Anti-MRSA (Methicillin-resistant Staphylococcus aureus) Substance
- Alternative Title
- 대황에서 메티실린계 내성 황색포도상구균에 대한 항균물질의 화학구조 구명 및 작용기작 해석
- Abstract
- This study was performed to isolate antibacterial substances from brown seaweed, Eisenia bicyclis, and to elucidate the antibacterial mechanism against methicillin-resistant Staphylococcus aureus (MRSA). The antimicrobial activities of methanolic extract of E. bicyclis and its organic solvent soluble extracts including n-hexane (Hexane), dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (BuOH) and water (H2O) extracts were measured through disk diffusion and minimum inhibitory concentration assay. The anti-MRSA activity of E. bicyclis extracts was in the order of EtOAc-, DCM-, BuOH-, Hexane- and H2O-soluble extract. Moreover, the content of total polyphenolic compounds in each extract showed in accordance with each of the anti-MRSA activity, suggesting that the polyphenolic compounds are related with the anti-MRSA activity. Finally, six phlorotannins were isolated from the EtOAc-soluble extract, which exhibited the strongest anti-MRSA activity among the organic solvent-fractionated extracts. According to the comprehensive spectral analyses of fast-atom bombardment (FAB) mass and NMR profiles, the six compounds were identified to be eckol (EK), fucofuroeckol-A (FF), 7-phloroeckol (7-P), dioxinodehydroeckol (DD), phlorofucofuroeckol-A (PFF) and dieckol (DE), respectively. Among them, PFF showed the highest anti-MRSA activity compared to those of other phlorotannins tested. The MIC of PFF was determined to be in a range of 32 ug/mL against MRSA strains. Also, PFF was assessed its synergistic effects in combination with beta-lactams, ampicillin, penicillin and oxacillin, against MRSA, suggesting PFF will disturb a drug-resistant mechanism in MRSA cells. In MRSA strain, it has been well known that penicillin binding protein 2a (PBP2a) plays a key role to acquire a drug resistance against beta-lactam antibiotics. Therefore, it was investigated the effect of PFF on the expression of genes and protein related the drug resistance. PFF exhibited a significant direct inhibition on mRNA expression of mecI, mecR1 and mecA gene and synthesis of PBP2a protein in dose-dependent manner. These results provide a possible anti-MRSA mechanism of PFF. Thus, PFF interferes with the mRNA expression of PBP2a gene or regulator genes not the synthesis of protein.
It was also performed to select an effective microbial strain to enhance the anti-MRSA activity of E. bicyclis using microbial fermentation. For this purpose, several microorganisms isolated from traditional Korean fermented foods were innoculated and cultivated in E. bicyclis water extract. Ultimately, yeast strain YM-1 was selected for further study based on its TP compound contents and anti-MRSA activity, which were enhanced by microbial fermentation. The yeast strain YM-1 was identified as Candida utilis based on an analysis of its physiological characteristics. The fermentation of E. bicyclis water extract by C. utilis YM-1 resulted in the increase of anti-MRSA activity. The strongest activity was observed after 1 day of YM-1 fermentation and the EtOAc-soluble extract exhibited the strongest anti-MRSA activity. As expected, the yield of TP in EtOAc-soluble extract was increased by C. utilis YM-1 fermentation compared to that of unfermented extract, suggesting that the yeast fermentation mainly resulted in the break-down of EtOAc-soluble compounds in the E. bicyclis. HPLC analysis revealed that the EtOAc-soluble extract contained sizeable quantities of DE and PFF, which are known as anti-MRSA compounds in this study, respectively. The total content of phlorotannins (EK, DD, PFF and DE) identified in this study decreased in the EtOAc-soluble extract as progressed the fermentation by C. utilis YM-1. However, the content of PFF and DE was dramatically increased compared those of control after one day of fermentation. Among phlorotannins, PFF exhibits the strongest anti-MRSA activity and DE shows the second antibacterial activity against MRSA activity. These data strongly suggest that the anti-MRSA activity of E. bicyclis may be mediated by phlorotannins such as DE and PFF. During fermentation of the extract by C. utilis YM-1, no significant differences were observed in the proximate composition, including moisture, crude lipid, crude protein, and crude ash. Fermentation by C. utilis YM-1 also resulted in enhanced antioxidant activity of E. bicyclis water extract. Thus, fermentation by C. utilis YM-1 is an attractive strategy for developing value-added food ingredients.
In the current study, it was reported the isolation of PFF from E. bicyclis, which exhibited a remarkable anti-MRSA activity, and its ant-MRSA mechanism. It was also reported that microbial fermentation would be a good strategy to enhance biological activity such as anti-MRSA and antioxidative activity. The current findings, PFF as an anti-MRSA substance, will contribute to the development of an alternative phytotherapeutic agent against MRSA and with applications in the treatment of MRSA infections. Further study is needed to identify the rest of active compounds and to investigate the characteristics of the anti-MRSA activity.
- Issued Date
- 2012
- Awarded Date
- 2012. 2
- Type
- Dissertation
- Keyword
- antimicrobial activity Eisenia bicyclis methicillin-resistant staphylococcus aureus phlorotannins
- Publisher
- 부경대학교
- Affiliation
- 부경대학교
- Department
- 대학원 식품공학과
- Advisor
- 김영목
- Table Of Contents
- Table of contents
Abstract i
Table of Contents v
List of Tables x
List of Figures xiii
Chapter I. General Introduction 1
1. Background 2
2. An antibiotic resistance of Staphylococcus aureus 4
3. Mechanism of an antibiotic resistance S. aureus 4
4. Strategies for overcoming antibiotic resistance of bacteria 6
5. Enhancement of anti-MRSA activity through microbial fermentation 8
6. The objectives of this study 9
Chapter II. Isolation and Characterization of Anti-MRSA Substances from Eisenia bicyclis 11
1. Introduction 12
2. Materials and Methods 14
2.1. Raw materials and extraction 14
2.2. Microorganism and culture 14
2.3. Quantification of total phenolic contents 15
2.4. Disk diffusion assay 15
2.5. Detection of mecA gene 16
2.6. Measurement of minimum inhibitory concentrations 17
2.7. Isolation and purification of phlorotannins from E. bicyclis 17
2.8. Scanning electron microscopy 18
2.9. Synergic effects between phlorofucofuroeckol-A and β-lactams against MRSA 18
2.10. RNA isolation and RT-PCR analysis 19
2.11. Western blot analysis 20
2.12. Statistical analysis 21
3. Results and Discussion 22
3.1. Anti-MRSA activity of E. bicyclis extract 22
3.2. TP contents of the MeOH extract and its solvent fractions 22
3.3. Anti-MRSA activity of the E. bicyclis extracts 24
3.4. Determination of MIC of E. bicyclis extracts 27
3.5. Isolation of phlorotannins from E. bicyclis 29
3.6. Identification of compounds isolated from E. bicyclis 30
3.7. Spectroscopic properties of compounds 1-6 isolated from the EtOAc fraction 30
3.8. MIC of the phlorotannins derived from E. bicyclis against MRSA 40
3.9. Synergistic effects between PFF and β-lactams against MSSA and MRSA 46
3.10. Effect of PFF on MRSA cell morphology 49
3.11. Inhibitory activity of PFF on the expression of genes and the production of PBP2a related
in drug resistance 51
4. Summary 56
Chapter III. Screening of Microbial Strains for E. bicyclis Fermentation 58
1. Introduction 59
2. Materials and Methods 60
2.1. Plant materials and sample preparation 60
2.2. Microbial strains and strain selection for fermentation 60
2.3. Analysis of total phenolic contents 61
2.4. Physiological identification of isolated yeast strain 61
2.5. Analysis of proximate composition, pH and salinity 61
2.6. Analysis of free amino acids 62
2.7. DPPH radical-scavenging activity 62
2.8. Statistical analysis 63
3. Results and Discussion 64
3.1. Change of TP contents and DPPH radical-scavenging activity by microbial fermentation 64
3.2. Identification of yeast YM-1 strain 66
3.3. Physicochemical change of E. bicyclis extract by YM-1 fermentation 68
4. Summary 73
Chpater IV. Enhancement of anti-MRSA Activity of E. bicyclis by Microbial Fermentation 75
1. Introduction 76
2. Materials and Methods 79
2.1. Plant materials 79
2.2. Bacterial stains and medium 79
2.3. Sample preparation and microbial fermentation 79
2.4. Determination of cell growth 80
2.5. Extraction and fractionation of samples 80
2.6. Antibacterial activity assay 81
2.7. Determination of total polyphenolic contents 81
2.8. Evaluation of antibacterial substances against MRSA 81
2.9. Statistical analysis 82
3. Results and Discussion 83
3.1. Anti-MRSA activity of the fermented E. bicyclis extracts 83
3.2. Yield of the E. bicyclis residue extracts by C. utilis YM-1 fermentation 83
3.3. Microbial growth curve 85
3.4. Effect of C. utilis YM-1 fermentation on anti-MRSA activity of E. bicyclis 85
3.5. Change of MIC value of the fermented E. bicyclis extracts 88
3.6. Determination of TP contents of the fermented E. bicyclis extracts 94
3.7. Identification of anti-MRSA substances from the fermented E. bicyclis 96
4. Summary 104
Chapter V. Conclusions 106
References 109
Abstract by Korean Language 130
Acknowledgements 133
- Degree
- Doctor
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