Evolutionary and Pan-genome Analysis of Three Important Black-pigmented Periodontal Pathogens.

OBJECTIVE: To analyse the pan-genome of three black-pigmented periodontal pathogens: Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens. METHODS: Pan-genome analyses of 66, 33 and 5 publicly available whole-genome sequences of P. gingivalis, P. intermedia and P. nigrescens, respectively, were performed using Pan-genome Analysis Pipeline software (version 1.2.1; Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China). Phylogenetic trees were constructed based on the entire pan-genome and single nucleotide polymorphisms within the core genome. The distribution and abundance of virulence genes in the core and dispensable genomes were also compared in the three species. RESULTS: All three species possess an open pan-genome. The core genome of P. gingivalis, P. intermedia and P. nigrescens included 1001, 1514 and 1745 orthologous groups, respectively, which were mainly related to basic cellular functions such as metabolism. The dispensable genome of P. gingivalis, P. intermedia and P. nigrescens was composed of 2814, 2689 and 906 orthologous groups, respectively, and it was enriched in genes involved in pathogenicity or with unknown functions. Phylogenetic trees presented a clear separation of P. gingivalis, P. intermedia and P. nigrescens, verifying the reclassification of the black-pigmented species. Furthermore, the three species shared almost the same virulence factors involved in adhesion, proteolysis and evasion of host defences. Some of these virulence genes were conserved across species whereas others belonged to the dispensable genome, which might be acquired through horizontal gene transfer. CONCLUSION: This study highlighted the usefulness of pan-genome analysis to infer evolutionary cues for black-pigmented species, indicating their homology and phylogenomic diversity.

Fumarate and nitrite reduction by Prevotella nigrescens and Prevotella buccae isolated from Chronic Periodontitis patients.

OBJECTIVE: This study is an investigation of anaerobic nitrite and fumarate reduction/respiration abilities of two characterised Prevotella species namely Prevotella nigrescens (SS6B) and Prevotella buccae (GS6B) isolated from the periodontal pockets of chronic periodontitis (ChP) patients. METHODS: Isolation and identification of the periodontal bacteria from 20 patients showing clinical symptoms of ChP. Characterisation of anaerobic nitrite and fumarate reduction was done in P. nigrescens (SS6B) and P. buccae (GS6B) using reduction assays, inhibition assays with use of specific inhibitors, growth assays and enzyme activity assays. Degenerate PCR was used to detect and amplify nitrite reductase (nrfA) and fumarate reductase (frdA) gene sequences in these Prevotella isolates. In addition, molecular and in silico analysis of the amplified anaerobic reductase gene sequences was performed using NCBI conserved domain analysis, Interpro database and MegaX. RESULTS: We provided experimental evidence for presence of active nitrite and fumarate reductase activities through enzyme activity, reduction, inhibitor and growth assays. Moreover, we were able to detect presence of 505 bps nrfA gene fragment and 400 bps frdA gene fragment in these Prevotella spp. These fragments show similarity to multiheme ammonia forming cytochrome c nitrite reductases and fumarate reductases flavoprotein subunit, respectively. CONCLUSION: Anaerobic nitrite and fumarate respiration abilities in P. nigrescens and P. buccae isolates appear to be important for detoxification process and growth, respectively.

Reciprocal allostery arising from a bienzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens.

Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bienzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional interreliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. Here, we have further investigated the complex allosteric communication demonstrated by this bifunctional enzyme. We observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small-angle X-ray scattering (SAXS) experiments, we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual-function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.

Frequency of putative periodontal pathogens among type 1 diabetes mellitus: a case-control study.

OBJECTIVE: The aim of the present study is to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria viz., Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens. To fulfil the above objective, polymerase Chain reaction using the primers targeting 16S rRNA gene of the bacterial species was performed with the subgingival plaque collected from the permanent first molars of type 1 diabetic children and age matched healthy children. RESULTS: The prevalence of periodontal pathogens in diabetic and healthy children was 6% and 16% for E. corrodens, 18% and 36% for C. rectus, 2% and 2% for P. intermedia, 4% and 0%, for P. nigrescens respectively. Statistically, significant difference was not observed for the prevalence of all the four periodontal pathogens between type 1 diabetic and healthy children (P = 1.00). The results of the present study thus reveal a negative correlation of type I diabetes to periodontitis in association to Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens.

Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids.

Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthetic pathway of aromatic amino acids, and a chorismate mutase (CM), functioning at a branch of this pathway leading to the synthesis of tyrosine and phenylalanine. In this study, we characterized this P. nigrescens enzyme and found that its two catalytic activities exhibit substantial hetero-interdependence and that the separation of its two distinct catalytic domains results in a dramatic loss of both DAH7PS and CM activities. The protein displayed a unique dimeric assembly, with dimerization solely via the CM domain. Small angle X-ray scattering (SAXS)-based structural analysis of this protein indicated a DAH7PS-CM hetero-interaction between the DAH7PS and CM domains, unlike the homo-association between DAH7PS domains normally observed for other DAH7PS proteins. This hetero-interaction provides a structural basis for the functional interdependence between the two domains observed here. Moreover, we observed that DAH7PS is allosterically inhibited by prephenate, the product of the CM-catalyzed reaction. This allostery was accompanied by a striking conformational change as observed by SAXS, implying that altering the hetero-domain interaction underpins the allosteric inhibition. We conclude that for this C-terminal CM-linked DAH7PS, catalytic function and allosteric regulation appear to be delivered by a common mechanism, revealing a distinct and efficient evolutionary strategy to utilize the functional advantages of a bifunctional enzyme.

Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species.

Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1ß, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.

Phenotypic identification of periodontal Prevotella intermedia/nigrescens group isolates validated by MALDI-TOF mass spectrometry.

The accuracy of a phenotypic scheme to recognize periodontal Prevotella intermedia/nigrescens group clinical isolates on primary isolation culture plates was assessed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 84 fresh subgingival isolates from 23 chronic periodontitis patients were presumptively recognized on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens based on their dark-pigmented colony morphology, brick-red autofluorescence under long-wave ultraviolet light, and a negative fluorescence test for lactose production. The presumptive P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF MS analysis using Bruker MALDI Biotyper analytic software containing mass spectra for P. intermedia and Prevotella nigrescens in its reference library of bacterial protein profiles. Using a ≥1.7 log score agreement threshold, 60 (71.4%) of the presumptive P. intermedia/nigrescens clinical isolates were confirmed as either P. intermedia (25 isolates) or P. nigrescens (35 isolates). All isolates with a <1.7 log score were also identified as P. intermedia or P. nigrescens from the top choice designated on the MALDI Biotyper most likely species identification list. These MALDI-TOF MS findings document the ability of the phenotypic scheme to correctly recognize most periodontal P. intermedia/nigrescens group clinical isolates on primary isolation culture plates.

Population-Genomic Insights into Variation in Prevotella intermedia and Prevotella nigrescens Isolates and Its Association with Periodontal Disease.

High-throughput sequencing has helped to reveal the close relationship between Prevotella and periodontal disease, but the roles of subspecies diversity and genomic variation within this genus in periodontal diseases still need to be investigated. We performed a comparative genome analysis of 48 Prevotella intermedia and Prevotella nigrescens isolates that from the same cohort of subjects to identify the main drivers of their pathogenicity and adaptation to different environments. The comparisons were done between two species and between disease and health based on pooled sequences. The results showed that both P. intermedia and P. nigrescens have highly dynamic genomes and can take up various exogenous factors through horizontal gene transfer. The major differences between disease-derived and health-derived samples of P. intermedia and P. nigrescens were factors related to genome modification and recombination, indicating that the Prevotella isolates from disease sites may be more capable of genomic reconstruction. We also identified genetic elements specific to each sample, and found that disease groups had more unique virulence factors related to capsule and lipopolysaccharide synthesis, secretion systems, proteinases, and toxins, suggesting that strains from disease sites may have more specific virulence, particularly for P. intermedia. The differentially represented pathways between samples from disease and health were related to energy metabolism, carbohydrate and lipid metabolism, and amino acid metabolism, consistent with data from the whole subgingival microbiome in periodontal disease and health. Disease-derived samples had gained or lost several metabolic genes compared to healthy-derived samples, which could be linked with the difference in virulence performance between diseased and healthy sample groups. Our findings suggest that P. intermedia and P. nigrescens may serve as "crucial substances" in subgingival plaque, which may reflect changes in microbial and environmental dynamics in subgingival microbial ecosystems. This provides insight into the potential of P. intermedia and P. nigrescens as new predictive biomarkers and targets for effective interventions in periodontal disease.

Arthritis-induced alveolar bone loss is associated with changes in the composition of oral microbiota.

Rheumatoid arthritis (RA) and periodontitis (PD) are chronic inflammatory disorders that cause bone loss. PD tends to be more prevalent and severe in RA patients. Previous experimental studies demonstrated that RA triggers alveolar bone loss similarly to PD. The aim of this study was to investigate if arthritis-induced alveolar bone loss is associated with modification in the oral microbiota. Checkerboard DNA-DNA hybridization was employed to analyze forty oral bacterial species in 3 groups of C57BL/6 mice: control (n = 12; without any challenge); Y4 (n = 8; received oral inoculation of Aggregatibacter Actinomycetemcomitans strain FDC Y4) and AIA group (n = 12; chronic antigen-induced arthritis). The results showed that AIA and Y4 group exhibited similar patterns of bone loss. The AIA group exhibited higher counts of most bacterial species analyzed with predominance of Gram-negative species similarly to infection-induced PD. Prevotella nigrescens and Treponema denticola were detected only in the Y4 group whereas Campylobacter showae, Streptococcus mitis and Streptococcus oralis were only found in the AIA group. Counts of Parvimonas micra, Selenomonas Noxia and Veillonella parvula were greater in the AIA group whereas Actinomyces viscosus and Neisseira mucosa were in large proportion in Y4 group. In conclusion, AIA is associated with changes in the composition of the oral microbiota, which might account for the alveolar bone loss observed in AIA mice.

Detection and genetic characterization of ß-lactamases in Prevotella intermedia and Prevotella nigrescens isolated from oral cavity infections and peritonsillar abscesses.

A prospective analysis on ß-lactam resistance mechanisms and ß-lactamase prevalence was conducted on Prevotella intermedia and Prevotella nigrescens recovered from patients with chronic periodontitis and peritonsillar abscesses. Both phenotypic and genotypic methods were performed to characterize the ß-lactamases, their coding genes and their genetic contexts. Overall, ß-lactamase production was observed in 64% (16/25) P. intermedia and 23.8% (5/21) P. nigrescens (p < 0.01). Besides higher ß-lactamase production rates were observed in P. intermedia (8/16) than in P. nigrescens (2/16) recovered from chronic periodontitis, almost all isolates from peritonsillar abscesses were producers (8/9 and 3/3, respectively). cfxA, but not cepA and cblA, was detected in those isolates, which were previously categorized as ß-lactamase producers. CfxA producing isolates displayed higher ß-lactam MICs than non-producers in both species. The most frequent allele was cfxA2, followed by cfxA3 and a new allelic variant named cfxA6. The analysis of the downstream flanking region in the three cfxA variants revealed the association with mobA of Tn4555, suggesting their localization in a mobilizable element. ß-lactam resistance and cfxA carriage prevalence seems to be not only related to the bacterial species but also to the infection site.

Beta-lactamic resistance profiles in Porphyromonas, Prevotella, and Parvimonas species isolated from acute endodontic infections.

INTRODUCTION: Susceptibility to beta-lactamic agents has changed among anaerobic isolates from acute endodontic infections. The aim of the present study was to determine the prevalence of the cfxA/cfxA2 gene in Prevotella spp., Porphyromonas spp., and Parviomonas micra strains and show its phenotypic expression. METHODS: Root canal samples from teeth with acute endodontic infections were collected and Porphyromonas, Prevotella, and Parvimonas micra strains were isolated and microbiologically identified with conventional culture techniques. The susceptibility of the isolates was determined by the minimum inhibitory concentration of benzylpenicillin, amoxicillin, and amoxicillin + clavulanate using the E-test method (AB BIODISK, Solna, Sweden). The presence of the cfxA/cfxA2 gene was determined through primer-specific polymerase chain reaction. The nitrocefin test was used to determine the expression of the lactamase enzyme. RESULTS: Prevotella disiens, Prevotella oralis, Porphyromonas gingivalis, and P. micra strains were susceptible to benzylpenicillin, amoxicillin, and amoxicillin + clavulanate. The cfxA/cfxA2 gene was detected in 2 of 29 isolates (6.9%). Simultaneous detection of the cfxA/cfxA2 gene and lactamase production was observed for 1 Prevotella buccalis strain. The gene was in 1 P. micra strain but was not expressed. Three strains were positive for lactamase production, but the cfxA/cfxA2 gene was not detected through polymerase chain reaction. CONCLUSIONS: There is a low prevalence of the cfxA/cfxA2 gene and its expression in Porphyromonas spp., Prevotella spp., and P. micra strains isolated from acute endodontic infections. Genetic and phenotypic screening must be performed simultaneously to best describe additional mechanisms involved in lactamic resistance for strict anaerobes.

Biofilms of black tooth stains: PCR analysis reveals presence of Streptococcus mutans

This study investigated the presence of the black-pigmented bacteria Prevotella nigrescens and Prevotella intermedia, the non-black-pigmented bacteria Actinomyces spp and particularly the cariogenic pathogen Streptococcus mutans in the dental biofilms of patients with or without black extrinsic tooth stains, using the multiplex polymerase chain reaction (PCR) technique. Analysis of the dental biofilms of patients with (n=26) or without (n=26) black tooth stains was performed using duplex PCR for the 16S ribosomal RNA gene (P. nigrescens, P. intermedia, Actinomyces spp) and glucosyltransferase-I gene for S. mutans. P. nigrescens and S. mutans were the most frequent bacteria detected in both groups. The least frequently detected were P. intermedia and Actinomyces spp. The similar bacterial composition of dental biofilms of black tooth stains and healthy tooth surfaces indicates that black tooth stains are not free of cariogenic bacteria.

O objetivo deste estudo foi investigar a presença das bactérias pigmentadoras de negro Prevotella nigrescens e Prevotella intermedia, da não pigmentadora de negro Actinomyces spp e particularmente a bactéria cariogênica Streptococcus mutans, no biofilme dentário de pacientes com ou sem manchas dentárias extrínsecas negras, utilizando a técnica multiplex PCR (reação em cadeia da polimerase). Análises do biofilme dentário de pacientes com manchas (n=26) e sem manchas (n=26) foram realizadas utilizando a multiplex PCR para o gene 16S RNA ribosomal (P. nigrescens, P. intermedia, Actinomyces spp) e o gene glucosiltransferase-I para S. mutans. P. nigrescens e S. mutans foram as bactérias mais frequentemente detectadas em ambos os grupos. As menos frequentemente detectadas foram P. intermedia e Actinomyces spp. A similaridade entre a composição bacteriana dos biofilmes dentários das manchas dentárias extrínsecas negras e das superfícies dentárias sem manchas indicam que as manchas dentárias extrínsecas negras não estão livres de bactérias cariogênicas.

Genome-wide association study of periodontal pathogen colonization.

Pathological shifts of the human microbiome are characteristic of many diseases, including chronic periodontitis. To date, there is limited evidence on host genetic risk loci associated with periodontal pathogen colonization. We conducted a genome-wide association (GWA) study among 1,020 white participants of the Atherosclerosis Risk in Communities Study, whose periodontal diagnosis ranged from healthy to severe chronic periodontitis, and for whom "checkerboard" DNA-DNA hybridization quantification of 8 periodontal pathogens was performed. We examined 3 traits: "high red" and "high orange" bacterial complexes, and "high" Aggregatibacter actinomycetemcomitans (Aa) colonization. Genotyping was performed on the Affymetrix 6.0 platform. Imputation to 2.5 million markers was based on HapMap II-CEU, and a multiple-test correction was applied (genome-wide threshold of p < 5 × 10(-8)). We detected no genome-wide significant signals. However, 13 loci, including KCNK1, FBXO38, UHRF2, IL33, RUNX2, TRPS1, CAMTA1, and VAMP3, provided suggestive evidence (p < 5 × 10(-6)) of association. All associations reported for "red" and "orange" complex microbiota, but not for Aa, had the same effect direction in a second sample of 123 African-American participants. None of these polymorphisms was associated with periodontitis diagnosis. Investigations replicating these findings may lead to an improved understanding of the complex nature of host-microbiome interactions that characterizes states of health and disease.

Biofilms of black tooth stains: PCR analysis reveals presence of Streptococcus mutans.

This study investigated the presence of the black-pigmented bacteria Prevotella nigrescens and Prevotella intermedia, the non-black-pigmented bacteria Actinomyces spp and particularly the cariogenic pathogen Streptococcus mutans in the dental biofilms of patients with or without black extrinsic tooth stains, using the multiplex polymerase chain reaction (PCR) technique. Analysis of the dental biofilms of patients with (n=26) or without (n=26) black tooth stains was performed using duplex PCR for the 16S ribosomal RNA gene (P. nigrescens, P. intermedia, Actinomyces spp) and glucosyltransferase-I gene for S. mutans. P. nigrescens and S. mutans were the most frequent bacteria detected in both groups. The least frequently detected were P. intermedia and Actinomyces spp. The similar bacterial composition of dental biofilms of black tooth stains and healthy tooth surfaces indicates that black tooth stains are not free of cariogenic bacteria.

Gingival tissue transcriptomes in experimental gingivitis.

AIMS: We investigated the sequential gene expression in the gingiva during the induction and resolution of experimental gingivitis. MATERIAL AND METHODS: Twenty periodontally and systemically healthy non-smoking volunteers participated in a 3-week experimental gingivitis protocol, followed by debridement and 2-week regular plaque control. We recorded clinical indices and harvested gingival tissue samples from four interproximal palatal sites in half of the participants at baseline, Day 7, Day 14 and Day 21 (the "induction phase"), and at Day 21, Day 25, Day 30 and Day 35 in the other half (the "resolution phase"). RNA was extracted, amplified, reversed transcribed, amplified, labelled and hybridized using Affymetrix Human Genome U133Plus2.0 microarrays. Paired t-tests compared gene expression changes between consecutive time points. Gene ontology analyses summarized the expression patterns into biologically relevant categories. RESULTS: The median gingival index was 0 at baseline, 2 at Day 21 and 1 at Day 35. Differential gene regulation peaked during the third week of induction and the first 4 days of resolution. Leucocyte transmigration, cell adhesion and antigen processing/presentation were the top differentially regulated pathways. CONCLUSIONS: Transcriptomic studies enhance our understanding of the pathobiology of the reversible inflammatory gingival lesion and provide a detailed account of the dynamic tissue responses during the induction and resolution of experimental gingivitis.

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.

Prevalence of periodontal bacteria in saliva of Kuwaiti children at different age groups.

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, Tannerella forsythensis and Porphyromonas gingivalis and to a lesser extent Prevotella intermedia and Prevotella nigrescens, are Gram-negative species that are associated with destructive periodontitis. Studies from different parts of the world have shown variable detection rates of periodontal organisms. Hardly any data exist on their carriage in children living in the Middle East. This study was designed to determine the detection of these species in the oral cavity of 240 generally healthy Kuwaiti children, divided into five age groups: <6 years (n=40), 6-9 years (n=60), 10-12 years (n=40), 13-15 years (n=40) and 16-18 years (n=60). Saliva was used as the microbiological specimen, and the samples were analyzed by molecular methods using multiplex PCR. A total of 185 (77.1%) of the 240 children were colonized by at least one of the target periodontal bacteria. In all age groups, P. nigrescens was the most prominent and detected in saliva of 15%, 32%, 63%, 50%, and 47% of the children at the five age groups, respectively. P. gingivalis was detected only occasionally. Only few pathogens were found before the permanent dentition, i.e. at the age of <6 years. The highest carriage rates were from the groups between 6 and 15 years of age. The salivary carriage of the pathogens was essentially similar in the age groups of 10-12 years and 13-15 years. In conclusion, except for P. gingivalis, the examined periodontal pathogens are relatively common findings in Kuwaiti children and colonize the oral cavity from childhood onwards.

The identification of genes specific to Prevotella intermedia and Prevotella nigrescens using genomic subtractive hybridization.

Prevotella intermedia and Prevotella nigrescens, which are often isolated from periodontal sites, were once considered two different genotypes of P. intermedia. Although the genomic sequence of P. intermedia was determined recently, little is known about the genetic differences between P. intermedia and P. nigrescens. The subtractive hybridization technique is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains or species. We used subtractive hybridization to identify the DNA regions specific to P. intermedia ATCC 25611 and P. nigrescens ATCC 25261. Using this method, four P. intermedia ATCC 25611-specific and three P. nigrescens ATCC 25261-specific regions were determined. From the species-specific regions, insertion sequence (IS) elements were isolated for P. intermedia. IS elements play an important role in the pathogenicity of bacteria. For the P. intermedia-specific regions, the genes adenine-specific DNA-methyltransferase and 8-amino-7-oxononanoate synthase were isolated. The P. nigrescens-specific region contained a Flavobacterium psychrophilum SprA homologue, a cell-surface protein involved in gliding motility, Prevotella melaninogenica ATCC 25845 glutathione peroxide, and Porphyromonas gingivalis ATCC 33277 leucyl-tRNA synthetase. The results demonstrate that the subtractive hybridization technique was useful for distinguishing between the two closely related species. Furthermore, this technique will contribute to our understanding of the virulence of these species.

The gene coding for nigrescin produced by Prevotella nigrescens ATCC 25261.

AIM: To identify the gene that encodes nigrescin, a bacteriocin produced by Prevotella nigrescens ATCC 25261. METHODS AND RESULTS: Each open reading frame (ORF) of the nig gene cluster (nigA, nigB, nigC and nigD) was transferred into an expression vector. The recombinant proteins encoded by nigA, nigB, nigC and nigD were purified and assayed for bacteriocin activity against Porphyromonas gingivalis. The ORFs of the nig gene cluster in Pr. nigrescens ATCC 25261 were re-analysed. It revealed that the position of nig ORFs was similar to previously designated locations, except that the start codon of nigC was reassigned. The new nigC gene started at the nucleotide base position 2454 and stopped at position 3608 (the position designated is relative to the first nucleotide base of the nig locus) and putatively encoded a protein with a predicted molecular mass of 41.9 kDa. The N-terminal 6xHistidine-tag recombinant proteins of NigA, NigB, NigC and NigD were overexpressed in Escherichia coli BL21 star (DE3) and were purified using Ni-NTA resins. Only recombinant NigC showed inhibitory activity against P. gingivalis A244 with minimal inhibition concentration (MIC) of 40 microg ml(-1). CONCLUSION: These results indicate that nigC is the gene that encodes nigrescin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that indicates that the gene nigC codes for nigrescin, a bacteriocin produced by Pr. nigrescens ATCC 25261.