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.     

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.     

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.     

Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.     

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.     

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.     

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.     

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.     

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 .     

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.     

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.     

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.     

Hemolytic and hemagglutinating activities of Prevotella intermedia and Prevotella nigrescens

A total of 91 isolates of Prevotella intermedia or Prevotella nigrescens from subgingival sites were identified by PCR using primers specific for sequences of 16S rRNA. The hemolytic and hemagglutinating activities of the P. intermedia isolates exhibited significantly higher levels compared to those of the P. nigrescens isolates by quantitative analysis. The hemagglutinin gene (phg) was found in 23 of 26 P. intermedia isolates (88.5%), whereas it was found in only two of 44 isolates (4.5%) of P. nigrescens. The high hemolytic and hemagglutinating activities of P. intermedia may be involved in the pathogenicity of P. intermedia in the progression of periodontal disease.     

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

Abstract 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.     

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.     

酯酶试验与聚合酶链反应鉴别中间普氏菌的比较研究

目的 研究脂酶试验和常规生化方法与聚合酶链反应比较在鉴别中间普氏菌Pi中的敏感度和特异度。方法 对207株牙周临床分离的产黑色素G厌氧杆菌分别进行脂酶试验、常规生化法鉴定和16srRNA特异引物PCR鉴定。结果 207株实验菌中PCR鉴定出Pi97株,脂酶试验检测出Pi126株,其中有85株PCR阳性,其敏感度为87．6％,特异度为63．7％。常规生化方法和脂酶试验共同鉴定出Pi80株,其中有55株PCR为阳性,其敏感度为67％,特异度为86．4％。结论 脂酶与常规生化方法对Pi的鉴别能力低于PCR（ P＜0．05）,尚不能作为Pi菌种的可靠鉴定方法。     

Multilocus enzyme electrophoretic analysis and DNA-DNA reassociation of strains designated Prevotella intermedia

Strains of Prevotella intermedia which have been characterized previously by a variety of biochemical and chemical techniques were subjected to multilocus enzyme electrophoresis and DNA-DNA reassociation. Two separate groups of strains were discernible. One had high homology with the type strain ATCC 25611 DNA probe and electrophoretically fast migrating malate dehydrogenase (MDH) (3.8–4.0 cm) and glutamate dehydrogenase (GDH) (3.2–3.4 cm) bands. The other group of strains hybridized with the DNA probe of reference strain ATCC 33563 and possessed slower moving enzymes (MDH, 3.0 cm; GDH, 1.4–1.6 cm). These results indicate that strains currently identified as P. intermedia comprise at least two geno-species, and that the criteria used to define this species are inadequate.     

Classification and typing methods of black-pigmented Gram-negative anaerobes

Abstract Until recently, black-pigmented Gram-negative anaerobes were classified as ‘black-pigmented Bacteroides ’. At present, 11 distinct species are recognized in this group. Because of major differences with Bacteroides fragilis , the type species of the genus Bacteroides , new genera have been proposed: Porphyromonas for three asaccharolytic species, and Prevotella for the saccharolytic species. Typing methods have been developed for some species of black-pigmented Gram-negative anaerobes. These include biotyping and serotyping, but relatively few types can be distinguished with these methods. Recently, DNA restriction endonuclease analysis has been used for typing of P. gingivalis, Pr. intermedia and P. endodontalis strains. Great heterogeneity was observed within all three species. This typing method can be useful for epidemiological studies.     

Dextran sodium sulfate-induced inflammation alters the expression of proteins by intestinal Escherichia coli strains in a gnotobiotic mouse model

To identify Escherichia coli proteins involved in adaptation to intestinal inflammation, mice were monoassociated with the colitogenic E. coli strain UNC or with the probiotic E. coli strain Nissle. Intestinal inflammation was induced by treating the mice with 3.5% dextran sodium sulfate (DSS). Differentially expressed proteins in E. coli strains collected from cecal contents were identified by 2-dimensional difference gel electrophoresis. In both strains, acute inflammation led to the downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower concentrations of bacterial fermentation products in their cecal contents than control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterized protein YggE. NfuA expression was 3-fold higher in E. coli strains from DSS-treated than from control mice. Reporter experiments confirmed the induction of nfuA in response to iron deprivation, mimicking Fe-S cluster destruction by inflammation. YggE expression, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC. This was confirmed by in vitro reporter gene assays indicating that Nissle is better equipped to cope with oxidative stress than UNC. Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold-higher than those of UNC. Levels of indole resulting from the TnaA reaction were higher in control animals associated with E. coli Nissle. Because of its anti-inflammatory effect, indole is hypothesized to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle.