内氏放线菌尿素酶对牙菌斑生物膜酸碱平衡调节作用的初步研究

目的：初步探讨在牙菌斑生物膜天然环境中,内氏放线菌尿素酶能否发挥高效尿素水解反应,以及尿素水解对口腔环境中pH值的调节作用。方法：通过酶促反应动力学实验寻找内氏放线菌尿素水解的最适条件．监测尿素水解调节细菌产酸后的pH值变化。采用SPSS软件包,对酶促动力学实验数据进行线性回归与相关分析。结果：内氏放线菌尿素酶米氏常数Km=7．5mmol／L,在口腔中正常尿素浓度3-10mmol／L范围内,内氏放线菌尿素酶催化活性大约保持在最大活性的20％-63％;内氏放线菌尿素酶最适pH值=6．5,但是在牙菌斑临界pH5．0,尿素酶活性仍保持40％的最大活性;在口腔正常尿素浓度范围内,内氏放线菌尿素水解产物中和细菌产酸后,pH值不会下降到牙菌斑临界pH5．0以下。结论：在牙菌斑生物膜中,内氏放线菌尿素酶可以发挥高效尿素水解反应．尿素水解对口腔环境pH值具有明显的调节作用。     

Effect of the quorum‐sensing luxS gene on biofilm formation by Enterococcus faecalis

Enterococcus faecalis is the species of bacterium most frequently isolated from the root canals of teeth that exhibit chronic apical periodontitis refractory to endodontic treatment. In this study, we evaluated the effect of the S‐ribosylhomocysteine lyase (luxS) quorum‐sensing gene on E. faecalis biofilm formation by constructing a knockout mutant. The biofilms formed by both E. faecalis and its luxS mutant strain were evaluated using the MTT method. Important parameters that influence biofilm formation, including cell‐surface hydrophobicity and the nutrient content of the growth medium, were also studied. Biofilm structures were observed using confocal laser scanning microscopy (CLSM), and expression of biofilm‐related genes was investigated using RT‐PCR. The results showed that the luxS gene can affect biofilm formation, whereas it does not affect the bacterial growth rate. Deletion of the luxS gene also increased cell‐surface hydrophobicity. Biofilm formation was accelerated by the addition of increasing concentrations of glucose. The CLSM images revealed that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures, whereas the wild‐type strain appears confluent and more evenly distributed. All genes examined were up‐regulated in the biofilms formed by the luxS mutant strain. The quorum‐sensing luxS gene can affect E. faecalis biofilm formation.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

D-型氨基酸对细菌生物膜解离分散的作用

生物膜是细菌主要的生存方式,受营养等因素的限制其存在的寿命有限,成熟生物膜分泌的某些因子促使生物膜解离,恢复细菌的单细胞浮游状态。促进生物膜的解离分散是目前的研究热点和前沿方向,D-型氨基酸对细菌生物膜的解离分散作用是新近几年来受到关注的研究领域,本文就D-型氨基酸对细菌生物膜相关作用的研究作一综述。     

Effect of short chain fatty acids on human gingival epithelial cell keratins in vitro

Hemidesmosomal attachment of the junctional epithelial cells to the tooth and the ability of the attached cells to divide are essential features of the healthy dentogingival junction. Short chain fatty acids are bacterial metabolites associated with gingival inflammation and periodontal pockets. In vitro, short chain fatty acids have been shown to inhibit epithelial cell division and increase the density of their keratin filaments. This study examined these keratin changes by making use of human gingival keratinocyte cultures, gel electrophoresis and Western blot. Short chain fatty acids, butyrate and propionate, increased the relative amount of keratin proteins in the cells, most strikingly keratin K17. The distribution of K17 was further studied in a culture model for human junctional epithelium and in gingival biopsies. In butyrate-treated cultures of junctional epithelium, K17 expression was markedly increased and extended to the basal cells and to the cells mediating the attachment of the explant to the substratum. In clinically healthy gingiva, K17 was expressed predominantly in sulcular epithelium. The dividing basal cells and the cells attached to the tooth were negative. In advanced periodontitis, a strong reaction for K17 was localised to the pocket epithelium. The inhibition of epithelial cell division and the simultaneous upregulation of K17 in vitro, and the strong expression of this protein in detached pocket epithelium suggest a role for the short chain fatty acids in the degenerative process that leads to subgingival advancement of pathogens and, eventually, to periodontal pocket formation.     

镍铬合金析出的镍离子对内氏放线菌糖代谢和尿素代谢的影响

目的研究镍铬合金析出的镍离子对内氏放线菌糖代谢和尿素代谢的影响。方法分别用含0.260mg/L和0.625mg/L镍离子的BHI培养基厌氧培养内氏放线菌WVU45型菌株,计算pH的变化值,检测乳酸脱氢酶(LDH)含量,测定尿素酶活性。结果镍离子对内氏放线菌产酸和产乳酸脱氢酶的作用不明显(P>0.05),对尿素酶活性有促进作用(P<0.05)。结论镍铬合金析出的镍离子可以增加内氏放线菌尿素酶活性,促进尿素代谢。     

外源性右旋糖酐酶和氟化钠对粘性放线菌生物膜的影响

目的：探讨外源性右旋糖苷酶（dextranase ,Dex）和氟化钠（NaF）对粘性放线菌生物膜的影响。方法用结晶紫（crystal violet,CV）染色法测量不同浓度外源性Dex和NaF作用下粘性放线菌生物膜量;用扫描电镜观察不同实验组生物膜的形态和结构。结果外源性Dex和NaF的浓度升高,对粘性放线菌生物膜形成的抑制作用增强;在实验条件下,0.25 U/mL Dex与40/80μg/mL NaF联用对生物膜形成的抑制作用明显强于二者单独作用。结论外源性Dex和NaF能够抑制粘性放线菌生物膜的形成,且在一定条件下,它们对粘性放线菌生物膜的形成表现出协同抑制作用。