Properties of hemagglutination by Prevotella melaninogenica

Although Prevotella melaninogenica belongs to the commensal oral microbiota, some strains possess putative virulence factors. For example, we have previously described fimbriated, hemagglutinating strains of P. melaninogenica, isolated from patients with periodontal disease. The aim of this investigation was to compare some chemical and physical properties of hemagglutination (HA) of P. melaninogenica with those of other pigmented gram-negative anaerobes. HA of 13 P. melaninogenica strains proved to be considerably weaker than that of the major periodontal pathogen, Porphyromonas gingivalis. Vigorous shaking reduced HA of shaken cells but the shaken supernatant had the same hemagglutinating activity as non-shaken cells. The hemagglutinating agent on P. melaninogenica seemed to be a protein, which can be separated from the cell and binds to lactose-, galactose-, and raffinose-containing carbohydrates on the erythrocytes. Adherence to epithelial cells did not differ significantly between the hemagglutinating and non-hemagglutinating strains of P. melaninogenica. Although P. melaninogenica is able to agglutinate erythrocytes, this potential virulence factor is of a considerably lower magnitude than that of major periodontal pathogens.     

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.     

Occurrence of plasmids in black-pigmented Gram-negative anaerobes

Abstract Black-pigmented Gram-negative anaerobes are causative agents of pyogenic infections and are closely linked to various forms of periodontal diseases. Whereas many studies have shown a high incidence of plasmids in intestinal Bacteroides spp., there have been only a few reports of plasmid analyses in pigmented Gram-negative anaerobes. According to previous reports and confirmed in this study, plasmids can be present in Porphyromonas asaccharolytica, Prevotella intermedia, Pr. melaninogenica , and B. levii but have not been detected in P. gingivalis or other black-pigmented species. There were no correlations between plasmids and phenotypes such as resistance to antibiotics or bacteriocinogenicity. The highest carriage rate was found in isolates from cases of chronic otitis media, but the relationship between this site of infection and a high incidence of plasmids could be incidental. The size of plasmids ranged from 1.5 to 29 MDa. Plasmids with molecular weight > 10 MDa were described for the first time in these organisms. Repeated plasmid analyses showed that the plasmid patterns were generally stable.     

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.     

Pigmented Prevotella species in the periodontally healthy oral cavity

Abstract Pigmented Prevotella spp. have been connected with oral infections as well as being part of the healthy gingival flora. The aim of this study was to determine the presence of pigmented Prevotella spp. in saliva and gingival crevice samples from periodontally healthy adults. Twelve Caucasian female subjects (mean age 28 years, range 21–36 years) with no pockets ≥ 4 mm, nor bleeding after probing were selected for this study. Paraffin-stimulate saliva was collected first; then, a pooled subgingival bacterial sample was taken with a sterile curette from mesiobuccal surfaces of all first molars. The samples were inoculated on to non-selective and selective media and incubated anaerobically. The most frequent species isolated were Pr. melaninogenica, Pr. intermedia and Pr. loescheii , found in 11, ten and nine subjects, respectively. The mean percentages of the total cultivable anaerobic microflora in salivary/subgingival samples were 14.7/0.6 for Pr. melaninogenica , 3.1/5.3 for Pr. intermedia and 2.6/1.2 for Pr. loescheii. Pr. denticola was found in one saliva sample and Pr. corporis , in two subgingival samples only. The number of different pigmented Prevotella spp. in the same mouth was 2–4 (mean 2.75). In conclusion, Pr. melaninogenica, Pr. intermedia and Pr. loescheii seem to be common microorganisms in the periodontally healthy oral cavity.     

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.     

Black-pigmented Gram-negative anaerobes in genito-urinary tract and pelvic infections

Abstract Black-pigmented Gram-negative anaerobes are part of the normal vaginal flora and contribute to a range of superficial and deep genital infections. Prevotella melaninogenica is found in moderate numbers (10⁴⁻⁶ cfu/g) in healthy women; the numbers and detection rates increase in anaerobic vaginosis (where it may be a significant contributor to changed microbial metabolic activity that gives the signs and symptoms of this condition) and in other vaginal infective conditions. Pr. melaninogenica is also part of the mixed flora in deep pelvic infections: endometritis, post-partum and post-abortal uterine infections; salpingitis and tubo-ovarian abscesses; PID and pelvic abscesses. Porphyromonas asaccharolytica is probably not a vaginal commensal, but may be isolated from patients with vaginal or pelvic disease. It is more specifically associated with superficial abscesses (e.g. Bartholin's abscess) and ulcers of the genitalia and perineum. P. asaccharolytica was the commonest species isolated from men with genital ulcers of various primary causes and ranging in severity from superficial balanitis/balanoposthitis to synergic gangrene.     

Contribution of periodontal pathogens on tongue dorsa analyzed with real-time PCR to oral malodor

Oral malodor is considered to originate primarily from tongue microbiota populations. However, the relationship between oral malodor and tongue microbiota remains unclear. In this study, tongue periodontal pathogens were analyzed via real-time PCR, and the association between oral malodor and tongue periodontal pathogens, including Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola, was examined. The subject population consisted of 29 individuals with and 10 healthy persons without oral malodor. Oral malodor was assessed by organoleptic test and volatile sulfur compound (VSC) levels as measured by gas chromatography. Real-time PCR was conducted for anaerobes in tongue biofilm samples employing a LightCycler system; furthermore, bacterial proportion served as a quantitative parameter. Among the five anaerobes, only T. forsythia displayed higher proportions in malodor subjects than corresponding values in healthy controls. Proportions of P. intermedia and P. nigrescens correlated strongly with hydrogen sulfide concentration. Proportions of P. gingivalis and P. nigrescens also exhibited strong correlation with methyl mercaptan concentration. The correlation coefficient between the proportion of the total of the five anaerobes and total VSC level (r = 0.88) was greater than that between bacterial proportion and organoleptic score (r = 0.29). When a linear regression analysis was performed utilizing the proportion of each of the five periodontal pathogens as an independent variable, the explanatory power of these independent variables revealed 81% for total VSC level and 16% for organoleptic score. These results suggest that these five periodontal pathogens on tongue dorsa may contribute greatly to VSC production.     

Black-pigmented Gram-negative anaerobes in periodontitis

Abstract Black-pigmented Gram-negative anaerobes have been associated with periodontal disease and tooth loss since they were first isolated by Burdon in 1928. Porphyromonas gingivalis , which is usually not isolated from children, adolescents or adults with no periodontal breakdown, has been recognized as one of the most important periodontopathogens. Its presence is strongly correlated with deep periodontal pockets, which are assumed to be its main habitat. Correlations have been shown also with attachment loss, clinical inflammation and serum antibody levels, indicating an aetiological role in the periodontal disease. Their pathogenicity in animal models resembling periodontal disease is documented. They are frequently isolated from periodontal abscesses. The relationship between Prevotella intermedia and periodontal disease is not clear. It is frequently isolated from advanced periodontitis, often as the only black-pigmented Gram-negative anaerobic species; however, the prevalence in adults with no periodontal breakdown is high. It is found frequently in periodontal abscesses and in acute necrotizing and ulcerative gingivitis. Serogroup I is found predominantly in deep periodontal pockets, whereas all serogroups (I–III) are found in shallow pockets and gingivitis. No conclusive difference in pathogenicity between serogroups has been found. Pr. melaninogenica, Pr. denticola and Pr. loescheii are frequently found in the gingival crevice in preschool children and other age groups with gingivitis, but are seldom found in deep periodontal pockets.     

INTERACTIONS BETWEEN UV‐B EXPOSURE AND PHOSPHORUS NUTRITION. I. EFFECTS ON GROWTH,PHOSPHATE UPTAKE,AND CHLOROPHYLL FLUORESCENCE1

The interactive effects of P starvation and exposure to UV radiation on growth rates, quantum efficiency of PSII electron transport, and P‐uptake capacity of the chlorophyte microalga Dunaliella tertiolecta Butcher are presented. Ultraviolet radiation did not in itself cause marked changes in growth rate, though it did induce changes in the effective quantum yield of PSII. Depriving cells of phosphate resulted in significant changes in all parameters examined. The decline of growth rate and fluorescence parameters after P starvation was significantly faster in the presence of UV radiation. Ultraviolet radiation also stimulated the magnitude of the transient changes in chl fluorescence (nutrient‐induced fluorescence transient) exhibited by P‐starved cells after resupply of that nutrient.     

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.     

Antilysozyme activity of anaerobic bacteria from fecal microflora in man

For the first time anaerobic bacteria of the fecal microflora in man have been found be capable of inactivating lysozyme. The presence of this antilysozyme sign has been noted in both Gram-positive anaerobes (Bifidobacterium, Propionibacterium, Eubacterium, Actinomyces israelii) and in Gram-negative anaerobes (Bacteroids, Prevotella melaninogenica). The expression of antilysozyme activity in the anaerobes under study has been determined. The possible biological role of this sign of the indigenous intestinal microflora has been discussed.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

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.     

Resistance of selected strains of Pseudomonas aeruginosa to low-intensity ultraviolet radiation.

The resistances of 10 strains of Pseudomonas aeruginosa and other microorganisms to an ultraviolet (UV) intensity of 100 muW/cm2 were determined. Organisms were exposed in 2- or 15-ml saline suspensions contained in uncapped polyethylene bottles for increasing periods of time, and the surviving fractions were enumerated. Decimal reduction times were calculated by regression analysis, using the least-squares method. The 10 strains of P. aeruginosa, compared with Micrococcus radiodurans and Candida albicans, were very susceptible to low-intensity UV radiation. Results from this study showed that a UV intensity of 100 muW/cm2 penetrated saline suspensions up to 40 mm deep sufficiently to kill high levels of microbial cells, especially P. aeruginosa cells. These results allowed us to design a system for determining and monitoring the sterilization capability of low-intensity UV radiation. In our particular case, UV proved to be an efficient mode for sterilizing saline suspensions of P. aeruginosa in polyethylene bottles. The significance and application of these findings with regard to supporting UV as a sterilant are discussed.     

Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry

Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells.     

Induction of oxidative DNA damage in anaerobes

We compared oxidative DNA damage in strictly anaerobic Prevotella melaninogenica, aerotolerant anaerobic Bacteroides fragilis, and facultative anaerobic Salmonella typhimurium after exposure to O2 or H2O2. Using HPLC with electrochemical detection, we measured 8-hydroxydeoxyguanosine (8OHdG) as a damage marker. O2 induced 8OHdG in P. melaninogenica but not in B. fragilis, which shows catalase activity, or in S. typhimurium. In P. melaninogenica, with catalase, O2 induced less 8OHdG; superoxide dismutase had no effect; with glucose and glucose oxidase, O2 induced more 8OHdG. H2O2 also markedly increased 8OHdG. O2 was suggested to induce 8OHdG through H2O2. O2 or H2O2 decreased survival only in P. melaninogenica. Highly sensitive to oxidative stress, P. melaninogenica could prove useful for investigating oxidative DNA damage.     

Emended descriptions of Prevotella denticola, Prevotella loescheii, Prevotella veroralis, and Prevotella melaninogenica.

During studies of human periodontal disease, a number of bacterial strains were encountered that, on the basis of results of standard biochemical tests, appeared to be Prevotella buccalis, Prevotella denticola, Prevotella melaninogenica, or Prevotella loescheii. However, use of the standard biochemical tests, cellular fatty acid analyses, and the polyacrylamide gel electrophoresis patterns of soluble proteins resulted in conflicting identifications of these strains. The results of tests for cellobiose fermentation, inulin fermentation, and pigment production were responsible for most of the discordant results. Cellular fatty acid analyses in which the Microbial Identification System was used did not differentiate these strains from validly described species, even though separate library entries were created for them. DNA reassociation determinations in which the S1 nuclease procedure was used showed that cellobiose fermentation and pigment production are variable among strains of P. melaninogenica and P. denticola and that fermentation of xylan is not a reliable characteristic for differentiating P. buccalis from Prevotella veroralis. In contrast to previous indications, most strains of P. veroralis do not ferment xylan. These species can be differentiated by DNA-DNA reassociation and by cellular fatty acid analysis, using the Microbial Identification System, but differentiation by currently described phenotypic characteristics is not reliable. Similarly, P. loescheii and the genetically distinct (but closely related) D1C-20 group cannot be distinguished reliably from each other or from P. veroralis, P. denticola, and P. melaninogenica on the basis of currently described phenotypic tests other than cellular fatty acid composition or, for some species, electrophoretic patterns of soluble whole-cell proteins.     

Oxygen induces mutation in a strict anaerobe, Prevotella melaninogenica

Strict anaerobes are highly sensitive to oxygen, but the mutagenicity of oxygen in strict anaerobes has not been well understood. Prevotella melaninogenica, a strict anaerobe, is susceptible to oxygen and shows an increase in oxidative DNA damage upon exposure to oxygen. In this study, we have investigated the mutagenicity of oxygen and the types of mutations induced by oxygen. Exposure to oxygen decreased cell survival and increased the levels of 8-oxo-deoxyguanosine (8-oxodG). The frequency of rifampicin-resistant mutants was markedly increased after exposure to oxygen. After sequencing a 254-bp fragment of the rpoB gene, which encodes the beta subunit of bacterial RNA polymerase, a target molecule of rifampicin, we found that most mutants induced by oxygen had GC to TA transversions, a signature of 8-oxodG. In addition, all detected single-nucleotide changes would lead to amino acid changes that confer rifampicin resistance. These results indicate that oxygen is mutagenic in a strict anaerobe, P. melaninogenica, and its mutagenic characteristics could be analyzed with this experimental system.     

Rapid and simple detection of eight major periodontal pathogens by the loop-mediated isothermal amplification method

Loop-mediated isothermal amplification (LAMP) was applied to develop a rapid and simple detection system for eight periodontal pathogens: Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia. Primers were designed from the 16S ribosomal RNA gene for each pathogen, and the LAMP amplified the targets specifically and efficiently under isothermal condition at 64 degrees C. To simplify the manipulation of LAMP examination, boiled cells and intact cells suspended in phosphate-buffered saline (PBS) were tested as templates besides extracted DNA template. The detection limits were 1-10 cells per tube using extracted DNA template. However, LAMP methods using boiled cells and intact cells required 10-100 and 100-1000 cells per tube, respectively. LAMPs for A. actinomycetemcomitans, P. gingivalis and P. intermedia were then applied to clinical plaque samples, and the method demonstrated equal or higher sensitivity compared with the conventional real-time PCR method. These findings suggest the usefulness of the LAMP method for the rapid and simple microbiological diagnosis of periodontitis, and the possibility of LAMP examination without the DNA extraction step.