• Korean Journal of Microbiology
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• v.55 no.1
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• pp.58-60
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• 2019

Anaerobic Gram-negative bacterium Prevotella intermedia is a part of normal flora of the oral cavity and associated with various types of oral and systemic diseases. We present here a draft genome sequence of P. intermedia ATCC 15032, originally isolated from cervicofacial actinomycosis. The genome is 2,848,426 bp in length and has a GC content of 43.45%. The genome includes 2,358 protein-coding genes, 5 rRNAs, and 43 tRNA. The sequence information will provide important clues in understanding the genome diversity within the bacterial species, and genetic basis for phenotypic differences among P. intermedia strains.

• International Journal of Oral Biology
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• v.40 no.4
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• pp.205-210
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• 2015

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase ${\beta}$-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC $25611^T$. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

• Restorative Dentistry and Endodontics
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• v.22 no.1
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• pp.413-427
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• 1997

Porphyromonas endodontalis and Prevotella intermedia are black-pigmented anaerobic gram negative rods which have been isolated from infected root canals and submucous abscesses of endodontic origin. And they are associated with clinical symptoms such as pain, percussion, and foul odor. It has been reported that there are 3 serotypes according to capsule membrane in P. endodontalis and 2 DNA homology groups and 3 serotypes in P. intermedia, but there is no data available regarding genetic diversity for the species P. endodontalis and P. intermedia. The purpose of this study is to investigate genetic diversities between individual strains of P. endodontalis and P. intermedia which are indistinguishable by serotyping and biotyping using bacterial DNA restriction endonuclease analysis. 45 teeth with at least one clinical symptoms, with single canal, and with pulp necrosis were sampled. For sampling bacteria, access cavity was prepared after disinfecting tooth and its surroundings. Then the paper point was inserted to the apex of the canal, leave there for 15 seconds, and finally it was placed into PRAS Ringer's solution and PBS solution. P. endodontalis and P. intermedia were identified by biochemical test and IIF after subculturing black and brown colonies which were produced after 7 days of incubation on BAP in anaerobic chamber. P. endodontalis and P. intermedia strains were grown in BHI broth and whole genomic DNA was extracted by phenol-chloroform extraction technique and digested by restriction endonuclease, Eco RI and Pst I. The resulting DNA fragments were separated by agarose gel electrophoresis, stained with EtBr and photographed under UV light. The results were as follows : 1. In both P. endodontalis and P. intermedia, different serotypes could be found within a root canal of same patient. 2. There were obvious genetic heterogeneity within a patient and within a serotype in both P. endodontalis and P. intermedia. 3. P. endodontalis serotype c, isolated from different patients, exhibited limited genotypic diversity.

• Journal of Periodontal and Implant Science
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• v.40 no.3
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• pp.119-124
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• 2010

Purpose: In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-$\alpha$ production in differentiated THP-1 cells, a human monocytic cell line. Methods: LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-$\alpha$ and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-$\alpha$ and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis. Results: Leptin enhanced P. intermedia LPS-induced TNF-$\alpha$ production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-$\alpha$ expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-$\alpha$ production was not mediated by the leptin receptor. Conclusions: The ability of leptin to enhance P. intermedia LPS-induced TNF-$\alpha$ production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

• Journal of Periodontal and Implant Science
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• v.41 no.3
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• pp.157-163
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• 2011

Purpose: Curcumin is known to exert numerous biological effects including anti-inflammatory activity. In this study, we investigated the effects of curcumin on the production of interleukin-6 (IL-6) by murine macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory periodontal disease, and sought to determine the underlying mechanisms of action. Methods: LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time polymerase chain reaction to detect IL-6 mRNA expression. $I{\kappa}B-{\alpha}$ degradation, nuclear translocation of NF-${\kappa}B$ subunits, and STAT1 phosphorylation were characterized via immunoblotting. DNA-binding of NF-${\kappa}B$ was also analyzed. Results: Curcumin strongly suppressed the production of IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW 264.7 cells. Curcumin did not inhibit the degradation of $I{\kappa}B-{\alpha}$ induced by P. intermedia LPS. Curcumin blocked NF-${\kappa}B$ signaling through the inhibition of nuclear translocation of NF-${\kappa}B$ p50 subunit. Curcumin also attenuated DNA binding activity of p50 and p65 subunits and suppressed STAT1 phosphorylation. Conclusions: Although further study is required to explore the detailed mechanism of action, curcumin may contribute to blockade of the host-destructive processes mediated by IL-6 and appears to have potential therapeutic values in the treatment of inflammatory periodontal disease.

• Journal of Periodontal and Implant Science
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• v.35 no.1
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• pp.21-30
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• 2005

Matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes and implicated in the remodeling and degradation of extracellular matrix under both physiological and pathological conditions. Connective tissue degradation in periodontal diseases is thought to be due to excessive MMP activities over their specific inhibitors. The effects of lipopolysaccharide (LPS) from Prevotella intermedia, one of the major putative pathogens of periodontitis, on the expression of mRNA for MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human gingival and periodontal ligament fibroblasts were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNAs encoding MMP-1, -2, -3, -10, and -14 was increased in human gingival fibroblasts treated with p. intermedia LPS, whereas MMP-11 and TIMP-2 mRNA expression was decreased in these cells stimulated with LPS. P. intermedia LPS increased the MMP-1, -2, -10, -11, and -14 mRNA expression and decreased TIMP-1 and -2 mRNA expression in human periodontal ligament fibroblasts. These findings imply that P. intermedia LPS may play an important role in the connective tissue degradation in periodontitis.

• Restorative Dentistry and Endodontics
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• v.22 no.2
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• pp.693-701
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• 1997

In 1992, Prevotella intermedia was shown to be comprised of another spoecies now known as Prevotella nigrescens. Strain ATCC 33563 is now designated the type strain of P. nigrescens while strain ATCC 25611 is remains the type strain of P. intermedia. The purpose of this study was to find the differences in protein profiles of P. intermedia and P. nigrescens, using sodium dodecyl sulfate polyacrylamide gel electrophoresis, which can be used for differentiation of those two species. A partial amino acid sequence of the 18.6 kDa protein band, which was specific in P. nigrescens, was also determined. The cellular proteins were extracted from the cell pellets of pure cultures of P. intermedia. and P. nigrescens by either sonication or being shaken continuously for 20 min at $21^{\circ}C$ with 1 % SDS or being boiled for 3 min with 1 % SDS. SDS-PAGE was performed according to the method of laemmli using either 12% (w/v) gels or 18% (w/v) gels. Results were as follows ; 1. The similar electrophoretic protein profiles were shown by 3 cellular protein extraction methods for each strain. (Fig. 1 and 2) 2. the 18.6 kDa band which was specific only in P. nigrescens could be used for the differentiation of P. intermedia. and P. nigrescens. (Fig. 1 and 2, Table 1) 3. A total of 4 different tryptic fragments from the 18.6 kDa protein were sequenced. the resulting amino acid sequences were fragment 1.GNPVNIGGEW, 2.FNVVR, 3.NYLT-VAPY, and 4.GGDNVTTYQVLPEIGYN. By comparison to the sequences of known proteins in the Swiss-Prot database and PIR database. 90 % matching between fragment 1 and serine hydroxymethyl transferase(P24060) in the Swiss-Prot, and 90% matching between fragment 1 and glycine hydroxymethyl transferase(S15203) in the PIR were shown, but the identity and function of the 18.6 kDa protein remains unknown.

• Journal of Periodontal and Implant Science
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• v.43 no.4
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• pp.191-197
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• 2013

Purpose: Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action. Methods: LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory ${\kappa}B$ $(I{\kappa}B)-{\alpha}$ degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting. Results: Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of $I{\kappa}B-{\alpha}$ induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling. Conclusions: Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-${\kappa}B$ and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease.

• Journal of Chest Surgery
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• v.33 no.5
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• pp.440-444
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• 2000

A 54-year-old male patient was admitted due to dyspnea with chest pain and reddish swelling in the right lteral neck and right upper which developed 2 or 3 days age. He was treated with acupuncture in the neck about one week ago prior to admission. CT scans of the chest showed density of air and abnormal soft tissue which suggested abscess of the anterior mediastinum and subcutaneous tissue. He underwent cervical and mediastinal drainage with closed thoracostomy and antibiotic therapy. Black-pigmente anaerobic nonspore-forming gram-negative bacilli were isolated from the mediastinal pus and were identified as Prevotella intermedia/nigrescens upon performing biochemical tests and API rapid ID 32A (bioMeriux, France) kit. He underwent decortication of the right side because of loculated empyema on 41st postoperative day. He was fully recovered and discharged on the 82nd hospital day. This was thought to be another case of descending necrotizing mediastinitis not caused by peritonsilar abscess but by cellulitis developed after acupuncture. Early deterction of mediastinits and aggressive drainage of mediastinal abscess are important.

• Journal of the korean academy of Pediatric Dentistry
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• v.33 no.2
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• pp.304-310
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• 2006

Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$, and screened for recombinant clones using heminbinding activity by plating onto hemin-containing agar. Approximately 5,000 recombinant E. coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phonotype. The 2.5kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHI, BglII, EcoRI, HindIII and PstI-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity It needs further investigation to clarify the mechanisms of heme uptake in P. intermedia.