变异链球菌绿色荧光蛋白报告株在双菌种生物膜研究的应用

doi:10.3877/cma.j.issn.1674-1366.2019.03.002

中华口腔医学研究杂志(电子版) ›› 2019, Vol. 13 ›› Issue (03) : 136 -143. doi: 10.3877/cma.j.issn.1674-1366.2019.03.002

所属专题：文献；

基础研究

变异链球菌绿色荧光蛋白报告株在双菌种生物膜研究的应用

李晓岚¹, 王肖¹, 凌均棨¹^,^†(

), 胡晓莉¹, 邓动梅²

^1. 中山大学光华口腔医学院·附属口腔医院，广东省口腔医学重点实验室，广州　510055
^2. 荷兰阿姆斯特丹大学牙科研究中心预防牙科　1081LA

收稿日期:2019-02-14 出版日期:2019-06-01
通信作者: 凌均棨

Application of green fluorescent protein reporter system in Streptococcus mutans for study on dual-species biofilms

Xiaolan Li¹, Xiao Wang¹, Junqi Ling¹^,^†(), Xiaoli Hu¹, Dongmei Deng²

^1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
^2. Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam, Amsterdam, 1081LA, Netherlands

Received:2019-02-14 Published:2019-06-01
Corresponding author: Junqi Ling
About author:
Corresponding author: Ling Junqi, Email: lingjq@mail.sysu.edu.cn
Supported by:
National Natural Science Foundation of China(11772361, 81400505)

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引用本文：

李晓岚, 王肖, 凌均棨, 胡晓莉, 邓动梅. 变异链球菌绿色荧光蛋白报告株在双菌种生物膜研究的应用[J]. 中华口腔医学研究杂志(电子版), 2019, 13(03): 136-143.

Xiaolan Li, Xiao Wang, Junqi Ling, Xiaoli Hu, Dongmei Deng. Application of green fluorescent protein reporter system in Streptococcus mutans for study on dual-species biofilms[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2019, 13(03): 136-143.

目的

探讨变异链球菌（S.mutans）绿色荧光蛋白（GFP）报告株在单、双菌种生物膜的形成、代谢和抗药性研究的应用。

方法

采用基因重组技术构建S.mutans GFP报告株C67-1 pDM15和UA159 pDM15；通过荧光显微镜和荧光光度计，评估报告株转化效率、生长能力和荧光表达规律；构建S.mutans GFP报告株与戈登链球菌（S.gordonii）的单、双菌种生物膜，比较单、双菌种生物膜中S.mutans的生物膜形态、糖代谢活力及氯已定（CHX）作用下的抗药性差异。数据运用单因素方差分析、Pearson相关性分析与独立样本t检验进行统计学分析。

结果

S.mutans GFP报告株可稳定地表达gfp基因，生长能力和生物膜形成能力与野生株相似；生物膜加入0.2%的葡萄糖后荧光量迅速增加，可检测细菌代谢改变，4 h的相对荧光增长值（ΔRLU）可反映生物膜量，二者显著相关（r=0.9818~0.9985，P<0.001）；S.gordonii改变S.mutans C67-1和UA159的生物膜结构，抑制UA159菌株生物膜的形成；剩余荧光增长率[ΔRLU（%）]反映耐药能力，单菌种生物膜C67-1和UA159的ΔRLU（%）相似，分别为（70.2 ± 8.0）%和（72.3 ± 7.9）%（t=-0.521，P=0.630），在双菌种生物膜中S.mutans的ΔRLU（%）发生改变，与各自单菌种生物膜相比差异有统计学意义：C67-1升高至（85.6 ± 4.3）%（t=-2.872，P=0.045），UA159降低至（41.2 ± 10.1）%（t=3.551，P=0.024）。

结论

S.gordonii对S.mutans生物膜形成能力和抗药性的改变具有亚型差异。GFP报告株可作为多菌种生物膜定量与空间结构研究的模式菌。

关键词: 变异链球菌, 戈登链球菌, 绿色荧光蛋白报告株, 双菌种生物膜, 抗药性

Objective

To investigate the application of green fluorescent protein (GFP) reporter system in Streptococcus mutans (S.mutans) in the formation, metabolism and antimicrobial resistance of single - and dual - species biofilms.

Methods

The genomic reporter strains C67-1 pDM15 and UA159 pDM15 were constructed by gene recombination technique. The transformation efficiency, growth curve and fluorescence expression of the reporter strains were detected by fluorescent microscopy, spectrophotometer and fluorophotometer. Single- and dual-species biofilms were formed by GFP S.mutans and Streptococcus gordonii (S.gordonii) . Glucose metabolism activity and chlorhexidine (CHX) resistance were evaluated by related light unit (RLU) . Morphology of the biofilms was observed under fluorescent microscope. The data were analyzed by One-Way ANOVA, Pearson correlation analysis and independent samples t-test.

Results

The GFP plasmids were effectively expressed in S.mutans strains. Their growth ability and biofilm formation were similar to those of wild-type strains. Upon addition 0.2% of glucose, the fluorescence intensity in the biofilm increased significantly. GFP synthesis can be used as a metabolic activity indicator. A significant correlation was obtained between the relative fluorescence change value (ΔRLU) within 4 h and the biomass (r=0.9818~0.9985, P<0.001) . S.gordonii altered the biofilm structure of C67-1 and UA159 in the dual-species biofilm, significantly inhibited the biofilm formation of UA159. The residual fluorescence growth rate[ΔRLU (%) ]indicating antimicrobial resistance showed that ΔRLU (%) of single-species biofilm C67-1 and UA159 were close, which were (70.2 ± 8.0) % and (72.3 ± 7.9) % respectively (t=-0.521, P=0.630) . Compared with the single-species biofilm, the ΔRLU (%) of S.mutans changed significantly in the dual-species biofilms: C67-1 increased to (85.6 ± 4.3) % (t=-2.872, P=0.045) , while UA159 reduced to (41.2 ± 10.1) % (t=3.551, P=0.024) .

Conclusions

The biofilm formation and antimicrobial resistance of S.mutans displayed distinct strain dependence when co-cultured with S.gordonii. GFP reporter can be used as a strain model for quantification and spatial structure research in multi-species biofilms.

Key words: Streptococcus mutans, Streptococcus gordonii, Green fluorescent protein reporter, Dual-species biofilm, Antimicrobial resistance

图1 变异链球菌（S.mutans）绿色荧光蛋白（GFP）报告株生物膜显微镜图像（荧光显微镜与死活菌染色　高倍放大）A、B和C分别为8 h的C67-1 pDM15生物膜荧光显微镜图像、Syto 64死活菌染色显微镜图像和ImageJ软件重叠A和B图像；D、E和F分别为8 h的UA159 pDM15生物膜荧光显微镜图像、Syto 64死活菌染色显微镜图像和ImageJ软件重叠D和E图像；G、H和I分别为24 h的C67-1 pDM15生物膜荧光显微镜图像、Syto 64死活菌染色显微镜图像和ImageJ软件重叠G和H图像；J、K和L分别为24 h的UA159 pDM15生物膜荧光显微镜图像、Syto 64死活菌染色显微镜图像和ImageJ软件重叠J和K图像

图2 变异链球菌绿色荧光蛋白报告株与野生株生长曲线

表1 48 h生物膜变异链球菌野生株、绿色荧光蛋白报告株与对照株生物膜形成能力比较

报告株	活菌计数（log₁₀ CFU/mL）			F值	P值	生物膜总量（OD₆₀₈）			F值	P值
报告株	野生株	pDM15菌株	pVA838菌株	F值	P值	野生株	pDM15菌株	pVA838菌株	F值	P值
C67-1 pDM15	8.02 ± 0.33	8.00 ± 0.56	7.92 ± 0.65	0.168	0.849	2.25 ± 0.20	2.15 ± 0.09	2.14 ± 0.13	1.032	0.412
UA159 pDM15	7.21 ± 0.34	7.12 ± 0.23	7.23 ± 0.26	0.008	0.992	1.18 ± 0.19	1.24 ± 0.13	1.12 ± 0.25	1.139	0.381

表1 48 h生物膜变异链球菌野生株、绿色荧光蛋白报告株与对照株生物膜形成能力比较

报告株	活菌计数（log₁₀ CFU/mL）			F值	P值	生物膜总量（OD₆₀₈）			F值	P值
报告株	野生株	pDM15菌株	pVA838菌株	F值	P值	野生株	pDM15菌株	pVA838菌株	F值	P值
C67-1 pDM15	8.02 ± 0.33	8.00 ± 0.56	7.92 ± 0.65	0.168	0.849	2.25 ± 0.20	2.15 ± 0.09	2.14 ± 0.13	1.032	0.412
UA159 pDM15	7.21 ± 0.34	7.12 ± 0.23	7.23 ± 0.26	0.008	0.992	1.18 ± 0.19	1.24 ± 0.13	1.12 ± 0.25	1.139	0.381

表2 0.2%葡萄糖溶液对变异链球菌绿色荧光蛋白报告株4 h生物膜相对荧光增长值（ΔRLU₄）的影响

报告株	G+组	G-组	t值	P值
C67-1 pDM15	195.74 ± 5.06	50.86 ± 6.53	42.92	<0.001
UA159 pDM15	77.48 ± 2.96	14.23 ± 2.61	39.24	<0.001

表2 0.2%葡萄糖溶液对变异链球菌绿色荧光蛋白报告株4 h生物膜相对荧光增长值（ΔRLU₄）的影响

报告株	G+组	G-组	t值	P值
C67-1 pDM15	195.74 ± 5.06	50.86 ± 6.53	42.92	<0.001
UA159 pDM15	77.48 ± 2.96	14.23 ± 2.61	39.24	<0.001

图3 0.2%葡萄糖溶液作用下变异链球菌绿色荧光蛋白报告株生物膜荧光表达曲线　A：C67-1报告株；B：UA159报告株；pDM15和pVA838分别为加入质粒；G+为加入0.2%葡萄糖溶液组；G-为不加入0.2%葡萄糖溶液组

图4 0.2%葡萄糖溶液作用下变异链球菌绿色荧光蛋白报告株相对荧光增长值（ΔRLU）与生物量间效应曲线　A：2 h；B：4 h

图5 变异链球菌（S.mutans）绿色荧光蛋白（GFP）报告株单菌种和双菌种生物膜　A：S.mutans单菌种生物膜和S.mutans+戈登链球菌（S.gordonii）双菌种生物膜条件下C67-1 pDM15与UA159 pDM15的4 h相对荧光增长值（ΔRLU₄），^aP<0.05；B~C：S.mutans单菌种生物膜和S.mutans+S.gordonii双菌种生物膜的C67-1 pDM15荧光显微镜图像（低倍放大）；D~E：S.mutans单菌种生物膜和S.mutans + S.gordonii双菌种生物膜的UA159 pDM15荧光显微镜图像（低倍放大）

图6 0.006%氯已定（CHX）作用4 h下单、双菌种生物膜变异链球菌（S.mutans）生物膜剩余荧光增长率[ΔRLU（%）] ^aP<0.05

[1]	Zaura E, Keijser BJ, Huse SM, et al. Defining the healthy "core microbiome" of oral microbial communities[J]. BMC Microbiol, 2009(9): 259. DOI: 10.1186/1471-2180-9-259.
[2]	Kuramitsu HK, He X, Lux R, et al. Interspecies interactions within oral microbial communities[J]. Microbiol Mol Biol Rev, 2007, 71(4): 653-670. DOI: 10.1128/MMBR.00024-07.
[3]	Marsh PD, Moter A, Devine DA. Dental plaque biofilms: communities, conflict and control[J]. Periodontol 2000, 2011, 55(1): 16-35. DOI: 10.1111/j.1600-0757.2009.00339.x.
[4]	Donoghue HD, Newman HN. Effect of glucose and sucrose on survival in batch culture of Streptococcus mutans C67-1 and a noncariogenic mutant, C67-25[J]. Infect Immun, 1976, 13(1): 16-21.
[5]	Deng DM, Liu MJ, ten Cate JM, et al. The VicRK system of Streptococcus mutans responds to oxidative stress[J]. J Dent Res, 2007, 86(7): 606-610. DOI: 10.1177/154405910708600705.
[6]	Zhang M, He LB, Exterkate RA, et al. Biofilm lagers affect the treatment outcomes of NaF and Nano-hydroxyapatite[J]. J Dent Res, 2015, 94(4): 602-607. DOI: 10.1177/0022034514565644.
[7]	Cieplik F, Zaura E, Brandt BW, et al. Microcosm biofilms cultured from different oral niches in periodontitis patients[J]. J Oral Microbiol, 2018, 11(1): 1551596. DOI: 10.1080/20022727.2018.1551596.
[8]	Li X, Hoogenkamp MA, Ling J, et al. Diversity of Streptococcus mutans strains in bacterial interspecies interactions[J]. J Basic Microbiol, 2014, 54(2): 97-103. DOI: 10.1002/jobm.201200457.
[9]	Jefferson KK. What drives bacteria to produce a biofilm？[J]. FEMS Microbiol Lett, 2004, 236(2): 163-173. DOI: 10.1016/j.femsle.2004.06.005.
[10]	Wang BY, Deutch A, Hong J, et al. Proteases of an early colonizer can hinder Streptococcus mutans colonization in vitro[J]. J Dent Res, 2011, 90(4): 501-505. DOI: 10.1177/0022034510388808.
[11]	Park SC, Park Y, Hahm KS. The role of antimicrobial peptides in preventing multidrug-resistant bacterial infections and biofilm formation[J]. Int J Mol Sci, 2011, 12(9): 5971-5992. DOI: 10.3390/ijms12095971.
[12]	Kara D, Luppens SB, Cate JM. Differences between single- and dual - species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine[J]. Eur J Oral Sci, 2006, 114(1): 58-63. DOI: 10.1111/j.1600-0722.2006.00262.x.
[13]	Kuramitsu HK, Wang BY. The whole is greater than the sum of its parts: dental plaque bacterial interactions can affect the virulence properties of cariogenic Streptococcus mutans[J]. Am J Dent, 2011, 24(3): 153-154. DOI: 10.1111/j.1460-9568.2005.04493.x.
[14]	Keren I, Kaldualu N, Spoering A, et al. Persister cells and tolerance to antimicrobials[J]. FEMS Microbiol Lett, 2004, 230(1): 13-18. DOI: 10.1016/S0378-1097(03)00856-5.
[15]	Pihl M, Davies JR, Chávez de Paz LE, et al. Differential effects of Pseudomonas aeruginosa on biofilm formation by different strains of Staphylococcus epidermidis[J]. FEMS Immunol Med Microbiol, 2010, 59(3): 439-446. DOI: 10.1111/j.1574-695X.2010.00697.x.

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