南京地区临床分离大肠杆菌耐药性基因多样性及相关特性的研究
摘要
细菌感染曾严重威胁着人类的生命和健康。自Fleiming发现青霉素后60余年来,人们不断地从微生物次级代谢产物中提取出众多的抗生素,并开发出半合成抗生素。近年来,抗生素药物的广泛和不合理应用所形成的强选择压力使细菌耐药性逐年上升,特别是多重耐药菌株的出现和耐药性的快速传播已经成为临床感染性疾病治疗的难题。
细菌的耐药性可由染色体或质粒上相关耐药性基因介导,耐药性质粒可通过接合方式在不同菌种之间传递,使受体细菌成为耐药性菌株,这是细菌获得耐药性的主要方式。本实验将从接合基因水平转移角度研究南京临床大肠杆菌耐药性产生机制及相关基因的多样性。
选取2008年3月到8月南京军区总院临床分离培养的200株大肠杆菌,采用平板涂布方法测定对8种不同抗生素耐药情况,结果显示94%的分离菌株具有超过4种抗生素耐药性,未得到不耐受或仅耐受1种抗生素菌株。其中超过90%的菌株对Amp, Nal耐药,Chi, Spe耐药率相对较低,但也分别超过了30%和50%。
接合实验来研究耐药基因的水平情况,筛选其中KmS或StrS的菌株75株,通过与SM10λpir (KmR)或Bw20676 (StrR)接合实验发现,大部分菌株耐药基因可通过接合方式发生水平转移,其中Amp, Tet, Nal, Str耐药基因发生水平转移概率较Chl, Spe,Gm高,分别占可接合菌株的91.69%,54.70%,53.08%,33.77%。并且多种不同的耐药基因可转移至同一受体菌中。
对可发生水平转移菌株,其耐药基因水平转移方式通过电击转化实验来确定,得到的接合子提质粒后电击转化实验发现,90%以上Amp耐药基因通过质粒方式转移,其它的Tc, Str耐药基因也部分通过质粒方式转移,其比率为可发生水平转移菌株的20.62%和5.52%。对可同时转移多种耐药基因至同一受体菌的转化子进一步实验发现,其多种耐药基因可同时携带于同一质粒,或多个不同质粒分别携带不同的耐药性。并在实验中发现一株同时携带有Amp, Tet, Chl, Str多种耐药性质粒菌株。
由于大部分的Amp耐药基因可通过质粒方式转移,实验设计通过PCR的方法检测菌株TEM型,SHV型及CTX-M型β-内酰胺酶基因分型及耐药基因携带情况。结果显示我们研究的南京地区大部分大肠杆菌对氨苄青霉素耐药的机制主要是由TEM和CTX-M型β-内酰胺酶的产生,对200株大肠杆菌PCR发现,其中TEM, CTX-M检出率分别为36.5%和35.5%。SHV型PCR扩增未发现阳性标本。对耐受Amp抗生素程度不同的菌株序列比对发现,其基因型相同,推测其耐药程度与β-内酰胺酶表达量有关。
Bacterial infections were serious threat to human life and health. Since the discovery of penicillin by Fleiming ore than 60 years, people extracted a large number of antibiotics from microbial secondary metabolites and developed semi-synthetic antibiotics. In recent years, inadequate selection and abuse of antimicrobials may lead to resistance in various bacteria and make the treatment of bacterial infections more difficult. In particular the emergence of multidrug resistant strains and the rapid spread of antimicrobial resistance has become a problem of clinical infectious disease.
Antibiotic-resistant gene transfer in bacteria is mediated through chromosome or plasmid. Much of the genomic information that affects antimicrobial resistances is acquired by plasmid, and conjugation is generally considered to play a major role in this process. In this study, we will investigate diversity of antibiotics resistant genes in clinical isolated E.coli in Nanjing.
200 multi-resistant E.coli clinical isolates were collected in the Military hospital of Nanjing from March to August in 2008. Antimicrobial resistance for the isolates were determined by spreading strains to antimicrobial plates. the research showed that 94% of isolates were resistant to more than 4 antimicrobials tested, no one was resistant to less than 1 of the 8 antimicrobials tested. The resistance to 8 antimicrobial agents for 200 clinical isolates. more than 90% of which resistant to ampicillin and nalidixic acid. The rate resistant chloramphenicol and spectinomycin are relatively lower, but more than 30% and 50% were found.
To determine whether resistance was transferable, conjugations were performed. Multidrug-resistant E.coli isolates recovered from patients in the military hospital of Nanjing as donor strains and Kanamycin-resistant E. coli SM10λpir or streptomycin resistant E. coli Bw20676 were used as recipient strains.The antimicrobial resistance of different resistant strains was transferred to E.coli. Antimicrobial resistance, such as ampicillin (91.69%), tetracycline (54.70%), nalidixic acid (53.08%) and streptomycin (33.77%), could be transferred from a donor to a recipient cell via conjugation with high ratio. Chloramphenicol, spectinomycin and gentamicin resistance was transferred from donor strains rare.
If the resistant genes is due to acquisition of plasmids which was assayed in transformation experiments. We found that more than 90% ampicillin resistance mediated by plasmid, and parts of tetracycline and streptomycin resistance by acquiring gene on plasmid with ratio of 20.62% and 5.52%. Antimicrobial susceptibility in E.coli transconjugants found that different gene can transfer to the same recipient strains,
Therefore, we supposed that plasmids and other mobilizable molecules may account for the resistant via conjugation, and clinical human E.coli isolates which could transfer their different mobilizable molecules to recipient together or one mobilizable molecule with multidrug resistance. Plasmid encoded resistance to ampicillin, chloramphenicol, tetracycline and streptomycin discoveried in the study.
As most of the ampicillin resistance gene transfer via plasmids, TEM, SHV and CTX-M gene conferring P-lactamase were detected by polymerase chain reaction (PCR) The results showed that TEM and CTX-Mβ-lactamase is the principal mechanism of ampicillin antiobiotic of Escherichia coli isolates from Nanjing. PCR found that most of strains all contained TEM(36.5%) and CTX-M(35.5%). No SHV was detected in these isolates. Different strains with differernt ability to resistant ampicillin have the same genotype, Therefore, we supposed that discrepancy of this resistance related withβ-lactamase expression.
细菌的耐药性可由染色体或质粒上相关耐药性基因介导,耐药性质粒可通过接合方式在不同菌种之间传递,使受体细菌成为耐药性菌株,这是细菌获得耐药性的主要方式。本实验将从接合基因水平转移角度研究南京临床大肠杆菌耐药性产生机制及相关基因的多样性。
选取2008年3月到8月南京军区总院临床分离培养的200株大肠杆菌,采用平板涂布方法测定对8种不同抗生素耐药情况,结果显示94%的分离菌株具有超过4种抗生素耐药性,未得到不耐受或仅耐受1种抗生素菌株。其中超过90%的菌株对Amp, Nal耐药,Chi, Spe耐药率相对较低,但也分别超过了30%和50%。
接合实验来研究耐药基因的水平情况,筛选其中KmS或StrS的菌株75株,通过与SM10λpir (KmR)或Bw20676 (StrR)接合实验发现,大部分菌株耐药基因可通过接合方式发生水平转移,其中Amp, Tet, Nal, Str耐药基因发生水平转移概率较Chl, Spe,Gm高,分别占可接合菌株的91.69%,54.70%,53.08%,33.77%。并且多种不同的耐药基因可转移至同一受体菌中。
对可发生水平转移菌株,其耐药基因水平转移方式通过电击转化实验来确定,得到的接合子提质粒后电击转化实验发现,90%以上Amp耐药基因通过质粒方式转移,其它的Tc, Str耐药基因也部分通过质粒方式转移,其比率为可发生水平转移菌株的20.62%和5.52%。对可同时转移多种耐药基因至同一受体菌的转化子进一步实验发现,其多种耐药基因可同时携带于同一质粒,或多个不同质粒分别携带不同的耐药性。并在实验中发现一株同时携带有Amp, Tet, Chl, Str多种耐药性质粒菌株。
由于大部分的Amp耐药基因可通过质粒方式转移,实验设计通过PCR的方法检测菌株TEM型,SHV型及CTX-M型β-内酰胺酶基因分型及耐药基因携带情况。结果显示我们研究的南京地区大部分大肠杆菌对氨苄青霉素耐药的机制主要是由TEM和CTX-M型β-内酰胺酶的产生,对200株大肠杆菌PCR发现,其中TEM, CTX-M检出率分别为36.5%和35.5%。SHV型PCR扩增未发现阳性标本。对耐受Amp抗生素程度不同的菌株序列比对发现,其基因型相同,推测其耐药程度与β-内酰胺酶表达量有关。
Bacterial infections were serious threat to human life and health. Since the discovery of penicillin by Fleiming ore than 60 years, people extracted a large number of antibiotics from microbial secondary metabolites and developed semi-synthetic antibiotics. In recent years, inadequate selection and abuse of antimicrobials may lead to resistance in various bacteria and make the treatment of bacterial infections more difficult. In particular the emergence of multidrug resistant strains and the rapid spread of antimicrobial resistance has become a problem of clinical infectious disease.
Antibiotic-resistant gene transfer in bacteria is mediated through chromosome or plasmid. Much of the genomic information that affects antimicrobial resistances is acquired by plasmid, and conjugation is generally considered to play a major role in this process. In this study, we will investigate diversity of antibiotics resistant genes in clinical isolated E.coli in Nanjing.
200 multi-resistant E.coli clinical isolates were collected in the Military hospital of Nanjing from March to August in 2008. Antimicrobial resistance for the isolates were determined by spreading strains to antimicrobial plates. the research showed that 94% of isolates were resistant to more than 4 antimicrobials tested, no one was resistant to less than 1 of the 8 antimicrobials tested. The resistance to 8 antimicrobial agents for 200 clinical isolates. more than 90% of which resistant to ampicillin and nalidixic acid. The rate resistant chloramphenicol and spectinomycin are relatively lower, but more than 30% and 50% were found.
To determine whether resistance was transferable, conjugations were performed. Multidrug-resistant E.coli isolates recovered from patients in the military hospital of Nanjing as donor strains and Kanamycin-resistant E. coli SM10λpir or streptomycin resistant E. coli Bw20676 were used as recipient strains.The antimicrobial resistance of different resistant strains was transferred to E.coli. Antimicrobial resistance, such as ampicillin (91.69%), tetracycline (54.70%), nalidixic acid (53.08%) and streptomycin (33.77%), could be transferred from a donor to a recipient cell via conjugation with high ratio. Chloramphenicol, spectinomycin and gentamicin resistance was transferred from donor strains rare.
If the resistant genes is due to acquisition of plasmids which was assayed in transformation experiments. We found that more than 90% ampicillin resistance mediated by plasmid, and parts of tetracycline and streptomycin resistance by acquiring gene on plasmid with ratio of 20.62% and 5.52%. Antimicrobial susceptibility in E.coli transconjugants found that different gene can transfer to the same recipient strains,
Therefore, we supposed that plasmids and other mobilizable molecules may account for the resistant via conjugation, and clinical human E.coli isolates which could transfer their different mobilizable molecules to recipient together or one mobilizable molecule with multidrug resistance. Plasmid encoded resistance to ampicillin, chloramphenicol, tetracycline and streptomycin discoveried in the study.
As most of the ampicillin resistance gene transfer via plasmids, TEM, SHV and CTX-M gene conferring P-lactamase were detected by polymerase chain reaction (PCR) The results showed that TEM and CTX-Mβ-lactamase is the principal mechanism of ampicillin antiobiotic of Escherichia coli isolates from Nanjing. PCR found that most of strains all contained TEM(36.5%) and CTX-M(35.5%). No SHV was detected in these isolates. Different strains with differernt ability to resistant ampicillin have the same genotype, Therefore, we supposed that discrepancy of this resistance related withβ-lactamase expression.
引文
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