合肥市部分医院产CTX-M型超广谱β-内酰胺酶细菌的耐药性分析及其分子生物学特征
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摘要
目的
了解产CTX-M型酶的大肠埃希菌和肺炎克雷伯菌对14种抗菌素的耐药性，以指导临床用药。
了解合肥市部分医院CTX-M酶的基因型，研究新的CTX-M酶的生化及耐药特性。材料与方法菌株来源：
1999年至2000年收集的合肥市四家大型医院的临床产ESBLs大肠埃希菌和肺炎克雷伯菌，共98株。
方法
以98株ESBLs~+菌株总DNA为模板，使用bla_(CTX-M)通用引物进行PCR扩增以筛选出CTX-M~+菌株；再将CTX-M~+菌株与E．coliC600共孵，行转移接合试验；采用琼脂平皿稀释法对野生株与接合子进行MIC测定。
根据通用引物扩增结果，设计两对全编码区扩增引物，为方便后续试验，5｀端分别加EcoRI和BamHI酶切位点。
将全编码区PCR产物，交送生物公司进行直接序列分析，结果至Genbank进行比对，确定CTX-M酶的基因型。
在EcoRI和BamHI酶切之后，将新基因型全编码基因克隆入表达载体pHSG_(398)，转化于感受态细胞E．coliDH_(5α)，用琼脂平皿稀释法测定14种抗生素的MIC值。
采用超声破碎法进行产新酶细菌的接合子的粗酶提取，等电聚焦电泳测定其pI。
采用碱裂解法抽取产新酶的细菌的质粒，PstI消化，进行质粒分析。
采用脉冲场凝胶电泳(pulsed field gel electrophoresis，PFGE)分析产新酶细菌的同源性。
结果
Objective
To study the resistance of the CTX-M-producing Escherichia coli and Klebsiella pneumoniae strains against 14 antibiotics, and offer the resistance data to clinical therapy.
To investigate the genotypes and epidemiology of CTX-M enzyme carrier, Escherichia coli and Klebsiella pneumoniae isolates, in several hospitals of Hefei.
To study the biochemical properties of the 6 novel CTX-M enzymes and the resistance of the novel enzyme producing strains. Materials and Methods Isolates
Totally 98 strains of ESBLs-producing Escherichia coli and Klebsiella pneumoniae were collected between 1999 and 2000 from four hospitals in Hefei. Method
Adopting PCR methods, universal primers of bla_(CTX-M) were used for CTX-M~+ isolates screening; matting method was used between CTX-M~+ isolates and E.coli C600 to transfer resistance plasmid; agar dilution method was used to determine MICs of 14 antibiotics against wild-type isolates and the transconjugants.
According to the universal primers PCR results, two pairs of entire coding gene primers were designed, and to convenient the consequence experiments, endonucleotide cleavage site of EcoRI and BamHI were added at 5' terminal of the upstream and downstream of the two groups of primers.
The sequence was determined by direct sequencing of PCR products, carried out by the dideoxy chain termination procedure of Sanger on an ABI377 automatic sequencer (Invotrigen Biocompany, Shanghai, China). Then blastn program was used to ascertain the genotype at Genbank.
After digestion by EcoRI and BamHI, the whole ORF amplicon was linked into the vector pHSG398 by T_4DNA lingase. And then, the recombinant plasmid was introduced into the
了解产CTX-M型酶的大肠埃希菌和肺炎克雷伯菌对14种抗菌素的耐药性，以指导临床用药。
了解合肥市部分医院CTX-M酶的基因型，研究新的CTX-M酶的生化及耐药特性。材料与方法菌株来源：
1999年至2000年收集的合肥市四家大型医院的临床产ESBLs大肠埃希菌和肺炎克雷伯菌，共98株。
方法
以98株ESBLs~+菌株总DNA为模板，使用bla_(CTX-M)通用引物进行PCR扩增以筛选出CTX-M~+菌株；再将CTX-M~+菌株与E．coliC600共孵，行转移接合试验；采用琼脂平皿稀释法对野生株与接合子进行MIC测定。
根据通用引物扩增结果，设计两对全编码区扩增引物，为方便后续试验，5｀端分别加EcoRI和BamHI酶切位点。
将全编码区PCR产物，交送生物公司进行直接序列分析，结果至Genbank进行比对，确定CTX-M酶的基因型。
在EcoRI和BamHI酶切之后，将新基因型全编码基因克隆入表达载体pHSG_(398)，转化于感受态细胞E．coliDH_(5α)，用琼脂平皿稀释法测定14种抗生素的MIC值。
采用超声破碎法进行产新酶细菌的接合子的粗酶提取，等电聚焦电泳测定其pI。
采用碱裂解法抽取产新酶的细菌的质粒，PstI消化，进行质粒分析。
采用脉冲场凝胶电泳(pulsed field gel electrophoresis，PFGE)分析产新酶细菌的同源性。
结果
Objective
To study the resistance of the CTX-M-producing Escherichia coli and Klebsiella pneumoniae strains against 14 antibiotics, and offer the resistance data to clinical therapy.
To investigate the genotypes and epidemiology of CTX-M enzyme carrier, Escherichia coli and Klebsiella pneumoniae isolates, in several hospitals of Hefei.
To study the biochemical properties of the 6 novel CTX-M enzymes and the resistance of the novel enzyme producing strains. Materials and Methods Isolates
Totally 98 strains of ESBLs-producing Escherichia coli and Klebsiella pneumoniae were collected between 1999 and 2000 from four hospitals in Hefei. Method
Adopting PCR methods, universal primers of bla_(CTX-M) were used for CTX-M~+ isolates screening; matting method was used between CTX-M~+ isolates and E.coli C600 to transfer resistance plasmid; agar dilution method was used to determine MICs of 14 antibiotics against wild-type isolates and the transconjugants.
According to the universal primers PCR results, two pairs of entire coding gene primers were designed, and to convenient the consequence experiments, endonucleotide cleavage site of EcoRI and BamHI were added at 5' terminal of the upstream and downstream of the two groups of primers.
The sequence was determined by direct sequencing of PCR products, carried out by the dideoxy chain termination procedure of Sanger on an ABI377 automatic sequencer (Invotrigen Biocompany, Shanghai, China). Then blastn program was used to ascertain the genotype at Genbank.
After digestion by EcoRI and BamHI, the whole ORF amplicon was linked into the vector pHSG398 by T_4DNA lingase. And then, the recombinant plasmid was introduced into the
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21. http://www.ncbi.nlm.nih.gov/entrez
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21. http://www.ncbi.nlm.nih.gov/entrez
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