Stenotrophomonas maltophilia 誘發L1及L2乙內醯胺酶基因表現之探討

Abstract

Stenotrophomonas maltophilia has assumed an increasingly important role as a nosocomial pathogen in compromised patients.。S. maltophilia isolates are resistant to many clinically useful antibiotics，with β-lactam resistance mediated through two β-lactamases，L1 and L2，whose expression is induced when cells are exposed to β-lactam antibiotics。Little is known about the induction of L1 and L2 β-lactamases。A clinical S. maltophilia isolate, S. maltophilia N, was obtained in China Medical University Hospital. The PCR amplicons containing L1 and L2 ??-lactamase genes, respectively, were sequenced and analyzed. The L1- and L2- mutants of S. maltophilia N were constructed by gene replacement strategy, respectively. A reporter gene, xylE, is inserted into the L1 and L2 gene, down-strand the promoter of L1 and L2 gene and with the same orientation as that of L1 and L2 gene, respectively. The induction of L1 and L2 gene promoters can be monitored by analyzing the catechol 2,3-dioxygenase (C23O) activities in different induced conditions. The studies in the thesis were designed to elucidate the factors that affect the induction of L1 and L2 gene promoters. The induction potency of cefuroxime against NL1- and NL2- is independent on the type of carbon source in growth medium and the addition of 5% human serum. Clavulanic acid acts as an inducer toward the L1 and L2 gene promoters, and its induction potency against L1 and L2 genes is concentration dependent. The susceptibility test of S. maltophilia N against different combined regimens of ??-lactams/clavulanate was evaluated.The factors affected the effect of combination regimens were elucidated using the isogeneic mutants, NL1- and NL2-, as the assayed systems. To be a successful combined regimen, three important elements must be considered: (1) The hydrolytic ability of L1 toward the selected ??-lactam. (2) The minimal concentration of clavulanate needed to inhibit the activity of induced L2 enzyme. (3) The occurrence of induction synergy by the combination of ??-lactam and clavulanate.