基于重水拉曼技术的白色念珠菌对变异链球菌代谢活性和耐药性影响的研究

Effects of Candida albicans on the metabolic activity and drug resistance of Streptococcus mutans based on D_(2)O-labeled single-cell Raman microspectroscopy

目的评价氟化钠(NaF)对变异链球菌的抑菌效能;白色念珠菌代谢产物对变异链球菌代谢活性和NaF作用下的耐药性影响。方法分别采用肉汤稀释法和重水拉曼技术,参考最低抑菌浓度(MIC)及最小代谢活性抑制浓度(MIC-MA)指标,评价NaF对变异链球菌生长及代谢的抑制效果;采用重水拉曼技术,检测不同浓度白色念珠菌上清液对变异链球菌代谢活性的影响;采用重水拉曼技术,检测在加入白色念珠菌上清液的混合培养体系中变异链球菌对NaF耐药性的变化,评价两菌互作对于耐药性的影响。结果NaF对变异链球菌的MIC为0.4 g·L^(-1),MIC-MA为1.2 g·L^(-1)。在MIC下,该菌生长受到完全抑制,但仍具有很高的代谢活性,存在再次恢复毒力的可能;只有达到3×MIC,即MIC-MA,该菌的生长和代谢才能得到完全抑制。当白色念珠菌上清液OD_(600)≥0.5时,变异链球菌的代谢活性明显高于纯培养体系,且在不同时间点差异均存在统计学意义(P<0.05)。混合体系中NaF对变异链球菌的抑菌效能较纯培养降低,且在不同时间点差异均存在统计学意义(P<0.05),直到4×MIC,即1.6 g·L^(-1),该菌的代谢才能得到完全抑制。结论重水拉曼技术适用于评价药物对细菌代谢活性的影响;混合体系中一定浓度(OD_(600)≥0.5)的白色念珠菌上清液中的代谢产物可增强变异链球菌的代谢活性,降低变异链球菌对于NaF的药物敏感性。

Objective To evaluate the susceptibility of Streptococcus mutans(S.mutans)to NaF and the effects of Candida albicans(C.albicans)metabolites on the metabolic activity and drug resistance of S.mutans.Methods The minimum inhibitory concentration(MIC)and MIC based on metabolic activity(MIC-MA)of NaF against S.mutans were measured through the broth dilution test and D;O-labeled single-cell Raman microspectroscopy,respectively,to explore the inhibitory effect of NaF on the growth and metabolism of S.mutans.The effects of different concentrations of C.albicans supernatant on the metabolic activity of S.mutans were evaluated by using D;O-labeled single-cell Raman microspectroscopy.The correlation between C.albicans and S.mutans was explored through assessing the drug resistance of S.mutans against NaF in a mixed system containing C.albicans supernatant by using D;O-labeled singlecell Raman microspectroscopy.Results The MIC and MIC-MA of NaF against S.mutans were 0.4 g·L^(-1)and 1.2 g·L^(-1),respectively.At the MIC level,S.mutans showed completely inhibited growth but retained high metabolic activity.Therefore,S.mutans may regain its virulence.At the MIC-MA level,the metabolic activity of S.mutans was inhibited only when the NaF concentration reached 3×MIC.At the concentration of OD_(600)≥0.5,the C.albicans supernatant significantly promoted the metabolic activity of S.mutans at different time points(P<0.05).In the mixed system,the susceptibility of S.mutans to NaF decreased,and statistical differences at different time points were observed(P<0.05).The metabolic activity of S.mutans was inhibited completely only at the concentration of 4×MIC(1.6 g·L^(-1)).Conclusion D;O-labeled single-cell Raman microspectroscopy is suitable for evaluating the effects of drugs on bacterial metabolic activity.In the mixed system,the metabolites in the C.albicans supernatant at the concentration of OD_(600)≥0.5 significantly promoted the metabolic activity of S.mutans and reduced the susceptibility of S.mutans to NaF.