초록 ▼

최근에 새롭게 항생제 내성유전체(antibiotic resistome: D' Costa et al. Science 311:374-7)가 주장되었지만 추가적인 연구 결과가 필요한 실정임. 특히, 항생제 또는 내성균이 유입되지 않은 곳 (심해지역 sediment 또는 marine subsurface
sediment)에서의 배양과 무관한(culture-independent) 연구 결과가 미흡함. 따라서 해양내(심해지역 sediment 또는 marine subsurface sediment) 항생제 내성유전체 동정 및 기능(내성단백질의

최근에 새롭게 항생제 내성유전체(antibiotic resistome: D' Costa et al. Science 311:374-7)가 주장되었지만 추가적인 연구 결과가 필요한 실정임. 특히, 항생제 또는 내성균이 유입되지 않은 곳 (심해지역 sediment 또는 marine subsurface
sediment)에서의 배양과 무관한(culture-independent) 연구 결과가 미흡함. 따라서 해양내(심해지역 sediment 또는 marine subsurface sediment) 항생제 내성유전체 동정 및 기능(내성단백질의 생화학적 특성) 연구가 필요함.
Metagenomic library의 90,823 clones로부터 disk diffusion test 및 pulse-field gel electrophoresis를 이용하여 다재내성인 60 clones의 선별, 임상에서 중요한 cephalosporines인 ceftazidime, cefotaxime에 내성을 나타내는 ESBLs(extended-spectrum β-lactamases) 유전자의 존재를 확인, 항생제 내성 유전자의 검출을 위하여 제작한 bla Detetction Kit 사용으로 항생제 내성 유전자의 존재를 확인 후 Next Generation Sequencing (NGS)통하여 선별 된 4종 유전자 PNG0004, PNG0018-1, PNG0018-2, PNG0018-3 중 가용성 단백질로 대량 순수분리 된 PNG0018-2 및 PNG0018-3을 298K에서 단백질 결정화. PNG0018-2만 결정을 획득, 회절 데이터(space group P21과 C2)를 획득하였으며, P21은 2.14 Å, C2는 2.24 Å으로 획득, PNG0018-2의 결정으로부터 얻은 회절데이터를 이용하여
molecular replacement방법으로 구조분석을 시도한 결과 지금까지 연구되어진 구조의 sequence identity가 20%이하로 분석이 어려움. 다양한 phasing 방법으로부터 회절데이터를 획득하였으나 High molecules per asymmetric unit(8), 높은 Met 함량으로, 구조분석이 지연됨. PNG0018-2의 구조분석을 위한 해결책으로 Sm 또는 Gd를 사용하여 SAD data 를 획득, Molecules per asymmetric unit가 1~2개의 새로운 결정 획득하여 구조 분석, Se-Met의 개수를 줄이기 위해서 PNG0018-2(386 amino acids)에서 beta-lactamase domain(219 amino acids: truncated mutant)만 구조분석, 그리고 PNG0018-2에 있는 18개 Met 중 일부를 Ala으로 mutation하여 결정 획득하여 구조 분석하는 다양한 구조분석 방법으로 분석 중임. 구조분석이 어려운 만큼 novel enzyme의 가능성이 큼.

Abstract ▼

Ⅳ. R&D R esults
1. Identification of antibiotic resistome in the marine
-A total of 90,823 metagenomic library clones were obtained.
-Resistance (R) prevalence for each specific antibiotic: benzylpenicillin (11.89%), ampicillin (0.07%), cephalothin (17.48%), cefotetan (4.11%), cefoxitin (5.

Ⅳ. R&D R esults
1. Identification of antibiotic resistome in the marine
-A total of 90,823 metagenomic library clones were obtained.
-Resistance (R) prevalence for each specific antibiotic: benzylpenicillin (11.89%), ampicillin (0.07%), cephalothin (17.48%), cefotetan (4.11%), cefoxitin (5.96%), ceftazidime (0.86%), cefotaxime (0.23%), imipenem (0.004%), and aztreonam(0.005%)
-The frequency, tendency, and diversity of resistance prevalence is new result that did not appeared in previous reports.
-It is the first detection of ESBL (causing a severe problem in the clinical) that has an important implication.
-MICs were determined in resistant clones: some clones appeared resistance for ampicillin and piperacillin; and other clones showed resistance for ceftazidime, cefotaxime, and aztreonam.
2. Investigation of horizontal transfer of resistance genes and the role of resistome
in the marine through analysis of antibiotic extended resistome
-Selection of 60 clones resistant against more than 5 antibiotics and acquisition of average 35 Kbp sequence information through analysis of surrounding parts of resistomes selected from 60 clones
-We found 5 or more Transfer Element (TE).
-We proved the existence of antibiotic extended resistomes in the marine and horizontal transfer of resistance genes would be caused by these TEs.
-Discovery of TEM-116, CTX-M-15, OXA-2, ACT-2, and the aac(6')-I e-aph(2" )-I a genes using bla Detection Kit
-Discovery of TEM-116 like, CTX-M-15 like, and OXA-210 like genes which were not reported previously (substitution of a few amino acid through evolution). Particularly, 4 kinds of novel antibiotic resistance genes were found through the NGS and these genes could not be detected by bla Detection Kit due to their novelty.
-Antibiotic resistome plays an important role as reservoir of variety resistance genes.
3. Expression and characterization of proteins encoded antibiotic resistome in the marine
-Three genes (PNG0018-1, PNG0018-2, PNG0018-3; similar to class B bla) and one gene (similar to class C) was founded from antibiotic resistance genes identified by NGS
-One (PNG0018-3) gene among three Class B genes was expressed in E. Coli BL21(DE3) as a soluble form
-PNG0004 (Class C), PNG0018-1 (Class B), and PNG0018-2 (Class B) were expressed in E . coli BL21(DE3) as insoluble forms. To solve the problem of insoluble forms, we checked expression condition of resistant proteins.
- Analysis results of biochemica characteristics of resistance protein for substrates or inhibitors showed that it could hydrolyze third-generation cephalosporins (ceftazidime and cefotaxime) and fourth-generation cephalosporin(cefepime). It could also hydrolyze imipenem and meropenem., showing class B enzymatic characteristics.
4. Crystallization and analysis of expressed resistant protein
-Two genes (PNG0018-2 and PNG0018-3) among four genes (PNG0018-1, PNG0018-2, PNG0018-3, and PNG0004) were expressed as soluble forms and then they were purified.
-PNG0018-2 was only crystallized.
-We obtained the diffraction data from five crystals of PNG0018-2 (through a variety of phasing methods)
-Structural analysis is delayed because of high molecules (8) per asymmetric unit and a high content of Met (using the various methods for analysis of structure in present)
-From 404 clinical isolates, the accuracy of bla Detetction Kit was verified.
When it applied to the real field (hospital or environmental samples), the Kit could exactly determine resistance gene type.