临床产酶大肠埃希氏菌、肺炎克雷伯氏菌对β-内酰胺酶抑制剂敏感性降低的特征研究

Characterization of β-lactamase inhibitors reduced susceptibility in clinical isolates of Escherichia coli and Klebsiella pneumoniae

目的 探讨临床产β-内酰胺酶大肠埃希氏菌及肺炎克雷伯氏菌对氨苄西林 /舒巴坦 (ABPC/SB)、阿莫西林 /舒巴坦 (AMPC/ SB)敏感性降低的特征。方法 琼脂二倍稀释法、酶动力学参数及等电点测定、质粒提取和转化。结果 临床产酶 3株大肠埃希氏菌 Ec1、Ec2、Ec3及 4株肺炎克雷伯氏菌 Kp1、Kp2、Kp3、Kp4对 ABPC/ SB、AMPC/ SB的敏感性明显降低 (MIC≥ 12 8mg/ L ) ,(1) Ec1、Ec2、Ec3及 Kp1对所试头孢菌素类及碳青霉烯类敏感 ;仅见一约 35 kb的质粒 ;含有单一β-内酰胺酶 (p I,5 .4 ) ,与 TEM- 1相比 ,各酶活性降低 ,对β-内酰胺类抗生素的相对水解率下降 ,对 AMPC的 Km值比 TEM- 1高 2～ 4倍 ,三唑巴坦 (TB)、舒巴坦 (SB)、克拉维酸 (CA)对各酶的 IC50 比 TEM- 1高 4～ 32 0倍 ,表明此 4株菌对青霉素类亲和力明显降低 ,对酶抑制剂的敏感性显著下降 ;(2 ) Kp2、Kp3、Kp4与 Kp1不同 ,对 ABPC/ SB、AMPC/ SB和头孢菌素类均耐药 ,含有多个质粒 ,显示多个β-内酰胺酶 ,p I值分别为 5 .4、7.6、8.2等 ;而 4株质粒转化菌株所提取β-内酰胺酶的 p I值均为 5 .4 ,且均仅见一条约 35 kb的质粒 ,对 ABPC/ SB、AMPC/ SB获得了耐药 ,但仍对头孢菌素类、碳青霉烯类敏感。结论 本研究中大肠埃希氏菌。

Objective To investigate three Escherichia coli and four Klebsiella pneumoniae with reduced susceptibility to ampicillin/sulbactam (ABPC/SB) and amoxicillin/sulbactam (AMPC/SB) (MIC≥128mg/L). Methods Susceptibility testing to β-lactams,isoelectric focusing electrophoresis analysis,β-lactamase kinetic parameters determination,plasmid extraction and transformation were performed in this study. Results There were two different resistance phenotypes. (1) TEM-1 and all sonic extracts from E. coli Ec1、Ec2、Ec3 and Klebsiella pneumoniae Kp1 tested producing a single β-lactamase band,focusing at pI of 5.4. The relative rates of hydrolysis of β-lactams by the β-lactamase responsible of ABPC/SB and AMPC/SB resistance generally appeared lower than those observed with TEM-1;the enzymes activities were decreased;The four enzymes K_m values of AMPC were 2～4 fold higher than that of TEM-1;accordingly,the IC_ 50 of CA,SB,TB were 4～320 fold higher than TEM-1. These indicated that the affinity of the four isolates producing β-lactamases with AMPC were significantly decreased. The electrophoresis plasmid profile showed the all four strains consisted of a similar 35kb plasmid DNA. This resistance phenotypes is similar to usual resistant profile had been reported as a characteristic of IRTs which were susceptible by the cephalosporins and carbapenems. (2) Klebsiella pneumoniae Kp2,Kp3 and Kp4 revealed a complex resistant phenotype,conferring resistance to AMPC/SB,ABPC/SB and the cephalosporins. The results of isoelectric focusing electrophoresis analysis presented multiple β-lactamases from Kp2,Kp3 and Kp4 focusing at pI 5.4,7.6,8.2. Kp4 harbored seven plasmids,and Kp2,Kp3 harbored three plasmids,and each had a plasmid at approximately 35kb which was resemble to Kp1 mentioned above. In plasmid transformation experiments,All transformants in E.coli DH5α contained a 35kb plasmid DNA,And at same site as donor′s isolates. Each transformant produced only a β-lactamases of pI 5.4,conferred resistance to AMPC/SB,but remained susceptible to cephalosporins and carbapenems. Conclusions These data suggested clinical isolates of Escherichia coli and Klebsiella pneumoniae produced plasmid-mediated inhibitor resistance TEM β-lactamases.