乳酸乳球菌对致龋菌生长及生物膜形成的作用

The effect of Lactococcus lactis on the growth and biofilm formation of cariogenic bacteria

目的研究乳酸乳球菌(Ll)对4种致龋菌生长及生物膜形成的影响,为Ll龋病防治提供基础。方法厌氧条件下培养Ll、变异链球菌(Sm)、内氏放线菌(An)、黏放线菌(Av)和嗜酸乳杆菌(La),琼脂扩散实验检测Ll对4种致龋菌的抑菌圈直径(IZD);琼脂板竞争实验检测Ll与4种致龋菌的拮抗相互作用;4种致龋菌单独培养生物膜为单独培养组,Ll分别与其共培养生物膜为共培养组,结晶紫染色法(CVS)及扫描电镜(SEM)检测Ll对4种致龋菌生物膜形成的影响。采用SPSS 20.0统计软件数据分析。Ll对4种致龋菌的IZD整体分析采用单因素方差分析,Tukey′s HSD检验进行组间多重比较,采用Student′s t检验对细菌生物膜量进行比较。结果琼脂扩散实验结果显示,Ll对4种致龋菌均表现出一定的抑制作用。Ll对An的IZD最大,为(11.97±0.40)mm,对Sm的IZD最小,为(4.67±0.32)mm。Ll对4种致龋菌的IZD两两比较,差异有统计学意义(F=241.22,P<0.001)。琼脂板竞争实验显示,Ll对La、An、Av表现为竞争性抑制作用;CVS结果显示,Ll与Sm、An共培养形成的双菌种生物膜的生物膜量分别为(30.66±0.48)、(10.27±0.04),Sm、An单独培养形成的单菌种生物膜的生物膜量分别为(80.26±2.55)、(73.98±0.35),Sm、An双菌种生物膜的生物膜量与单菌种生物膜的生物膜量比较差异有统计学意义(t_(Sm)=19.090,PSm<0.001,t_(An)=183.3,PAn<0.001)。SEM结果显示,Ll与Sm或An共培养形成的双菌种生物膜明显变薄。结论Ll可抑制Sm、An、Av和La生长,并减少Sm、An生物膜的形成。

Objective To study the effects of Lactococcus lactis(Ll)on the growth and biofilm formation of 4 kinds of cariogenic bacteria,and to provide the basis for the application in caries prevention and control.Methods Ll,Streptococcus mutans(Sm),Actinomyces naeslundii(An),Actinomyces viscosus(Av)and Lactobacillus acidophilus(La)were cultured under anaerobic conditions.A spot-on-lawn assay was used to evaluate the inhibition of Ll for these 4 kinds of cariogenic bacteria.The antagonistic interaction between Ll and these cariogenic bacteria was detected by plate competition test.The four kinds of cariogenic bacteria cultured separately were the single-cultured group,Ll co-cultured with 4 cariogenic bacteria separately were the co-cultured group.Crystal violet staining(CVS)and scanning electron microscopy(SEM)was used to evaluate the effect of Ll on the biofilm formation of the 4 kinds of cariogenic bacteria.SPSS 20.0 statistical software was used for data analysis.The data from the measurement of the inhibitory-zone diameters(IZD)of Ll against the 4 kinds of cariogenic bacteria were compared and statistically analyzed with One-Way analysis of variance(ANOVA)and tukey′s HSD test,and the amount of bacterial biofilm was compared by Student′s t test.Results Spot-on-lawn assay showed that Ll inhibited the growth of the 4 kinds of cariogenic bacteria.Ll had the largest IZD for An,(11.97±0.40)mm,and the smallest for Sm,(4.67±0.32)mm.Ll showed statistically significant difference in the IZD of the 4 kinds of cariogenic bacteria(F=241.22,P<0.001).The plate competition test showed that Ll showed competitive inhibition against La,An and Av.CVS showed that the biofilm quantity of Sm and An co-cultured with Ll was(30.66±0.48)and(10.27±0.04),respectively.The biofilm quantity of Sm and An single-cultured was(80.26±2.55)and(73.98±0.35),respectively.For Sm and An biofilm,there was statistical difference in the biofilm quantity between co-cultured and single-cultured groups(t_(Sm)=19.090,PSm<0.001,t_(An)=183.3,PAn<0.001).The SEM showed that Sm and An showed significantly thinner biofilm in coexistence with Ll than in the absence of Ll.Conclusion Ll could inhibit the growth of Sm,An,Av,La,and reduce the formation of Sm,An biofilms.