的Tc, Str耐藥基因也部分通過質(zhì)粒方式轉(zhuǎn)移,其比率為可發(fā)生水平轉(zhuǎn)移菌株的20.62%和5.52%。對(duì)可同時(shí)轉(zhuǎn)移多種耐藥基因至同一受體菌的轉(zhuǎn)化子進(jìn)一步實(shí)驗(yàn)發(fā)現(xiàn),其多種耐藥基因可同時(shí)攜帶于同一質(zhì)粒,或多個(gè)不同質(zhì)粒分別攜帶不同的耐藥性。并在實(shí)驗(yàn)中發(fā)現(xiàn)一株同時(shí)攜帶有Amp, Tet, Chl, Str多種耐藥性質(zhì)粒菌株。 由于大部分的Amp耐藥基因可通過質(zhì)粒方式轉(zhuǎn)移,實(shí)驗(yàn)設(shè)計(jì)通過PCR的方法檢測(cè)菌株TEM型,SHV型及CTX-M型β-內(nèi)酰胺酶基因分型及耐藥基因攜帶情況。結(jié)果顯示我們研究的南京地區(qū)大部分大腸桿菌對(duì)氨芐青霉素耐藥的機(jī)制主要是由TEM和CTX-M型β-內(nèi)酰胺酶的產(chǎn)生,對(duì)200株大腸桿菌PCR發(fā)現(xiàn),其中TEM, CTX-M檢出率分別為36.5%和35.5%。SHV型PCR擴(kuò)增未發(fā)現(xiàn)陽性標(biāo)本。對(duì)耐受Amp抗生素程度不同的菌株序列比對(duì)發(fā)現(xiàn),其基因型相同,推測(cè)其耐藥程度與β-內(nèi)酰胺酶表達(dá)量有關(guān)。
[Abstract]:Bacterial infection has been a serious threat to human life and health. Since the discovery of penicillin by Fleiming for more than 60 years, many antibiotics have been extracted from microbial secondary metabolites and semi-synthetic antibiotics have been developed. In recent years, the strong selective pressure caused by the extensive and irrational use of antibiotic drugs has led to the increase of bacterial resistance year by year, especially the emergence of multidrug resistant strains and the rapid spread of drug resistance, which has become a difficult problem in the treatment of clinical infectious diseases. Drug resistance of bacteria can be mediated by related genes on chromosomes or plasmids. Drug resistant plasmids can be transferred between different strains by conjugation, which makes the receptor bacteria become resistant strains, which is the main way for bacteria to obtain drug resistance. In this study, the mechanism of drug resistance and the diversity of related genes in clinical Escherichia coli in Nanjing were studied from the perspective of horizontal transfer of conjugate genes. From March to August 2008, 200 strains of Escherichia coli were isolated and cultured in Nanjing military region General Hospital, and the resistance to 8 different antibiotics was determined by plate coating method. The results showed that 94% of the isolates had more than 4 antibiotic resistance. No intolerance or only one antibiotic strain was obtained. More than 90% of the strains were resistant to Amp, Nal and the rate of Spe resistance was relatively low, but more than 30% and 50%, respectively. The level of drug resistance gene was studied by conjugation test. 75 strains of KmS or StrS were screened. It was found that most of the drug resistance genes could be transferred horizontally by conjugation with SM10 位 pir (KmR) or Bw20676 (StrR). The probability of horizontal transfer of Amp, Tet, Nal, Str resistance gene was higher than that of Chl, Spe,Gm, accounting for 91.69% of the conjugable strains, 54.70% and 53.08%, 33.77% respectively. And many different drug resistance genes can be transferred to the same receptor bacteria. The way of horizontal transfer of drug resistance genes was determined by electroporation experiment. After the plasmids were extracted from the conjugate, it was found that more than 90% of Amp resistance genes were transferred by plasmid. Other Tc, Str resistance genes were also partially transferred by plasmid, the rates of which were 20.62% and 5.52% of those that could be transfered horizontally. Further experiments on transformants that can transfer multiple drug resistance genes to the same receptor bacteria at the same time showed that their multidrug resistance genes could be carried in the same plasmid at the same time, or different plasmids could carry different drug resistance at the same time. One strain carrying multiple Amp, Tet, Chl, Str resistance plasmids was also found in the experiment. Because most of the Amp resistant genes can be transferred by plasmid, PCR was designed to detect the genotyping of TEM and CTX-M 尾 -lactamases and the carrying of drug resistance genes. The results showed that the mechanism of ampicillin resistance of most Escherichia coli in Nanjing was mainly produced by TEM and CTX-M 尾 -lactamases, and 200 strains of Escherichia coli PCR were found. The positive rate of TEM, CTX-M was 36.5% and 35.5%.SHV type PCR was not found. It was found that the genotypes of the strains with different antibiotic resistance to Amp were the same, and the degree of drug resistance was related to the expression of 尾 -lactamases.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378
本文編號(hào)：2208334
[Abstract]:Bacterial infection has been a serious threat to human life and health. Since the discovery of penicillin by Fleiming for more than 60 years, many antibiotics have been extracted from microbial secondary metabolites and semi-synthetic antibiotics have been developed. In recent years, the strong selective pressure caused by the extensive and irrational use of antibiotic drugs has led to the increase of bacterial resistance year by year, especially the emergence of multidrug resistant strains and the rapid spread of drug resistance, which has become a difficult problem in the treatment of clinical infectious diseases. Drug resistance of bacteria can be mediated by related genes on chromosomes or plasmids. Drug resistant plasmids can be transferred between different strains by conjugation, which makes the receptor bacteria become resistant strains, which is the main way for bacteria to obtain drug resistance. In this study, the mechanism of drug resistance and the diversity of related genes in clinical Escherichia coli in Nanjing were studied from the perspective of horizontal transfer of conjugate genes. From March to August 2008, 200 strains of Escherichia coli were isolated and cultured in Nanjing military region General Hospital, and the resistance to 8 different antibiotics was determined by plate coating method. The results showed that 94% of the isolates had more than 4 antibiotic resistance. No intolerance or only one antibiotic strain was obtained. More than 90% of the strains were resistant to Amp, Nal and the rate of Spe resistance was relatively low, but more than 30% and 50%, respectively. The level of drug resistance gene was studied by conjugation test. 75 strains of KmS or StrS were screened. It was found that most of the drug resistance genes could be transferred horizontally by conjugation with SM10 位 pir (KmR) or Bw20676 (StrR). The probability of horizontal transfer of Amp, Tet, Nal, Str resistance gene was higher than that of Chl, Spe,Gm, accounting for 91.69% of the conjugable strains, 54.70% and 53.08%, 33.77% respectively. And many different drug resistance genes can be transferred to the same receptor bacteria. The way of horizontal transfer of drug resistance genes was determined by electroporation experiment. After the plasmids were extracted from the conjugate, it was found that more than 90% of Amp resistance genes were transferred by plasmid. Other Tc, Str resistance genes were also partially transferred by plasmid, the rates of which were 20.62% and 5.52% of those that could be transfered horizontally. Further experiments on transformants that can transfer multiple drug resistance genes to the same receptor bacteria at the same time showed that their multidrug resistance genes could be carried in the same plasmid at the same time, or different plasmids could carry different drug resistance at the same time. One strain carrying multiple Amp, Tet, Chl, Str resistance plasmids was also found in the experiment. Because most of the Amp resistant genes can be transferred by plasmid, PCR was designed to detect the genotyping of TEM and CTX-M 尾 -lactamases and the carrying of drug resistance genes. The results showed that the mechanism of ampicillin resistance of most Escherichia coli in Nanjing was mainly produced by TEM and CTX-M 尾 -lactamases, and 200 strains of Escherichia coli PCR were found. The positive rate of TEM, CTX-M was 36.5% and 35.5%.SHV type PCR was not found. It was found that the genotypes of the strains with different antibiotic resistance to Amp were the same, and the degree of drug resistance was related to the expression of 尾 -lactamases.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378
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