| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 97 === Fourteen clinical isolate of Acinetobacter genomic species 13TU were used in
this study. These isolates are imipenem-resistant strains（A22、A25、A1245、Ah34）
and imipenem-susceptible strains （A5、A6、A10、A13、A14、A15、A17、A18、
A19、A21）strains were used in this study.
All imipenem-resistant strains and one imipenem-susceptible strain A5 contain
class D β-lactamase gene, blaOXA-58 using multiplex PCR. An insertion sequence,
ISAba3, located in the upstream of blaOXA-58 in the all above strains. Also the mRNA
of blaOXA-58 gene of these strains are similar.
Subsequently, we detected the class B β-lactamase may also exist in the
imipenem-resistant strains but not imipenem-susceptible strains using MBLs E-test
strip. Furthermore, PCR showed blaIMP-1 gene present in the four imipenem-resistant
strains, and RT-PCR results demonstrated that the RNA expression levels in these
strains were similar. To elucidate the contribution of OXA-58 and IMP-1 in imipenem
resistance, we constructed pBAD /Myc-His-OXA-58 and the pBAD /Myc-His-IMP-1
clones and then introduced into E.coli Top10 system. The MIC value of pBAD
/Myc-His-IMP-1 strain was 8-folds increased compared to pBAD /Myc-His-OXA-58
was 3-folds increased. Besides, the enzyme activity assay of the OXA-58 and IMP-1
using the CENTA as the substrate, the results showed that the activity of IMP-1 has
about 2-folds higher than those of OXA-58. Then, we purified the periplasmic
fraction of clinical isolate and was analyzed using the SDS-PAGE. The results showed
that a protein band about 27kDa was revealed in the imipenem-resistant strains but
not in the imipenem-susceptible. The result of mass spectrometry demonstrated that
only IMP-1 was identified in these 27KDa protein bands. Taken together, these results
6
suggest that IMP-1 rather than to OXA-58 hydrolyze imipenem and resulted in
imipenem resistence of A22, A25, A1245 and Ah34.
|
|---|
