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.

Isolates of Piscirickettsia salmonis from Scotland and Ireland show evidence of clonal diversity.

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.

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.

Conscious sedation experiences in graduate pediatric dentistry programs.

PURPOSE: Conscious sedation is a behavior modification adjunct taught in all postgraduate pediatric dental residency programs. It has been a decade since the last survey was done specifically related to didactic and clinical aspects of conscious sedation in postgraduate pediatric dental programs. The aim of the study was to determine the clinical and didactic experiences associated with conscious sedation in these programs and to compare some of the findings to those collected a decade ago. METHODS: A 31-item survey similar to that of a decade ago was constructed and sent to all pediatric dentistry program directors of accredited postgraduate and residency programs in the United States. The items covered several didactics including didactic topics, sedative agents, monitoring, and emergency policy among others. A follow-up mailing was done involving those who had not responded 6 weeks following the initial mailing. RESULTS: Fifty-four of 58 (93%) program directors returned the 31-item survey. The following are highlighted findings. Conscious sedation among residency programs was achieved most commonly with a combination of sedative agents used with N2O. Midazolam was more popular than chloral hydrate. The oral route was the predominant route of administration. More lecture hours were spent on conscious sedation than 10 years ago. The pre-cordial stethoscope, pulse oximeter, and blood pressure cuff were the most commonly used monitors. Sedative agent and anticipated depth of sedation were the factors most often considered in choosing monitors used during the sedation of a patient. The capnograph was being used more frequently than it was 10 years ago. Programs did not report an increase in sedation emergencies but practiced emergency drills more often and had increased numbers of individuals certified in Advanced Cardiac Life Support (ACLS) or Pediatric Advanced Life Support (PALS). The percent of the total patient population which required sedation is about 1-20%, with most directors reporting an increase in the numbers of sedations done in the past few years. CONCLUSIONS: While many factors remained unchanged or slightly modified when compared to the survey done a decade ago, the results of this study suggest that there has been significant changes in several key factors including the most frequently used sedative (i.e., midazolam) and increased preparation in the area of emergency preparedness.

Compomers as Class II restorations in primary molars.

A variety of alternatives to amalgam are now available for use in class II restorations in primary teeth, including glass ionomer, composites, and intermediate materials such as compomer and resin modified glass ionomers (RMGI). The purpose of the present study was to evaluate the clinical performance of two compomers, Hytac and Dyract, and to compare these results to those reported for other intracoronal restorative materials. Evaluation after 24 months shows Hytac and Dyract to have performed well and comparably as class II restorations in primary teeth. The low failure rate, even in a population with a high caries increment, suggests that compomers are a suitable alternative to amalgam or other, tooth-colored materials when used as class II restorations in primary teeth.

Combined use of growth rate correlated and growth rate independent promoters for recombinant glucoamylase production in Fusarium venenatum.

Fusarium venenatum JeRS 325, a strain which produces recombinant glucoamylase under control of a growth rate independent promoter was transformed with a plasmid carrying the Aspergillus niger glucoamylase gene under control of its own growth rate correlated promoter. Some disruption of the original recombinant genes occurred and at pH 5.8 the double transformant did not produce as much glucoamylase as JeRS 325 in batch culture. However, the double transformant still produced as much glucoamylase as JeRS 325 in fed-batch cultures, illustrating the potential for the combined use of growth rate independent and dependent promoters to improve production of recombinant proteins in fed-batch culture systems.

Dental caries. An infectious and transmissible disease.

Of the infectious diseases that affect humans, dental caries may be the most prevalent. Pediatric primary medical care providers are usually the first health care providers to examine the oral cavity of a child and so need to be able to recognize suspicious dental lesions. This article provides information on recognizing caries and reviews the epidemiology, pathogenesis, and treatment of caries and odontogenic infections.

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.

The Effect of organic nitrogen sources on recombinant glucoamylase production by Aspergillus niger in chemostat culture.

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 +/- 0.01 h(-1), pH 5.4). In cultures supplemented with l-alanine, l-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA.

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.

High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods.

The development and application of a novel, sensitive TaqMan fluorescent probe-based product enhanced RT test (F-PERT) for the detection of retrovirus are described. The assay allows discrimination between the amplification signals generated by genuine positive signals that result from retroviral RT activity and the RT-like activity from DNA polymerases. The RT-like activity from DNA polymerases was suppressed by the addition of activated calf-thymus DNA with no reduction in the RT activity. A linear relationship between threshold cycle (C(T)) and the number of virus particles was demonstrated, allowing quantification of retroviruses in unknown samples. The F-PERT assay was able to detect a wide range of retroviral RT activities, including that from porcine endogenous retrovirus (PoERV), murine leukaemia virus (MLV), simian foamy virus (SFV), simian immunodeficiency virus (SIVmac) and squirrel monkey retrovirus (SMRV). The detection limit of SMRV, MLV and PoERV was approximately 100 virion particles and the test was able to detect at least 10(2) molecules of purified RT enzyme. RT activity was not detected in cellular lysates and supernatants from MRC-5, BT, VERO, or Raji cells, whereas RT activity was detected in C1271, Mus dunni, K-Balb, BHK-21, CHO-K1, SP2/0-Ag14 and NSO cell supernatants. RT activity was also detected in the Spodoptera cell line Sf9.

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.