Species-specificity of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens

Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P. intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P. nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens. These isolates were not recognised by mAbs 39BI1.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.     

Incidence of Prevotella intermedia and Prevotella nigrescens in periodontal health and disease

The incidence of black-pigmented rods (BPRs), especially Prevotella intermedia and Prevotella nigrescens, in periodontal health and disease were examined. Furthermore, the degradative enzyme activities of P. intermedia were compared among the strains from periodontal health and disease. Microbiological specimens were collected from subgingival crevice or periodontal pocket by paper point. The BPRs were found in 71.1% of periodontally healthy subjects (n=45), and in 47.1% of healthy sites (n=34) and 87.8% of active sites (n=41) among periodontally diseased patients. Porphyromonas gingivalis was detected only in active sites of periodontally diseased patients (17.8% of 180 strains). P. intermedia was the predominant BPR in both healthy and active sites (37.3 and 41.7%, respectively) of the patients. However, P. nigrescens was the predominant BPR (70.5% of 173 strains) in periodontally healthy subjects. The enzyme activities of esterase, esterase-lipase, acid-phosphatase and alpha-fucosidase of P. intermedia strains isolated from active sites in patients were significantly higher (P<0.05) than those of healthy subjects. The results suggest that P. intermedia might increase the activity of degradative enzymes under a certain condition and support the progression of periodontitis.     

The lipopolysaccharide of Porphyromonas gingivalis is not antigenically cross-reactive with that of other species

Large numbers of Porphyromonas, Prevotella, and Bacteroides strains were screened by 3 monoclonal antibodies (MAbs) and 8 rabbit antisera raised against Porphyromonas gingivalis, in order to detect any possible recognition of non-P. gingivalis surface antigens by these immunoreagents. All three MAbs, which were LPS-specific, extensively recognized LPS from 10 P. gingivalis strains in immunoblotting, whereas they recognized none of the 34 non-P. gingivalis strains. Rabbit antisera were similarly specific for P. gingivalis cells in immunofluorescence and with LPS in grid-blotting, but several of them recognized LPS from one Prevotella melaninogenica and 5 Prevotella intermedia strains in Western blotting. Since several pre-immune sera and an irrelevant serum raised to a Streptococcus species recognized up to 5 of these preparations, we exclude that the reactions were due to antigens shared by P. gingivalis and Prevotella. Rather, we consider that they were false-positive reactions due to natural antibodies, stimulated in a non-specific manner upon immunization with P. gingivalis, in animals whose immune systems were sensitized to Prevotella species before immunization.     

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 Fusobacterium warrants further investigation.     

Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.     

Occurrence of Prevotella nigrescens and Prevotella intermedia in infections of endodontic origin

The occurrence of Prevotella intermedia and Prevotella nigrescens in endodontic infections was studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell protein to distinguish between the species. Previous studies have shown an association between black-pigmented bacteria (BPB) and endodontic infections and that Prevotella intermedia (previously known as Bacteroides intermedius) was the most commonly isolated BPB. Recently, however, strains identified as P. intermedia were shown to in fact be composed of two separate species, P. intermedia and P. nigrescens. Fifty-six strains of BPB isolated from endodontic infections and previously identified as P. intermedia were used in this study. Following SDS-PAGE, P. nigrescens showed a unique 18.6 kDa band that was used to differentiate P. nigrescens from P. intermedia. Of the 56 strains of BPB, 41 (73.2%) were identified as P. nigrescens and 15 (26.8%) as P. intermedia. This study confirms that P. nigrescens, and not P. intermedia, is the BPB most often isolated from infections of endodontic origin.     

Infusion therapy with milrinone in the home care setting for patients who have advanced heart failure

The pathogenicity of 14 isolates identified as Prevotella intermedia or Prevotella nigrescens by serogrouping using monoclonal antibodies was compared in a tissue cage model in rabbits. Seven strains from periodontal abscesses, 5 strains from deep periodontal pockets and 2 strains from gingivitis were tested in the animal model comprising 6 Teflon tissue cages implanted on the back each of 34 rabbits. A total of 10(5)-10(8) cells of P. intermedia or P. nigrescens strains were inoculated alone or together with either Actinobacillus actinomycetemcomitans or Streptococcus mitis. Five strains of Porphyromonas gingivalis were used as a reference. The infectivity was recorded as pus formation and log viable count in aspirated material for 3, 7 and 14 days. None of the Prevotella strains inoculated in monoculture survived more than 3 days, and they had no capacity to produce abscess. P. intermedia or P. nigrescens strains in combination with A. actinomycetemcomitans produced abscesses in 33-100% and with S. mitis in 42-100%. No difference in abscess formation or log viable count in samples after 14 days was recorded between serogroup I (P. intermedia) and serogroup II and III (P. nigrescens). The infectivity of P. intermedia or P. nigresceas strains did not differ whether they were isolated from periodontal abscess, periodontal pocket or gingivitis. P. intermedia and P. nigrescens strains produced abscesses in combination with a facultative anaerobic strain and appears to have a similar pathogenicity in the wound chamber model in rabbits.     

LDLR, GYPA, HBGG, D7S8 and GC allele and genotype frequencies in the northwest Italian population

Anaerobic culture is employed routinely in the primary isolation of periodontal pathogenic bacteria. However, little or no data exist on the relative abilities of the Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.), the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.), and the AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) systems to grow important periodontal species, including Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Bacteroides forsythus, Eubacterium species, Campylobacter species, Fusobacterium species, and Peptostreptococcus micros. A total of 78 specimens from advanced periodontitis lesions were collected anaerobically, plated on enriched blood agar medium, and incubated at 35 degrees C for 5 to 7 days in each anaerobic culture system. The three culture systems were equally efficient in isolating Porphyromonas gingivalis and Prevotella intermedia/nigrescens. The Coy anaerobic chamber yielded the highest proportional recoveries of Campylobacter (P = 0.0001; nonparametric analysis of variance) and Eubacterium (P = 0.009). The Coy anaerobic chamber and the GasPak system demonstrated higher proportional recoveries of Bacteroides forsythus (P = 0.0006) and Peptostreptococcus micros (P = 0.0001) than the AnaeroPack system. The AnaeroPack system was most efficient in growing Fusobacterium species (P = 0.0001). Overall, the Coy anaerobic chamber and the GasPak system showed the highest proportional recoveries of putative periodontal pathogens, but the recoveries by the various anaerobic test systems varied considerably from sample to sample.     

Validity of epidemiological classification: study of asymptomatic properties of a discriminant criterion

Aiming to assess the presence of selected anaerobic microorganisms in root canals of human teeth with chronic apical periodontitis. 25 central and lateral upper incisors presenting with radiographic evidence of chronic apical periodontitis were studied. The pulp chamber was opened under aseptic conditions and samples of the root canal content were collected with sterile absorbent paper points, which were placed and dispersed in test tubes containing reduced transport medium RTT. Aliquots were dried on glass slides and stained by indirect immunofluorescence for detection of Actinomyces viscosus, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia. The results showed a positive indirect immunofluorescence reaction in 24 of the 25 samples. Fourteen were positive for the specie Actinomyces viscosus, 12 for Prevotella intermedia, 10 for Fusobacterium nucleatum and 4 for Porphyromonas gingivalis. A semiquantitative assay was easily implemented for assessment of degree of infection by the organisms in individual cases.     

Reduced serum IgG reactivities with bacteria from dental plaque in HIV-infected persons with periodontitis

Serum samples were obtained from 44 HIV-seropositive (HIV+) and 37 HIV-seronegative (HIV-) persons that were grouped according to periodontal status. Serum IgG and IgA reactivities towards Streptococcus mutans, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis. Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum were measured by means of ELISA. HIV+ persons with chronic marginal periodontitis showed significantly lower IgG reactivities to the periodontal pathogens A. actinomycetemcomitans, P. gingivalis, P. intermedia and F. nucleatum as compared with their HIV- counterparts (p < 0.05). Specific serum IgA reactivities were similar in the two periodontitis groups, except for P. nigrescens where the HIV+ group with chronic marginal periodontitis had lower values than their systemically healthy counterparts (p < 0.05). The results indicate that HIV infection affects the humoral serum immune responses against bacteria in dental plaque; the depressed antibody responses may contribute to the increased susceptibility for periodontal infections in HIV-infected patients.     

Evidence for the absence of hyaluronidase activity in Porphyromonas gingivalis

The aim of the present study was to evaluate the ability of Porphyromonas gingivalis to degrade hyaluronic acid. No hyaluronidase activity was detected using a turbidimetric method, whereas a standard plate assay showed a positive reaction for P. gingivalis. We postulated that the high proteolytic activity of P. gingivalis may account for this observation. A modified plate assay was designed to avoid false-positive reactions caused by proteolytic bacteria. The new assay, based on the formation of a water-insoluble salt between hyaluronic acid and the polyanion cetylpyridinium chloride, indicated that P. gingivalis does not have hyaluronidase activity. By this modified plate method, it was found that among 24 different oral bacterial species tested, Propionibacterium acnes and Prevotella oris were the only species that possess hyaluronidase activity.     

In vitro antimicrobial susceptibility of Porphyromonas gingivalis to azithromycin, a novel macrolide

The in vitro antimicrobial susceptibility of Porphyromonas gingivalis to azithromycin, a new macrolide antibiotic of a new class known as azalides, was investigated by the agar dilution method on Brucella agar. Eighty-two P. gingivalis strains, 79 recent oral isolates, 1 nonoral isolate and 2 reference strains were included in the study. Azithromycin was highly effective against P. gingivalis. All strains were inhibited at 1.0 microgram/ml of azithromycin or less. The minimal inhibitory concentrations were 0.25 microgram/ml for 50% and 0.5 microgram/ml for 90%. These in vitro data as well as the favorable pharmacokinetics of azithromycin indicate that this new oral macrolide might be a good candidate for future clinical trials aiming to eradicate P. gingivalis from refractory periodontitis.     

Immune response to anaerobic bacteria in patients with peritonsillar cellulitis and abscess

The role of four oral organisms (Fusobacterium nucleatum. Prevotella intermedia, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) was investigated in 19 children with peritonsillar abscess, and 17 with peritonsillar cellulitis. Antibody titers to these organisms were measured by enzyme- linked immunosorbent assay in the patient, as well as in 32 control patients. Serum levels in the patients were determined at day 1 and 42-56 days later. Significantly higher antibody levels to F. nucleatum and P. intermedia were found in the second serum sample of patients with peritonsillar cellulitis or abscess, as compared to their first sample or the levels of antibodies in controls. A total of 136 bacterial isolates, 100 anaerobic and 36 aerobic were isolated from the 19 peritonsillar abscesses. Anaerobic bacteria were found in all abscesses, and they were mixed with aerobic bacteria in 5 (26%). F. nucleatum was recovered in 14 (74%) abscesses and P. intermedia was isolated in 13 (68%). The elevated antibody levels to F. nucleatum and P. intermedia, known oral pathogens, suggest a pathogenic role for these organisms in peritonsillar infections.     

Binding and utilization of human transferrin by Prevotella nigrescens

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.     

Investigations into the occurrence and the antibiotic susceptibility of gram negative anaerobes of the genera Bacteroides, Prevotella, Porphyromonas and Fusobacterium in specimens obtained from diseased animals

From different samples of 247 diseased animals (cattle, sheep, goat, horse, pig, dog, cat, rodent, zoo-animals), 410 strains of gram-negative anaerobes were cultured. 297 isolates (72.4%) could be differentiated to the species level by using cultural-biochemical methods, gaschromatography and cell-wall-lipidanalysis. They belonged to 29 different species. For an additional 113 strains (27.6%) only the genus could be determined. Bacteria belonging to the genus Fusobacterium occurred with the highest isolation rates (36% of all strains) in the samples examined, followed by Bacteroides spp. (26.1%), Prevotella spp. (19.9%) and Prophyromonas spp. (17.8%). Fusobacterium necrophorum was the single species isolated most frequently. Antibiotic susceptibility tests by E-test were performed on 100 strains belonging to the the above mentioned genera. Of these strains 18% were resistant to penicillin and 20% to tetracycline. The resistant strains belonged mainly to the Bacteroides fragilis-group. Resistant rates to most other antimicrobial agents tested were Amoxicillin in combination with clavulanic acid: 1%, Chloramphenicol: 3%, Clindamycin:8%. All 100 selected strains proved to be susceptible to Metronidazol.     

Increased activity of a new chlorofluoroquinolone, BAY y 3118, compared with activities of ciprofloxacin, sparfloxacin, and other antimicrobial agents against anaerobic bacteria

A total of 435 clinical isolates of anaerobes were tested with a broth microdilution method to determine the activity of BAY y 3118 compared with those of other agents against anaerobic bacteria. All strains of Bacteroides capillosus, Prevotella spp., Porphyromonas spp., Fusobacterium spp., Clostridium spp., Eubacterium spp., Peptostreptococcus spp., and Veillonella parvula were susceptible (MICs of < or = 2 micrograms/ml) to BAY y 3118. Against the 315 strains of the Bacteroides fragilis group, five strains required elevated MICs (> or = 4 micrograms/ml) of BAY y 3118. Only imipenem and metronidazole were active against all anaerobes. Overall, BAY y 3118 was more active than ciprofloxacin, sparfloxacin, piperacillin, cefotaxime, and clindamycin against the test isolates.     

In vitro activity of Bay 12-8039, a new 8-methoxyquinolone, compared to the activities of 11 other oral antimicrobial agents against 390 aerobic and anaerobic bacteria isolated from human and animal bite wound skin and soft tissue infections in humans

The in vitro activity of Bay 12-8039, a new oral 8-methoxyquinolone, was compared to the activities of 11 other oral antimicrobial agents (ciprofloxacin, levofloxacin, ofloxacin, sparfloxacin, azithromycin, clarithromycin, amoxicillin clavulanate, penicillin, cefuroxime, cefpodoxime, and doxycycline) against 250 aerobic and 140 anaerobic bacteria recently isolated from animal and human bite wound infections. Bay 12-8039 was active against all aerobic isolates, both gram-positive and gram-negative isolates, at < or = 1.0 microg/ml (MICs at which 90% of isolates are inhibited [MIC90s < or = 0.25 microg/ml) and was active against most anaerobes at < or = 0.5 microg/ml; the exceptions were Fusobacterium nucleatum and other Fusobacterium species (MIC90s, > or = 4.0 microg/ml) and one strain of Prevotella loeschii (MICs, 2.0 microg/ml). In comparison, the other quinolones tested had similar in vitro activities against the aerobic strains but were less active against the anaerobes, including peptostreptococci, Porphyromonas species, and Prevotella species. The fusobacteria were relatively resistant to all the antimicrobial agents tested except penicillin G (one penicillinase-producing strain of F. nucleatum was found) and amoxicillin clavulanate.     

Low back pain in college athletes. A prospective study correlating lower extremity overuse or acquired ligamentous laxity with low back pain

This study compared the presence of 6 periodontopathic bacteria in whole saliva and subgingival plaque of 202 subjects. The test bacteria were identified using a 16S rRNA-based PCR detection method. Each study subject contributed a whole saliva sample and a paper point sample pooled from the deepest periodontal pocket in each quadrant of the dentition. The kappa test revealed a fair agreement between the presence of Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola in whole saliva and periodontal pocket samples (kappa > 0.4). The McNemar test showed that the differences between sample types were due to a more frequent detection of the 3 organisms in whole saliva than in periodontal pocket samples (P < 0.01). Prevotella nigrescens also was detected more frequently in whole saliva than in periodontal pocket samples (P < 0.01; McNemar test). Although little agreement between samples was found for Actinobacillus actinomycetemcomitans and Bacteroides forsythus (kappa < or = 0.4), neither whole saliva nor pocket samples showed better detection for these 2 species (P < 0.01, McNemar test). The results indicate that whole saliva is superior to pooled periodontal pocket samples to detect P. gingivalis, P. intermedia, P. nigrescens, and T. denticola in the oral cavity. The detection of oral A. actinomycetemcomitans and B. forsythus with reasonably good accuracy may require both whole saliva and periodontal pocket samples.     

Classification of black-pigmented anaerobes by pyrolysis mass spectrometry (PMS) and conventional tests (CTs)

Clinical (66: dental 53; vaginal 4; wound 9) and reference (5*) strains of pigmented Gram-negative anaerobic bacilli were examined in pyrolysis mass spectrometry (PMS) and conventional tests (CTs). The strains were identified in CTs as: Prevotella intermedia (48*); Pr. melaninogenica (1); Pr. corporis (7); Porphyromonas asaccharolytica (12*); P. endodontalis (1*) and P. gingivalis (2*). Numerical classification based on CTs resolved five clusters comprising strains identified as (I) Pr. corporis, (II) Pr. melaninogenica, (III) Pr. intermedia, (IV) P. gingivalis and (V) P. asaccharolytica and P. endodontalis. Numerical classification based on PMS showed a similar division, with decreasing homogeneity in the order Pr. intermedia, Pr. corporis, P. asaccharolytica, in agreement with the ordering of homogeneity for these species in CTs. PMS clusters corresponding the Porphyromonas spp. were clearly distinct from those of Prevotella spp. PMS and CT classifications disagreed on cluster membership for only six of the strains. PMS identification from blind challenge sets agreed with conventional identification for 64 of 67 strains.     

Molecular analysis of rifampin-resistant Mycobacterium tuberculosis isolated from Korea by polymerase chain reaction-single strand conformation polymorphism sequence analysis

The production of and sensitivity to bacteriocin-like activity among 44 strains of black-pigmented anaerobes isolated from periodontal sites were evaluated by both an overlay and an agar diffusion method. The species studied were Porphyromonas gingivalis, Prevotella intermedia and the closely related species Pr. nigrescens. Pr. intermedia strains (90%) produced bacteriocin-like activity against Pr. nigrescens and all Pr. nigrescens were active against Pr. intermedia. Both species showed a high degree of activity against P. gingivalis, whereas only one P. gingivalis strain produced bacteriocin-like activity against either of the other two species. Both Pr. nigrescens and Pr. intermedia showed some activity (40% and 20%, respectively) against other strains of the same species. Such bacteriocin production might be expected to influence the distribution of these black-pigmented species in vivo. Of 224 periodontal sites sampled, only 2.6% yielded mixed cultures of black-pigmented species and of these only one strain, a P. gingivalis isolate, produced bacteriocin-like activity against any of the other strains isolated from these sites. These data support the concept that local production of bacteriocin-like activity in vivo may contribute to the selection of the black-pigmented bacterial profile in subgingival sites.