Quantitative detection of periodontopathogens by real-time PCR

Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10(2) to 10(8) and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples.     

Quantification of Subgingival Bacterial Pathogens at Different Stages of Periodontal Diseases

Anaerobic gram-negative oral bacteria such as Treponema denticola, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Campylobacter rectus, and Fusobacterium nucleatum are closely associated with periodontal diseases. We measured the relative population (bacterial levels) of these oral pathogens in subgingival tissues of patients at different stages of Korean chronic periodontal diseases. We divided the individuals into those with chronic gingivitis (G), moderate periodontitis (P1), severe periodontitis (P2), and normal individuals (N) (n?=?20 for each group) and subgingival tissue samples were collected. We used real-time PCR with TaqMan probes to evaluate the change of periodontal pathogens among different stages of periodontitis. Bacterial levels of A. actinomycetemcomitans and C. rectus are significantly increased in individuals with chronic gingivitis and moderate periodontitis, but unchanged in severe periodontitis patients. These results suggest that analyzing certain bacterial levels among total oral pathogens may facilitate understanding of the role of periodontal bacteria in the early stages of periodontitis.     

Distribution of Archaea in Japanese patients with periodontitis and humoral immune response to the components

There is controversy regarding the existence of archaeal pathogens. Periodontitis is one of the human diseases in which Archaea have been suggested to have roles as pathogens. This study was performed to investigate the distribution of Archaea in Japanese patients with periodontitis and to examine the serum IgG responses to archaeal components. Subgingival plaque samples were collected from 111 periodontal pockets of 49 patients (17 with aggressive periodontitis and 32 with chronic periodontitis), and 30 subgingival plaque samples were collected from 17 healthy subjects. By PCR targeting the 16S rRNA gene, Archaea were detected in 15 plaque samples (13.5% of total samples) from 11 patients (29.4% of patients with aggressive periodontitis and 18.8% of patients with chronic periodontitis). Archaea were detected mostly (14/15) in severe diseased sites (pocket depth >/=6 mm), while no amplicons were observed in any samples from healthy controls. Sequence analysis of the PCR products revealed that the majority of Archaea in periodontal pockets were a Methanobrevibacter oralis-like phylotype. Western immunoblotting detected IgG antibodies against M. oralis in eight of the 11 sera from patients. These results suggest the potential of Archaea (M. oralis) as an antigenic pathogen of periodontitis.     

FAM5C Contributes to Aggressive Periodontitis

Aggressive periodontitis is characterized by a rapid and severe periodontal destruction in young systemically healthy subjects. A greater prevalence is reported in Africans and African descendent groups than in Caucasians and Hispanics. We first fine mapped the interval 1q24.2 to 1q31.3 suggested as containing an aggressive periodontitis locus. Three hundred and eighty-nine subjects from 55 pedigrees were studied. Saliva samples were collected from all subjects, and DNA was extracted. Twenty-one single nucleotide polymorphisms were selected and analyzed by standard polymerase chain reaction using TaqMan chemistry. Non-parametric linkage and transmission distortion analyses were performed. Although linkage results were negative, statistically significant association between two markers, rs1935881 and rs1342913, in the FAM5C gene and aggressive periodontitis (p = 0.03) was found. Haplotype analysis showed an association between aggressive periodontitis and the haplotype A-G (rs1935881-rs1342913; p = 0.009). Sequence analysis of FAM5C coding regions did not disclose any mutations, but two variants in conserved intronic regions of FAM5C, rs57694932 and rs10494634, were found. However, these two variants are not associated with aggressive periodontitis. Secondly, we investigated the pattern of FAM5C expression in aggressive periodontitis lesions and its possible correlations with inflammatory/immunological factors and pathogens commonly associated with periodontal diseases. FAM5C mRNA expression was significantly higher in diseased versus healthy sites, and was found to be correlated to the IL-1β, IL-17A, IL-4 and RANKL mRNA levels. No correlations were found between FAM5C levels and the presence and load of red complex periodontopathogens or Aggregatibacter actinomycetemcomitans. This study provides evidence that FAM5C contributes to aggressive periodontitis.     

Single nucleotide polymorphisms associated with aggressive periodontitis and severe chronic periodontitis in Japanese

Periodontitis is a common inflammatory disease causing destruction of periodontal tissues. It is a multifactor disease involving genetic factors and oral environmental factors. To determine genetic risk factors associated with aggressive periodontitis or severe chronic periodontitis, single nucleotide polymorphisms (SNPs) in multiple candidate genes were investigated in Japanese. We studied 134 patients with aggressive periodontitis, 117 patients with severe chronic periodontitis, and 125 healthy volunteers without periodontitis, under case-control setting, and 310 SNPs in 125 candidate genes were genotyped. Association evaluation by Fisher's exact test (p < 0.01) revealed statistically significant SNPs in multiple genes, not only in inflammatory mediators (IL6ST and PTGDS, associated with aggressive periodontitis; and CTSD, associated with severe chronic periodontitis), but also in structural factors of periodontal tissues (COL4A1, COL1A1, and KRT23, associated with aggressive periodontitis; and HSPG2, COL17A1, and EGF, associated with severe chronic periodontitis). These appear to be good candidates as genetic factors for future study.     

口腔细菌对铜绿假单胞菌黏附及侵入肺上皮细胞能力的影响

目的 通过比较铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入细胞的能力,探讨细菌间相互作用在呼吸道感染的最初阶段的作用机制.方法 应用培养法和抗生素保护法检测铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入肺上皮细胞的能力.结果 铜绿假单胞菌与口腔细菌共同作用于肺上皮细胞,牙龈卟啉单胞菌和伴放线放线杆菌降低了铜绿假单胞菌的黏附能力,却增强了其侵入能力;而铜绿假单胞菌能够影响口腔细菌对肺上皮细胞的黏附,同时增强口腔细菌侵入肺上皮细胞的能力.结论 口腔细菌,尤其是牙周可疑致病菌主要通过增强铜绿假单胞菌对肺上皮细胞的侵入而影响呼吸道感染过程.     

Association of generalized aggressive periodontitis and ectrodactyly-ectodermal dysplasia-cleft syndrome

Ectrodactyly-ectodermal dysplasia-cleft (EEC) syndrome is an autosomal dominant disorder characterized by the triad of ectrodactyly, ectodermal dysplasia, and facial clefting. Even though literature has documented the association of various genetic disorders with aggressive periodontitis, the periodontal manifestations in patients with EEC syndrome have never been addressed. This case report presents the periodontal status of three patients in a family with EEC syndrome. The presence of generalized aggressive periodontitis was noticed in these patients. EEC syndrome could be a new addition to the group of genetic disorders associated with aggressive periodontitis.     

牙龈卟啉杆菌的分离培养与鉴定

牙龈卟啉杆菌（Ｐｏｒｐｈｙｒｏｍａｎｕｓｇｉｎｇｉｖａｌｉｓ．Ｐｇ）与成年人慢性牙周炎有密切关系。我们对７６例慢性成人牙周炎龈下菌斑中的Ｐｇ菌进行了分离培养鉴定,共分离出Ｐｇ５５株。从表型特征上看Ｐｇ与其它２株卟啉杆菌极为相似,经ＤＮＡ同源性测定Ｐｇ菌具有高度同源性、且Ｐｇ抗血清特异性较强,血清学手段可作为辅助鉴定该菌的简便方法。本文还对Ｐｇ菌超显微结构进行了研究。     

亚甲基蓝/光化学法辅助治疗慢性牙周炎对牙龈卟啉单胞菌的影响

目的研究亚甲基蓝/光化学法辅助治疗慢性牙周炎对牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)检出率的变化。方法经伦理委员会同意后,本研究拟筛选出45例慢性牙周炎患者(每位患者经过详细牙周检查及X线检查后确认为牙周炎,有4个以上≥5 mm的牙周袋并分布在2个以上口腔区域),随机分3组:其中A组在SRP之后接受1次PERIOWAVE光化学治疗,B组在SRP后接受1次PERIOWAVE治疗以及在6周后再一次接受光化学治疗,而SRP组仅接受SRP治疗,各组患者取治疗前后相同4个位点龈下菌斑,聚合酶链式反应(Poly-merase chain reaction,PCR)检测Pg,观察Pg菌的检出率,采用卡方检验,并计算χ2值。结果 SRP组、A组、B组3组各组治疗后均较治疗前检出率降低(P<0.001),A组与SRP组治疗后比较差异无统计学意义[A组:40%(24/60),SRP组:45%(27/60),χ2=1.4815,P>0.05],B组与SRP治疗后比较检出率降低[SRP组:45%(27/60),B组:20%(12/60)χ2=8.547,P<0.01];B组与A组治疗后比较检出率降低[A组:40%(24/60),B组:20%(12/60),χ2=5.7143,P<0.05]。结论亚甲基蓝/光化学法辅助治疗慢性牙周炎后牙龈卟啉单胞菌的检出率较治疗前降低;结合临床试验结果,尚可认为2次激光疗效较1次激光好。     

Non-radioactively labelled DNA probes for the detection of periodontopathogenic Prevotella and Porphyromonas species

Abstract Dioxigenin-labelled synthetic DNA probes directed against the 16S rRNA were used for the direct detection of the periodontopathogenic bacteria Prevotella intermedia and Porphyromonas gingivalis in subgingival plaque by applying a DNA-RNA dot-blot hybridization procedure. The test was evaluated with 134 plaque samples from 26 patients with adult periodontitis or rapidly progressive periodontitis. The lower limit of detection was 10⁴–10⁵ bacteria/specimen. A semiquantitative assessment of the two species in each sample and in the corresponding periodontal site was achieved by this technique. It is possible to examine 80–90 samples within two days with low material costs.     

Arterial Stiffness and Pulse Wave Reflection Are Increased in Patients Suffering from Severe Periodontitis

Aim

This single blind cross-sectional study compared the vascular health of subjects suffering from severe chronic periodontitis, severe aggressive periodontitis and periodontal healthy controls by evaluating pulse wave velocity (PWV), augmentation index (AIx) and pulse pressure amplification (PPA).

Material and Methods

In a total of 158 subjects, 92 suffering from severe periodontitis and 66 matched periodontal healthy controls, PWV, AIx, central and peripheral blood pressure were recorded using an oscillometric device (Arteriograph).

Results

Subjects suffering from severe chronic or aggressive periodontitis exhibited significantly higher PWV (p = 0.00004), higher AIx (p = 0.0049) and lower PPA (p = 0.028) than matched periodontal healthy controls.

Conclusions

The results of this study confirm the association between periodontal inflammation and increased cardiovascular risk shown by impaired vascular health in case of severe periodontitis. As impaired vascular health is a common finding in patients suffering from severe periodontal disease a concomitant routine cardiovascular evaluation may be advised.     

Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rDNA libraries

The oral bacterial flora in the saliva from two patients with periodontitis and from a periodontally healthy subject were compared using a sequence analysis of 16S rDNA libraries without cultivation. 16S rDNAs were amplified from salivary DNA by PCR and cloned. Randomly selected clones were partially sequenced. On the basis of sequence similarities, the clones were classified into several clusters corresponding to the major phylum of the domain Bacteria. The major phylum in the libraries was the low G+C Gram-positive bacteria. There was no clonal sequence affiliated with periodontopathic bacteria in the salivary sample from the healthy subject, while a number of periodontal pathogens such as Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis and Treponema socranskii were detected in the salivary samples from the patients with periodontitis. In addition, a number of previously uncharacterized and uncultured microorganisms were recognized. These organisms may have some role in periodontal disease. This study reveals some potential for a molecular-biological technique to analyze the oral microflora associated with periodontal disease, including previously uncharacterized and uncultured microorganisms, without cultivation.     

Periodontal microflora of HIV infected patients with periodontitis

The aim of this study was to determine the microbial profile of periodontal lesions in HIV seropositive patients and to compare it with rapidly progressing periodontal lesions in systemically healthy patients. The subgingival microflora of 20 CDC II, 20 CDC III, 20 CDC IV/V and 20 systemically healthy patients with rapidly progressing periodontitis was examined. Four sites with greatest probing depth in each patient were selected for microbiological sampling. The samples were cultured aerobically and anaerobically for bacterial isolation using selective and non-selective media. Isolates were characterized to species level by conventional biochemical tests and various identification kits. The microflora of periodontitis lesions within the three stages of the HIV infection was similar to that of progressing periodontitis in systemically healthy adults including Campylobacter rectus, Capnocytophaga spp., Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Selenomonas spp. and Peptostreptococcus micros. However, HIV seropositive periodontitis lesions harboured a range of exogenous pathogens rarely associated with common types of periodontitis including Staphylococcus aureus, Enterobacter cloaca, Pseudomonas aeruginosa, Candida albicans, Enterococcus faecalis, Enterococcus avium, Clostridium difficile, Aspergillus fumigatus, Klebsiella pneumoniae and Mycoplasma incognitum. The lack of immune effector and regulatory cells in HIV infected patients could in fact explain the increase of some opportunistic pathogens and the characteristic and rapidly progressing nature of the periodontal disease in these patients.     

Actinobacillus actinomycetemcomitans serotype-specific genotypes and periodontal status in Brazilian subjects

Periodontitis is associated with members of the oral microbiota, such as Actinobacillus actinomycetemcomitans. To our knowledge, this is the first study to evaluate, by PCR, the occurrence of the six known bacterium serotypes that included subjects with and without periodontitis. Our group comprised 49 Brazilian subjects. We studied 146 bacterial isolates from 23 patients with aggressive or chronic periodontitis and 26 subgingival specimens from subjects with or without periodontitis, all originating in our collection. Serotypes b and c were observed in similar frequencies, and no subject harboured d, e, or f serotype strains. Around 78% subjects had single-serotype infection. Mixed infection was seen only in aggressive periodontitis patients. An association between serotype b and healthy periodontium and between serotype c and chronic periodontitis was observed. Our results diverge from those previously reported, which may be explained by specific distribution patterns in distinct populations. The association of different serotypes with the same periodontal status or conversely of a serotype with different periodontal conditions indicates that organism serotyping should not be used as a sole reliable marker for predicting the outcome of the infection. Evaluation of factors involved in human oral cavity colonization by subsets of A. actinomycetemcomitans is essential for elucidating organism-host-environment relationships.     

侵袭性牙周炎伴错牙合畸形患者牙周-正畸联合治疗前后血清SAA、leptin的变化及与牙周指标和辅助性T细胞亚群的相关性分析

摘要 目的：探讨侵袭性牙周炎伴错牙合畸形患者牙周-正畸联合治疗前后血清淀粉样蛋白A（SAA）、瘦素（leptin）的变化及与牙周指标和辅助性T细胞（Th）亚群的相关性。方法：选择2020年6月-2022年8月解放军总医院京中医疗区黄寺门诊部口腔科收治的80例侵袭性牙周炎伴错牙合畸形患者（牙周炎组）和65例于口腔门诊检查的健康志愿者（对照组）。所有患者均接受牙周-正畸联合治疗,治疗前后分别检测血清SAA、leptin水平以及外周血中Th1、Th2、Th17细胞占比,并评估牙周指标变化。Pearson相关性分析血清SAA、leptin水平与牙周指标以及外周血中Th1、Th2、Th17细胞占比的相关性。结果：牙周炎组治疗前血清SAA、leptin水平,外周血Th1、Th17细胞占比,出血指数（SBI）、菌斑指数（PLI）、附着丧失（AL）、牙周探诊深度（PD）高于对照组（P＜0.05）,外周血Th2细胞占比低于对照组（P＜0.05）。牙周炎组治疗后血清SAA、leptin水平,外周血Th1、Th17细胞占比,PLI、SBI、AL、PD较治疗前降低（P＜0.05）,外周血Th2细胞占比较治疗前增高（P＜0.05）。牙周炎组血清SAA、leptin与PLI、SBI、AL、PD,外周血Th1、Th17细胞占比呈正相关,与外周血Th2细胞占比呈负相关（P＜0.05）。结论：侵袭性牙周炎伴错牙合畸形患者血清SAA、leptin水平增高,经牙周-正畸联合治疗后下降,高水平SAA、leptin与牙周组织破坏程度以及Th亚群紊乱有关,检测血清SAA、leptin水平可评估侵袭性牙周炎牙周组织破坏程度以及细胞免疫状态。     

Association of interleukin-4 and interleukin-17F polymorphisms in periodontitis in Dravidian ethnicity

BACKGROUND:

Complex network of pro and anti-inflammatory cytokines are known to act in inflamed periodontal tissue. This study explores the distribution of interleukin (IL)-4 (+33 C/T) and IL-17F (7383A/G, 7488A/G) gene polymorphism in chronic and aggressive periodontitis subjects of Dravidian ethnicity.

MATERIALS AND METHODS:

This case control study consisted of 124 periodontitis individuals comprising of 63 chronic and 61 aggressive periodontitis subjects as cases, and control group consisted of 101 healthy subjects. All subjects were genotyped for IL-4 + 33C/T, IL-17F 7383A/G, 7488A/G by polymerase chain reaction amplification followed by TaqMan assay for IL-4 + 33C/T, restriction enzyme digestion and gel electrophoresis for IL-17F 7383A/G and sequencing for IL-17F 7488A/G.

RESULTS:

IL-4 + 33C/T was significantly associated with periodontitis (P < 0.05) at both allelic and genotypic level. In subgroup analysis also significant difference (P < 0.05) in allelic distribution between aggressive periodontitis and control group for loci IL-4 + 33C/T was noted. However, there was a lack of association between IL-17F 7383A/G and IL-17F 7488A/G with periodontitis and its sub-groups at both allelic and genotypic levels.

CONCLUSIONS:

In Malayalam speaking Dravidian population IL-4 + 33C/T loci appears to be an important risk factor for periodontal disease with a leaning towards aggressive periodontitis. The association between IL-17F at 7383A/G and 7488A/G loci with either chronic or an aggressive periodontitis could not be ascertained.     

Oxidative damage of periodontal tissue in the rat periodontitis model: effects of a high-cholesterol diet

Studies suggest an association between consumption of a high-cholesterol diet and periodontitis. We addressed the mechanism by which high dietary cholesterol could be detrimental to periodontal health in a rat model. Feeding a high-cholesterol diet augmented the effects of bacterial pathogens and their products (e.g., lipopolysaccharide and proteases) on production of pro-inflammatory cytokines in fibroblasts. High dietary cholesterol also increased mitochondrial 8-hydroxydeoxyguanosine in the periodontal tissues. These results suggest that excessive tissue oxidative damage induced by high dietary cholesterol could potentiate pro-inflammatory cytokine production by fibroblasts stimulated with bacterial pathogens.     

Periodontitis in Cardiovascular Disease Patients with or without Marfan Syndrome -A Possible Role of Prevotella intermedia-

Background

Although periodontitis is a risk factor for cardiovascular disease (CVD), the influence of periodontitis on Marfan syndrome (MFS) with CVD is unclear. The aim of this study was to assess the relationship between periodontal bacterial burden and MSF with CVD.

Methods and Results

The subjects were patients with MFS with CVD (n = 47); age and gender matched non-MFS CVD patients (n = 48) were employed as controls. Full-mouth clinical measurements, including number of teeth, probing of pocket depth (PD), bleeding on probing (BOP) and community periodontal index (CPI) were recorded. We also evaluated the existence of three periodontal pathogens, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia using polymerase chain reaction assays. Serum antibody titers against the pathogens were also measured. We revealed that MFS with CVD patients had periodontitis more frequently than the age and gender matched non-MFS CVD control subjects. MFS with CVD patients had significantly severer periodontitis, fewer remaining teeth and deeper PD compared to the non-MFS CVD controls. Furthermore, the serum antibody titer level against Prevotella intermedia was significantly lower in MFS plus CVD patients compared to the non-MFS CVD patients.

Conclusion

Periodontitis may influence the pathophysiology of cardiovascular complications in MFS patients. A specific periodontal pathogen might be a crucial therapeutic target to prevent CVD development.     

Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.