产金属β-内酰胺酶嗜麦芽寡养单胞菌的研究

Research on resistant mechanism of Stenotrophomonas maltophilia producing metallo a-lactamases

目的探讨嗜麦芽寡养单胞菌所产金属β-内酰胺酶的酶学特性及分子生物学特征。方法采用纸片协同法筛选产金属β-内酰胺酶菌株;测定产酶株所产β-内酰胺酶对亚胺培南的稳定性和丝氨酸β-内酰胺酶、金属β-内酰胺酶抑制剂的抑酶保护效应;检测金属β-内酰胺酶的等电点;用PCR法检测金属β-内酰胺酶全基因,并测序。结果纸片协同法筛选产金属β-内酰胺酶菌株4株,药敏试验显示对多种抗菌药物耐药。酶稳定性试验显示,4株产酶菌产碳青霉烯水解酶。酶抑制试验表明,克拉维酸对这4株菌所产酶无抑酶保护效应,而金属β-内酰胺酶抑制剂EDTA对4株菌所产酶有抑制作用,且酶活性能被ZnCl2复活。等电聚焦显示,2株菌(710、750)产的碳青霉烯水解酶pI为6.6,1株菌(603)的pI为6.2,EDTA抑制试验证实均为金属β-内酰胺酶。PCR结果显示,以4株细菌基因组DNA为模板,用所设计的金属β-内酰胺酶全基因引物进行PCR扩增,2株(750,710)扩增结果为阳性,进行序列分析,均为867bp。经GeneBank网上同源性比较,发现与金属酶L1的编码基因blaS(注册号AJ291672.1)的核苷酸序列同源性均为99%。结论嗜麦芽寡养单胞菌710和750号产L1型金属β-内酰胺酶;603号可能产非L1型金属β-内酰胺酶。

Objective To study the enzymology and molecular characteristics of metallo a-lactamase (MBL) in Stenotrophomonas maltophilia. Methods The st rains producing MBL of S.maltophilia were screened by double disk synergy,and th e stabillity of 11 antimicrobial agents to MBL were studied with spectrophotomer trical method and the inhibitory activity of a-lactamases inhibitors to MBL was observed.The pI of unrepeated MBL-positive strains were determined by is oelectric focusing.Finally, the blaMBL gene was amplified by PCR and seq uenced. Results Four MBL positive strains of S.maltophilia detected by dou ble disk synergy, their hydrolytic activity was inhibited by EDTA,but not a-lactamase inhibitors clavulanate, and they were partially reversible by the addition of zinc cation.Strain 710 and 750 of unrepeated MBL p ositive strains had a-lactamase of pI 6.6 and strain 603 had a-lactamase s of pI 6.2 and pI 8.4. Bands with pI 6.6 and pI 6.2 could be inhibited by EDTA were classified as MBL. The 867-bp DNA fragment of MBL encoding gene was amplified from both strains 710 and 750 by PCR and sequenced. Results Resu lts showed that the gene was 99% homologous to blaS of MBL L1 (AJ291672.1).