五倍子水提取物对菌斑生物膜影响的体外研究
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摘要
菌斑生物膜是粘附于牙齿表面的微生物团体，是龋病发生的始动因子。生物膜中细菌的基因表达和表型明显不同于浮游细菌，对抗生素具有较强的抵抗力。正常情况下，生物膜内部的微生态处于平衡状态。若摄糖频率增加，菌斑更多时间处于低pH状态，利于致龋菌的增殖，平衡向脱矿方向转变，导致龋病发生。因此，去除或控制菌斑生物膜的形成，维持菌斑生物膜的稳态对预防龋病的发生具有重大的意义。中药五倍子富含鞣酸，具有很好的抑菌特性。动物实验及体外研究均表明五倍子可有效防龋，效果与2％氟化物相当，但有关五倍子对菌斑生物膜的影响尚无详细研究。本实验应用人工口腔建立菌斑生物膜模型，观察五倍子水提取物对菌斑生物膜的消除作用以及对菌斑生物膜形成和组成的影响；并通过釉质磨片获取口内菌斑生物膜模型，观察五倍子水提取物对菌斑生物膜活性的影响，旨在进一步探讨五倍子的防龋机理。
实验一 五倍子水提取物对菌斑生物膜的消除作用
本实验对各实验菌的最小抑菌浓度(MIC)进行了测定，并进一步应用人工口腔、菌落计数技术观察了五倍子水提取物对菌斑生物膜的消除作用。结果显示五倍子水提取物对变链菌、粘放菌、血链菌、口腔链球菌的生长均有抑制作用，其中对变链菌、粘放菌和血链菌的MIC为64mg/ml，口腔链球菌为8mg/ml；不同浓度的五倍子水提取物对变形链球菌、粘性放线菌、血链球菌、口腔链球菌形成的单一细菌菌斑生物膜均有一定的消除作用，随
第四军医大学硕士学位论文
着浓度的提高，釉质表面的细菌数逐渐减少，呈浓度依赖性。与
最小抑菌实验相比，发现五倍子水提取物即使采用大于MIC的
浓度，也不能把釉质表面形成的生物膜完全消除，釉质表面仍有
细菌生长。说明与浮游细菌相比，消除生物膜中的细菌需要较大
浓度的五倍子水提取物，这可能与菌斑生物膜的特性有关。
实验二五倍子水提取物对菌斑生物膜形成的影响
本实验应用人工口腔、菌落计数和扫描电镜技术观察五倍子
水提取物对菌斑生物膜形成的影响。结果显示，应用较小浓度的
五倍子水提取物(6mg/m1)处理后的釉质表面其细菌量明显少于
对照组，细菌散在分布于釉质表面，未见细菌团块的堆积;对照
组釉质表面细菌呈块状堆积，可见栅栏状结构和谷穗状结构的形
成。说明五倍子水提取物具有显著的抗生物膜形成作用。
实验三五倍子水提取物对菌斑生物膜组成的影响
本实验应用人工口腔和细菌的选择性培养技术观察五倍子
水提取物对菌斑生物膜组成的影响，从生态学的角度探讨五倍子
的防龋机理。结果显示，应用较小浓度的五倍子水提取物
(6mg/ml)处理后，生物膜中变形链球菌和粘性放线菌的比例明
显降低，血型链球菌和口腔链球菌的比例有所升高。说明五倍子
水提取物可防止变形链球菌在生物膜中的选择性生长，减少菌斑
微生物向有害的方向转变。
实验四五倍子水提取物对口内菌斑生物膜活性的影响
本实验在建立口内菌斑生物膜模型的基础上，应用EB/FDA
染色和CLSM技术观察五倍子水提取物对口内生物膜活性的影
响。结果显示五倍子水提取物处理后，生物膜内细菌的活性明
显低于对照组。提示五倍子水提取物对生物膜中的细菌具有良
好的杀伤效应。
Plaque biofilms, a well-organized microbial community on dental surface, is a major etiological factor of caries. Compared with planktonic bacteria, the bacteria in biofilms have the different gene expression and phenotype, as well as have an increased resistance to antimicrobials. In normal conditions, the microbial ecology of plaque biofilms is in a state of equilibrium, and the bacteria components of biofilms remain relatively stable. If the intake of suger is too frequent, the biofilms will be in a low pH in most of time, which will benefit the proliferation of S.mutans and Lactobacillus, and further lead to caries. So the control on the formation of plaque biofilms and the maintenance of microcosm homeostasis play an important role in preventing caries. Gallnut contains tannin abundantly and can be used as an effective drug in the prevention of caries. The aim of the our research was to investigate the effects of gallnut water extract on the plaque biofilms in vitro, thereby to explore its mechanism of
preventing caries.
1. The role of of gallnut water extract in eradicating plaque biofilms
In this part, the minimal inhibitory concentrations(MIC) of all experimental strains were determined, and the role of gallnut water extract in eradicating plaque was further investigated by using the artificial mouth and the technology of colony counting. The results
showed that gallnut water extract could inhibit the growth of S.mutans, A.viscosus, S.sanguis and S.oralis. The MIC of S.mutans was 64mg/ml, which was same as A.viscosus and S.sanguis. The MIC of S.oralis was 8mg/ml; The plaque biofilms formed by S.mutans, A.viscosus, S.sanguis and S.oralis could be eradicated effectively by different concentrations of gallnut water extract respectively. With the increasing concentration, the number of bacteria was reduced gradually, and the effect was dose-dependent. Compared with the experiment in determining the MIC, it was found that even though higher concentration than MIC of gallnut water extract was adopted, the plaque biofilms couldn't be eradicated completely. It suggested that the eradication of plaque biofilms need a higher concentration of gallnut water extract comparing with the planktonics, which might correlate with the propert of plaque biofilms.
2. The effects of gallnut water extract on the formation of plaque biofilms
The artificial mouth, the technology of colony counting and scanning electron microscope(SEM) were used to investigate the effects of gallnut water extract on the formation of plaque biofilms. The results of colony counting showed that the amount of bacteria on enamel fragments treated with gallnut water extract was less than that of control team, and the difference between two groups was significant. The observation with SEM revealed that enamel fragments treated with gallnut water extract had no biofilm formed, the bacteria were distributed scatteredly, while the mature biofilm occurred on the control enamel fragments . So we can draw a conclusion that Chinese nutgall can inhibit the formation of oral
biofilm significantly.
3. The effects of gallnut water extract on the composition of plaque biofilms
The artificial mouth and the technology of selective culture of
bacteria were used to observe the effects of gallnut water extract on the composition of plaque biofilms. The results showed that gallnut water extract could reduce the proportion of S.mutans and A.vicosus, and increase the proportion of S.sanguis and S.oralis, It suggested that gallnut water extract can prevent the selective growth of S.mutans in biofilms, and can reduce the harmful conversion of microbial.
4. The effects of gallnut water extract on the vitality of plaque biofilms model in the oral cavity
The technology of EB/FDA staining and CLSM were used to observe the effects of gallnut water extract on the vitality of of plaque biofilms on the basis of establishing a plaque biofilms model in the oral cavity. The results showed that gallnut water extract could decrease the vatility of plaque b
实验一 五倍子水提取物对菌斑生物膜的消除作用
本实验对各实验菌的最小抑菌浓度(MIC)进行了测定，并进一步应用人工口腔、菌落计数技术观察了五倍子水提取物对菌斑生物膜的消除作用。结果显示五倍子水提取物对变链菌、粘放菌、血链菌、口腔链球菌的生长均有抑制作用，其中对变链菌、粘放菌和血链菌的MIC为64mg/ml，口腔链球菌为8mg/ml；不同浓度的五倍子水提取物对变形链球菌、粘性放线菌、血链球菌、口腔链球菌形成的单一细菌菌斑生物膜均有一定的消除作用，随
第四军医大学硕士学位论文
着浓度的提高，釉质表面的细菌数逐渐减少，呈浓度依赖性。与
最小抑菌实验相比，发现五倍子水提取物即使采用大于MIC的
浓度，也不能把釉质表面形成的生物膜完全消除，釉质表面仍有
细菌生长。说明与浮游细菌相比，消除生物膜中的细菌需要较大
浓度的五倍子水提取物，这可能与菌斑生物膜的特性有关。
实验二五倍子水提取物对菌斑生物膜形成的影响
本实验应用人工口腔、菌落计数和扫描电镜技术观察五倍子
水提取物对菌斑生物膜形成的影响。结果显示，应用较小浓度的
五倍子水提取物(6mg/m1)处理后的釉质表面其细菌量明显少于
对照组，细菌散在分布于釉质表面，未见细菌团块的堆积;对照
组釉质表面细菌呈块状堆积，可见栅栏状结构和谷穗状结构的形
成。说明五倍子水提取物具有显著的抗生物膜形成作用。
实验三五倍子水提取物对菌斑生物膜组成的影响
本实验应用人工口腔和细菌的选择性培养技术观察五倍子
水提取物对菌斑生物膜组成的影响，从生态学的角度探讨五倍子
的防龋机理。结果显示，应用较小浓度的五倍子水提取物
(6mg/ml)处理后，生物膜中变形链球菌和粘性放线菌的比例明
显降低，血型链球菌和口腔链球菌的比例有所升高。说明五倍子
水提取物可防止变形链球菌在生物膜中的选择性生长，减少菌斑
微生物向有害的方向转变。
实验四五倍子水提取物对口内菌斑生物膜活性的影响
本实验在建立口内菌斑生物膜模型的基础上，应用EB/FDA
染色和CLSM技术观察五倍子水提取物对口内生物膜活性的影
响。结果显示五倍子水提取物处理后，生物膜内细菌的活性明
显低于对照组。提示五倍子水提取物对生物膜中的细菌具有良
好的杀伤效应。
Plaque biofilms, a well-organized microbial community on dental surface, is a major etiological factor of caries. Compared with planktonic bacteria, the bacteria in biofilms have the different gene expression and phenotype, as well as have an increased resistance to antimicrobials. In normal conditions, the microbial ecology of plaque biofilms is in a state of equilibrium, and the bacteria components of biofilms remain relatively stable. If the intake of suger is too frequent, the biofilms will be in a low pH in most of time, which will benefit the proliferation of S.mutans and Lactobacillus, and further lead to caries. So the control on the formation of plaque biofilms and the maintenance of microcosm homeostasis play an important role in preventing caries. Gallnut contains tannin abundantly and can be used as an effective drug in the prevention of caries. The aim of the our research was to investigate the effects of gallnut water extract on the plaque biofilms in vitro, thereby to explore its mechanism of
preventing caries.
1. The role of of gallnut water extract in eradicating plaque biofilms
In this part, the minimal inhibitory concentrations(MIC) of all experimental strains were determined, and the role of gallnut water extract in eradicating plaque was further investigated by using the artificial mouth and the technology of colony counting. The results
showed that gallnut water extract could inhibit the growth of S.mutans, A.viscosus, S.sanguis and S.oralis. The MIC of S.mutans was 64mg/ml, which was same as A.viscosus and S.sanguis. The MIC of S.oralis was 8mg/ml; The plaque biofilms formed by S.mutans, A.viscosus, S.sanguis and S.oralis could be eradicated effectively by different concentrations of gallnut water extract respectively. With the increasing concentration, the number of bacteria was reduced gradually, and the effect was dose-dependent. Compared with the experiment in determining the MIC, it was found that even though higher concentration than MIC of gallnut water extract was adopted, the plaque biofilms couldn't be eradicated completely. It suggested that the eradication of plaque biofilms need a higher concentration of gallnut water extract comparing with the planktonics, which might correlate with the propert of plaque biofilms.
2. The effects of gallnut water extract on the formation of plaque biofilms
The artificial mouth, the technology of colony counting and scanning electron microscope(SEM) were used to investigate the effects of gallnut water extract on the formation of plaque biofilms. The results of colony counting showed that the amount of bacteria on enamel fragments treated with gallnut water extract was less than that of control team, and the difference between two groups was significant. The observation with SEM revealed that enamel fragments treated with gallnut water extract had no biofilm formed, the bacteria were distributed scatteredly, while the mature biofilm occurred on the control enamel fragments . So we can draw a conclusion that Chinese nutgall can inhibit the formation of oral
biofilm significantly.
3. The effects of gallnut water extract on the composition of plaque biofilms
The artificial mouth and the technology of selective culture of
bacteria were used to observe the effects of gallnut water extract on the composition of plaque biofilms. The results showed that gallnut water extract could reduce the proportion of S.mutans and A.vicosus, and increase the proportion of S.sanguis and S.oralis, It suggested that gallnut water extract can prevent the selective growth of S.mutans in biofilms, and can reduce the harmful conversion of microbial.
4. The effects of gallnut water extract on the vitality of plaque biofilms model in the oral cavity
The technology of EB/FDA staining and CLSM were used to observe the effects of gallnut water extract on the vitality of of plaque biofilms on the basis of establishing a plaque biofilms model in the oral cavity. The results showed that gallnut water extract could decrease the vatility of plaque b
引文
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56 Dunne WM Jr, Mason EO Jr, Kaplan SL. Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm. Antimicrob Agents Chemother. 1993, 37:2522-2526
57 Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol. 1986,32 ( Pt 5):1297-304
58 Tuomanen E, Durack DT, Tomasz A. Antibiotic tolerance among clinical isolates of bacteria. Antimicrob Agents Chemother. 1986,30(4):521-7
59 Brown MR, Allison DG, Gilbert P. Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J Antimicrob Chemother. 1988,22(6):777-80
60 Wentland EJ, Stewart PS, Huang CT, McFeters GA. Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm. Biotechnol Prog. 1996 May-Jun,12(3):316-21
61 Evans DJ, Allison DG, Brown MR, Gilbert P. Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. J Antimicrob Chemother. 1991 Feb,27(2):177-84
62 Duguid IG, Evans E, Brown MR, Gilbert P. Growth-rate-in dependent killing by ciprofloxacin of biofilm-derived Staph ylococcus epidermidis; evidence for cell-cycle dependency. J Antimicrob Chemother. 1992 Dec, 30(6):791-802
63 Duguid IG, Evans E, Brown MR, Gilbert P. Effect of biofilm culture upon the susceptibility of Staphylococcus epidermidis to tobramycin. J Antimicrob Chemother. 1992 Dec,30(6): 803-10
64 Desai M, Buhler T, Weller PH, Brown MR. Increasing resistance of planktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacin and ceftazidime during exponential growth. J Antimicrob Chemother. 1998 Aug,42(2): 153-60
65 Brooun A, Liu S, Lewis K. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2000,44(3):640-6
66 Jaffe A, Chabbert YA, Semonin O. Role of porin proteins OmpF and OmpC in the permeation of beta-lactams. Antimicrob Agents Chemother. 1982,22(6):942-8
67 A.P. Pugsley, C.A. Schnaitman, Outer membrane proteins of Escherichia coli.Ⅶ. Evidence that bacteriophage-directed protein 2 functions as a pore. J. Bacteriol. 1978, 133:1181-1189
68 Prigent-Combaret C, Vidal O, Dorel C, Lejeune P. Abiotic
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53 Stewart PS, Roe F, Rayner J, Elkins JG, Lewandowski Z, Ochsner UA, Hassett DJ. Effect of catalase on hydrogen peroxide penetration into Pseudomonas aeruginosa biofilms. Appl Environ Microbiol. 2000,66(2):836
54 Anderl JN, Franklin MJ, Stewart PS. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother. 2000,44:1818-1824
55 B Hoyle. Pseudomonas aeruginosa biofilm as a diffusion ba rrier to piperacillin. Antimicrob Agents Chemother. 1992,36: 2054-2056
56 Dunne WM Jr, Mason EO Jr, Kaplan SL. Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm. Antimicrob Agents Chemother. 1993, 37:2522-2526
57 Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol. 1986,32 ( Pt 5):1297-304
58 Tuomanen E, Durack DT, Tomasz A. Antibiotic tolerance among clinical isolates of bacteria. Antimicrob Agents Chemother. 1986,30(4):521-7
59 Brown MR, Allison DG, Gilbert P. Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? J Antimicrob Chemother. 1988,22(6):777-80
60 Wentland EJ, Stewart PS, Huang CT, McFeters GA. Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm. Biotechnol Prog. 1996 May-Jun,12(3):316-21
61 Evans DJ, Allison DG, Brown MR, Gilbert P. Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate. J Antimicrob Chemother. 1991 Feb,27(2):177-84
62 Duguid IG, Evans E, Brown MR, Gilbert P. Growth-rate-in dependent killing by ciprofloxacin of biofilm-derived Staph ylococcus epidermidis; evidence for cell-cycle dependency. J Antimicrob Chemother. 1992 Dec, 30(6):791-802
63 Duguid IG, Evans E, Brown MR, Gilbert P. Effect of biofilm culture upon the susceptibility of Staphylococcus epidermidis to tobramycin. J Antimicrob Chemother. 1992 Dec,30(6): 803-10
64 Desai M, Buhler T, Weller PH, Brown MR. Increasing resistance of planktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacin and ceftazidime during exponential growth. J Antimicrob Chemother. 1998 Aug,42(2): 153-60
65 Brooun A, Liu S, Lewis K. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2000,44(3):640-6
66 Jaffe A, Chabbert YA, Semonin O. Role of porin proteins OmpF and OmpC in the permeation of beta-lactams. Antimicrob Agents Chemother. 1982,22(6):942-8
67 A.P. Pugsley, C.A. Schnaitman, Outer membrane proteins of Escherichia coli.Ⅶ. Evidence that bacteriophage-directed protein 2 functions as a pore. J. Bacteriol. 1978, 133:1181-1189
68 Prigent-Combaret C, Vidal O, Dorel C, Lejeune P. Abiotic
surface sensing and biofilm-dependent regulation of gene expression in Eschericia coli. J Bacteriol. 1999, 181:5993-6002