Cultivation of Peptidiphaga gingivicola from subgingival plaque: The first representative of a novel genus of Actinomycetaceae.

A novel bacterium was isolated from the subgingival plaque of a patient with periodontal disease. Bacterial strain BA112T is a facultative Gram-positive coccus. It metabolizes alanine, arginine, glycine, histidine, leucine, proline, serine and tyrosine, but does not appear to use carbohydrates. Urease, esculin, indole, catalase and nitrate reduction tests were all negative. Major cellular fatty acids were C18:0 , C12:0 , C16:0 , C18:1 w9c and C20:0 . The genome was sequenced and is 2.4 Mbp in length and has 64% GC content. Based on phylogenetics of the 16S rRNA sequence and concatenated alignments of 37 conserved proteins, BA112T belongs to the family Actinomycetaceae but is located on a branch of the tree without currently named members. Based on our phenotypic and phylogenetic studies, we propose that BA112T is the first known representative of a new genus, for which the name Peptidiphaga gingivicola gen. nov., sp. nov. is proposed. The type strain is BA112T .

Filifactor alocis interactions with gingival epithelial cells.

An association between the gram-positive anaerobe Filifactor alocis and periodontal disease has recently emerged; however, possible pathogenic mechanisms have not been investigated. In this study we examined the responses of primary cultures of gingival epithelial cells (GECs) to infection with F. alocis. Secretion of the pro-inflammatory cytokines interleukin-1ß, interleukin-6 and tumor necrosis factor-α from GECs was stimulated by F. alocis infection. F. alocis also induced apoptosis in GECs through pathways that involved caspase-3 but not caspase-9. Apoptosis was coincident with inhibition of mitogen-activated protein kinase kinase (MEK) activation. These results show that F. alocis has characteristics in common with established periodontal pathogens and has the potential to contribute to periodontal tissue destruction.

New bacterial species associated with chronic periodontitis.

Recent investigations of the human subgingival oral flora based on ribosomal 16S cloning and sequencing have shown many of the bacterial species present to be novel species or phylotypes. The purpose of the present investigation was to identify potential periodontal pathogens among these newly identified species and phylotypes. Species-specific ribosomal 16S primers for PCR amplification were developed for detection of new species. Associations with chronic periodontitis were observed for several new species or phylotypes, including uncultivated clones D084 and BH017 from the Deferribacteres phylum, AU126 from the Bacteroidetes phylum, Megasphaera clone BB166, clone X112 from the OP11 phylum, and clone I025 from the TM7 phylum, and the named species Eubacterium saphenum, Porphyromonas endodontalis, Prevotella denticola, and Cryptobacterium curtum. Species or phylotypes more prevalent in periodontal health included two uncultivated phylotypes, clone W090 from the Deferribacteres phylum and clone BU063 from the Bacteroidetes, and named species Atopobium rimae and Atopobium parvulum.

Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria.

Accurate quantitation of the number of cells of individual bacterial species in dental plaque samples is needed for understanding the bacterial etiology of periodontitis. Real-time PCR offers a sensitive, efficient, and reliable approach to quantitation. Using the TaqMan system we were able to determine both the amount of Porphyromonas gingivalis and the total number of bacterial cells present in plaque samples. Using species-specific primers and a fluorescent probe, detection of DNA from serial dilutions of P. gingivalis cells was linear over a large range of DNA concentrations (correlation coefficient = 0.96). No difference was observed between P. gingivalis DNA alone and the same DNA mixed with DNA isolated from dental plaque, indicating that P. gingivalis levels can be determined accurately from clinical samples. The total number of cells of all bacterial species was determined using universal primers and a fluorescent probe. Standard curves using four different bacterial species gave similar results (correlation coefficient = 0.86). Levels of both P. gingivalis and total bacteria were determined from a series of human plaque samples. High levels of P. gingivalis were observed in several of the samples from subjects with periodontitis and none of those from healthy subjects. Real-time quantitative PCR provided a sensitive and reliable method for quantitating P. gingivalis. In addition, it allowed the determination of the total number of bacterial cells present in a complex sample so that the percentage of P. gingivalis cells could be determined.

Phylogeny of Porphyromonas gingivalis by ribosomal intergenic spacer region analysis.

Periodontitis has been associated with the presence of Porphyromonas gingivalis, and previous studies have shown phenotypic differences in the pathogenicities of strains of P. gingivalis. An accurate and comprehensive phylogeny of strains of P. gingivalis would be useful in determining if there is an evolutionary basis to pathogenicity in this species. Previous phylogenies of P. gingivalis strains based on random amplified polymorphic DNA (RAPD) analysis and multilocus enzyme electrophoresis (MLEE) show little agreement. While the 16S ribosomal gene is the standard for phylogenetic reconstruction among bacterial species, it is insufficiently variable for this purpose. In the present study, the phylogeny of P. gingivalis was constructed on the basis of the sequence of the most variable region of the ribosomal operon, the intergenic spacer region (ISR). Heteroduplex analysis of the ISR has been used to study the variability of P. gingivalis strains in periodontitis. In the present study, typing by heteroduplex analysis was compared to ISR sequence-based phylogeny and close agreement was observed. The two strains of P. gingivalis whose heteroduplex types are strongly associated with periodontitis were found to be closely related and were well separated from strains whose heteroduplex types are less strongly associated with disease, suggesting a relationship between pathogenicity and phylogeny.

Acquisition and colonization stability of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in children.

The presence of Porphyromonas gingivalis has been shown to be a risk factor for periodontitis in adults, and Actinobacillus actinomycetemcomitans has been implicated as a pathogen in early-onset periodontitis. Both species have been shown to establish stable colonization in adults. In cross-sectional studies, both A. actinomycetemcomitans and P. gingivalis have been detected in over one-third of apparently healthy children. Information on the stability of colonization with these organisms in children could help to elucidate the natural history of the development of periodontitis. For this purpose, samples previously collected from a cohort of 222 children between the ages of 0 and 18 years and previously examined for the presence of P. gingivalis with a PCR-based assay were examined for the presence of A. actinomycetemcomitans. It was detected in 48% of subjects and, like P. gingivalis, was found at similar frequencies among children of all ages (P = 0.53), suggesting very early initial acquisition. One hundred one of the original subjects were recalled after 1 to 3 years to determine the continuing presence of both A. actinomycetemcomitans and P. gingivalis. The prevalence of both species remained unchanged at resampling. However, in most children both species appeared to colonize only transiently, with random concordance between the results of the first and second sampling. Stability of colonization was unrelated to age for A. actinomycetemcomitans, but P. gingivalis was more stable in the late teenage years.

Identification of Porphyromonas gingivalis strains by heteroduplex analysis and detection of multiple strains.

Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of Porphyromonas gingivalis. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of P. gingivalis for identification. We identified 22 distinct but closely related heteroduplex types of P. gingivalis in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which P. gingivalis was detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains of P. gingivalis in large numbers of samples.

Porphyromonas gingivalis strain variability and periodontitis.

To determine if there is variability in virulence among strains of Porphyromonas gingivalis in human periodontitis, their distribution in a group of subjects with clear indicators of periodontitis and in a healthy, age-matched control group was examined. The presence of heteroduplex types of P. gingivalis in the two groups was determined with a PCR-based assay. This assay relied on detection of polymorphisms in the ribosomal internal spacer region (ISR). ISR fragments generated by PCR with P. gingivalis-specific primers were hybridized to fragments from reference strains, and the formation of heteroduplexes from the hybridization of nonidentical sequences was observed by polyacrylamide gel electrophoresis. Characteristic fingerprints from comparison with a panel of reference strains allowed the identification of heteroduplex types in clinical samples. One hundred thirty adults with periodontitis and 181 controls were sampled. With this approach, 11 heteroduplex types of P. gingivalis were detected in the population. Sufficient numbers were available for statistical analysis of six of these types. Heteroduplex type hW83 was found to be very strongly associated with periodontitis (P = 0.0000), and two additional types, h49417 and hHG1691, were also significantly associated with disease. The remaining types, h23A4, h381, and hA7A1, were detected more frequently in subjects with periodontitis than in healthy subjects, but the difference was not significant. These data indicate that virulence in human periodontitis varies among strains of P. gingivalis, and they identify an apparently highly virulent subgroup.

Sequencing of the ribosomal intergenic spacer region for strain identification of Porphyromonas gingivalis.

The ribosomal intergenic spacer regions (ISRs) of 19 laboratory strains and 30 clinical samples of Porphyromonas gingivalis were amplified by PCR and sequenced to provide a strain identifier. The ISR is a variable region of DNA located between the conserved 16S and 23S rRNA genes. This makes it an ideal locus for differentiation of strains within a species: primers specific for the conserved flanking genes were used to amplify the ISR, which was then sequenced to identify the strain. We have constructed a P. gingivalis ISR sequence database to facilitate strain identification. ISR sequence analysis provides a strain identifier that can be easily reproduced among laboratories and catalogued for unambiguous comparison.

Prevalence of Porphyromonas gingivalis and periodontal health status.

Periodontitis is a common, progressive disease that eventually affects the majority of the population. The local destruction of periodontitis is believed to result from a bacterial infection of the gingival sulcus, and several clinical studies have provided evidence to implicate Porphyromonas gingivalis. If P. gingivalis is a periodontal pathogen, it would be expected to be present in most subjects with disease and rarely detected in subjects with good periodontal health. However, in most previous studies, P. gingivalis has not been detected in the majority of subjects with disease, and age-matched, periodontally healthy controls were not included for comparison. The purpose of the study reported here was to compare the prevalence of P. gingivalis in a group with periodontitis to that of a group that is periodontally healthy. A comprehensive sampling strategy and a sensitive PCR assay were used to maximize the likelihood of detection. The target sequence for P. gingivalis-specific amplification was the transcribed spacer region within the ribosomal operon. P. gingivalis was detected in only 25% (46 of 181) of the healthy subjects but was detected in 79% (103 of 130) of the periodontitis group (P < 0.0001). The odds ratio for being infected with P. gingivalis was 11.2 times greater in the periodontitis group than in the healthy group (95% confidence interval, 6.5 to 19.2). These data implicate P. gingivalis in the pathogenesis of periodontitis and suggest that P. gingivalis may not be a normal inhabitant of a periodontally healthy dentition.

Concordance of Porphyromonas gingivalis colonization in families.

Periodontitis is a widespread disease that appears to be due to a specific bacterial infection. Several species of bacteria have been investigated as potential pathogens, and particularly strong evidence links the presence of Porphyromonas gingivalis with indicators of periodontitis. Information concerning the transmission of P. gingivalis between human contacts may be important in determining risk factors for disease and developing preventive strategies. A few small studies have provided some evidence of transmission between related individuals, but no large-scale study of families that would reflect the typical transmission of this pathogen in the population has been reported. The purpose of this study was to investigate the transmission of P. gingivalis within randomly selected, extended families. The colonization status of 564 members of multigeneration families was determined, and the degree of concordance observed among members of these families was then compared to that expected to occur based on the prevalence of colonization in the population studied. A PCR assay was used for detection of P. gingivalis. Concordance in colonization was more frequently observed within entire families (P = 0.0000) and for spouses (P < 0.001), children and their mothers (P < 0.001), children and their fathers (P < 0.01), adults and their mothers (P < 0.005), and siblings (P < 0.05) than would be expected if P. gingivalis were randomly distributed in the population studied. Results showed that contact with an infected family member substantially increased the relative risk of colonization in these intrafamilial pairs. This indicates that P. gingivalis is commonly transmitted by contact with an infected family member.

Age and prevalence of Porphyromonas gingivalis in children.

The acquisition of Porphyromonas gingivalis was examined in a cross-sectional study of 198 subjects from 0 to 18 years of age using a PCR-based assay. P. gingivalis was detected in the oral cavities of 37% of subjects and at similar frequencies among subjects of all ages. These data indicate that P. gingivalis may be acquired in the first days of life.

Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria.

Strain identification of Actinobacillus actinomycetemcomitans using the polymerase chain reaction.

A molecular assay that distinguishes among strains of the periodontal pathogen Actinobacillus actinomycetemcomitans was developed by identifying DNA restriction site polymorphisms in the highly variable transcribed spacer region between the 16S and 23S ribosomal genes. The polymerase chain reaction (PCR) was used to amplify this region from genomic DNA using primers within conserved regions of the 16S and 23S genes. This amplified region was digested using a series of restriction enzymes and electrophoresed. Examination of restriction fragment length polymorphisms obtained by separate digestion with RsaI and NciI allowed the 7 strains examined to be divided into 4 genetic groups. This assay provides a more precise and reproducible method of strain identification than whole genomic methods and should be useful as a method for studying the epidemiology of A. actinomycetemcomitans strains in human subjects. The genetic variability detected supplies strong evidence that direct sequence analysis of the region could provide extremely precise and potentially definitive identification of strains.

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

Transcription of the mouse dihydrofolate reductase gene proceeds unabated through seven polyadenylation sites and terminates near a region of repeated DNA.

We observed equimolar transcription throughout the 35-kilobase mouse dihydrofolate reductase structural gene. Transcription termination occurred within a discrete region (900 base pairs) located 1 kilobase beyond the last of seven functional polyadenylation sites and near a repetitive DNA sequence element. The results imply that a distinct genetic signal may be associated with the process of transcription termination.

Dihydrofolate reductase gene expression in cultured mouse cells is regulated by transcript stabilization in the nucleus.

Methotrexate-resistant cells, which contain a 500-fold amplification of dihydrofolate reductase (DHFR) genes, were used as a model system for studying the regulation of DHFR gene expression during growth stimulation. We have shown that a threefold increase in DHFR mRNA levels following growth stimulation results from a corresponding increase in DHFR mRNA production (i.e., delivery to the cytoplasm) and is not the result of a change in DHFR mRNA half-life. We previously showed that the increase in DHFR mRNA production during growth stimulation is not accompanied by an increase in the relative rate of transcription of the DHFR gene. This suggested that changes in DHFR mRNA production during growth stimulation are due to changes in the stability of DHFR transcripts in the nucleus. Using continuous labeling experiments in vivo comparing the stability of DHFR RNA with specific reference sequences, we show that in growing cells most DHFR transcripts were converted to mRNA, whereas in resting cells the majority of DHFR transcripts were rapidly degraded in the nucleus. There was no significant difference in the rate of processing and transport of stable DHFR transcripts. Therefore, changes in the stability of DHFR RNA in the nucleus control the amount of mRNA available for translation in the cytoplasm.

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.

Control of dihydrofolate reductase messenger ribonucleic acid production.

We used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.