국내 젖소 유방염 유래 황색포도상구균의 항생제 내성 및 분자유전학적 특성|RISS 상세보기

다국어 초록 (Multilingual Abstract)

Staphylococcus aureus(S. aureus) belongs to the group of major
contagious mastitis pathogens, whereas the coagulase nagative
staphylococci(CNS) are also capable of causing opportunistic bovine
mastitis. Many of these strains are resistant to penicillin or ampicillin
because of long-term use of β-lactam antibiotics in agricultural and
healthcare settings. Based on the simple and highly specific coagulase
genotyping by PCR-RFLP used for discriminating among S. aureus
strains, the relationship between phenotypic antibiogram and the
polymorphism of coagulase gene was determined in this study. We
investigated antibiogram and coagulase gene diversity in staphylococcal
enterotoxin (StE)-producing Staphylococcus aureus isolated from raw milk
samples of cows infected with mastitis from 140 dairy farms in Korea
between 1997 and 2004. Of the 696 S. aureus isolates collected in this
study, 164 isolates (23.6%) produced one or more staphylococcal enterotoxins (A to D), and 19 isolates (2.7%) were methicillin-resistant.
The percentage of StE-producing S. aureus (SES) isolates resistant to
methicillin, kanamycin, neomycin, amikacin, and tetracycline was greater
than that of non-SES. Ten coagulase genotype patterns were observed,
including 4 main types comprising I (25.4%), II (13.9%), VII (13.2%), and
VIII (17.8%). More than 4 S. aureus types were isolated from each of 82
dairy farms in different geographic locations, and only 1 coagulase
genotype pattern was observed in 39 of the herds (47.6%). There was
no significant correlation between coagulase genotypes harbored by S.
aureus and their specific StE type. The percentage of isolates producing
major StE types (A, B, AC, and ABCD) and being resistant to cephalothin
and methicillin was greater among the S. aureus isolates with the 4
predominant coagulase genotypes (I, II, VII, and VIII) than among the
isolates harboring the 6 rare coagulase types (III, IV, V, VI, IX, and X).
Also, this study was investigated for in vitro susceptibility to 10
antimicrobial agents and β-lactamase production of 98 isolated from
bovine subclinical mastitis samples of 26 Korean dairy herds in 2003.
Most of isolates were resistant to penicillin (79.6%) and ampicillin
(79.6%), but susceptible to cephalothin (98.0%), kanamycin (81.6%),
gentamicin (90.8%), erythromycin (100%), and tetracycillin (89.8%) by
agar diffusion test. Seventy nine (80.6%) isolates of isolates were also
positive for β-lactamase production and all these strains showed the
penicillin and ampicillin resistance. The levels of cephalothin and
gentamicin resistance of β-lactamase producing strains were higher than
β-lactamase non-producing strains. The results of drug susceptibility obtained from 17 dairy farms had different from each farms. The
staphylococci strains(835 S. aureus and 763 CNS) were isolated from
3,047 bovine mastitic milk samples from 153 dairy farms in 8 provinces
from 1997 to 2004 in Republic Korea. Twenty-one(2.5%) S. aureus and
19(2.4%) CNS strains were resistant to methicillin〔oxacillin minimum
inhibitory concentration(MIC)≥4 ㎍/㎖〕. The mecA gene was also found
in 13 methicillin- resistant S. aureus (MRSA) and 12 methicillin-resistant
CNS(MRCNS) isolates with a significantly higher detection rate of the
mecA gene in MRSA with high MIC(≥16 ㎍/㎖) compared with those MIC
≤8 ㎍/㎖. MRSA and MRCNS were also more resistant to other
antibiotics (ampicillin, cephalothin, kanamycin, and gentamycin) than
methicillin-susceptible staphylococci. Among 10 different coa PCR-RFLP
patterns (A to J) in 706 S. aureus strains, the main types were A
(26.9%), B (17.0%), G(10.5%) and H (15.4%), with the frequent
observation of A and H types (6 and 10 isolates) in MRSA. This study
indicates that major epidemic S. aureus clones may be spread between
different dairy farms, and the profile of coa genotype can applied
epidemiological investigations and control of bovine mastitis, particularly
one caused by MRSA with specific prevalent coa types.

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국문 초록 (Abstract)

젖소 목장에서 유방염은 유량감소, 유질저하, 치료비 증가 등으로 가장 많은 경제적 피해를 주는 질병이다. 유방염은 다양한 미생물에 의해 발생되며 그 중 Staphylococcus aureus는 전염성 세균으로 젖소의 체내에 상존하며 유방 내로 침입하여 유즙내 백혈구에 의해 탐식되어도 백혈구 내에서 계속 생존한다. 또한 S. aureus는 다양한 enterotoxin을 산생하기 때문에 치즈, 버터 등과 같은 유제품에 오염되어 식중독을 일으킬 수 있다. 본 연구에서는 국내 젖소의 유방염에서 분리한 S. aureus의 유전형 분포를 조사하기 위해 1997년부터 2004년까지 7년간 총 5개 지역의 140개 목장에서 채취한 유방염 의심 시료에서 분리한 총 696개의 S. aureus 균주를 분리하였다. 동 분리주에 대해 PCR-RFLP를 이용한 coagulase 타이핑 결과 126bp와 670bp 사이에서 1개에서 4개의 분절을 보유하는 총 10가지의 PCR-RFLP 양상이 관찰되었으며 이 중 4개의 우세 타입이 약 70.3%를 차지하는 것으로 나타났다. 특정 유전형과 독소 타입의 연관성을 비교한 결과, 본 연구에서는 관찰되지는 않았지만 우세하게 분포하는 4개의 유전형(I, II, VII, VIII)이 장독소 산생능(A, B, AC, ABCD)이 잘 관찰되지 않는 6개의 유전형(III, IV, V, VI, IX, X)에 비해 연관성이 유의성이 있는 것으로 나타났다(P < 0.05).
또한 분리주에 대한 항생제 감수성 검사를 실시하고 β-lactam계 항생제 내성균에 대한 β-lactamase 산생능을 조사하였다. 국내 유방염 의심 시료에서 분리한 S. aureus 98균주에 대해 항생제 감수성 검사를 실시한 결과, 모든 균주가 oxacillin, vancomycin, erythromycin, amikacin에 대해서 감수성이 있는 것으로 나타났다. 또한 kanamycin, gentamicin, tetracycline에서도 내성이 낮아 전반적으로 비교적 감수성이 높은 것으로 나타났다. 그러나 erythromycin에 대해서는 43.9%로 비교적 감수성이 낮았다.
β-lactam계 항생제의 주된 내성기전인 β-lactamase 산생여부와 penicillin 및 ampicillin 이외의 다른 항생제 내성과의 관계를 비교 분석한 결과 β-lactamase 산생균주가 비 산생균주에 비하여 상대적으로 cephalothin 및 gentamicin 항생제 내성에 영향을 주는 것으로 나타났다.
메티실린 내성 포도상구균 (MRSA, Methicillin Resistant Staphylococcus areus)에 의한 원내 감염은 병원이나 지역사회에서 심각한 문제가 되고 있으며 메티실린에 내성인 coagulase에 음성인 포도상구균 (MRCNS, Methicillin Resistant Coagulase-Negative Staphylococci)도 중요한 원내 감염균으로 인식되고 있다. 젖소 유방염의 경우 감염균 중 MRSA의 비율이 높을 가능성이 있어 내성의 파악과 신속한 검출을 위한 방법이 요구된다. 본 연구에서는 Polymerase Chain Reaction(PCR) 법을 이용하여 methicillin 내성 유전자를 검출하고 유방염 감염우로부터 MRSA 와 MRCNS의 분리율과 내성 현황을 파악하고자 하였다. 1997년부터 2004년까지 유방염 우유로부터 MRSA 분포를 조사한 결과, 623균주에서 총 21균주의 MRSA(2.5%)가 분리되었으며, CNS 763균주 중 19균주(3.4%)가 메티실린에 내성을 나타내었다. mecA 유전자의 검출에서 단일 PCR법과 본 연구의 multiplex PCR 법을 비교하면 동일한 발현으로 검출법에 의한 차이는 없었고, 기존의 PCR 법으로 mecA 유전자만을 검출하는데 2.5시간이 소요되는데 비해 multiplex PCR법은 1.5시간내에 mecA 및 coa 2가지 유전자를 동시에 검출할 수 있어 메티실린 내성과 CNS 균주를 신속하게 동정할 수 있음을 확인하였다. PCR-RFLP 결과 나타난 10가지의 양상 중 4가지 유전형(A, B, G, H형)은 많은 목장에 분포하고 있었으며, 조사 대상 목장의 대부분이 오직 한 개의 단일 유전형을 보유하고 있는 것으로 조사되었다. MRSA 균주에서는 대부분 A와 H 유전형이 검출되었으며 D, F, I 그리고 J 유전형은 검출되지 않았다. 본 연구는 젖소 유방염에서 분리한 포도상구균으로 부터 multiplex PCR법을 통하여 PBP2'을 코딩 하는 mecA 유전자와 S. aureus의 병원성과 관련있는 coa 유전자를 함께 검출할 수 있는 방법을 확립하고자 하였으며, 국내 목장에서 가장 널리 사용되고 있는 10종의 항생제에 대한 내성 검사를 실시하고 S. aureus의 β-lactamase 산생능을 조사하였다. 또한, 젖소 유방염 유래 S. aureus의 coagulase 유전자의 유전형 분석과 enterotoxin 생산능을 통해 목장별 분리균간의 유전적인 연관성과 유전형들의 특성을 조사하고 감염경로를 밝힘으로써 국내 유방염 방제 전략을 세우는 자료가 될 수 있을 것으로 생각된다.

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젖소 목장에서 유방염은 유량감소, 유질저하, 치료비 증가 등으로 가장 많은 경제적 피해를 주는 질병이다. 유방염은 다양한 미생물에 의해 발생되며 그 중 Staphylococcus aureus는 전염성 세균으...

목차 (Table of Contents)

그림 및 표차례 ······································································································1
국문요약 ················································································································4
1. 제1장 서론 ·······································································································7
참고문헌 ········································································································14
2. 제2장 Staphylococcus aureus의 항생제내성 양상 및 coagulase 유전

그림 및 표차례 ······································································································1
국문요약 ················································································································4
1. 제1장 서론 ·······································································································7
참고문헌 ········································································································14
2. 제2장 Staphylococcus aureus의 항생제내성 양상 및 coagulase 유전
자 다양성 조사 ·························································································20
서론 ················································································································20
재료 및 방법 ··································································································23
결과 ··················································································································27
고찰 ··················································································································35
참고문헌 ··········································································································40
3. 제3장 Staphylococcus aureus의 β-lactamase 산생능 조사 ············· 49
서론 ················································································································49
재료 및 방법 ··································································································51
결과 ··················································································································54
고찰 ················································································································61
참고문헌 ········································································································65
4. 제4장 Methicillin 내성 Staphylococci 특성분석 ··································· 69
서론 ··················································································································69
재료 및 방법 ··································································································71
결과 ··················································································································75
고찰 ··················································································································83
참고문헌 ··········································································································87
5. 제 5 장 결론 ································································································· 92
6. Abstract ··········································································································97
7. 감사의 글 ·····································································································100