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研究生:劉馥郁
研究生(外文):Fu-YuLiu
論文名稱:研究低濃度雙氧水如何調控具碳青黴烯抗藥性大腸桿菌的藥物敏感性
論文名稱(外文):The study of how low-concentration H2O2 regulates drug sensitivity of Carbapenem-resistant Escherichia coli
指導教授:王德華
指導教授(外文):Tak-Wah Wong
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:57
中文關鍵詞:大腸桿菌抗藥性大腸桿菌雙氧水傷口照護氧化壓力
外文關鍵詞:E. colidrug resistant-E.coliH2O2wound careoxidative stress
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抗藥性大腸桿菌 (CREC) 為革蘭氏陰性菌的一種,因為大部分可用的抗生素都已經對CREC沒有效果,目前已經被美國食品藥物管理局列為最危急的菌株之一。3% H2O2 是一般在傷口治療常用的殺菌劑,而我們先前研究發現0.1% H2O2 可以用來抑制革蘭氏陽性菌的抗藥性金黃色葡萄球菌 (MRSA) 生長達2 logs且能夠減低細菌的抗藥性。在這個研究中,我們想知道低濃度的H2O2 作用在CREC上能不能夠有相同的效果,本研究蒐集了臨床3株對碳青黴烯敏感的大腸桿菌以及9株CREC來進行測試,測試的方法是以平板計數法來看殺菌效果;以最小抑菌濃度測試以及紙錠擴散法來看藥物敏感性。實驗結果發現0.1% H2O2 可以抑制CREC達7 logs,而以0.0075% H2O2 則抑制所有CREC達1~2 logs。且在這個濃度下,對於人類纖維母細胞也是相對安全,可達60%以上的存活率。另一方面,9株CREC中有4株(44%)經過一次0.0075% H2O2 處理後的子代和5株(55%)經過重複0.0075% H2O2處理後的CREC都對抗生素變得比較敏感。這種表現型在CREC子代中一直持續到第十代,所以推測可能在未知的基因或是抗藥性基因上有所改變。經過H2O2處理後的CREC,其抗藥性基因cmy2和ompC基因並未發現有所改變。除了基因以外,功能蛋白的表現也經由成功大學貴儀中心高解析Orbitrap質譜儀串聯液相層析暨晶片電泳分析系統(CE/LC-MS)來進行檢測,從中發現了經過低濃度的H2O2處理後,與tRNA合成相關的蛋白Threonylcarbamoyl-AMP synthase以及外排幫浦蛋白AcrA以及AcrB表現下降,推測這些蛋白可能與抗藥性改變相關。總結來說,低濃度的H2O2能夠有效的抑制CREC生長並且減少細菌的抗藥性。
Carbapenem-resistant Escherichia coli (CREC), a Gram-negative bacterium, is listed as an urgent threat to health care by the U.S. Centers for Disease Control and Prevention (CDC) because it does not respond to nearly all available antibiotics. Three percentage of H2O2 is a traditional antiseptic in wound care. Our previous study had shown that a low concentration of H2O2 (0.1%) inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive aerobic bacterium, by 2 logs and also reduced the carbapenem-resistance. In this study, we hypothesized that low-concentration H2O2 has similar effects on CREC. Clinical isolates of 3 carbapenem-sensitive strains and 9 carbapenem resistant strains were examined. The growth inhibition by H2O2 treatment was analyzed with the plate count. The drug sensitivity of CREC was evaluated with MIC value and the disk diffusion test. Treating CREC by 0.1% H2O2 for 30 minutes inhibited the growth of all 12 E. coli isolates by 7 logs and exposing CREC to a lower concentration of H2O2 (0.0075%) for 30 minutes inhibited their growth by 1-2 logs. This low-concentration H2O2 was relatively safe to human fibroblasts with more than 60% cell survived after treatment. After one H2O2 treatment, 4 out of 9 (44%) CREC cell passages reduced their carbapenem-resistance to antibiotics. Exposing CREC repeatedly with this low-concentration H2O2 reduced carbapenem-resistance, instead of tolerance in 5/9 (55%) CREC strains. This phenotype persisted for at least 10 passages, which suggests that novel and/or persistent gene mutations might have occurred after H2O2 treatment. The mechanistic studies on resistant genes, cmy2 and ompC, of 2 CREC strains showed no obvious changes after treatments. Additionally, the expression of functional proteins was detected by NCKU Instrument Center Orbitrap CE/LC-MS. The results showed decrease of threonylcarbamoyl-AMP synthase, a protein involve in tRNA synthesis, and efflux proteins AcrA and AcrB in treated CREC. These proteins might play roles in the drug resistance alteration after H2O2 treatments. In conclusion, a very low-concentration H2O2 treatment has high efficacy in growth inhibition and reduced drug resistance in CREC.
中文摘要 I
Abstract II
Table of contents III
Abbreviations V
I. Introduction 1
II. Materials and Methods 3
2.1. Bacteria 3
2.2. H2O2 oxidative stress 3
2.3. Antibiotic susceptibility tests 3
2.4. Human cell viability 4
2.5. Bacterial growth curve 4
2.6. Polymerase chain reaction 5
2.7. Multiplex polymerase chain reaction 5
2.8. Biofilm formation assay 6
2.9. Protein extraction and purifications 6
2.10. Protein Digestion 6
2.11. Statistics 7
III. Results 8
3.1. Growth inhibition by low-concentration H2O2 8
3.2. Cytotoxicity of low-concentration H2O2 on human fibroblasts 8
3.3. Low-concentration H2O2 treatment reduced carbapenem-resistance of CREC 8
3.4. The altered phenotype persisted for at least 10 passages 9
3.5. No drug tolerance after repeated low-concentration H2O2 treatments 9
3.6. H2O2 damaged multiple cell organelles in CREC 10
3.7. Low-concentration H2O2 treatment reduced CREC growth 11
3.8. Reduction in biofilm formation after H2O2 treatments 11
3.9. No obvious cmy2 gene and ompC gene alterations after low-concentration H2O2 treatments 11
3.10. Protein expressions after low-concentration H2O2 treatments 11
IV. Discussions 13
V. References 15
VI. Table 19
Table 1. The reduction of MIC in CREC/0111 and CREC/1018, respectively after 3 repeated H2O2 treatments. 19
VII. Figures 20
Figure 1. The antimicrobial effect of different H2O2 concentrations on E. coli. 21
Figure 2. The antimicrobial effects of different H2O2 concentrations on CREC/0111 and CREC/1018.. 24
Figure 3. The cytotoxicity of different H2O2 concentrations on fibroblast. 25
Figure 4. Cells in the subculture passages (P) (four-ninth) of CREC strains treated with 0.0075% H2O2 became less carbapenem-resistance.. 30
Figure 5. CREC became less carbapenem-resistance after cell passages. 32
Figure 6. Repeated H2O2 treatments decreased carbapenem-resistance of five-ninth of CREC strains. 37
Figure 7. CREC became more carbapenem -sensitive after five repeated treatments with 0.0075% H2O2. 39
Figure 8. The decrease in carbapenem-resistance of treated CREC was a generic phenomenon. 42
Figure 9. 0.0075% H2O2 might damage multiple organelles in CREC. 43
Figure10. 0.0075% of H2O2 decreased the growth rate of CREC. 44
Figure11. Biofilm formation between treated and untreated CREC. 45
Figure12. PCR Multiplex of CREC treated with 0.0075% H2O2 showed no gene deletion. 48
Figure13. Different protein expressions after H2O2 treatments. 50
VIII. Supplementary 53
Supplementary Table S1. The primer used in PCR 53
Supplementary Figure S1. Repeated H2O2 treatments decreased carbapenem-resistance of five-ninth of CREC strains. 56
Supplementary Figure S2. The growth rate of three clinical strains of carbapenem-sensitive E. coli. 57
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