一株携带新的突变ampC酶基因的弗劳地枸橼酸杆菌的研究

Gene sequence and biochemical characterization of ACT-1 from Citrobacter freundii,a new ampC-type plasmid-mediated β-lactamase with molecular variants

目的探讨1株携带新的突变ampC基因的弗劳地枸橼酸杆菌分子生物学特征的改变及其与该突变位点的关系。方法对该株携带突变ampC基因的细菌克隆其编码基因后进行序列分析,并使用Southern杂交的方法定位该基因。测定其野生株、接合子与重组菌对12种抗菌药物的最低抑菌浓度(MICs),并对其编码蛋白进行等电聚焦电泳和酶动力学的检测。结果该细菌所产AmpC酶第147位氨基酸发生改变,由赖氨酸变为苏氨酸。Southern杂交结果表明,其编码基因位于质粒上。接合子、重组菌及野生株都对头孢西丁的MICs值均较高(>128mg/L)。等电聚焦电泳检测该AmpC酶的等电点为8.4。结论发现一株携带新的质粒介导ACT型ampC突变基因的弗劳地枸橼酸杆菌,其分子生物学特性与以往报道有一定区别。

Objective To determine the change of the characteristics of molecular biology in Citrobacter freundii which carring a new mutant gene, and find relationship with the change in mutation. Methods Clone the mutation ampC gene and made sequence analysis on it. Southern hybridization was done to detect the mutation gene whether located on plasmid. MICs was determine about the clincal stain, the transconjugant and the transformant by 12 antimicrobial agents. Results Sequence analysis revealed that the coding gene differed from blaACT-1 by four mutations, and one of them led to amino acid substitution ： threonine for lysine at position 147. Southern hybridization showed that the coding gene was located on plasmid. All the clincal stain, the transconjugant and the transformant exhibi- ted high MICs to cefoxitin（ 〉 128 mg/L）. The isoelectric point （pI） of the AmpC β-lactamase was estimated to be 8.4. Conclusion We identify a new type of ampC gene which is mediated by plasmid, and its biochemical characterization is not similar with the same type reported before.