Prevalence of P.intermedia and P.nigrescens and preparation of their hemolytic toxins

本研究的目的是确定分布的假定牙周致病菌中间普雷沃菌和黑普雷沃菌在牙周健康和疾病患者。牙周病患者的黑色素厌氧革兰氏阴性杆菌（BPR）比健康人的样本数量更多。BPR被鉴定为P.intermedia和P. nigrescens。牙周组织中仅发现中间变形杆菌和变黑变形杆菌的比例分别为47.1%和11.8%，而健康人中仅发现中间变形杆菌和变黑变形杆菌的比例分别为23.1%和38.5%。另外41.2%和38.5%的菌株同时具有中间疫霉和变黑疫霉，以中间疫霉为主.结果表明，中间变形杆菌与牙周病的关系可能比变黑变形杆菌更密切。溶血素在血琼脂平板上产生清晰的β溶血带。分离和纯化过程涉及 ...更多信息 硫酸铵和聚乙二醇沉淀以及在DEAE-Sephacel和CM-Sepharose上的离子交换层析。溶血素的最适pH为7.5。溶血活性被还原剂刺激。胰蛋白酶或热处理导致溶血活性的完全抑制。阳离子和EDTA对酶活性无影响。结果表明，中间对虾β-溶血素属于巯基活化毒素家族，而另一种溶血物质是从中间对虾培养上清中提取的，经硫酸铵分级沉淀、Sephadex LH-20和Wakogel C-200柱层析分离得到。溶血物质在薄层色谱（TLC）上显示0.97 Rf值，使用氯仿：甲醇：水（65：35：5）作为展开剂。糖定量分析和薄层色谱分析表明，溶血物质不含胆碱、氨基、磷酸基和碳水化合物。60℃加热30 min和胰蛋白酶处理对溶血耐受性无影响。溶血物质可被认为是简单脂质或衍生脂质。少

The purpose of this study was to determined the distribution of the putative periodontal pathogen Prevotella intermedia and Prevotella nigrescens in periodontal health and disease patients. Black-pigmented anaerobic Gram-negative rods (BPRs) were more numerous in samples from patients with periodontal disease than in those from healthy individuals. The BPRs were identified as P.intermedia and P.nigrescens. Sites exhibiting only P.intermedia or P.nigrescens were found in 47.1 % and 11.8 % of periodontal and 23.1 % and 38.5 % of healthy subjects, respectively. The remaining 41.2 % and 38.5 % possessed both P.intermedia and P.nigrescens, the former of which was predominant. These results suggested that P.intermedia may be associated with periodontal disease more than P.nigrescens.The hemolysin from P.intermedia has been purified from culture supernatant and characterized. The hemolysin produced clear β-hemolytic zone on blood agar plate. The isolation and purification procedure involved a … More mmonium sulfate and polyethylene glycol precipitations and ion-exchange chromatographies on DEAE-Sephacel and CM-Sepharose. The optimal pH of the hemolysin was 7.5. Hemolytic activity was stimulated by reductants. Trypsin or heat treatment resulted in complete inhibition of hemolytic activity. Cation and EDTA did not affect the activity. These results indicate that the β-hemolysin from P.intermedia, appeared to belong to a family of toxins known as the thiol-activated toxin.The other hemolytic substance was extracted and separated from culture supernatant by ammonium sulfate fractionation, Sephadex LH-20 and Wakogel C-200 column chromatographies. The hemolytic substance showed 0.97 Rf value on thin layer chromatography (TLC) using chloroform : methanol : water (65 : 35 : 5) as a developing solvent. Quantitative analysis of sugar ant TLC analysis showed that the hemolytic substance did not consist of choline, amino group, phosphate group and carbohydrate. The hemolytic stubstance was not affected by 60℃ for 30 minutes heating and trypsin treatment. The hemolytic substance may be regarded as simple lipid or derived lipid. Less