Endopeptidase activities of selected Porphyromonas spp., Prevotella spp. and Fusobacterium spp. of oral and non-oral origin

The ability of three Porphyromonas spp., seven Prevotella spp., seven Fusobacterium spp. and two related Bacteroides spp. (B. levii and B. macacae) to degrade an extensive range of synthetic endo-, amino- and diamino peptidase substrates linked to the fluorescent leaving group 7-amido-4-methylcoumarin (NHMec) was investigated. Many more species than was previously recognized exhibited peptidase activities, albeit at lower levels than those already described for Porphyromonas gingivalis. Detection of chymotrypsin-like activity was dependent on which of three NHMec-linked substrates was used, but all species exhibited degradative activity with at least one of these substrates. Elastase-like activity was detected in all species though not all species reacted with each of the elastase substrates. Glycylprolyl peptidase activity was detected in all of the species tested with the exception of F. mortiferum, F. gonidiaformans, F. naviforme and F. necrophorum. While the detection of peptidase activities does not appear to be useful for the differentiation of species within the genera Bacteroides and Prevotella, its ability to differentiate species of the genus Porphyromonas or Fosobacterium further investigation.     

Characterisation of black-pigmented anaerobes isolated from diseased and healthy periodontal sites

Prevotella intermedia has recently been re-defined and a new species, Prevotella nigrescens has been proposed. However, there is little data available on the incidence of these new species in periodontal health or disease. Black-pigmented anaerobes isolated from diseased and healthy subgingival sites were identified by serotyping, SDS-PAGE and physiological tests. In adult periodontitis subjects, 64% of active sites, 35.7% of inactive sites and 38.5% of healthy sites yielded black-pigmented anaerobes. Of these, Porphyromonas gingivalis was found in 11% of active and 5% of healthy sites in diseased patients, Prevotella intermedia in 15.5% of active and 20.5% of healthy sites, Prevotella nigrescens in 37.7% of active and 11.5% of healthy sites and Prevotella denticola in 3% of active and 1% of healthy sites. In healthy subjects, 50% of sites yielded black-pigmented anaerobes. P. gingivalis was not found in healthy subjects but P. intermedia was found in 18% and P. nigrescens in 31% of sites. SDS-PAGE proved to be a useful method for routinely differentiating P. intermedia and P. nigrescens and two sub-types of the latter species were detected on the basis of band pattern. Only one P. nigrescens sub-type was found in any given individual and one type, typified by ATCC 25261, was more commonly found in deep pockets. However, overall both P. nigrescens and P. intermedia as species were just as frequently found at healthy sites as diseased sites. Thus, these species, in contrast to P. gingivalis , appear to be common commensals but they may act as opportunistic pathogens.     

Kaempferol Inhibits P. intermedia Lipopolysaccharide‐Induced Production of Nitric Oxide Through Translational Regulation in Murine Macrophages: Critical Role of Heme Oxygenase‐1‐Mediated ROS Reduction

Background: Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage‐like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. Methods: NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real‐time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase‐1 (HO‐1) mRNA expression. iNOS and HO‐1 protein expression and phosphorylation of c‐Jun N‐terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate. Results: Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS‐stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO‐1 expression in LPS‐activated cells. Inhibition of HO‐1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS‐induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS‐induced NO. Conclusions: Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS‐stimulated RAW264.7 cells at the translational level via HO‐1‐mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.     

Relationship between salivary antioxidant capacity and phases of the menstrual cycle

Kawamoto A, Sugano N, Motohashi M, Matsumoto S, Ito K. Relationship between salivary antioxidant capacity and phases of the menstrual cycle. J Periodont Res 2012; 47: 593–598. © 2012 John Wiley & Sons A/S Background and Objective: Reactive oxygen species and free radicals are involved in the pathogenesis of periodontal disease. Previous studies have shown that the stage of the menstrual cycle is associated with the levels of gingival inflammation and discomfort. This study examined changes in salivary antioxidant activities, clinical parameters and bacterial levels during the menstrual cycle. Material and Methods: The study group consisted of 16 women with periodontitis and 12 healthy women. Clinical and bacterial measurements were performed for all subjects during the ovulatory and follicular phases. Results: Salivary antioxidant activity during the ovulatory phase was significantly lower than during the follicular phase in the women with periodontitis. The antioxidant activity in all subjects during the ovulatory phase was negatively correlated with Prevotella intermedia (r = ?0.430; p = 0.023) and total bacterial counts (r = ?0.496; p = 0.007); however, these correlations were not significant for subjects in the follicular phase. Conclusion: This study showed that salivary antioxidant capacity decreased, while bleeding on probing and P. intermedia increased, over the course of the menstrual cycle in women with periodontitis. Antioxidant capacity could be involved in the pathogenesis of periodontitis.     

Indonesian children fecal microbiome from birth until weaning was different from microbiomes of their mothers

ABSTRACT

Gastrointestinal (GI) microbiota play an important role in human health and wellbeing and the first wave of gut microbes arrives mostly through vertical transmission from mother to child. This study has undertaken to understand the microbiota profile of healthy Southeast Asian mother-infant pairs. Here, we examined the fecal, vaginal and breast milk microbiota of Indonesian mothers and the fecal microbiota of their children from less than 1 month to 48 months old. To determine the immune status of children and the effect of diet at different ages, we examined the level of cytokines, bile acids in the fecal water and weaning food frequency. The fecal microbiota of the children before weaning contained mainly Bacteroides and Bifidobacterium, which presented at low abundance in the samples of mothers. After weaning, the fecal microbiome of children was mainly of the Prevotella type, with decreasing levels of Bifidobacterium, thus becoming more like the fecal microbiome of the mother. The abundance of infant fecal commensals generally correlated inversely with potential pathogens before weaning. The fecal Bifidobacterium in children correlated inversely with the consumption of complex carbohydrates and fruits after weaning. The specific cytokines related to the proliferation and maturation of immunity were found to increase after weaning. A decreasing level of primary bile acids and an increase of secondary bile acids were observed after weaning. This study highlights the change in the GI microbiota of infants to adult-type microbiota after weaning and identifies diet as a major contributing factor.     

慢性牙周炎口腔变黑普雷沃氏菌分离鉴定及促进食管鳞癌演进

目的分离、鉴定口腔变黑普雷沃氏菌(Prevotella nigrescens,P.nigrescens),探讨其对食管鳞癌是否有促进作用。方法胰豆胨肝粉琼脂(GAM)培养基厌氧原代培养慢性牙周炎患者龈沟液,挑选黑色菌落分离纯化;革兰染色、16S rRNA基因测序鉴定分离的单克隆细菌;该细菌感染食管鳞癌细胞NE6-T后细胞生物学性状变化。结果慢性牙周炎患者龈沟液厌氧原代培养120 h后可见多个菌落,其中灰黑色菌落经连续划线法分离纯化为黑色、圆形光滑单克隆菌落。该细菌革兰染色阴性、串珠状排列,16S rDNA序列与P.nigrescens F0103同源性为99.78%(P.nigrescens LY01)。LY01感染促进NE6-T细胞体外增殖、迁移、侵袭和裸鼠皮下荷瘤生长,并诱导Ki67表达上调和p-STAT3激活。结论慢性牙周炎口腔P.nigrescens可能促进了食管鳞癌发展。     

牙周源性牙周牙髓联合病变常见病原菌的分布及其意义

目的研究牙周源性牙周牙髓联合病变常见病原菌分布及其相关性,为临床治疗提供依据。方法选择2018年1月至2020年6月于新疆维吾尔自治区人民医院口腔科就诊的牙周源性牙周牙髓联合病变患者43例43颗牙作为实验组,重度牙周炎患者41例41颗牙作为对照组。分别采集根管内组织和龈下菌斑,构建含有8种待测细菌基因片段的重组质粒,建立定量标准,应用实时荧光定量PCR技术检测福赛斯坦纳菌(Tannerella forsythia,Tf)、牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、具核梭杆菌(Fusobacterium nu?cleatum,Fn)、中间普氏菌(Prevotella intermedia,Pi)、齿垢密螺旋体(Treponema denticola,Td)、消化性链球菌(Digestive streptococcus,Ds)、粪肠球菌(Enterococcus faecalis,Ef)、牙髓卟啉单胞菌(Porphyromanus endodontics,Pe)数量。结果实验组根管内组织与其龈下菌斑中Ds、Pe数量差异无统计学意义(Ρ>0.05),其余6种病原菌数量差异均有统计学意义(P<0.05);实验组与对照组龈下菌斑中Ds数量差异无统计学意义(P=0.241),其余7种病原菌数量差异均有统计学意义(P<0.05);实验组根管内组织与其龈下菌斑中Ef、Pe、Pg、Td、Tf细菌数量密切相关,Ef(r=0.347,Ρ<0.05)、Pe(r=0.363,Ρ<0.05)、Pg(r=0.437,Ρ<0.01)、Td(r=0.471,Ρ<0.01)、Tf(r=0.679,Ρ<0.01)。结论牙周源性牙周牙髓联合病变常见病原菌在根管内组织中数量低于龈下菌斑但根管内病原菌数量与龈下菌斑密切相关,临床治疗期间在控制牙周组织感染的同时,还应重视牙髓组织感染的控制。     

Multiplex polymerase chain reaction assay for simultaneous detection of black-pigmented Prevotella species in oral specimens

Prevotella species are a major component of the oral microflora and some have been implicated in various forms of periodontal disease. Despite the importance of understanding the prevalence of these organisms in the oral microflora, no rapid, simultaneous detection system for these species has been reported. This study developed a multiplex polymerase chain reaction assay for the simultaneous detection of four oral black-pigmented Prevotella species in various oral specimens. This assay will be useful for determining the prevalence of these organisms in the oral ecosystem. Furthermore, this assay system should prove a useful tool for analyzing the role of black-pigmented Prevotella species in the mouth.     

Invasion of vascular cells in vitro by Porphyromonas endodontalis

AIM: The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. METHODOLOGY: Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. RESULTS: Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. DISCUSSION: Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.