In vitro antimicrobial activity and mode of action of erythorbyl laurate, a novel antimicrobial agent, against Staphylococcus aureus

다국어 초록 (Multilingual Abstract)

Abstract

Based on the results of susceptibility screening (spot-on-lawn assay) and bactericidal activity, it was found that erythorbyl laurate (6-O-lauroyl-erythorbic acid) had effect on gram positive bacteria. For evaluating antimicrobial activity of erythorbyl laurate against gram positive bacteria, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed by broth micro-dilution method. With sub-MICs of erythorbyl laurate, gram positive bacteria showed increased lag time (Δλ) and decreased maximum specific growth rate (μmax) compared to control without erythorbyl laurate treatment. S. aureus showed maximum crystal violet uptakes at 1.2-1.6 mM of erythorbyl laurate. Samples treated with positive control, nisin (40 mg/mL), of which mechanism was induction of pore formation on membranes increased permeability similar to maximum crystal violet uptake levels of each bacteria samples treated with erythorbyl laurate. Exposure of S. aureus to erythorbyl laurate at various concentrations showed altered cytoplasmic membrane rupture, and these were assessed by using the Live/Dead BacLight viability kit. Relative live percentage of Staphylococcus aureus reached 0% below 1.6 mM erythorbyl laurate. Morphological analysis was performed by EF-TEM, and inhibitory and bactericidal concentrations indicated that erythorbyl laurate addition caused dissolution of the cytoplasmic space, significant roughing of the membrane, and cytoplasmic convolution. In combination with other antimicrobial agents, nisin, kanamycin, and erythromycin showed great synergistic effect with erythorbyl laurate with 0.281, 0.256, and 0.266 of fractional inhibitory concentration (FIC) index, respectively. Transcriptional changes of S. aureus treated with erythorbyl laurate showed significantly different patterns compared to those of sample without erythorbyl laurate treatment, including genes related with amino acid synthesis, cell envelope, protein function, cellular processes, regulatory function, and signal transduction.

국문 초록 (Abstract)

Erythorbic acid는 L-ascorbic acid의 이성질체로서 항산화제로 흔히 쓰이며, 상대적으로 L-ascorbic acid에 비하여 경제적인 측면을 가지고 있다. 이러한 이유로 선행연구에서는 erythorbic acid와 항균성을 가지는 중쇄 지방산인 lauric acid를 기질로서 lipase에 의한 esterification을 통하여 erythorbyl laurate를 합성하였고, 이로 인해 항산화성과 항균성을 동시에 지닌 유화제로 사용할 수 있게 되었다. 현재 erythorbyl laurate의 기능성을 분석하여 이를 산업에 이용하고자 다양한 연구가 수행되고 있다. 본 연구는 erythorbyl laurate의 항균성 메커니즘을 규명하고자 다양한 생리화학적 실험법과 전사 분석을 위한 RNA-Seq 기법을 이용하여 대표적인 유해 미생물 중 하나인 Staphylococcus aureus에 대한 erythorbyl laurate의 효과를 검증하였다. Erythorbyl laurate는 그람 양성균에 특이적으로 효과를 보였고, 처리한 erythorbyl laurate의 농도가 증가할수록 유도기(Δλ)는 증가하고 최대비성장률(μmax)은 감소하는 경향을 보였다. Crystal violet과 SYTO 9, propidium iodide를 이용한 실험에서는 erythorbyl laurate에 의한 세포막 손상으로 인해 막 투과성이 변한다는 것을 확인하였다. 형광 현미경과 EF-TEM을 이용하여 이에 대한 형태학적 분석을 수행한 결과, erythorbyl laurate로 인해 세포 내 물질의 방출과 세포 표면의 손상 등의 물리적인 변화가 일어남을 관찰할 수 있었다. 또한 다양한 항균제와의 상승효과에 관해 연구한 결과, erythorbyl laurate는 nisin, kanamycin, erythromycin과 뛰어난 상승효과를 보였다. RNA-Seq 기법을 이용한 전사체 분석에서는 아미노산 합성, 단백질 기능, 세포막과 세포벽의 합성, 신호전달체계와 같은 다양한 세포 내 대사에 관여하는 유전자의 발현 차이를 관찰할 수 있었다. 이러한 결과를 바탕으로 erythorbyl laurate가 세포벽에 자극을 주고 세포막의 손상을 일으키며, 이로 인해 균체의 다양한 대사체계가 영향을 받는다는 것을 알 수 있었다.
본 연구는 효소적 합성을 통해 생산된 erythorbyl laurate를 다기능성 식품첨가물로서 산업에 적용하기 위한 기초 연구로, erythorbyl laurate의 항균력을 검증하고 메커니즘을 밝힘으로써 식품뿐만 아니라 그람 양성균을 제어할 필요가 있는 다양한 분야에서 활용될 수 있는 가능성을 제시하는 것에 의의가 있다.

목차 (Table of Contents)

• Contents
• Abstract •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• І
• Contents ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• ІІІ
• List of tables•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• VІІ
• List of figures ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• VІІІ
• 1. Introduction ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 1
• 2. Materials and methods ••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 3
• 2-1. Cell culture ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 3
• 2-2. Zone of growth inhibition assay ••••••••••••••••••••••••••••••••••••••••• 3
• 2-3. Bactericidal and bacteriostatic activity ••••••••••••••••••••••••••••••••• 4
• 2-3-1. Bactericidal activity of erythorbyl laurate•••••••••••••••••••••••• 4
• 2-3-2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) •••••••••••••••••••••••••••••• 5
• 2-3-3. Increase in lag time (Δλ) and maximum specific growth rate (μmax) ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 6
• 2-4. Crystal violet assay •••••••••••••••••••••••••••••••••••••••••••••••••••••••• 6
• 2-5. Cell rupture assessment by fluorescence ••••••••••••••••••••••••••••••• 7
• 2-5-1. Fluorescence intensity of SYTO 9 and propidium iodide ••••• 7
• 2-5-2. Fluorescent microscopy •••••••••••••••••••••••••••••••••••••••••••• 9
• 2-6. Energy filtered transmission electron microscopy (EF-TEM) •••••• 9
• 2-7. Synergistic effect with antibiotics and food additives •••••••••••••• 10
• 2-8. Transcriptional analysis by RNA-Seq ••••••••••••••••••••••••••••••••• 11
• 2-8-1. Extraction and purification of RNA from S. aureus ••••••••••• 11
• 2-8-2. cDNA library construction ••••••••••••••••••••••••••••••••••••••• 12
• 2-8-3. RNA-Seq data analysis•••••••••••••••••••••••••••••••••••••••••••• 13
• 2-9. Qauntitative real-time RT-PCR •••••••••••••••••••••••••••••••••••••••• 14
• 3. Results and discussion ••••••••••••••••••••••••••••••••••••••••••••••••••••••• 15
• 3-1. Susceptibility screening••••••••••••••••••••••••••••••••••••••••••••••••• 15
• 3-2. Bactericidal and bacteriostatic activity of erythorbyl laurate •••••• 17
• 3-2-1. Bactericidal activity of eythorbyl laurate••••••••••••••••••••••• 17
• 3-2-2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) •••••••••••••••••••••••••••••• 20
• 3-2-3. Bacteriostatic activity of erythorbyl laurate •••••••••••••••••••• 23
• 3-3. Change in cell permeability •••••••••••••••••••••••••••••••••••••••••••• 27
• 3-4. Assessment of cell rupture by fluorescence •••••••••••••••••••••••••• 30
• 3-4-1. Evaluation of the percentage of S. aureus with damaged membrane •••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 30
• 3-4-2. Visualized images of cell rupture evidence by fluorescence microscopy •••••••••••••••••••••••••••••••••••••••••••••••••••••••• 33
• 3-5. Morphological characterization of bacterial cell rupture by erythorbyl laurate by EF-TEM ••••••••••••••••••••••••••••••••••••••• 36
• 3-6. Synergistic effect with antibiotics and food additives •••••••••••••• 39
• 3-7. Transcriptional analysis by RNA-Seq ••••••••••••••••••••••••••••••••• 42
• 3-8. Validation of RNA-Seq data validation by qRT-PCR ••••••••••••••• 53
• 4. References ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 54
• 국문초록 ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 58