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과제정보

과제정보
과제명 수산 환경 중의 항생제 내성균 모니터링
기관명 식품의약품안전처 담당부서 식품의약품안전평가원 연구기획조정과
전화번호 연구기간 2004-04-14 ~ 2004-11-30
연구분야 식품안전
개요 수산 환경 중의 항생제 내성균 모니터링

계약정보

과제정보
수행기관 소비자보호원 [기타]
수행연구원 정윤희 계약일자 2004-04-14
계약방식 일반경쟁입찰에 의한 계약 계약금액 50,000,000원

연구결과 정보

과제정보
제목 수산 환경 중의 항생제 내성균 모니터링
연구보고서
목차
■ 요약문 ·················································································································································· 1 ■ Summary ············································································································································ 5 제 1장 서론 ············································································································································ 29 제 1절 연구과제의 중요성 ·············································································································· 29 1. 항생제 내성균의 문제 ··········································································································· 29 2. 연구개발의 필요성 ··················································································································· 32 3. 연구의 범위 ······························································································································· 34 제 2장 국내?외 기술개발 현황 ············································································································ 35 제 3장 연구 개발 수행 내용 및 결과 ······························································································ 39 제 1절 샘플 채취 대상 및 방법 ···································································································· 39 1. 검체채취 대상 ··························································································································· 39 2. 검체채취 방법 ··························································································································· 43 제 2절 내성균 모니터링 대장균주의 선정 ·················································································· 47 제 3절 대상균주의 분리 방법 ········································································································ 47 1. 장구균(Enterococcus faecium/faecalis) ··········································································· 47 2. 황색포도상구균(Staphylococcus aureus) 분리 ································································ 49 3. 대장균(E. coli) 분리 ················································································································ 49 4. 비브리오균(V. cholerae V. parahaemolyticus, V. vulnificus) 분리 ························ 50 5. 살모넬라균(Salmonella spp.) 분리 ······················································································ 50 제 4절 분리 대상 균주의 검출률 ·································································································· 51 1. 장구균(Enterococcus faecium/faecalis) ··········································································· 51 2. 황색포도상구균(Staphylococcus aureus) ·········································································· 59 3. 대장균(E. coli) ·························································································································· 63 4. 비브리오균(V. cholerae V. parahaemolyticus, V. vulnificus) 분리 ························ 67 5. 살모넬라균(Salmonella spp.) ································································································ 69 6. 균종 별 검출률 비교 ··············································································································· 70 제 5절 항생제 감수성 시험 ············································································································ 73 1. Disk Diffusion Testing ········································································································· 73 제 6절 항생제 감수성 결과 ············································································································ 74 1. 장구균(Enterococcus faecium/faecalis) ··········································································· 74 2. 황색포도상구균(Staphylococcus aureus) ······································································· 115 3. 대장균(E. coli) ······················································································································· 132 4. 비브리오균(V. parahaemolyticus) ···················································································· 148 제 7절 PFGE(Pulse Field Gel Electrophoresis)를 이용한 Molecular subtyping ······ 159 1. 실험방법 ··································································································································· 159 2. 실험결과 ··································································································································· 160 제 4장 연구개발 목표 달성도 및 대외 기여도 ············································································ 162 제 5장 연구개발결과의 활용성과 및 계획 ·················································································· 165 1. 계량적 성과 ····························································································································· 165 2. 성과내용기술 ··························································································································· 165 3. 활용계획 ··································································································································· 167 제 6장 기타 중요변경사항 ·············································································································· 168 제 7장 참고문헌 ·································································································································· 169
 

■ 요약문 ·················································································································································· 1
■ Summary ············································································································································ 5
제 1장 서론 ············································································································································ 29
제 1절 연구과제의 중요성 ·············································································································· 29
1. 항생제 내성균의 문제 ··········································································································· 29
2. 연구개발의 필요성 ··················································································································· 32
3. 연구의 범위 ······························································································································· 34
제 2장 국내?외 기술개발 현황 ············································································································ 35
제 3장 연구 개발 수행 내용 및 결과 ······························································································ 39
제 1절 샘플 채취 대상 및 방법 ···································································································· 39
1. 검체채취 대상 ··························································································································· 39
2. 검체채취 방법 ··························································································································· 43
제 2절 내성균 모니터링 대장균주의 선정 ·················································································· 47
제 3절 대상균주의 분리 방법 ········································································································ 47
1. 장구균(Enterococcus faecium/faecalis) ··········································································· 47
2. 황색포도상구균(Staphylococcus aureus) 분리 ································································ 49
3. 대장균(E. coli) 분리 ················································································································ 49
4. 비브리오균(V. cholerae V. parahaemolyticus, V. vulnificus) 분리 ························ 50
5. 살모넬라균(Salmonella spp.) 분리 ······················································································ 50
제 4절 분리 대상 균주의 검출률 ·································································································· 51
1. 장구균(Enterococcus faecium/faecalis) ··········································································· 51
2. 황색포도상구균(Staphylococcus aureus) ·········································································· 59
3. 대장균(E. coli) ·························································································································· 63
4. 비브리오균(V. cholerae V. parahaemolyticus, V. vulnificus) 분리 ························ 67
5. 살모넬라균(Salmonella spp.) ································································································ 69
6. 균종 별 검출률 비교 ··············································································································· 70
제 5절 항생제 감수성 시험 ············································································································ 73
1. Disk Diffusion Testing ········································································································· 73
제 6절 항생제 감수성 결과 ············································································································ 74
1. 장구균(Enterococcus faecium/faecalis) ··········································································· 74
2. 황색포도상구균(Staphylococcus aureus) ······································································· 115
3. 대장균(E. coli) ······················································································································· 132
4. 비브리오균(V. parahaemolyticus) ···················································································· 148
제 7절 PFGE(Pulse Field Gel Electrophoresis)를 이용한 Molecular subtyping ······ 159
1. 실험방법 ··································································································································· 159
2. 실험결과 ··································································································································· 160
제 4장 연구개발 목표 달성도 및 대외 기여도 ············································································ 162
제 5장 연구개발결과의 활용성과 및 계획 ·················································································· 165
1. 계량적 성과 ····························································································································· 165
2. 성과내용기술 ··························································································································· 165
3. 활용계획 ··································································································································· 167
제 6장 기타 중요변경사항 ·············································································································· 168
제 7장 참고문헌 ·································································································································· 169
초록
금번 수산 환경중의 항생제 내성균 모니터링 실험 대상은 육상 수조식 양식장 100샘 플, 해상 가두리 양식장 64샘플, 내수면 양식장 110 샘플과 양식어가 소비자에게 전달되 는 과정 중에서 물고기를 운반 하는 차량에서 항생제의 투입이 있는지를 간접적으로 알 아보기 위한 산지 횟집 21곳과 수도권 횟집 20곳 등 총 41곳 41샘플, 또 어민의 손에서 분리한 균과 일반인의 손에서 분리한 균의 내성률의 차이가 있는지를 알아보기 위하여 수도권의 일반인 20명의 손, 총 335개였다. 이 대상 샘플에서 E. faecalis, E. faecium, E. coli, Salmonella spp, Vibrio spp, S. aureus을 검사하였다. 그 결과 E. faecalis는 229주, E. faecium은 94주, S. aureus는 65 주, E. coli는 58주, V. parahaemolyiticus는 139주를 분리하였다. 균의 검출률을 보면 E. faecalis가 가장 높은 검출률을 보이고 E. faecium의 검출률 도 높게 나타났다. S. aureus는 E. faecalis와 함께 어민 손에서 많이 검출되었다. E. faecalis는 양식어에서 55.2%, E. faecium은 양식어에서 32.8%, S. aureus는 어민 손에서 41.4%, E. coli는 양식어에서 15.5% 검출되었다. V. parnfahaemolyticus는 내수면 양식장 에서는 검출되지 않았으며, 육상 수조식 양식장과 가두리 양식장에서 검출되었다. 분리된 균에서 항생제 감수성 시험을 실시하였다. E. faecalis는 전체 분리된 229주 중 205주 89.5%가 한 종류 이상의 항생제에 내성을 가지고 있고, 4가지 계열의 서로 다른 항생제에 내성이 있는 균주인 다제 내성균은 30주 13.1% 이었다. 양식어에서 분리된 균들이 횟집이나 어민, 일반인 손에서 분리된 균들보 다 다제 내성률이 높게 나타났다. 분리된 균 중 내성률이 높은 항생제는 Tetracycline(TE) 64.2%, Rifampin(RA) 49.8%, Erythromycin(E) 31.8%, Chloramphenicol(C) 20.9%, sulfamethoxazole/Trimethopenem (SXT) 12.9%, Ciprofloxacin(CIP) 10.0%, Gentamycin(GM) 0.5%, Penicillin(P) 0.5%이었다. 토양에서 분 리된 균이 다른 채취지역보다 내성을 보이는 항생제 수가 적게 나타나고, 지하수에서 분 리한 균은 그 수가 적어 의미 있는 데이터로 보기에는 어려움이 있지만 분리된 균에 있 어서는 항생제 내성이 획득되어 있었으며, 특히 지하수에는 토양을 제외한 다른 채취지 역에서 분리한 균에서 보이는 내성 패턴과 마찬가지로 내성 항생제의 종류가 많은 것으 로 나타났다. E. faecium은 전체 분리된 94주 중 89주 94.7%가 항생제 1종류 이상에 내성을 가지 고 있고, 다제 내성균은 5주 5.3%로 낮은 검출률을 보였다. 육상 수조식 물고기에서 분 리한 1균주와 어민 손에서 분리한 3균주만이 다제 내성균이었다. 분리된 균 중 내성률 이 높은 항생제는 Tetracycline(TE) 55.2%, Rifampin(RA) 52.9%, Erythromycin(E) 49.4%, Ciprofloxacin(CIP) 20.7%, Penicillin(P) 17.2%, sulfamethoxazole /Trimethopenem (SXT) 3.4%, Ampicillin(AMP) 1.1%, Chloramphenicol(C) 1.1% 으로 나타나 E. faecalis와 유사한 결과를 보였다. S. aureus는 전체 분리된 65주 중 63주 96.9%가 항생제 1종류 이상에 내성을 가지 고 있고, 다제 내성균은 5균주 7.7% 검출되었다. 내수면 양식장 물고기 1균주와 산지, 수 도권 횟집 수조 환경에서 각각 1균주씩, 어민 손 2균주, 일반인 손에서 1균주의 다제 내 성균이 검출되었다. 분리된 균 중 내성률이 높은 항생제는 Penicillin(P) 89.7%, Ampicillin(AMP) 89.7%, Tetracycline(TE) 25.6%, Amikacin(AK) 17.9%, Oxacillin(OX) 5.1%, Gentamycin(GM) 5.1%, Erythromycin(E) 2.6%으로 나타났다. E. coli는 전체 분리된 58주 중 29주 50%가 항생제 1종류 이상에 내성을 가지고 있 고, 다제 내성균은 15개 25.9% 검출되었다. 육상 수조식 물고기에서 검출된 4균주 모두 와 내수면 양식장에서 검출된 균중에서 3균주(30%), 어민 손에서 분리한 1균주(14.3%)가 다제 내성균이었다. 분리된 균 중 내성률이 높은 항생제는 Tetracycline(TE) 47.3%, Cefazolin(KF) 32.7%, Ciprofloxacin(CIP) 25.5%, Enrofloxacin(ENR) 25.5%, Norfloxacin(NOR) 25.5%, Ampicillin(AMP) 18.2%, Streptomycin(S) 18.2%, sulfamethoxazole/Trimethopenem(SXT)9.1% 이고 Chloramphenicol (C), Ampicillin /Sulbactam(SAM), Aztreonam(ATM)은 5% 미만이었다. V. parahaemolyticus은 전체 분리된 139주 중 109주 78.4%가 항생제 1종류 이상에 내성을 가지고 있고, 다제 내성균은 검출되지 않았다. 분리된 균 중 내성률이 높은 항생 제는 Amikacin(AK) 10.2%, Ampicillin(AMP) 71.6%으로 나타났으며 그 외의 항생제는 채취지역에 상관없이 분리된 균에서 내성이 획득된 것이 없었다. 바다유래 세균인 비브 리오균의 경우 내성률이 높지 않은 것으로 나타났다. 산지 횟집의 물과 수도권 횟집의 물에서 분리된 균의 항생제 감수성 결과 수도권에 비해 산지 횟집에서 분리한 균의 항생제 내성률이 대체로 높았다. 항생제 사용에 노출 되어 있을 것으로 예상되는 어민의 손과 그렇지 않은 일반인 손에서 분리한 균의 항생제 감수성시험 결과 균종에 따라 다소 차이는 있으나 어민 손에서 분리한 균의 내성률이 높 을 것이라는 예상과 달리 일반 손에서 분리한 균의 내성률이 다소 높거나 비슷했다. 향 후 좀 더 많은 샘플을 가지고 집중적으로 연구할 필요가 있다. 이번 실시한 수산 환경에서 분리한 균과 2003년 축산 환경에서 분리한 균의 항생제 감수성시험 결과를 비교해 보면, E. faecalis와 E. faecium의 경우 축산환경에서는 한 가 지 이상의 항생제에 내성을 가지고 있는 균이 82.1%이며 다제 내성률은 6.2%였는데 수 산 환경에서 분리한 균은 91%, 10.8%으로 나타났고, S. aureus 는 축산환경에서 분리한 균이 한 가지 이상의 항생제에 내성을 가지고 있는 균이 78.6%이며, 다제 내성률은 11.9%였는데, 수산 환경에서는 96.9%, 7.7%였다. E. coli는 축산환경에서 분리한 균이 한 가지 이상의 항생제에 내성을 가지고 있는 균이 69.4%, 다제 내성률은 19.2%였는데, 수산 환경에서 분리한 균은 50%와 25.9% 으로 나타나 균에 따라 축산환경에서 분리한 균과 수산 환경에서 분리한 균의 내성 정도가 서로 차이가 있었다. 2003년 9월 기준 소, 돼지, 닭에 사용하는 항생제 및 합성항균제의 양은 952,072kg이며, 수산용은 123,862 kg 으로 수치상으로 비교할 때는 수산용이 축산용의 13%밖에 되지는 않는다. 그러나 전체 산업규모를 비교할 수 없어 항생제 사용이 어느 산업에서 더 사용되는지는 알 수 없었 다. 또한 시험결과에 있어서도 축산이나 수산이나 내성률에 있어서도 뚜렷한 차이점을 찾지 못했다. 다행인 것은 수산 환경에서 분리된 균 중에는 VRE, VRSA, MRSA 등과 같은 슈퍼박테리아는 검출되지 않았다. 또한 일부 균에 대해서 직접 환경적 연관이 있는 균과 그렇지 않은 균에 대하여 PFGE를 이용한 상호 연관 관계를 본 결과 서로 연관 관계가 있는 것으로 나타났다. 그 러나 이 부분은 향후 좀 더 많은 균을 가지고 계속적인 연구가 필요하다. 이번 연구 사업에서 분리된 균들은 수산 환경에서 분리된 내성균이 많지 않은 국내 현실을 감안할 때, 향후 다양한 분자유전학적 방법을 적용하여 채취특성에 따른 특이성 과 연관성을 연구하는데 좋은 재료가 될 것이다.
 

금번 수산 환경중의 항생제 내성균 모니터링 실험 대상은 육상 수조식 양식장 100샘
플, 해상 가두리 양식장 64샘플, 내수면 양식장 110 샘플과 양식어가 소비자에게 전달되
는 과정 중에서 물고기를 운반 하는 차량에서 항생제의 투입이 있는지를 간접적으로 알
아보기 위한 산지 횟집 21곳과 수도권 횟집 20곳 등 총 41곳 41샘플, 또 어민의 손에서
분리한 균과 일반인의 손에서 분리한 균의 내성률의 차이가 있는지를 알아보기 위하여
수도권의 일반인 20명의 손, 총 335개였다.
이 대상 샘플에서 E. faecalis, E. faecium, E. coli, Salmonella spp, Vibrio spp, S.
aureus을 검사하였다. 그 결과 E. faecalis는 229주, E. faecium은 94주, S. aureus는 65
주, E. coli는 58주, V. parahaemolyiticus는 139주를 분리하였다.
균의 검출률을 보면 E. faecalis가 가장 높은 검출률을 보이고 E. faecium의 검출률
도 높게 나타났다. S. aureus는 E. faecalis와 함께 어민 손에서 많이 검출되었다. E.
faecalis는 양식어에서 55.2%, E. faecium은 양식어에서 32.8%, S. aureus는 어민 손에서
41.4%, E. coli는 양식어에서 15.5% 검출되었다. V. parnfahaemolyticus는 내수면 양식장
에서는 검출되지 않았으며, 육상 수조식 양식장과 가두리 양식장에서 검출되었다.
분리된 균에서 항생제 감수성 시험을 실시하였다.
E. faecalis는 전체 분리된 229주 중 205주 89.5%가 한 종류 이상의 항생제에 내성을
가지고 있고, 4가지 계열의 서로 다른 항생제에 내성이 있는 균주인 다제 내성균은 30주
13.1% 이었다. 양식어에서 분리된 균들이 횟집이나 어민, 일반인 손에서 분리된 균들보
다 다제 내성률이 높게 나타났다. 분리된 균 중 내성률이 높은 항생제는
Tetracycline(TE) 64.2%, Rifampin(RA) 49.8%, Erythromycin(E) 31.8%,
Chloramphenicol(C) 20.9%, sulfamethoxazole/Trimethopenem (SXT) 12.9%,
Ciprofloxacin(CIP) 10.0%, Gentamycin(GM) 0.5%, Penicillin(P) 0.5%이었다. 토양에서 분
리된 균이 다른 채취지역보다 내성을 보이는 항생제 수가 적게 나타나고, 지하수에서 분
리한 균은 그 수가 적어 의미 있는 데이터로 보기에는 어려움이 있지만 분리된 균에 있
어서는 항생제 내성이 획득되어 있었으며, 특히 지하수에는 토양을 제외한 다른 채취지
역에서 분리한 균에서 보이는 내성 패턴과 마찬가지로 내성 항생제의 종류가 많은 것으
로 나타났다.
E. faecium은 전체 분리된 94주 중 89주 94.7%가 항생제 1종류 이상에 내성을 가지
고 있고, 다제 내성균은 5주 5.3%로 낮은 검출률을 보였다. 육상 수조식 물고기에서 분
리한 1균주와 어민 손에서 분리한 3균주만이 다제 내성균이었다. 분리된 균 중 내성률
이 높은 항생제는 Tetracycline(TE) 55.2%, Rifampin(RA) 52.9%, Erythromycin(E)
49.4%, Ciprofloxacin(CIP) 20.7%, Penicillin(P) 17.2%, sulfamethoxazole
/Trimethopenem (SXT) 3.4%, Ampicillin(AMP) 1.1%, Chloramphenicol(C) 1.1% 으로
나타나 E. faecalis와 유사한 결과를 보였다.
S. aureus는 전체 분리된 65주 중 63주 96.9%가 항생제 1종류 이상에 내성을 가지
고 있고, 다제 내성균은 5균주 7.7% 검출되었다. 내수면 양식장 물고기 1균주와 산지, 수
도권 횟집 수조 환경에서 각각 1균주씩, 어민 손 2균주, 일반인 손에서 1균주의 다제 내
성균이 검출되었다. 분리된 균 중 내성률이 높은 항생제는 Penicillin(P) 89.7%,
Ampicillin(AMP) 89.7%, Tetracycline(TE) 25.6%, Amikacin(AK) 17.9%, Oxacillin(OX)
5.1%, Gentamycin(GM) 5.1%, Erythromycin(E) 2.6%으로 나타났다.
E. coli는 전체 분리된 58주 중 29주 50%가 항생제 1종류 이상에 내성을 가지고 있
고, 다제 내성균은 15개 25.9% 검출되었다. 육상 수조식 물고기에서 검출된 4균주 모두
와 내수면 양식장에서 검출된 균중에서 3균주(30%), 어민 손에서 분리한 1균주(14.3%)가
다제 내성균이었다. 분리된 균 중 내성률이 높은 항생제는 Tetracycline(TE) 47.3%,
Cefazolin(KF) 32.7%, Ciprofloxacin(CIP) 25.5%, Enrofloxacin(ENR) 25.5%,
Norfloxacin(NOR) 25.5%, Ampicillin(AMP) 18.2%, Streptomycin(S) 18.2%,
sulfamethoxazole/Trimethopenem(SXT)9.1% 이고 Chloramphenicol (C), Ampicillin
/Sulbactam(SAM), Aztreonam(ATM)은 5% 미만이었다.
V. parahaemolyticus은 전체 분리된 139주 중 109주 78.4%가 항생제 1종류 이상에
내성을 가지고 있고, 다제 내성균은 검출되지 않았다. 분리된 균 중 내성률이 높은 항생
제는 Amikacin(AK) 10.2%, Ampicillin(AMP) 71.6%으로 나타났으며 그 외의 항생제는
채취지역에 상관없이 분리된 균에서 내성이 획득된 것이 없었다. 바다유래 세균인 비브
리오균의 경우 내성률이 높지 않은 것으로 나타났다.
산지 횟집의 물과 수도권 횟집의 물에서 분리된 균의 항생제 감수성 결과 수도권에
비해 산지 횟집에서 분리한 균의 항생제 내성률이 대체로 높았다. 항생제 사용에 노출
되어 있을 것으로 예상되는 어민의 손과 그렇지 않은 일반인 손에서 분리한 균의 항생제
감수성시험 결과 균종에 따라 다소 차이는 있으나 어민 손에서 분리한 균의 내성률이 높
을 것이라는 예상과 달리 일반 손에서 분리한 균의 내성률이 다소 높거나 비슷했다. 향
후 좀 더 많은 샘플을 가지고 집중적으로 연구할 필요가 있다.
이번 실시한 수산 환경에서 분리한 균과 2003년 축산 환경에서 분리한 균의 항생제
감수성시험 결과를 비교해 보면, E. faecalis와 E. faecium의 경우 축산환경에서는 한 가
지 이상의 항생제에 내성을 가지고 있는 균이 82.1%이며 다제 내성률은 6.2%였는데 수
산 환경에서 분리한 균은 91%, 10.8%으로 나타났고, S. aureus 는 축산환경에서 분리한
균이 한 가지 이상의 항생제에 내성을 가지고 있는 균이 78.6%이며, 다제 내성률은
11.9%였는데, 수산 환경에서는 96.9%, 7.7%였다. E. coli는 축산환경에서 분리한 균이
한 가지 이상의 항생제에 내성을 가지고 있는 균이 69.4%, 다제 내성률은 19.2%였는데,
수산 환경에서 분리한 균은 50%와 25.9% 으로 나타나 균에 따라 축산환경에서 분리한
균과 수산 환경에서 분리한 균의 내성 정도가 서로 차이가 있었다. 2003년 9월 기준 소,
돼지, 닭에 사용하는 항생제 및 합성항균제의 양은 952,072kg이며, 수산용은 123,862 kg
으로 수치상으로 비교할 때는 수산용이 축산용의 13%밖에 되지는 않는다. 그러나 전체
산업규모를 비교할 수 없어 항생제 사용이 어느 산업에서 더 사용되는지는 알 수 없었
다. 또한 시험결과에 있어서도 축산이나 수산이나 내성률에 있어서도 뚜렷한 차이점을
찾지 못했다. 다행인 것은 수산 환경에서 분리된 균 중에는 VRE, VRSA, MRSA 등과
같은 슈퍼박테리아는 검출되지 않았다.
또한 일부 균에 대해서 직접 환경적 연관이 있는 균과 그렇지 않은 균에 대하여
PFGE를 이용한 상호 연관 관계를 본 결과 서로 연관 관계가 있는 것으로 나타났다. 그
러나 이 부분은 향후 좀 더 많은 균을 가지고 계속적인 연구가 필요하다.
이번 연구 사업에서 분리된 균들은 수산 환경에서 분리된 내성균이 많지 않은 국내
현실을 감안할 때, 향후 다양한 분자유전학적 방법을 적용하여 채취특성에 따른 특이성
과 연관성을 연구하는데 좋은 재료가 될 것이다.
제작일 2004-11-30
발행년도 2004년

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