Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species.

Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1ß, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.

The influence of oral bacteria on epithelial cell migration in vitro.

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.

Revisión de la periodontitis crónica: evolución y su aplicación clínica / Chronic periodontitis review. Evolution and clinic application

El objetivo de este trabajo es realizar una descripción exhaustiva de la periodontitis crónica. La periodontitis crónica está causada por infecciones mixtas producidas por un biofilm de bacterias sibgingivales. No existe un único patógeno que sea el responsable de la etiopatologia de la periodontitis. La respuesta inmune de anticuerpos a P. intermedia, B. forsythus y T. denticola también ha sido demostrada en este tipo de pacientes (AU)

The aim of this issue is to make an exhaustive description of the chronic periodontitis. Chronic periodontitis is caused by mixed infections with the subgingival microbiota being organized as a biofilm. The antibodyreponse to ha P. intermedia, B. forsythus and T. denticola have also been demonstrated in this kind of patient (AU)

Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts.

BACKGROUND AND OBJECTIVE: Lipopolysaccharide is thought to be a major virulence factor of pathogens associated with periodontal diseases and is believed to stimulate bone resorption in vivo. Although Prevotella nigrescens has been implicated in periodontitis, its role in osteoclastogenesis has not been reported. In this study, we investigated the effects of lipopolysaccharide from P. nigrescens on the formation of osteoclasts and the production of cytokines related to osteoclast differentiation. MATERIAL AND METHODS: Mouse bone marrow mononuclear cells were cultured in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL), with or without lipopolysaccharide. Bone marrow mononuclear cells were also cocultured with calvarial osteoblastic cells in the presence or absence of lipopolysaccharide. Osteoclast formation was determined by tartrate-resistant acid phosphatase cytochemistry. The production of osteoprotegerin (OPG), M-CSF, tumor necrosis factor alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and prostaglandin E2 (PGE2) was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: P. nigrescens lipopolysaccharide inhibited osteoclast differentiation from bone marrow mononuclear cells cultured in the presence of M-CSF and RANKL. However, in the coculture system, P. nigrescens lipopolysaccharide stimulated osteoclastogenesis. Notably, P. nigrescens lipopolysaccharide decreased OPG production but increased TGF-beta secretion. In addition, treatment with P. nigrescens lipopolysaccharide increased PGE2 production during the late stage of the culture period. There was no difference in M-CSF and TNF-alpha production. CONCLUSION: These results demonstrate that P. nigrescens lipopolysaccharide stimulates osteoclastogenesis in the coculture system by decreasing the production of OPG and increasing the production of TGF-beta and PGE2. Through the mechanisms involving these factors, P. nigrescens lipopolysaccharide may cause alveolar bone resorption in periodontal diseases.

Prevalence of periodontopathic bacteria in aggressive periodontitis patients in a Chilean population.

BACKGROUND: Actinobacillus actinomycetemcomitans is considered a major etiologic agent of aggressive periodontitis (AgP). Other periodontopathic bacteria such as Porphyromonas gingivalis are also suspected of participating in aggressive periodontitis although the evidence to support this is controversial. The aim of the present study was to determine the prevalence of eight periodontopathic bacteria in Chilean patients with AgP. METHODS: Subgingival plaque samples were collected from 36 aggressive, 30 localized, and six generalized periodontitis patients. Samples from 17 advanced chronic periodontitis (CP) patients were taken as controls. Samples collected from the four deepest periodontal pockets in each patient were pooled in prereduced transport fluid (RTF) and cultured. Periodontal bacteria were primarily identified by colony morphology under stereoscopic microscope and rapid biochemical tests. The identity of some bacterial isolates was confirmed by colony polymerase chain reaction (PCR). RESULTS: AgP showed a significatively higher prevalence of C. rectus than CP (P = 0.036). The only statistical difference found was for C. rectus. Patients with AgP showed a higher, but not statistically significant, prevalence of P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. A similar prevalence in both groups of patients was observed for F. nucleatum and P. intermedia/nigrescens, and A. actinomycetemcomitans was less prevalent in AgP than CP patients. In localized AgP, P. intermedia/nigrescens, E. corrodens, F. nucleatum, and P. micros were the more prevalent pathogens in contrast to generalized AgP patients who harbored A. actinomycetemcomitans, P. gingivalis, and Capnocytophaga sp. as the most prevalent bacteria. CONCLUSIONS: C. rectus, P. gingivalis, E. corrodens, P. micros, and Capnocytophaga sp. were the most predominant periodontopathic bacteria of AgP in this Chilean population, but the only statistical difference found here between AgP and CP was for C. rectus, suggesting that the differences in clinical appearance may be caused by factors other than the microbiological composition of the subgingival plaque of these patients. In this study, the prevalence of A. actinomycetemcomitans was much lower than that of P. gingivalis.

Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR.

he aim of this study was to investigate the presence of four black-pigmented bacteria, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens, in endodontic infections by culture and polymerase chain reaction (PCR) analyses. Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Microbiological strict anaerobic techniques were used for serial dilution, plating, incubation, and identification. For PCR detection, the samples were analyzed using species-specific primers of 16S rDNA and the downstream intergenic spacer region. Culture and PCR detected the test species in 13/100 and 50/100 of the study teeth, respectively. The organisms were cultured from 11/50 (22%) of primarily infected root canal samples and from 2/50 (4%) of secondary root canal samples. PCR detection identified the target species in 32/50 (64%) and 18/50 (36%) of primary and secondary infections, respectively. P. gingivalis was rarely isolated by culture methods (1%), but was the most frequently identified test species by PCR (38%). Similarly, P. endodontalis was not recovered by culture from any tooth studied, but was detected by PCR in 25% of the sampled teeth. PCR-based identification also showed higher detection rates of P. intermedia (33%) and P. nigrescens (22%) than culture (13%). In conclusion, P. gingivalis, P. endodontalis, P. intermedia, and P. nigrescens were identified more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment. Also, a higher frequency of black-pigmented species was detected by PCR than by culture.

A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis.

Prevotella nigrescens, a gram-negative black-pigmented anaerobic rod, has frequently been isolated from periodontitis and periapical periodontitis lesions. We have isolated an exopolysaccharide-producing P. nigrescens, strain 22, from a chronic periodontitis lesion. The purpose of this study was to determine the chemical composition and function of the exopolysaccharide associated with this clinical isolate. The chemical composition and structure of the purified exopolysaccharide from strain 22 were determined by high performance liquid chromatography and methylation analysis. To define the biological function of this exopolysaccharide, a chemically induced exopolysaccharide nonproducing mutant, strain 328, which was derived from strain 22, was established. The biological effects of exopolysaccharide were determined by comparing the ability of strain 22, strain 328 or heat-killed strain 22 to form abscesses in mice and to interfere with the phagocytic activity of peripheral blood polymorphonuclear leukocytes. Chemical analysis showed that isolated exopolysaccharide consisted of mannose (521.6 microg/mg), glucose (25.6 microg/mg), fructose (65.8 microg/mg), galactose (12.5 microg/mg), arabinose (6.2 microg/mg), xylose (3.2 microg/mg), rhamnose (6.1 microg/mg), and ribose (0.6 microg/mg). Methylation analysis of exopolysaccharide indicated that the linkages of mannose were primarily (1-->2, 1-->6) (1-->2) (1-->6), and (1-->3). Strain 22 and, to a lesser extent, its heat-killed counterpart induced greater abscess formation in mice than strain 328, even though the enzymatic profile of strain 22 was similar to that of strain 328. The ability of strain 328 to induce abscess formation was restored by adding the purified exopolysaccharide isolated from strain 22 to the cell suspension of strain 328. Exopolysaccharide alone failed to induce abscess formation in mice. Further, strain 328 but not the untreated or heat-killed strain 22, was phagocytosed by polymorphonuclear leukocytes both in the presence and in the absence of opsonic factors. The results suggest that these polysaccharides isolated from strain 22, which primarily consisted of mannose, may play a key role in the development of the chronic inflammatory lesion from which this strain was isolated.