| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 102 === Acinetobacter baumannii is a non-fermenting Gram negative bacterium and one of the leading pathogens of nosocomial infection worldwide. The carbapenem resistance has been developed in a extremely speed among A. baumannii strains, which has severely compromised the therapeutic options. Therefore, new antimicrobials that are effective against CRAb (carbapenem resistant A. baumannii) are urgently needed. Recently, several researches have shown that overproduction of carbapenem hydrolyzing class D beta-lactamases (CHDLs) by itself can confer a high level of carbapenem resistance in A. baumannii, and these CHDLs are found in nearly all CRAb. Inhibition of CHDLs is among one of the effective strategies to settle CRAb infection. This approach often identifies chemicals that target on the active site of the enzymes.
We have firstly identified the plasmid bearing ISAba1-blaOXA-51-like has become one of the major carbapenem resistance determinants in A. baumannii in Taiwan. Insertion sequence ISAba1 acts as a strong promoter to enhance the expression of blaOXA-51-like. However, the carbapenem hydrolyzing characteristics of OXA-51-like carbapenemase have not been elucidated. We have found that different OXA-51-like enzymes contribute to carbapenem resistance in a different extent. The amino acid sequences are the same between ISAba1-blaOXA-66 and ISAba1-blaOXA-82 except for a Leu167Val amino acid substitution. The transformants carrying the ISAba1-blaOXA-82 would exhibit a much higher level of carbapenem resistance than those carrying ISAba1-blaOXA-66. In this study, we investigated the mechanism of difference in contribution to meropenem resistance between two blaOXA-51-like alleles. The minimum inhibitory concentrations (MIC) of two A.baumannii isolates, ATCC15151 (pYMAb2 ISAba1-blaOXA-66) and ATCC15151 (pYMAb2 ISAba1-blaOXA-82), were 2 and 24 μg/mL for meropenem respectively. The former was susceptible; the latter was resistant to antibiotic. The results of western blotting were shown that oxacillinase produced by ATCC15151 (pYMAb2 ISAba1-blaOXA-66) was approximately 1.5-2 times quantity of the OXA-82 with the short-time reaction (2 - 8 hr). ATCC15151 (pYMAb2 ISAba1-blaOXA-82) produced about 1.2 times quantity of oxacillinase than OXA-66 with the long-time reaction (8 - 16 hr). Hydrolytic efficiency of OXA-66 (kcat/Km = 0.04 μM-1sec-1) was greater than OXA-82 (kcat/Km = 0.01 μM-1sec-1) in Q-TOF LC/MS spectra, enzyme kinetics. The zone of inhibition with modified Hodge test showed OXA-82 hydrolyzed more meropenem than OXA-66. There was no statistically significant difference with mRNA expression between two strains. There was no different structure with molecular modeling between OXA-66 and OXA-82. The strain was possessed the lower MIC value but the oxacillinase was exhibited higher catalytic efficiency for antibiotics. The resistant strain produced more quantity of enzyme to resist and tolerate higher concentration of antibiotic. The results were elaborated the reason why the class D OXA-51-like group strains ATCC15151 (pYMAb2 ISAba1-blaOXA-82) was possessed higher MIC value than ATCC15151 (pYMAb2 ISAba1-blaOXA-66).
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