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

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

Abstract

Abstract:

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

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.

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References 15

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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

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

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

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

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