세포염색에 기반한 카바페넴 내성 진단법 개발과 차아염소산나트륨에 대한 살모넬라 내성의 이해
Development of Cell-staining Diagnostics for Carbapenem Resistance and Understanding of Salmonella Resistance against NaClO
- 주제(키워드) cell-staining , carbapemen resistance , sodium hypochlorite , transcriptome
- 발행기관 아주대학교
- 지도교수 윤현진
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029353
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Repetitive exposure to sublethal stressors stimulates bacteria to develop resistance mechanisms against hostile environmental stressors. Representative stressors that pathogens encounter during host infections and environmental persistence include antibiotics and bactericidal chemicals. In this study, as an effort to cope with bacterial resistance against diverse antimicrobial agents, two studies were conducted: development of rapid diagnostics for antibiotic resistance and comprehensive transcriptomic understanding of bacterial resistance against disinfectants. Carbapenems are a class of beta-lactam antibiotics with a broad spectrum of antimicrobial activity. Due to their sturdy structures resistant to most beta-lactamases, they have been regarded as one of the last-resort antibiotics to combat multidrug-resistant bacterial infections. However, the emergence of carbapenem resistance increases predominantly in pathogen isolates associated with nosocomial infections. To prevent spread of carbapenem resistance in early stages, it is imperative to develop rapid diagnostic tests that substantially reduce the time and cost in determining resistance against carbapenems. Thus, a staining-based diagnostic method was devised, which was applicable to four different pathogens of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa, all of which are liable to acquire carbapenem resistance. Regardless of the resistance mechanisms among bacterial species and strains, double staining with propidium iodide (PI) and alamar blue (AB) identified resistant bacteria with an averaged accuracy of 79.81% in 7 h after imipenem treatments in 378 clinical isolates. Among the tested four species, A. baumannii showed the highest diagnostic accuracy. The PI and AB-mediated method identified 65 resistant A. baumannii strains with 98.46 % accuracy. This method could be utilized as promising diagnostics with high-throughput efficacy and low costs. The food industry is using a variety of control technologies such as heat, sodium hypochlorite, quaternary ammonium, and hydrogen peroxide treatment for the purpose of suppressing the deterioration of food quality and the appropriate sterilization considering economic efficiency. Among them, sodium hypochlorite (NaClO) as a reactive chlorine species (RCS) is the most common disinfectant in food process. However, many studies have shown that sub-lethal stressors stimulate food poisoning bacteria to acquire multiple and cross-resistance, which enable them to survive even under various sterilization techniques and antibiotics. In an effort to understand the mechanism of resistance of food poisoning bacteria against control technology, transcriptomic approaches were applied to Salmonella enterica serovar Typhimurium under sub-lethal conditions of NaClO. In response to NaClO treatments, Salmonella, a typical food poisoning bacterium, changed the expression of 2563 genes significantly. These differentially expressed genes (DEGs) included 1343 up-regulated genes and 1220 down-regulated genes. These DEGs might be implicated in bacterial resistance against NaClO. Based on qRT-PCR validation, 18 DEGs were selected and 12 mutant strains lacking these 18 genes or operons (ΔmdlAB, ΔrclRABC, ΔramRA, Δfur, ΔycfR, ΔargD_1-astADBE, ΔydgFE, ΔmarBARC, ΔbdcRA, ΔtrpHLEDCBA, ΔzraP-hydHG, ΔsoxSR) were constructed individually to examine their roles in Salmonella resistance against NaClO treatments. PI-mediated screening test identified 5 deletion mutants (ΔrclRABC, Δfur, ΔargD_1-astADBE, ΔmarBARC, ΔbdcRA) attenuated in NaClO treatments, indicating that these deleted operons might be required for bacterial resistance against NaClO. These genes could be exploited as control targets to prevent the development of NaClO resistance in Salmonella. This result not only provides new sights into how bacteria survive NaClO stress but also contributes to the foundation for the development of control technology that can prevent the resistance of foodborne bacteria.
more목차
Chapter Ⅰ. Development of Propidium iodide and Alamar blue-mediated Rapid Diagnostics for Detection of Carbapenem-Resistant Bacteria
Ⅰ-1. Introduction ························································· 1
Ⅰ-2. Materials and Methods ··········································· 4
Ⅰ-2.1 Bacterial strains and isolates ··································· 4
Ⅰ-2.2 PI and AB-mediated resistance test ··························· 4
Ⅰ-2.3 Microdilution antibiotic susceptibility test ··················· 5
Ⅰ-3. Results ································································ 6
Ⅰ-3.1 Differentiation between dead and live bacteria using single
staining with PI or AB ·········································· 6
Ⅰ-3.2 Optimization of a double staining-mediated diagnostic test
for carbapenem resistance ······································ 8
Ⅰ-3.3 Validation of the PI and AB-mediated diagnostic method
using clinical isolates ··········································· 9
Ⅰ-4. Discussion ··························································· 27
Chapter Ⅱ. Compressive Understanding of Salmonella Response to NaClO treatments using transcriptomics
Ⅱ-1. Introduction ······················································· 30
Ⅱ-2. Materials and Methods ·········································· 31
Ⅱ-2.1 Bacterial wild type and growth condition ··················· 31
Ⅱ-2.2 NaClO treatment for RNA-Seq ······························· 31
Ⅱ-2.3 RNA extraction ················································· 31
Ⅱ-2.4 RNA-Sequencing and analysis ······························· 32
Ⅱ-2.5 Quantitative real-time PCR analysis ························· 32
Ⅱ-2.6 Construction of mutants ······································· 36
Ⅱ-2.7 Viability screening using Propidium Iodide (PI) staining ··39
Ⅱ-3. Results ······························································· 40
Ⅱ-3.1. Optimization of a sublethal condition of Salmonella under
NaClO treatments ············································· 40
Ⅱ-3.2. Comprehensive transcriptomic analysis of Salmonella
treated with NaClO ··········································· 42
Ⅱ-3.3. Exploring genes related to NaClO resistance in Salmonella
············································································ 45
Ⅱ-3.4. To test the effects of DEGs on Salmonella growth without
NaClO stressor ················································ 49
Ⅱ-3.5. Screening genes required for Salmonella resistance against
NaClO treatment ·············································· 51
Ⅱ-4. Discussion ·························································· 55
Reference ··································································· 59
Abstract in Korean ······················································· 65
