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

Abstract 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.     

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Abstract Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii , Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.     

Purification and biochemical characterization of gentisate 1,2-dioxygenase from Klebsiella pneumoniae M5a1

Abstract 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 serotypes 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. nigrescens .     

RAPD fingerprinting for the distinction of Prevotella intermedia sensu stricto from Prevotella nigrescens

A collection of 70 oral strains including reference strains and clinical isolates identified as Prevotella intermedia sensu lato was constituted to cover a large clinical and geographical diversity. Electrophoresis of the enzyme malate dehydrogenase allowed the identification of the 70 study strains as Prevotella intermedia sensu stricto (n= 36), Prevotella nigrescens (n= 31) and three unclassified strains. By using four primers, DNA fingerprints were generated from 20 strains as random amplified polymorphic DNA (RAPD). Matching co-migrating amplicon positions by pairwise comparison allowed the clustering of the fingerprints as two groups coincident with the P. intermedia/P. nigrescens assignment by enzyme electrophoresis of malate dehydrogenase. Our data suggest that isolates identified asP. intermedia sensu lato by conventional criteria can be speciated asP. intermedia sensu stricto or P. nigrescens by RAPD fingerprinting.     

Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens

Abstract The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed. Lf bound readily to whole cells of each species apparently via a high-affinity site and one or more low-affinity sites. P. gingivalis showed a lower affinity for Lf than the other two species ( P < 0.001). Virtually all strains of P. gingivalis completely degraded Lf under the conditions employed, whereas P. intermedia and P. nigrescens showed only partial degradation. These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P. gingivalis , is then degraded by cell-associated proteases. This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease. There was no association between the ability to degrade Lf and whether the strains had orginated from healthy or diseased oral sites.     

Susceptibility of Prevotella intermedia/Prevotella nigrescens (and Porphyromonas gingivalis) to propolis (bee glue) and other antimicrobial agents

Black-pigmented gram-negative anaerobes such as Porphyromonas gingivalis and Prevotella intermedia are suspected pathogens in adult periodontitis, whereas Prevotella nigrescens has been associated with health. Antimicrobial resistance among bacteria from this group has been reported in the past decade. This research aimed to evaluate and compare the susceptibility profile of 17 P. intermedia/P. nigrescens isolates recovered from patients with periodontitis and three reference strains to six antimicrobials, prescribed in dentistry in Brazil, and propolis (bee glue). The antimicrobial agents tested were tetracycline, penicillin, clindamycin, erythromycin, metronidazole, meropenem and six ethanolic extracts of propolis (EEPs) from Brazil. The reference strains P. gingivalis ATCC 33277 and P. intermedia ATCC 25611 were used for determination of minimum bactericidal concentration (MBC) and for time-kill assay to the EEPs. All of the strains were susceptible to penicillin, erythromycin, meropenem, metronidazole and 95% of them (n=19) to tetracycline. Thirty six percent (n=7) of the P. intermedia/P. nigrescens strains tested were resistant to clindamycin. As for propolis activity, all strains were susceptible and the minimum inhibitory concentration values ranged from 64 to 256 microg/mL. For the reference strains P. gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611 the MBC was 256 microg/mL and death was observed within 3 h of incubation for P. gingivalis and within 6 h for P. intermedia. The action of propolis (bee glue) against suspected periodontal pathogens suggests that it may be of clinical value.     

Synergy in biofilm formation between Fusobacterium nucleatum and Prevotella species

The formation of biofilm by anaerobic, Gram-negative bacteria in the subgingival crevice plays an important role in the development of chronic periodontitis. The aim of this study was to characterize the role of coaggregation between Fusobacterium nucleatum and Prevotella species in biofilm formation. Coaggregation between F. nucleatum and Prevotella species was determined by visual assay. Effect of co-culture of the species on biofilm formation was assessed by crystal violet staining. Effect of soluble factor on biofilm formation was also examined using culture supernatant and two-compartment co-culture separated by a porous membrane. Production of autoinducer-2 (AI-2) by the organisms was evaluated using Vibrio harveyi BB170. Cells of all F. nucleatum strains coaggregated with Prevotella intermedia or Prevotella nigrescens with a score of 1-4. Addition of ethylenediamine tetraacetic acid or l-lysine inhibited coaggregation. Coaggregation disappeared after heating of P. intermedia or P. nigrescens cells, or Proteinase K treatment of P. nigrescens cells. Co-culture of F. nucleatum ATCC 25586 with P. intermedia or P. nigrescens strains increased biofilm formation compared with single culture (p < 0.01); co-culture with culture supernatant of these strains, however, did not enhance biofilm formation by F. nucleatum. Production of AI-2 in Prevotella species was not related to enhancement of biofilm formation by F. nucleatum. These findings indicate that physical contact by coaggregation of F. nucleatum strains with P. intermedia or P. nigrescens plays a key role in the formation of biofilm by these strains.     

Distinction of Prevotella intermedia and Prevotella nigrescens from endodontic bacteremia through their fatty acid contents

Prevotella nigrescens has recently been recognized as a new species distinct from Prevotella intermedia. The distinction is based largely on DNA-DNA hybridization, electrophoretic migration of malate and glutamate dehydrogenase, and peptidase and lipase activities of type strains. Gas chromatography of cellular fatty acids can be a useful adjunct for characterization and identification of bacterial species. In the present study, cellular fatty acid profiles were determined for seven strains of P. intermedia and six strains of P. nigrescens. Six of these 13 strains were isolated from the root canal and blood of three patients during endodontic therapy of teeth with Asymptomatic apical periodontitis. The bacteria were cultivated anaerobically in 10 mL prereduced anaerobically sterilized peptone-yeast extract-glucose broth for 24 h. Dried cells of each isolate were methanolysed and their fatty acid contents determined by the Microbial Identification System software package by MIDI. The data were treated by principal component analysis, which distinguished P. nigrescensfromP. intermedia. Cellular fatty acid profiles of these strains of the species in blood matched the profiles of their respective root canal isolates, as demonstrated by Euclidean Distance Square assessment. This suggested that the organisms in the root canal had spread to the bloodstream during endodontic treatment.     

Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level

Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.     

Characterization of a bacteriocin produced by Prevotella nigrescens ATCC 25261

AIMS: To characterize the antimicrobial activity produced by Prevotella nigrescens ATCC 25261, and to evaluate its safety on cultured gingival fibroblasts. METHODS AND RESULTS: An antimicrobial activity was obtained from purifying the culture supernatant of Pr. nigrescens ATCC 25261. Purification of the active compound was achieved with ammonium sulphate precipitation followed by anion-exchange and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was relatively homogeneous, showing a protein with an approximate molecular weight of 41 kDa. The antimicrobial compound, named nigrescin, exhibited a bactericidal mode of action against Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces spp. Nigrescin was stable in a pH range between 6.5 and 9.5, at 100 degrees C for 10 min, and resistant to lyophilization. But its activity was lost after proteinase K treatment. Despite at very high concentrations beyond the minimum inhibitory concentration (MIC), nigrescin was not toxic to the gingival fibroblasts. CONCLUSION: Nigrescin is a novel bacteriocin produced by Pr. nigrescens ATCC 25261. It exhibits antimicrobial activity against species that are implicated in periodontal diseases. The absence of toxicity on the gingival fibroblasts suggests the possibility in using of nigrescin for an application in periodontal treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Novel evidence on nigrescin would make Pr. nigrescens ATCC 25261 attractive in biotechnological applications as an antimicrobial agent in clinical dentistry.     

Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.     

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.     

An examination of 'unidentified' Prevotella (formerly PINLO) using RAPD-PCR and partial 16S rRNA gene sequencing

Prevotella intermedia- and Prevotella nigrescens-like organisms (PINLO) have been described as organisms which are phenotypically and biochemically similar to P. intermedia and P. nigrescens and the species P. pallens was created to include some of them. Other PINLO groups which do not fit the definition of P. pallens exist, and in this study these 'unidentified' Prevotella sp. were compared with P. corporis, P. intermedia, P. nigrescens and P. pallens using commercial identification kits, GLC, RAPD-PCR and partial 16S rRNA gene sequencing. The Rapid ID 32 A and the RapID ANA II system both identified all 'unidentified' Prevotella as P. intermedia. Similarly they gave this identification to all the species tested (with the exception of P. corporis using the RapID ANA II system) clearly demonstrating biochemical similarities. Gas liquid chromatography (GLC) analysis of the volatile end-products of fermentation could not distinguish between strains. RAPD-PCR using arbitrary primer L10 demonstrated intra-species homogeneity within PINLO strains with amplification profiles which differed from other Prevotella species tested. Cluster analysis of the amplification profiles confirmed species divisions and yielded a distinct 'unidentified' Prevotella cluster. Comparison of partial 16S rDNA sequences displayed 98% sequence similarity between the 'unidentified' Prevotella strains, although 2 strains, HST 1156 and HST 2160 displayed 100% identity. The highest similarity between groups was seen between 'unidentified' Prevotella strains and P. corporis (approximately 94% similarity). The DNA techniques used here confirm that 'unidentified' Prevotella strains are distinct from the other species of Prevotella tested, including P. pallens. Partial 16S rDNA sequence comparisons suggested a close relationship with P. corporis.     

Isolation and characterization of hemolysin activated by reductant from Prevotella intermedia

The hemolysin from Prevotella intermedia was partially purified from culture supernatant and then characterized. The hemolysin produced a clear beta-hemolytic zone on a blood agar plate. Hemolytic activity was 2.5-fold greater in culture supernatant compared to that cell-associated. The isolation and purification procedure involved ammonium sulfate and polyethylene glycol precipitations and ion-exchange chromatographies on DEAE-Sephacel and CM-Sepharose. The activity of this hemolysin was stimulated by reductants such as cysteine, dithiothreitol, glutathione etc., and was lost upon oxidation. Trypsin or heat treatment resulted in complete inhibition of hemolytic activity. Ca(2+), Mg(2+) and EDTA did not affect the activity. The optimal pH of this hemolysin was 7.5.     

The influence of molecular oxygen exposure on the biology of Prevotella intermedia, with emphasis on its antibiotic susceptibility

AIM: This study focuses on investigating the molecular and physiological characteristics of Prevotella intermedia after molecular oxygen exposure (MOE) and the effect on drug susceptibility patterns. METHODS AND RESULTS: Samples of P. intermedia were used as parent strains: ATCC25611 and four clinical isolates. Strains adapted to oxidative stress by MOS were obtained by the enrichment technique. Drug susceptibility was evaluated by minimal inhibitory concentrations (MIC) using agar dilution. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to evaluate the genetic diversity of all strains and physiological analyses were made by sodiumdodecylsulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of crude, cell-free extracts. The genetic profile showed that lineages with altered MIC values were selected after MOE. Overall, we found significant decrease in drug susceptibility for the aero-strains against all tested antimicrobials (amoxicillin, amoxicillin+clavulanic acid, clindamycin, chloramphenicol, ertapenen and metronidazole). We also observed markedly different protein expression patterns between the parent and selected aero-strains. CONCLUSIONS: MOE induces changes in the genetic profile and protein expression patterns of P. intermedia that may also be linked to its drug resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of MOE on anaerobic bacterial physiology and behaviour may influence antimicrobial susceptibility patterns with potential consequences to antimicrobial chemotherapy.     

Antimicrobial activities of black-pigmented Gram-negative anaerobes

Abstract The antimicrobial activities of Prevotella intermedia and Porphyromonas gingivalis isolates were tested against other species of Gram-positive and Gram-negative anaerobes as well as against each other. Generally, Pr. intermedia possessed significantly higher antimicrobial activity than P. gingivalis . The strongest activity of P. gingivalis towards Gram-negative anaerobes was directed against Pr. intermedia . Cross-sensitivity between both species was observed with strains from different lesions. Antimicrobial activity towards strains of the same species was detected only with Pr. intermedia . No correlations were found between plasmid content and antimicrobial activity. It was concluded that the inhibitory potency of Pr. intermedia could be one reason for the high proportion of black-pigmented Gram-negative anaerobes in the subgingival flora of periodontitis lesions.     

Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov.

A total of 31 strains of Prevotella intermedia were subjected to DNA-DNA hybridization and were characterized by performing physiological tests and by performing a multilocus enzyme analysis, using malate dehydrogenase and glutamate dehydrogenase. All of the strains assigned to P. intermedia fermented glucose and sucrose, hydrolyzed starch but not esculin, and produced indole, acetic, isobutyric, isovaleric, and succinic acids as metabolic end products. The results of DNA reassociation experiments performed with the reference probe permitted separation of the strains into two well-defined homology groups. In addition, strains with DNAs that hybridized with DNA from strain ATCC 25611T (T = type strain) had high levels of peptidase activity and cleaved lipid substrates (4-methylumbelliferyl laurate and 4-methylumbellifelyl elaidate). Multilocus enzyme electrophoresis revealed two electromorphic profiles, one characteristic of strain ATCC 25611T and the other characteristic of strain ATCC 33563T. We propose that a new species, Prevotella nigrescens, should be created for the genetically distinct group of strains that hybridized with strain ATCC 33563T. Strain ATCC 33563 is designated the type strain of P. nigrescens.     

Aerotolerance of human clinical isolates of Prevotella spp

AIMS: To determine the influence of oxygen exposure and growth stages on oxygen tolerance for clinical and reference specimens of the genus Prevotella. METHODS AND RESULTS: Tolerance to oxygen exposure was evaluated along growth stages for a total of four Prevotella isolates constituted by two strains tested soon after isolation from periodontally compromised patients and two reference strains kept frozen (-86 degrees C) in our laboratory collection. Additionally, the recently recovered isolates were also assayed after 4 months exposed to oxygen during laboratory handling. On solid medium, recently recovered Prevotella specimens proved to be less tolerant to oxygen exposure soon after isolation than when exposed for 4 months to oxygen during laboratory handling. Oxygen resistance of reference strains maintained in the laboratory collection showed to be the highest when compared with the clinical isolates both before and after oxygen exposure. When oxygen tolerance was tested during growth, bacterial cells from the exponential phase were more tolerant. Differences were observed in protein patterns between clinical isolates soon after recovery and after laboratory handling as determined by SDS-PAGE of crude cell-free extracts. However, SOD activities were similar in all bacterial samples tested. CONCLUSIONS: The ability to adaptively change oxygen tolerance was observed for pathogenic specimens of Prevotella. This property may be considered a virulence factor. SIGNIFICANCE AND IMPACT OF THE STUDY: Adaptative aerotolerance of Prevotella as a factor for virulence and survival.