产ESBLs液化沙雷氏菌临床分离株的表型和基因型分析

Phenotype and genotype analyses of ESBLs producing Serratia liquefaciens CR04 clinical isolates

目的探索液化沙雷氏菌临床分离株CR04对第三代头孢菌素耐药的分子机制。方法采用琼脂二倍稀释法对临床分离的液化沙雷氏菌CR04株进行MIC检测,提取耐药菌β-内酰胺酶,并测定其酶活性。双纸片法筛选及确证实验鉴别耐药菌的ESBLs表型,进行结合传递实验并提取耐药菌质粒,进一步根据常见的质粒介导的β-内酰胺酶基因序列设计两对寡核苷酸引物,以耐药质粒为模板进行PCR扩增,并对其扩增产物进行DNA序列测定和同源性分析。结果头孢他啶对液化沙雷氏菌CR04株的MIC为512μg/ml,其酶活性为837.9U,双纸片法筛选及确证实验证明为产ESBLs耐药菌,结合传递实验表明产酶基因能传递给受体菌,从耐药菌中提取出大小约为10kb的质粒,以质粒DNA为模板进行PCR扩增,对扩增产物DNA测序结果进行分析表明,其核苷酸序列与已知的β-内酰胺酶blaSHV-5、blaSHV-1、blaSHV-2、blaSHV-7、blaSHV-8、blaSHV-12、blaSHV-24、blaSHV-34基因高度同源,其所推导的氨基酸序列与已知的β-内酰胺酶blaSHV-5、blaSHV-1、blaSHV-2、blaSHV-7、blaSHV-8、blaSHV-12、blaSHV-24、blaSHV-34相比较至少有30个氨基酸残基的差异。结论对临床分离的第三代头孢菌素耐药性液化沙雷氏菌CR04株进行详细的表型和基因型分析。

Aim To study the mechanism of drug-resistance of Gram-negative bacilli to the third generation of a-lactam antibiotics.Methods Serratia li quefaciens CR04 clinical isolates was identified using the API20-E system. MIC was determined with the standard agar dilution method.Conjungation experiment w as performed between clinical isolates and E.coli JM109.The enzyme activity of a-lactamase was detected. The plasmid was extracted by using alkaline lysis method.Two pairs of primers were designed to amplify the entire coding re gion of the a-lactamase gene.The PCR product was directly sequenced.The nuc leotide and deduced protein sequences were analysed with the BLAST sowftware of NCBI.Results MIC of ceftazidime for Serratia liquefaciens CR04 clinical iso lates was 512 ì g/ml.The enzyme activity of the a-lactamase was 837.9U.Tr ansconjugants were obtained through conjungation experiment.A 10kb plasmid was s uccessfully isolated.A gene fragment was obtained by PCR using the plasmid as te mplate.DNA Seqencing showed that the gene fragment contains 861 base pairs which is higly homologous to blaSHV-5,blaSHV-1,blaSHV-2,blaSHV-7,blaSHV-8,b laSHV-24 and blaSHV-34. Conclusions The phenotype and genotype of Serratia l iquefaciens CR04 clinical isolates were elucidated with bacteriological and mole cular biological methods.These results provided the basis for further study on t he mechanism of the drug-resistance of Gram-negative bacilii to the third ge neration of a-lactam antibiotics.