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FEMS Microbiology Letters Apr 1996Type strains and 62 clinical isolates of Prevotella intermedia and Prevotella nigrescens were typed with the use of genomic DNA fingerprints and rRNA gene probes. The... (Comparative Study)
Comparative Study
Type strains and 62 clinical isolates of Prevotella intermedia and Prevotella nigrescens were typed with the use of genomic DNA fingerprints and rRNA gene probes. The strains were further serotyped with monoclonal antibodies and characterized with SDS-PAGE, enzymatic activities, hemolysis and hemagglutination and coaggregation with Streptococcus and Actinomyces spp. P. intermedia and P. nigrescens were found to have distinct ribotype patterns which correspond to previously defined serotyupes I and II/III, respectively. No clear phenotypic difference related to hemolysis, hemagglutination and coaggregation with streptococcus and actinomyces species, or expression of aminopeptides and lipase was found between P. intermedia and P. migrescens.
Topics: Animals; Bacterial Adhesion; Hemagglutination; Hemolysis; Humans; In Vitro Techniques; Phenotype; Prevotella; Prevotella intermedia; RNA, Bacterial; RNA, Ribosomal; Rabbits; Serotyping; Species Specificity
PubMed: 8674976
DOI: 10.1111/j.1574-6968.1996.tb08140.x -
Oral Microbiology and Immunology Feb 1998A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the... (Comparative Study)
Comparative Study
A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P. intermedia sensu stricto and Prevotella nigrescens. Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest. PCR-DNA probe assays were used to speciate each strain as P. intermedia sensu stricto or P. nigrescens. By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016-0.064 microgram/ml range. In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5-96 micrograms/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016-0.38 microgram/ml, indicating a production of beta-lactamase as confirmed by nitrocefin tests. Of these beta-lactamase-producing strains, 20% (5/25) were identified as P. intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P. nigrescens. Our results provide support for the major role of P. nigrescens in the failure of therapy using beta-lactam antibiotics.
Topics: Anti-Bacterial Agents; DNA, Bacterial; Dose-Response Relationship, Drug; Microbial Sensitivity Tests; Polymerase Chain Reaction; Prevotella; Prevotella intermedia; beta-Lactamases
PubMed: 9573820
DOI: 10.1111/j.1399-302x.1998.tb00748.x -
Journal of Periodontal Research Aug 2006Lipopolysaccharide is thought to be a major virulence factor of pathogens associated with periodontal diseases and is believed to stimulate bone resorption in vivo....
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.
Topics: Alveolar Bone Loss; Animals; Bone Marrow Cells; Carrier Proteins; Cell Differentiation; Cells, Cultured; Coculture Techniques; Dinoprostone; Female; Glycoproteins; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Inbred ICR; Osteoblasts; Osteoclasts; Osteoprotegerin; Prevotella nigrescens; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transforming Growth Factor beta
PubMed: 16827722
DOI: 10.1111/j.1600-0765.2006.00876.x -
Journal of Clinical Periodontology Feb 2001The occurrence of Prevotella intermedia (Pi) and Prevotella nigrescens (Pn) in relation to natural gingivitis, gingival health and 14-day experimental gingivitis was...
AIM
The occurrence of Prevotella intermedia (Pi) and Prevotella nigrescens (Pn) in relation to natural gingivitis, gingival health and 14-day experimental gingivitis was investigated in 25 non-dental students.
MATERIALS AND METHODS
Samples were taken from the dorsum of the tongue, the tonsils (or tonsillar area), and the supra- and subgingival plaque.
RESULTS
The microbiological results show that 73% of the samples were positive for the bacterial species presumed to be Pi and/or Pn. In natural gingivitis, gingival health and in experimental gingivitis 25, 23 and 25 subjects were found to be positive for Pi and/or Pn, respectively. The results of the 889 isolates that were successfully purified and differentiated, show that almost all subjects were colonized with Pn whereas approximately half of the study population harboured Pi. These 2 species were isolated from both dental plaque and mucosal sites and were found to colonize the oral cavity simultaneously.
CONCLUSION
In natural gingivitis, at the start and after 14 days of experimental gingivitis, Pn was the predominant micro-organism.
Topics: Adolescent; Adult; Bacterial Typing Techniques; Colony Count, Microbial; Dental Plaque; Gingiva; Gingivitis; Humans; Mouth Mucosa; Prevotella; Prevotella intermedia
PubMed: 11168745
DOI: 10.1034/j.1600-051x.2001.028002189.x -
Infection and Immunity Feb 1999To survive and multiply within their hosts, pathogens must possess efficient iron-scavenging mechanisms. In the present study, we investigate the capacity of Prevotella...
To survive and multiply within their hosts, pathogens must possess efficient iron-scavenging mechanisms. In the present study, we investigate the capacity of Prevotella nigrescens and Prevotella intermedia to use various sources of iron for growth and characterize the transferrin-binding activity of P. nigrescens. Iron-saturated human transferrin and lactoferrin, but not ferric chloride and the iron-free form of transferrin, could be used as sources of iron by P. nigrescens and P. intermedia. Neither siderophore activity nor ferric reductase activity could be detected in P. nigrescens and P. intermedia. However, both species showed transferrin-binding activity as well as the capacity to proteolytically cleave transferrin. To various extents, all strains of P. nigrescens and P. intermedia tested demonstrated transferrin-binding activity. The activity was heat and protease sensitive. The capacity of P. nigrescens to bind transferrin was decreased when cells were grown in the presence of hemin. Preincubation of bacterial cells with hemin, hemoglobin, lactoferrin, fibrinogen, immunoglobulin G, or laminin did not affect transferrin-binding activity. The transferrin-binding protein could be extracted from the cell surface of P. nigrescens by treatment with a zwitterionic detergent. Subjecting the cell surface extract to affinity chromatography on an agarose-transferrin column revealed that it contained a protein having an estimated molecular mass of 37 kDa and possessing transferrin-binding activity. The transferrin-binding activity of P. nigrescens and P. intermedia may permit the bacteria to obtain iron for survival and growth in periodontal pockets.
Topics: Culture Media; Humans; Iron; Prevotella; Prevotella intermedia; Transferrin
PubMed: 9916061
DOI: 10.1128/IAI.67.2.576-580.1999 -
Journal of Medical Microbiology Nov 1999A specific 16S rDNA PCR and subsequent hybridisation reaction was designed to discriminate between strains of Prevotella intermedia (n = 15) and P. nigrescens (n = 15)....
A specific 16S rDNA PCR and subsequent hybridisation reaction was designed to discriminate between strains of Prevotella intermedia (n = 15) and P. nigrescens (n = 15). This technique was then used to detect the presence of these two bacterial species in acute suppurative oral infection. A total of 36 pus samples aspirated from 26 peri-apical abscesses, three root canals, three periodontal abscesses, two cases of refractory periodontitis, one cyst and one haematoma was examined. A portion of the pus sample was processed by PCR and the remainder of the specimen was subjected to routine culture. The PCR-based technique gave an identical pattern of detection of P. intermedia or P. nigrescens to that obtained by culture for 30 of the 36 specimens. Either P. intermedia or P. nigrescens was present in 14 samples and neither species was detected in 16 samples. In the remaining six samples the PCR method indicated the presence of one (n = 3) or both (n = 3) of the Prevotella species but neither or only one species was isolated by culture. It is concluded that the presence of P. intermedia and P. nigrescens in pus can be detected rapidly and specifically by direct PCR amplification of 16S rDNA. P. nigrescens was detected more frequently than P. intermedia in suppurative peri-apical infection both by culture and PCR.
Topics: Bacteroidaceae Infections; DNA Primers; DNA, Bacterial; DNA, Ribosomal; Humans; Nucleic Acid Hybridization; Periapical Abscess; Periodontal Abscess; Periodontitis; Polymerase Chain Reaction; Prevotella; Prevotella intermedia; Pulpitis; RNA, Bacterial; RNA, Ribosomal, 16S; Reproducibility of Results; Suppuration
PubMed: 10535646
DOI: 10.1099/00222615-48-11-1017 -
Oral Diseases Dec 1996Prevotella intermedia has been reported to be associated with periodontal disease whilst P. nigrescens has predominantly been isolated from more specific conditions and... (Comparative Study)
Comparative Study
OBJECTIVE
Prevotella intermedia has been reported to be associated with periodontal disease whilst P. nigrescens has predominantly been isolated from more specific conditions and healthy sites. The aim of the present study was to compare the enzyme activity of these species.
MATERIALS AND METHODS
Nine strains of P. intermedia and 12 strains of P. nigrescens were studied. Lipolytic, saccharolytic, nucleolytic and proteolytic activity was determined by traditional microbiological and chromogenic substrate methods.
RESULTS
All strains hydrolysed gelatine, casein, DNA and RNA. Lipase activity was produced by all strains except P. nigrescens ATCC 33563T. Lipolytic activity of P. nigrescens strains decreased as the environmental glucose concentration was increased. Only two strains, both P. intermedia, hydrolysed benzyl-arg-rho-nitroanilide. All strains hydrolysed alkaline rho-nitrophenolphosphate (except P. intermedia DAL100), produced glycylprolyl dipeptidase activity and demonstrated elastase-like activity. All but three strains (2 P. intermedia and I P. nigrescens) hydrolysed suc-ala-ala-pro-phe-rho-nitroanilide. Overall, no qualitatively analysed enzyme activity was exclusive to all strains of either species. Quantitatively analysed activity exhibited a high degree of variability both within and between species.
CONCLUSIONS
P. intermedia and P. nigrescens degrade natural and synthetic substrates, but intra- and interspecies activity is variable.
Topics: Alkaline Phosphatase; Chymotrypsin; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Glycosaminoglycans; Hot Temperature; Hydrolases; Lipase; Periodontal Diseases; Porphyromonas gingivalis; Prevotella; Prevotella intermedia; Species Specificity; Virulence
PubMed: 9171510
DOI: 10.1111/j.1601-0825.1996.tb00237.x -
Oral Microbiology and Immunology Apr 1997The gram-negative, anaerobic bacterium Prevotella intermedia plays an important role in the progression of periodontitis, whereas the etiological role of the closely...
The gram-negative, anaerobic bacterium Prevotella intermedia plays an important role in the progression of periodontitis, whereas the etiological role of the closely related but phenotypically indistinguishable species Prevotella nigrescens is controversial. To differentiate between these species properly, 16S rDNA/RNA directed, computer-optimized oligonucleotides were designed and tested with 26 P. intermedia, 26 P. nigrescens and a number of closely and more distantly related strains. The oligonucleotides were used as primers in a polymerase chain reaction and could be demonstrated to be species specific with a detection limit of 50 bacterial cells, which could also be detected when diluted 1:10(5) with different plaque bacteria. In addition, the described oligonucleotides were digoxigenin-labeled at the 3' end and used as DNA probes in a dot blot hybridization assay. This assay, although slightly less sensitive than the polymerase chain reaction-based method, gave species-specific reactions and also allowed (semi-)quantification of bacterial cells in clinical specimens.
Topics: Bacterial Typing Techniques; Base Sequence; DNA, Ribosomal; Molecular Sequence Data; Nucleic Acid Hybridization; Oligonucleotide Probes; Periodontitis; Polymerase Chain Reaction; Prevotella; Prevotella intermedia; RNA, Ribosomal, 16S; Species Specificity
PubMed: 9227136
DOI: 10.1111/j.1399-302x.1997.tb00627.x -
Journal of Clinical Microbiology Jul 1997The purpose of this study was to construct PCR-DNA probe assays specific for Prevotella intermedia sensu stricto and Prevotella nigrescens based on the ability of...
The purpose of this study was to construct PCR-DNA probe assays specific for Prevotella intermedia sensu stricto and Prevotella nigrescens based on the ability of randomly amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers. The strategy included four steps: (i) construction of first-generation DNA probes from a 850-bp RAPD marker for P. intermedia sensu stricto and a 1,300-bp RAPD marker for P. nigrescens, (ii) cloning and sequencing of each RAPD marker, (iii) designing of primer pairs flanking specific internal sequences of 754 bp for P. intermedia sensu stricto and of ca. 1,100 bp for P. nigrescens, and (iv) synthesis (by PCR amplification) and digoxigenin labeling of quantities of DNA probes 754 and ca. 1,100 bp in size. The PCR-DNA probe assays combine either PCR amplification of a 754-bp specific sequence in the genomic DNA of strains of P. intermedia sensu stricto and hybridization with the 754-bp digoxigenin-labeled probe or amplification of a ca. 1,100-bp sequence of P. nigrescens and hybridization with the ca. 1,100-bp probe. Specific hybridization was observed with the amplified DNAs from 25 strains of P. intermedia and 24 strains of P. nigrescens, and no reaction was observed with the PCR products from 20 foreign species. The PCR-DNA probe assays described here should allow a highly specific and sensitive detection of P. intermedia sensu stricto and P. nigrescens in mixed infections.
Topics: Bacterial Typing Techniques; Bacteroidaceae Infections; Base Sequence; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Prevotella; Prevotella intermedia
PubMed: 9196214
DOI: 10.1128/jcm.35.7.1876-1882.1997 -
Letters in Applied Microbiology Sep 2009To identify the gene that encodes nigrescin, a bacteriocin produced by Prevotella nigrescens ATCC 25261.
AIM
To identify the gene that encodes nigrescin, a bacteriocin produced by Prevotella nigrescens ATCC 25261.
METHODS AND RESULTS
Each open reading frame (ORF) of the nig gene cluster (nigA, nigB, nigC and nigD) was transferred into an expression vector. The recombinant proteins encoded by nigA, nigB, nigC and nigD were purified and assayed for bacteriocin activity against Porphyromonas gingivalis. The ORFs of the nig gene cluster in Pr. nigrescens ATCC 25261 were re-analysed. It revealed that the position of nig ORFs was similar to previously designated locations, except that the start codon of nigC was reassigned. The new nigC gene started at the nucleotide base position 2454 and stopped at position 3608 (the position designated is relative to the first nucleotide base of the nig locus) and putatively encoded a protein with a predicted molecular mass of 41.9 kDa. The N-terminal 6xHistidine-tag recombinant proteins of NigA, NigB, NigC and NigD were overexpressed in Escherichia coli BL21 star (DE3) and were purified using Ni-NTA resins. Only recombinant NigC showed inhibitory activity against P. gingivalis A244 with minimal inhibition concentration (MIC) of 40 microg ml(-1).
CONCLUSION
These results indicate that nigC is the gene that encodes nigrescin.
SIGNIFICANCE AND IMPACT OF THE STUDY
This is the first report that indicates that the gene nigC codes for nigrescin, a bacteriocin produced by Pr. nigrescens ATCC 25261.
Topics: Anti-Bacterial Agents; Bacteriocins; Chromatography, Affinity; Cloning, Molecular; Codon, Initiator; Codon, Terminator; DNA, Bacterial; Escherichia coli; Gene Expression; Genes, Bacterial; Microbial Sensitivity Tests; Molecular Sequence Data; Molecular Weight; Multigene Family; Porphyromonas gingivalis; Prevotella nigrescens; Recombinant Fusion Proteins; Sequence Analysis, DNA
PubMed: 19531060
DOI: 10.1111/j.1472-765X.2009.02657.x