중환자실의 임상검체로부터 분리된 methicillin 내성 Staphylococcus aureus의 독소유전자형 및 항생제 내성

다국어 초록 (Multilingual Abstract)

This study is aimed 1) to determine the toxin genes, 2) to estimate the correlation between toxin gene types and antibiotic resistance, and 3) to detect the prevalence of mupirocin resistant strains, from methicillin-resistant Staphylococcus aureus (MRSA).
Hospital-acquired MRSA (HA-MRSA) strains (20 strains from sputum, 11 from pus, 8 from urine, 4 from blood, 4 from tip, 3 from body fluid, and 2 from wound) were isolated from clinical specimens obtained from the intensive care unit of a hospital in Suncheon, Jeonnam, Korea from January to December, 2014. Identification and confirmation of isolates as MRSA was performed by physiological tests using Vitek system and oxacillin resistance with disc diffusion method. Genes encoding mecA, enterotoxin (SE), toxic shock syndrome toxin-1 (TSST-1), exfoliative toxin (ET) and Panton-Valentine leukocidin (PVL) were detected by multiplex PCR-mediated amplification using specific primers. The mupA gene was also detected with PCR. Antibiotic resistance of the MRSA was detected using both disc diffusion method and minimal inhibitory concentration test. Toxin genes, seg and sei, were present in most strains, 40 each (76.9%), followed by tst in 34
strains (65.4%). Other genes (eta, etb, sea, sed, see, seh, sej, pvl) were not detected. Peculiarly, none of 8 strains from urine samples did contain toxin genes. 40 MRSA strains (76.9%) had 2 or more toxin genes simultaneously, with the strain of 5 coexistent toxin-genes (seb, sec, seg,sei, tst) being the most common by 28 (53.8%) and 6 (11.5%) strains had, seg and sei genes, making it second most common. Also, as for coexistence of genes, not only was tst gene found with seg, sei genes in all the cases (100%) but also was seb gene found with tst, seg, and sei, sec gene with tst, seg, and sei, seg with sei, and sei with seg in all cases (100%). Coexistence of genes were 72.5 ~ 100%, showing high correlation among genes, seb, sec, seg, sei, and tst. To check the similarity of gene structure among strains, random amplified polymorphic DNA (RAPD) PCR analysis with 6 arbitrary primers was performed on 13 MRSA strains and 3 methicillin-sensitive Staphylococcus aureus (MSSA) strains isolated from clinical specimen. In the result, strain number 1, 2, 6 showed 100% correlation with 21, 28, 29, but as MRSA strains that had identical toxin-genotype showed different band form, it is assumed that there are different genotypes present. 3 MSSA strains showed low correlation with MRSA due to existence of different band appearance. As for antibiotics resistance, we could confirm that the strains showed 100% resistance to β-lactam drug and high resistance to most of antibiotics, including erythromycin and clindamycin. As strains that had particular toxin genes (seb, sec, seg, sei, tst) in multiple showed 100% resistance to ciprofloxacin, clindamycin, erythromycin, we could find that seb, sec, tst genes have close relationship to those antibiotics. It showed higher resistance to ciprofloxacin, clindamycin, erythromycin, tetracycline, compared to strains that had toxin genes independent from multiple toxin genes (seb,
sec, seg, sei, tst) or no genes, but lower resistance in gentamicin than average of other strains. Meanwhile, mupA gene was identified in all 4 strains with high resistance among 8 strains resistant to mupirocin and sec, seg, sei genes were each found in 6 strains (75.0 %). It was found that resistance to gentamicin was much higher in mucipirocin-resistant strains (100%) than susceptible strains (22.7%).

목차 (Table of Contents)

• I. 서 론 ········································································1
• 1. Methicillin 내성 Staphylococcus aureus(MRSA)의 특성···················1
• 2. MRSA 내성 기전·····························································5
• 3. Mupirocin 내성 기전························································7
• 4. Staphylococcus aureus 독소···················································8
• 5. 연구의 목적····································································9
• II. 재료 및 방법································································10
• 1. 대상 균주······································································10
• 1-1. Staphylococcus aureus 동정 및 분리································10
• 1-2. 전통적인 생화학적 검사···············································11
• 1-2-1. Catalase 시험························································11
• 1-2-2. Coagulase 시험······················································11
• 1-2-3. Manitol salt agar 시험·············································11
• 1-3. 자동화기기를 이용한 생화학적 검사··································12
• 2. Methicillin 내성 균주 (MRSA)의 판정·································14
• 2-1. 디스크확산법 및 최소억제 농도 (MIC)·····························14
• 3. 독소 유전자 유형 분석···················································15
• 3-1. Genomic DNA 추출···················································15
• 3-2. Multiplex PCR을 이용한 mecA와 독소 유전자 분석··············16
• 3-3. Mupirocin 내성 mupA 유전자 분석··································18
• 4. RAPD (Random Amplified Polymorphic DNA) PCR법에 의한 분자역
• 학적 분석········································································20
• 5. 항생제 감수성 검사·························································21
• III. 결 과······································································23
• 1. 대상 균주·····································································23
• 2. Methicillin 내성 균주 (MRSA)의 확인····································26
• 2-1. 항생제 내성 확인························································26
• 2-2. Mupirocin내성 MRSA의 분리율 ······································32
• 2-3. mecA 유전자 확인·····················································34
• 2-4. PCR에 의한 mupA 유전자 확인······································37
• 3. Staphylococcus aureus의 동정················································39
• 3-1. 독소 유전자 유형 분석··················································43
• 3-2. 검체별 독소 유전자와의 관계··········································44
• 3-3. Mupirocin 내성 및 감수성 균주와 독소 유전자와의 관계·········50
• 4. 분자역학적 검사 양상·······················································52
• 4-1. RAPD (Random Amplified Polymorphic DNA) PCR·············52
• 5. 항생제 감수성································································54
• 5-1. MRSA의 항생제 내성 분석·············································54
• 5-2. 독소 유전자형 MRSA의 항생제 내성 분석···························55
• 5-3. Mupirocin 내성 MRSA의 항생제 내성 분석·························59
• 5-4. Mupirocin 내성과 감수성균주의 항생제 내성 분석·················60
• Ⅳ. 고 찰········································································66
• Ⅴ. 요 약········································································72
• Ⅵ. 참고문헌···································································74