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龋齿是一种慢性进行性破坏性疾病,是由于口腔内共生菌和病原菌之间的生态失调而引起的。牙齿形成生物被膜(牙菌斑)后,牙齿残留的碳水化合物和糖经生物被膜内的细菌分解产生酸,导致牙齿表面的牙釉质脱落。生物被膜是由微生物群落在牙齿表面形成的生态环境,由细菌进行代谢产生的蛋白质、多糖和核酸组成的胞外聚合物(EPS)基质构成[1-2]。 变异链球菌(Streptococcus mutans)是口腔中常见的一种革兰氏阳性细菌,能引起口腔内微生物共生生态系统的失调。因此,变异链球菌也被认为是口腔内微生物形成生物被膜紧密相关的细菌之一[3]。变异链球菌通过复杂的生命活动定植于牙齿表面并形成生物被膜[4-5]。变异链球菌可以摄取口腔内残存的各种碳水化合物进行代谢活动,产生有机酸不断腐蚀口腔牙齿,导致龋齿的形成。变异链球菌代谢产酸是形成生物被膜中主要的毒力因素之一[6-7]。生物被膜表型在生理和功能上与浮游细菌有很大不同,生物被膜中的细菌表现出较低的代谢活性和生理机能,生物被膜结构可以作为细菌的物理屏障,限制抗生素渗透到生物被膜的深层。因此,在生物被膜中生长的细菌会增加其对抗生素的耐受性[8-9]。由于抗生素的滥用,导致常规疗法下细菌对抗生素的耐药性迅速增加,因此,迫切需要开发新型抗菌剂以抑制生物被膜的形成和降解成熟生物被膜。
近年来,使用天然植物产品治疗口腔疾病日益受到关注并被广泛研究[10]。茴香醛(p-methoxybezaldehyde)是广泛存在的一类天然产物,主要是从茴香、小茴香和大蒜中分离得到,目前已被广泛用于制药行业的抗菌药物生产[11-12]。《本草汇言》中记载,小茴香乃温中快气之药。最近研究结果表明,小茴香中主要成分之一茴香醛对许多微生物表现出很强的抗菌活性,包括金黄色葡萄球菌、念珠菌和酿酒酵母[13-15],在化妆品和药物的制备中也发挥着重要的作用。张冠楠等[16]的研究结果表明,茴香醛能改变金黄色葡萄球菌的菌体形态、细菌细胞膜的通透性;CHE等[17]的研究结果表明,茴香醛能改变细菌细胞壁完整性和细胞膜通透性发挥抑菌效果。然而,关于茴香醛对口腔致病菌中的变异链球菌抗菌活性尚未见报道。本研究旨在探讨茴香醛对口腔致病菌变异链球菌的抗菌效果及潜在的作用机制,以期将茴香醛开发为抗龋齿的天然药物。
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变异链球菌(S. mutans, ATCC25175)购自广东省微生物菌种保藏中心;茴香醛(货号A108958-5ml,阿拉丁)购自阿拉丁试剂(上海)有限公司;变异链球菌在脑心浸液肉汤 (BHI,中国北京陆桥技术有限公司)培养基中培养; Live/Dead BacLight Kit试剂盒(ThermoFisher,L7012)。
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采用GRENIER等[18]的二倍稀释法,将128 g·L−1茴香醛溶液和2.56 g·L−1洗必泰溶液稀释成系列溶液,测定茴香醛和洗必泰对变异链球菌的最小抑菌浓度(MIC)和最小杀菌浓度 (MBC)。变异链球菌培养至对数生长期后,无菌BHI 液体培养基稀释至2×105 cfu·mL−1,取100 μL稀释菌液加至含有不同浓度茴香醛的96孔板中,使孔中总体积为200 μL,细菌终浓度1×105 cfu·mL−1。菌液+PBS为阴性对照组,BHI液体培养基+PBS为空白对照组,洗必泰处理孔为阳性对照组。在37 ℃培养箱中培养24 h后观察不到混浊度的孔为茴香醛对变异链球菌的最低抑制浓度。
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将1.2实验组中未出现浑浊度的孔取100 μL涂布于BHI固体培养基上,在37 ℃培养箱中培养24 h后观察平板菌落生长情况,平板上无菌落形成视为茴香醛对变异链球菌完全杀灭,此浓度为最低杀菌浓度。每组设置3个复孔。
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根据CHEN等[13]的杀菌动力学测定方法,变异链球菌培养至对数生长期后,无菌BHI液体培养基稀释为2×10 6 cfu·mL−1的菌悬液。稀释菌悬液与不同浓度的茴香醛(终浓度为0.5×MIC,1×MIC和2×MIC)等体积混匀,置于37 ℃恒温水浴0、10 、20 、30 、40 、60、90 、120、180 min。0.2%洗必泰(CHX)为阳性对照,无菌PBS+菌液作为阴性对照。孵育至指定时间后,4 ℃下6 000 r·min−1离心10 min,弃上清液,使用无菌BHI重悬,根据需要稀释10倍,取50 µL涂布于无菌BHI固体培养基上,在37 ℃ 温育24 h后计算细菌菌落。实验重复3次并取平均值。
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参考WANG等[19]的方法,通过检测细菌核酸的释放量判断细菌细胞膜的完整性。将变异链球菌在37 ℃培养至对数生长期,无菌PBS稀释至2×108 cfu·mL−1,分别以终浓度1 ×,2×和4× MIC的茴香醛处理,细菌终浓度为1×108 cfu·mL−1。空白对照组以PBS处理。37 ℃下孵育0、1、2和4 h后, 4 ℃下6 000 r·min−1离心10 min。取上清液,OD260下测定吸光值。在进行DNA和RNA实验的同时,采用平板涂布法检测活细菌数目,方法同“1.4 时间杀菌曲线实验”平板涂布部分,实验重复3次并取平均值。
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细胞完整性可以通过测定上清液中细菌蛋白质的释放量来确定,上清液中蛋白质含量测定参考BRADFORD等[19]实验方法。变异链球菌培养至对数生长期(浓度为1× 108 cfu·mL−1)用茴香醛(1×MIC和2×MIC)处理。以PBS处理为空白对照。实验组和处理组均在37 ℃下孵育0、1、2和4 h,4 ℃下2 500 r·min−1离心5 min,取上清液,OD595下测定吸光值。实验重复3次并取平均值。
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参考钟亨任等[20]的方法,通过Live/Dead BacLight Kit试剂盒检测变异链球菌细胞膜的完整性。将变异链球菌培养至对数生长期,经PBS稀释至2×108 cfu·mL−1。用500 µL的茴香醛(终浓度为1×MIC,2×MIC或4×MIC)与等体积的稀释菌液在室温下共同孵育60 min。等体积的异丙醇和无菌0.9%NaCl处理稀释菌液分别作为阳性和阴性对照。离心收集变异链球菌,并与Live/Dead BacLight Kit试剂盒在避光条件下孵育15 min。孵育后离心去除残余荧光试剂,通过激光共聚焦扫描显微镜(CLSM,Leica,TCS-Sp8)观察细菌。通过BioFilmAnalyzer v.1.0软件计算细胞活力。
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参考HE等[21]测定茴香醛抑制变异链球菌形成生物被膜的活性。将变异链球菌培养至对数生长期后,含3%蔗糖的无菌BHI 液体培养基稀释至2×106 cfu·mL−1备用。将变异链球菌稀释液接种到96孔板上,茴香醛的终浓度分别为0.5×MIC,1×MIC和 2×MIC,37℃下培养24 h。以PBS (pH 7.2) 作为空白对照。变异链球菌形成生物被膜通过结晶紫测定。最小生物被膜抑制浓度(MBIC50)定义为至少抑制细菌50%生物被膜形成时的最低茴香醛浓度。
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将变异链球菌培养至对数生长期,含 3% 蔗糖的无菌BHI稀释至1×106 cfu·mL−1备用。将200 µL稀释菌液接种至96孔板中,在37 ℃下培养24 h后,轻轻将上清液去除,并取200 µL无菌液体培养基稀释的茴香醛(终浓度0.5×MIC,1×MIC和 2× MIC)加至各实验孔中。在37 ℃ 下孵育24 h,无菌 PBS (pH7.2) 用作空白对照,采用结晶紫法定量方法测定生物被膜的清除活性。
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变异链球菌表面疏水性变化采用微生物粘着碳烃化合物法进行测定。在37 ℃有氧条件下用相同浓度茴香醛(0.5×MIC,1MIC×和 2× MIC)分别孵育变异链球菌0 或30 min后离心,均用无菌PBS洗涤2次,并重悬于相同的缓冲液中。在550 nm处测量吸光度(记录为OD1),加入 20% (v/v) 二甲苯后剧烈摇动试管,将混合物静置直至水相与有机相分离,在550 nm处测量水相的吸光度(记录为OD2)。疏水性百分比由下式计算:H= (OD1−OD2)/OD2×100%。使用0 min (H1)和30 min (H2)值之间的差异确定最终疏水性。
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采用RT-qPCR方法评估茴香醛对变异链球菌毒力基因表达的影响。在37 ℃下将变异链球菌用不同浓度的茴香醛(0.25×MIC和0.5×MIC)孵育8 h,收集后按照Trizol™试剂(Invitrogen, CA, USA)说明进行总 RNA 提取。将纯化的 RNA 溶解于20 µL DEPC水中,并在−80 ℃下储存备用。cDNA合成试剂盒(NOVA, CA,中国)用于合成 cDNA,茴香醛对变异链球菌gtf基因 (gtf B, gtf C和gtf D) 表达的影响通过qRT-PCR测定,引物如表1所示。
表 1 引物序列
基因 引物序列 引物长度/bp recA-F CCTACGGGAGGCAGCAGTAG 100 recA-R CAACAGAGCTTTACGATCCGAAA gtf B-F CAACAGAGCTTTACGATCCGAAA 95 gtf B-R ACGAACTTTGCCGTTATTGTCA gtf C-F GTGCGCTACACCAATGACAGAG 107 gtf C-R GCCTACTGGAACCCAAACACCTA gtf D-F TGGCACCGCAATATGTCTCTTC 183 gtf D-R CAATCCGCAATAACCTGAATACCG
Antimicrobial and antibiofilm activity of p-methoxybezaldehyde against Streptococcus mutans
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摘要: 为研究茴香醛对变异链球菌的抗菌作用并阐明其抗菌机制,通过时间杀菌曲线、细胞膜完整性、细菌疏水性、生物被膜定量和RT-qPCR实验,测定茴香醛对变异链球菌的抗菌活性和抗生物被膜活性。结果表明,(1)茴香醛对变异链球菌的最小抑制浓度 (MIC)和最小杀菌浓度 (MBC)分别为4 g·L−1和8 g·L−1;(2)随着茴香醛浓度的增加,变异链球菌的核酸和蛋白质泄漏量逐渐增加;(3)激光共聚焦显微镜显示茴香醛在1×MIC的浓度下即可增加细菌细胞膜通透性破坏变异链球菌细胞膜的完整性;(4)茴香醛通过改变细菌细胞膜通透性发挥抑菌作用,通过改变变异链球菌的疏水性并下调基因表达发挥抗变异链球菌生物被膜活性。以上结果表明,茴香醛作为天然抗菌剂,具有进一步开发为抗龋齿药物的潜力。Abstract:
Objective The present study aimed to evaluate the antibacterial effect of p-methoxybezaldehyde on Streptococcus mutans, and to clarify its probable mechanism. Methods Determining the antibacterial and antibiofilm activity of p-methoxybezaldehyde against Streptococcus mutans by time-kill curve, cell membrane integrity, bacterial hydrophobicity, biofilm quantification and RT-qPCR experiments. Results The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of p-methoxybezaldehyde against Streptococcus mutans were 4 g·L−1 and 8 g·L−1. The result showed that the contents of protein and nucleic acid gradually increased with the increase of p-methoxybezaldehyde concentration. Confocal laser scanning microscopy revealed S. mutans at 1 × MIC concentration of p-methoxybezaldehyde, resulting in changed membrane integrity in contrast to control groups. Conclusion The antibiofilm activity of p-methoxybezaldehyde against S. mutans was determined by the crystal violet method. p-methoxybezaldehyde exerts S. mutans antibiofilm activity by changing the hydrophobicity of S. mutans and down-regulating gene expression.These results indicate the potential of p-methoxybezaldehyde as a natural antibacterial agent to be further developed as potential anti-caries agents. -
Key words:
- antimicrobial activity /
- biofilm /
- cell membrane /
- p-methoxybezaldehyde
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表 1 引物序列
基因 引物序列 引物长度/bp recA-F CCTACGGGAGGCAGCAGTAG 100 recA-R CAACAGAGCTTTACGATCCGAAA gtf B-F CAACAGAGCTTTACGATCCGAAA 95 gtf B-R ACGAACTTTGCCGTTATTGTCA gtf C-F GTGCGCTACACCAATGACAGAG 107 gtf C-R GCCTACTGGAACCCAAACACCTA gtf D-F TGGCACCGCAATATGTCTCTTC 183 gtf D-R CAATCCGCAATAACCTGAATACCG -
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