초록 ▼

○ 연구결과
가축 및 인체 주요 질병 원인체 분리 및 항생제 내성 분석을 통해 축산물에서의 수퍼박테리아 출현양상을 파악하였으며, 그와 관련된 임상균주의 특성도 분석하였다.
또한 축산물 중 항생제 내성세균과 인체 임상분리 균주의 출현양상 및 내성기전 검사를 통해 분자생물학적 상관성 분석, 유전자 패턴분석, 그리고 감염시 초기감염 치료를 위한 감수성 항생제를 조사하였다.
그리고 축산물 유래균주와 인체분리 균주의 상관성 분석을 통해 주요 항생제 내성 유전자 특이 검출기법을 확립하였으며, 그에 따른 내성균주의 성상, 내성유

○ 연구결과
가축 및 인체 주요 질병 원인체 분리 및 항생제 내성 분석을 통해 축산물에서의 수퍼박테리아 출현양상을 파악하였으며, 그와 관련된 임상균주의 특성도 분석하였다.
또한 축산물 중 항생제 내성세균과 인체 임상분리 균주의 출현양상 및 내성기전 검사를 통해 분자생물학적 상관성 분석, 유전자 패턴분석, 그리고 감염시 초기감염 치료를 위한 감수성 항생제를 조사하였다.
그리고 축산물 유래균주와 인체분리 균주의 상관성 분석을 통해 주요 항생제 내성 유전자 특이 검출기법을 확립하였으며, 그에 따른 내성균주의 성상, 내성유전자의 비교 분석을 통한 상호 관련성을 확인하였다.

Abstract ▼

Ⅳ. Results and proposals for its application
1. Results
A. Isolation and identification of indicative organisms (VRE, S. aureus and E. coli) and pathogenic bacteria (Salmonella spp., and Campylobacter spp.) from livestock, livestock products, environments and humans and investigation of their

Ⅳ. Results and proposals for its application
1. Results
A. Isolation and identification of indicative organisms (VRE, S. aureus and E. coli) and pathogenic bacteria (Salmonella spp., and Campylobacter spp.) from livestock, livestock products, environments and humans and investigation of their antibiotic resistance
1) Establishment of quick and simple isolation and identification method
a) Quick and simple isolation and identification method for Salmonella
(1) Establishment of detection method that includes culturing method which can reduce culture time compared with conventional method by introduction of a Salmonella test kit with monoclonal antibody which enables Salmonella to be isolated in 26 hours
25 g sample + BPW 200 ml
↓ 37℃, 7 h incubation
Enriched culture 200 ml + RV broth 200 ml
↓ 42℃, 19 h incubation
Test by PBM Salmonella MAb test kit
b) Quick and simple isolation and identification method for Methicillin resistant S. aureus (MRSA)
(1) MRSA-SCREEN Test for detecting PBP2a of MRSA. Detection of MRSA which couldn't be detected by PCR.
c) Quick and simple isolation and identification method for E. coli O157:H7
(1) Quick and simple isolation and identification of E. coli O157:H7 using Anti-O157 immune magnetic isolation
2) Isolation rate (Total No. of samples: 6915, Total No. of isolates: 2021)
a) S. aureus (Total No. of isolates/ Total No. of samples): 986/2931 (33.6%)
(1) Animal originated isolates: 786 strains
(2) Human clinical isolates: 200 strains
b) Salmonella spp. (Total No. of isolates/ Total No. of samples): 339/1292 (26.2%)
(1) Animal originated isolates: 229 strains
(2) Human clinical isolates: 110 strains
c) E. coli (Total No. of isolates/ Total No. of samples): 326/1034 (31.5%)
(1) Animal originated isolates: 233 strains
(2) Environment originated isolates: 71 strains
(3) Human clinical isolates: 22 strains
* EHEC: 45/1854 (2.42%)
d) Enterococcus faecium/faecalis (Total No. of isolates/ Total No. of samples): 177/1145 (15.5%)
(1) Animal originated isolates: 83 strains
(2) Human clinical isolates: 94 strains
e) Campylobacter spp. (Total No. of isolates/ Total No. of samples): 193/513 (37.6%)
(1) Animal originated isolates: 182 strains
(2) Human clinical isolates: 11 strains
B. Characterization of antibiotic resistant isolates originated from livestock, livestock products, environments and human hospitals
1) Detection of antibiotic resistant genes
a) PCR
(1) Salmonella spp. - quinolone resistant determining region (QRDR) in gyrA, detection of integron gene
(2) MRSA and S. aureus - detection of mecA, mecI, mecR, IS1272, ccrAccrB, Tn554, IS431, mer, pT181, pUB110, and staphylococcal enterotoxin (SE) genes
(3) VRE - detection of vanA, vanB, vanC1, vanC2/C3, vanS, ddl gene and Tn1546
(4) E. coli - detction of extended-spectrum β-lactamase (ESBL), aac, aphI, aphII, stx1, stx2, eaeA, rfbE0157, EhlyA, LTa, STb, VTc and VTed
b) Isoelectric focusing electrophoresis (IEF)
(1) Establishment of optimal condition for ESBL detection of Salmonella spp. and E. coli
c) Real time PCR (RT-PCR)
(2) Detection of gene for hydrolysis of hippurate in Campylobacter spp. and CeuE
2) Classification of Isolates according to their origin - Analysis of their genetic correlation
a) Overlapping PCR - vanA gene cluster mapping by detection of IS15421, IS1216, and IS1251
b) Plasmid transfer - Salmonella spp., VRE, and E. coli
c) RAPD - Salmonella spp., and E. coli
d) PFGE - Salmonella spp., MRSA, and Campylobacter spp.
C. Antibiotic resistance analysis for Salmonella spp. and comparison between livestock and human clinical isolates
1) Major isolates from different sources
a) Human clinical isolates - S. Enteritidis was major sertype.
b) Isolates of animals and livestocks - S. Typhimurium was major serotype.
2) Comparison of multi-drug resistance patterns
a) There were no significant differences between human and animal isolates although slightly high resistance patterns were observed in human isolates
3) Correlation between human clinical isolates and animal and livestock product isolates
a) PFGE analysis result - Among 17 subtypes, only one subtype had common band pattern between human and animal isolates
b) Antibiotic resistance gene analysis result - The genetic correlation between human and animal isolates, of which Salmonella spp. have been transferred from livestock or livestock products to human was not found
c) The probability of integron gene transfer from animal-originated Salmonella spp. to E. coli was confirmed in vitro.
d) Relatively high quinolone resistance rate of Salmonella spp. isolates were observed in animals and point mutation in gyrA sequences was also found.
e) The high quinolone resistance rate in animal-originated strains seemed to be affected by the frequent use of qiunolone antibiotics in animal husbandry.
f) Studies and monitoring on the antibiotic resistant Salmonella spp. should be continued.
D. Analysis of antibiotic resistance of MRSA and VISA, and comparison between human clinical and aniaml isolates
1) Isolation of MRSA and VISA in livestock and livestock products
a) VISA and VRSA were not isolated from livestocks product.
b) MRSA were isolated in low detection rate (3.6%). Most MRSA isolates were originated from mastitis-infected raw milk.
2) Comparison of multi-drug resistance patterns
a) Most MRSA originated from livestock products were resistant to about 5 antibiotics.
b) Human clinical isolates were resistant to more than 11 antibiotics among 14 antibiotics.
3) Analysis of correlation between human clinical isolates and animal isolates using SCCmec typing
a) Isolates from mastitis-infected milk were characterized as community-acquired MRSA
b) Most MRSA isolates from animals or human hospitals were confirmed as hospital-acquired MRSA
4) Analysis of correlation between human clinical isolates and animal isolates by PFGE
a) There was no homology between human clinical and animal isolates.
b) It seemed that the cross contamination between human and animals and the possibility of genetic mixing are low.
c) Point mutation of mecI gene was detected only in human isolates
d) According to the results of mecI gene mutation and ccarm 3001 analysis, the prudent use of antibiotics in human hospital is significantly important.
e) It is necessary to define the transfer routes between community- and hospital-acquired MRSA.
E. Analysis of antibiotic resistance of animal originated VRE and comparison of them with human clinical isolates
1) Isolation of VRE from livestock and livestock products
a) VanA VRE which showed high-level resistant to vancomycin and teicoplanin, were isolated in a large numbers from poultry (40%).
b) VRE have been continuously detected in livestock products since the use of avoparcin was banned in 1997.
c) VanC VRE which showed low-level resistant to vancomycin, were isolated in a low rate in healthy human
d) VanC VRE were isolated from cattle and pig as well as poultry.
2) Correlation between isolates of human origin and animal origin
a) PFGE pattern, vanA gene cluster and antimicrobial resistant pattern showed no genetic relatedness between human and animal isolates.
b) VanA isolates from animal hardly transferred vancomycin resistance gene to enterococci under the experimental conditions.
c) It is considered that vancomycin resistant determinant of VRE has not been transferred from animal to human.
d) It seems that van gene is not transferred from VRE to S. aureus based on the results that van gene from S. aureus isolated from the same livestock products which had vanA VRE was not detected.
e) VanC VRE have been recently increasing as a pathogen of human hospital infection in EU. Therefore, it is necessary to establish continuous monitoring and management program for the control of vanC VRE although there was no clinical report of vanC VRE in hospitals in Korea.
f) Isolation rate of VRE from livestocks was high, so it is required to examine the probability of transfer of resistance from animal to human. Also, efforts to prevent dissemination of vancomycin resistance should be continued.
F. Antimicrobial resistant patterns of E. coli and E. coli O157 and comparison of isolates from animal and human origin
1) Characterization of isolates and comparison of multi-drug resistant bacteria
a) A total of 36.8% of EHEC isolated from animal origin showed multi-drug resistance and there was no ESBL E. coli.
b) A total of 44.7% of E. coli isolates from animal origin were resistant to aminoglycosides.
c) Among E. coli isolates from human origin, a total of 7.3% produced ESBL and of which, 76.5% showed multi-drug resistance.
d) A total of 27.5% of ESBL producing E. coli of human isolates were resistant to quinolone.
e) High genetic diversity was found at gyrA of QRDR and subunit parC of topoisomerase IV in clinical E. coli isolates which produce ESBL with additonal mutations at gyrB locus.
2) Correlation between human and animal isolates
a) ESBL producing clinical E. coli isolates usually retained TEM type ESBL and this gene was transferred to animal originated E. coli under experimental condition
b) ESBL producing E. coli was also isolated from an environmental source such as Han river.
c) These results suggested that management and control of ESBL producing E. coli in human hospitals is very important.
d) There is no evidence of the transfer of E. coli from animal to human.
3) Multi-drug resistance and characteristics of E. coli O157 isolated from livestocks
a) E. coli O157 from livestocks could be a threat to human since a total of 62.2% of isolates had multi-drug resistant characteristics and most of the isolates were resistant to aminoglycoside antibiotics and produced toxins.
b) It is necessary that surveillance and monitoring of E. coli O157 should be continued because antibiotic resistant rate was remarkably higher than those in other countries.
c) Special cautious management and guideline for prudent use of aminoglycoside and fluoroquinolone in livestock husbandry should be established.
G. Analysis of antimicrobial resistance of Campylobacter spp. and comparison of isolates from animal and human origin
1) Antimicrobial resistance of Campylobacter spp. isolates
a) The results of antimicrobial resistance analysis of C. jejuni and C. coli isolated from animals showed high resistance to tetracycline and quinolone such as ciprofloxacin, nalidixic acid, etc..
b) Human Isolates showed lower resistance to antibiotics than animals.
2) Correlation between animal and human isolates
a) As the results of genetic analysis of isolates resistant to quinolone by PCR and sequencing, point mutation of gyrA gene which is a target to quinolone was detected.
b) There was no genetic relatedness between animal and human isolates based on PFGE analysis.
c) Antimicrobial feed additives might induce resistance of isolates to antibiotics since the same antimicrobials had been used as feed additives.
d) Continuous monitoring and surveillance of Campylobacter spp. which cause food borne diseases should be established to prevent the transfer of antibiotic resistant bacteria from animal to human.
2. Future application: Understanding of resistance transfer mechanism and establishment of preventive methods
A. Establishment of techniques for understanding of resistance transfer mechanisms
1) Establishment of complete analysis methods for antimicrobial resistance and its transfer mechanism using molecular techniques such as overlapping PCR, PFGE, RAPD, plasmid profiling and transconjugation.
B. Understanding of propensity of antimicobial resistance
1) Understanding of propensity of antimicrobial resistance of isolates such as Salmonella spp., MRSA, VRE, E. coli, E. coli O157:H7 and Campylobacter spp. from livestocks.
C. It is able to find the methods to prevent transfer of antimicrobial resistant bacteria based on these results.
1) Regulation and control of antimicrobials which are scientifically proven to induce antimicrobial resistance and transfer its resistance gene from animal to human or vice versa
2) Continuous development of substitutes to antimicrobials such as non-specific immunostimulator, probiotics, etc.
3) Distribution of standard guidelines for farmers and customers for the prudent use of antimicrobials

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 14
• CONTENTS ... 27
• 목차 ... 28
• 제 1 장 연구개발과제의 개요 ... 29
• 제1절 연구개발의 필요성 ... 29
• 제2절 국내·외 관련기술의 현황과 문제점 ... 39
• 제 2 장 연구개발수행 내용 및 결과 ... 41
• 제1절 연구수행방법 ... 41
• 제2절 Salmonella spp.의 내성기전과 연관 연구 ... 62
• 제3절 Staphylococcus aureus의 내성기전과 연관 연구 ... 83
• 제4절 Enterococcus spp.의 내성기전과 연관 연구 ... 96
• 제5절 E. coli의 내성기전과 연관 연구 ... 112
• 제6절 Campylobacter spp.의 내성기전과 연관 연구 ... 160
• 제 3 장 목표달성도 및 관련분야에의 기여도 ... 165
• 제1절 목표달성도 ... 165
• 제2절 관련분야에의 기여도 ... 170
• 제 4 장 연구개발결과의 활용계획 ... 172
• 제1절 활용계획 ... 172
• 제2절 발표 실적 ... 172
• 제 5 장 참고문헌 ... 177
• 끝페이지 ... 183