Propidium Iodide (PI) 및 Alamarblue (AB)에 기반한 항생제 내성균 신속진단법 개발
Propidium Iodide (PI) and Alamarblue (AB)-mediated Rapid Diagnostics for Antibiotic Resistant Bacteria
- 주제(키워드) fluorescence staining , bacterial viability , antibiotic resistant bacteria
- 발행기관 아주대학교
- 지도교수 윤현진
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000026794
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Antibiotics are considered as a cornerstone in the foundations of modern medicine. However, their abuse accelerated the emergence of antibiotics resistant bacteria, resulting in shortened lifespan of antibiotics. In an effort to abolish the antibiotic resistance propagation, I aimed to develop a rapid method for differentiating resistant bacteria in diverse antibiotics treatment using propidium iodide (PI) and alamarblue (AB). PI, a fluorescent nucleic acid-staining dye to detect damaged cell, and AB, a fluorescent dye to detect viable cells with reducing power, were used to determine bacterial viability. Because their availability has not been validated in diverse bacterial genera, prone to become resistant against antibiotics, I aimed to evaluate the applicability of PI and AB to differentiate resistant bacteria from susceptible or intermediate bacteria against antibiotic-treatment of imipenem. Imipenem is a kind of carbapenem antibiotics that have been generally applied to multi-drug resistant (MDR) bacteria. However, recently, carbapenem-resistant MDR pathogens occur world-wide and pose a serious threat to human health. Diagnostic criteria for antibiotic resistance using PI and AB were established using four different pathogenic gram (-) bacteria that frequently acquire antibiotics resistance, including Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. When the optimized diagnostic protocol was tested in 40 clinical isolates, all of them were successfully differentiated with an accuracy of over 90% compared with conventional method. Given that usage of PI and AB provides cost-effectiveness and economy of time, the devised method in this study has the potential for developing a rapid detection kit for antibiotics resistant bacteria.
more목차
1. Introduction 1
2. Materials and Methods 5
2.1 Bacterial strains and growth conditions 5
2.2 Chemicals 5
2.3 Equipment 6
2.4 Broth dilution method 6
2.5 Propidium Iodide (PI) and Alamarblue (AB)-mediated Rapid Diagnostics 6
3. Results 7
3.1 Determination of the target strain and antibiotics 7
3.2 Applicability of PI staining 12
3.3 Applicability of AB staining 15
3.4 Measurement of cell numbers after treating with different concentrations of imipenem 18
3.5 Optimization of reaction buffer or medium 24
3.6 Measurement of MIC50 of target strain 30
3.7 Establishment of final protocol using cell staining method 34
3.8 Determination of evaluation criteria for antibiotic resistance using PI and AB 36
3.9 Application of evaluation criteria into clinical isolates 39
4. Discussions 54
5. Reference 57
6. Abstract in Korea 61
