생명과학회지 (Journal of Life Science)

  • 제22권8호
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  • Pages.1114-1119
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  • 2012
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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돼지 정액에서 분리된 Stenotrophomonas maltophilia 확인을 위한 PCR 기법 개발 및 분리 균주의 항생제 감수성 양상

Establishment of PCR Conditions for the Identification of Stenotrophomonas maltophilia Isolated from Boar Semen and Antimicrobial Susceptibility Patterns of the Isolates

  • 정병열 (농림수산검역검사본부 세균질병과) ;
  • 박범수 (농림수산검역검사본부 세균질병과) ;
  • 김하영 (농림수산검역검사본부 세균질병과) ;
  • 변재원 (농림수산검역검사본부 세균질병과) ;
  • 김애란 (농림수산검역검사본부 세균질병과) ;
  • 전병윤 (농림수산검역검사본부 세균질병과) ;
  • 김인철 (국립축산과학원 양돈과) ;
  • 정기화 (경남과학기술대학교 동물소재공학과)
  • Jung, Byeong-Yeal (Bacteriology and Parasitology Division, Animal Plant and Fisheries Quarantine and Inspection Agency) ;
  • Park, Bum-Soo (Bacteriology and Parasitology Division, Animal Plant and Fisheries Quarantine and Inspection Agency) ;
  • Kim, Ha-Young (Bacteriology and Parasitology Division, Animal Plant and Fisheries Quarantine and Inspection Agency) ;
  • Byun, Jae-Won (Bacteriology and Parasitology Division, Animal Plant and Fisheries Quarantine and Inspection Agency) ;
  • Kim, Ae-Ran (Bacteriology and Parasitology Division, Animal Plant and Fisheries Quarantine and Inspection Agency) ;
  • Jeon, Albert Byung-Yun (Bacteriology and Parasitology Division, Animal Plant and Fisheries Quarantine and Inspection Agency) ;
  • Kim, In-Cheul (Swine Science Division, National Institute of Animal Science, Rural Development Administration) ;
  • Chung, Ki-Hwa (Department of Animal Resources Technology, Gyeongnam National University of Science and Technology)
  • Received : 2012.07.08
  • Accepted : 2012.08.22
  • Published : 2012.08.30
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Abstract

돼지 정액의 세균오염은 정자활력 감소나 수태율 저하 등을 유발하는데, 특히 Stenotrophomonas maltophilia는 자연환경에 널리 존재하여 비위생적으로 채취된 정액에 많이 오염될 뿐 아니라 사람에게도 병원성을 일으킨다. 본 연구에서는 S. maltophilia의 검사 시간 단축과 정확도를 높이고자 PCR 기법을 개발하였으며, 돼지 원정액에서 이들 세균의 오염율을 조사하고 유효 항생제를 선발하고자 하였다. 돼지 원정액에서 분리 빈도가 높은 18 균종을 대상으로 PCR을 적용한 결과, S. maltophilia에서만 445 bp의 특이 유전자 증폭산물이 확인되었다. 또한 순수 배양된 S. maltophilia는 $1.5{\times}10^3$ CFU/ml까지, 원정액에 혼합된 S. maltophilia에 대해서는 $1.5{\times}10^4$ CFU/ml까지 검출이 가능하였다. 2009년에 116건과 2011년 324건 등 총 440건의 원정액 중 26건(5.9%)에서 S. maltophilia가 분리되었으며, 여름과 가을철에 오염율이 상대적으로 높게 나타났다. 균분리법과 PCR간의 일치율은 98.9%, kappa value는 0.906이었으며, PCR의 민감도와 특이도는 각각 100%, 98.7%로 나타났다. 분리균주의 항생제 감수성 시험 결과, enrofloxacin (100%)과 florfenicol (92.3%)에 높은 감수성을 보였으나 amoxicillin/clavulanic acid, apramycin, ceftiofur, penicillin, spectinomycin에는 내성율이 높게 나타났다. 결론적으로 개발된 PCR기법은 민감도, 특이도, 일치율이 높아 균분리법을 보조하고 신속 정확하게 정액 내 오염세균을 확인할 수 있어 효율적인 정액관리가 가능할 것으로 기대된다.

Bacteria are frequently contaminated during the collection and processing procedures of boar semen. Of the contaminants, Stenotrophomonas (S.) maltophilia is a Gram-negative bacterium that is widely distributed in a variety of habitats. Although PCR assays have been developed for the detection of S. maltophilia, they cross-react with some species of Xanthomonas. In this study, we designed a primer set for the detection of S. maltophilia in order to target the chiA (GenBank accession no. NC_010943) gene. The specific PCR products were amplified from S. maltophilia only, not from other tested strains that are frequently found in semen. The detection limit of the PCR was $1.5{\times}10^3$ CFU/ml with pure-cultured S. maltophilia and $1.5{\times}10^4$ CFU/ml with S. maltophilia spiked in semen. Twenty-six (5.9%) S. maltophilia were isolated from 440 semen samples. The PCR results exhibited 98.9% agreement with a comparison of S. maltophilia isolation. Also, the sensitivity and specificity of the PCR were 100% and 98.7%, respectively. In the antimicrobial susceptibility test, S. maltophilia isolates were highly susceptible to enrofloxacin and florfenicol, while the majority of them were resistant to amoxicillin/clavulanic acid, apramycin, ceftiofur, penicillin, and spectinomycin. These results indicated that the PCR using the chiA gene was proven to be reliable and effective for the detection of S. maltophilia with high levels of sensitivity and specificity.

Keywords

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