Characterization of bla_(CTX-M) Gene in One Klebsiella pneumoniae Isolate from Sick Chickens in China  

作　　者：HU Gong-zheng HU Han LIU Bao-guang YUAN Li LIU Jian-hua PAN Yu-shan WU Hua CHEN Yu-xia 

机构地区：[1]College of Animal Husbandry and Veterinary Science, Henan Agricultural University [2]Department of Chemical and Biological Engineering, Zhejiang University

出　　处：《Journal of Integrative Agriculture》2012年第10期1714-1720,共7页农业科学学报（英文版）

基　　金：funded by the National Natural Science Foundation of China (31072170)

摘　　要：Two Klebsiella pneumoniae isolates （Kpcl and Kpc2） were obtained from liver samples of seven dead chickens and identified with Vitek-32 automated identification system. Antimicrobial susceptibilities were determined by the microdilution broth method. Detection of genes encoding class A β-lactamases was performed by PCR amplification, and cloning of the ESBL gene was by plasmid restriction and fragments ligation. Conjugation assay, transformation experiments and plasmid profile analysis were performed. The incompatibility group of ESBL-carrying plasmid was determined by the PCR-based replicon typing method. Lastly, the genetic environment was analysed by direct sequencing of the DNA surrounding the ESBL gene. The genes associated with tetracycline and gentamicin resistance were also sought by PCR. The results revealed that the ESBL phenotype-negative strain Kpc2 only showed resistance to ampicillin, amoxicillin, tetracycline, and doxycycline and carried bla TEM-1 and tet（A） genes. The ESBL-producing strain Kpcl exhibited multidrug resistant phenotype and harbored bla TEM-1 , bla CTX-M-14, tet（A）, tet（B）, and rmtB genes. K. pneumoniae Kpcl contained four plasmids with molecular sizes of approximately 59, 6.9, 2.8, and 1.6 kb, but only a 59-kb plasmid, carried bla TEM-1 and blac CTM-14 genes, was observed in its transconjugant. The incompatibility group of plasmid carrying blaCTX-M-14 gene could not be determined. The bla CTX-M-14 gene was flanked upstream by an ISEcpl insertion sequence and downstream by an IS903 element. This work shows that CTX-M-14 is present in K. pneumoniae isolates from chickens in China. The bla CTX -M-4 gene was associated with an upstream ISEcpl insertion sequence. Our results underline the need for continuous surveillance of the prevalence and evolution of this CTX-M-type β-lactamase in China.Two Klebsiella pneumoniae isolates （Kpcl and Kpc2） were obtained from liver samples of seven dead chickens and identified with Vitek-32 automated identification system. Antimicrobial susceptibilities were determined by the microdilution broth method. Detection of genes encoding class A β-lactamases was performed by PCR amplification, and cloning of the ESBL gene was by plasmid restriction and fragments ligation. Conjugation assay, transformation experiments and plasmid profile analysis were performed. The incompatibility group of ESBL-carrying plasmid was determined by the PCR-based replicon typing method. Lastly, the genetic environment was analysed by direct sequencing of the DNA surrounding the ESBL gene. The genes associated with tetracycline and gentamicin resistance were also sought by PCR. The results revealed that the ESBL phenotype-negative strain Kpc2 only showed resistance to ampicillin, amoxicillin, tetracycline, and doxycycline and carried bla TEM-1 and tet（A） genes. The ESBL-producing strain Kpcl exhibited multidrug resistant phenotype and harbored bla TEM-1 , bla CTX-M-14, tet（A）, tet（B）, and rmtB genes. K. pneumoniae Kpcl contained four plasmids with molecular sizes of approximately 59, 6.9, 2.8, and 1.6 kb, but only a 59-kb plasmid, carried bla TEM-1 and blac CTM-14 genes, was observed in its transconjugant. The incompatibility group of plasmid carrying blaCTX-M-14 gene could not be determined. The bla CTX-M-14 gene was flanked upstream by an ISEcpl insertion sequence and downstream by an IS903 element. This work shows that CTX-M-14 is present in K. pneumoniae isolates from chickens in China. The bla CTX -M-4 gene was associated with an upstream ISEcpl insertion sequence. Our results underline the need for continuous surveillance of the prevalence and evolution of this CTX-M-type β-lactamase in China.

关 键 词：Klebsiella pneumonia CTX-M-14 minimum inhibitory concentrations （MICs） genetic environment 

分 类 号：S858.31[农业科学—临床兽医学]