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세 가지의 ESBL 유전자를 가지고 있는 Klebsiella pneumoniae의 유전자접합체와 헝질전환체의 항생제내성분석

Analysis of Antibiotic Resistant Patterns in Conjugant and Transformant of Three ESBL gene Harboring Klebsiella pneumoniae

  • 김윤태 (부산가톨릭대학교 보건과학대학 임상병리학과)
  • Kim, Yun-Tae (Department of Clinical Laboratory Science, College of Health Science, Catholic University)
  • 발행 : 2007.10.30

초록

우리는 ESBL 유전자를 포함하는 세균의 항생제 내성 패턴을 조사하기 위해서 부산에 있는 임상실험실로부터 Klebsiella pneumoniae 한 균주를 수집하였다. 그 세균은 이중 디스크 확산법에 의해 ESBL을 생성하는 Klebsiella pneumoniae라는 것이 밝혀졌고 PCR과 DNA 염기서열분석을 통하여 세 가지의 ESBL gene (TEM-1, SHV-12, CTX-M-15)이 포함되어 있다는 것이 확인되었다. 그리고 그 세 가지 유전자의 특징을 알아내기 위해서 우리는 이 균주로부터 교차접합시험, 형질전환시험, 클로닝(SHV-12 gene)등을 이용하여 재조합균주를 배양하였다. 이렇게 각기 재조합된 균주들을 한천평판희석법을 이용하여 3세대 cephalosporin 항생제 (ceftazidime, cefotaxime, ceftriaxone)에 대한 최소억제농도의 측정하고 비교 분석하였다. Klebsiella pneumoniae (NO. 60031)의 MIC는 ceftazidime ($30\;{\mu}g/ml)$, cefotaxime ($30\;{\mu}g/ml$), ceftriaxone ($30\;{\mu}g/ml$)이 각각 ${\geq}256\;{\mu}g/ml,\;128\;{\mu}g/ml,\;128\;{\mu}g/ml$이었고, E. coli $RG176^{Na(r)}$에 교차접합 시킨 conjugant 균주의 MIC는 ceftazidime, cefotaxime, ceftriaxone이 각각 ${\geq}256\;{\mu}g/ml,\;64\;{\mu}g/ml,\;128\;{\mu}g/ml$이었다. E. coli $DH5{\alpha}$에 형질전환 시킨 transformant 균주의 MIC는 ceftazidime, cefotaxime, ceftriaxone이 각각 $128\;{\mu}g/ml,\;32\;{\mu}g/ml,\;32\;{\mu}g/ml$를 나타내었고, SHV-12 gene 만을 분리하여 E. coli $DH5{\alpha}$에 클로닝 시킨 균주는 ceftazidime, cefotaxime, ceftriaxone이 각각 $128\;{\mu}g/ml,\;8\;{\mu}g/ml,\;32\;{\mu}g/ml$을 나타내었다. 결국 conjugant 균주와 transformant 균주는 세 가지의 내성유전자를 가졌다는 공통점은 있으나 conjugant 균주의 MIC가 transformant 균주의 MIC보다 높게 나타나는 차이점을 보였다. 또한 SHV-12 gene 만을 분리하여 E. coli $DH5{\alpha}$에 클로닝 시킨 균주는 ceftazidime, ceftriaxone에 내성을 발현하였지만 cefotaxime에 대해서는 내성을 발현하지 않았다.

To investigate the antibiotic resistant patterns of the bacteria producing ESBL, we isolated one organism of Klebsiella pneumoniae from a clinical laboratory in Busan. The organism that produces ESBL gene was detected by double disk synergy test and the presence of three ESBL genes (TEM-1, SHV-12, CTX-M-15) was confirmed by polymerase chain reaction and DNA sequencing analysis. To analyse the characteristics of three ESBL genes, we performed transconjugation, transformation and cloning experiment with the organism. The MIC of Klebsiella pneumoniae was revealed that ceftazidime, cefotaxime and ceftriaxone were $256\;{\mu}g/ml,\;128\;{\mu}g/ml\;and\;128\;{\mu}g/ml$ respectively. The MIC of conjugant (E. coli $RG176^{Na(r)}$) af was revealed that ceftazidime, cefotaxime and ceftriaxone were $256\;{\mu}g/ml,\;64\;{\mu}g/ml\;and\;128\;{\mu}g/ml$ respectively. The MIC of transformant (E. cofi $DH5{\alpha}$) was revealed that ceftazidime, cefotaxime and ceftriaxone were $128\;{\mu}g/ml,\;32\;{\mu}g/ml,\;and\;32\;{\mu}g/ml$ respectively, The MIC of cloned organism of SHV-12 gene (E. coli $DH5{\alpha}$) was revealed that ceftazidime, cefotaxime and ceftriaxone were $128\;{\mu}g/ml,\;8\;{\mu}g/ml,\;and\;32\;{\mu}g/ml$ respectively. The results indicated that MIC of conjugant was higher than MIC of transformant and also SHV-12 gene were not resistant against cefotaxime antibiotic.

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