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Biomedical subjects

W G Wade

Publications and source records attributed to W G Wade.

At least 19 recordsLinked to original sources
PubMed2006

Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets.

INTRODUCTION: Molecular ecological analysis based on 16S rRNA gene sequence analysis is well established for the characterisation of complex bacterial communities. However, 'universal' PCR primers can introduce biases into the analysis of the species composition of clone libraries because of mismatches between the primer and target organism sequences. In this study, three universal primer sets were compared for the analysis of the microflora in subgingival plaque. METHODS: Three subgingival plaque samples were collected from two subjects with localised severe chronic periodontitis. Half of each sample was cultured while DNA was extracted from the remaining half and 16S rDNA amplified with universal primer pairs 27F, 1525R (A); 27F, 1492R (B) and 530F, 1525R (C). Amplified genes were cloned, sequenced and identified by comparison with 16S rRNA databases. RESULTS: 137 taxa were identified among 177 isolates and 417 clones sequenced. Of these, 86 were detected only by the molecular technique whereas 26 were found only by culture. Sequences from 81 taxa did not correspond to those of named species and of these, 38 were not represented in the nucleotide databases. 16S RNA genes for these 38 taxa were sequenced and deposited with GenBank. CONCLUSION: The use of three sets of universal primers allowed the identification of 38 novel bacterial phylotypes. There were marked differences in the composition of the libraries generated by the different primer sets. A combination of molecular and cultural techniques is recommended to maximise the coverage of detection of bacterial taxa in oral samples.

Bacteria↗
PubMed2004

Gram-positive anaerobic bacilli in human periodontal disease.

OBJECTIVE: The uncertain taxonomy of oral anaerobic gram-positive bacilli and their generally slow growing nature has limited the understanding of their role in periodontal disease. The current objective was to design and use species-specific oligonucleotide probes to investigate the relationship of selected gram-positive anaerobic bacilli to periodontal disease. METHODS: Plaque and clinical measurements were collected from 40 patients with periodontitis and from 40 matched controls. Oligonucleotide probes were designed for Bulleidia extructa, Eubacterium nodatum, Mogibacterium timidum and Slackia exigua and used to probe nucleic acids extracted from the samples with a chemiluminescent detection method. Species were quantified as absent or present at low (approximately 10(3)-10(4) cells), medium (approximately 10(4)-10(5) cells) or high levels (approximately 10(5)-10(6) cells). RESULTS: M. timidum and B. extructa were detected in only three and four samples, respectively. The level of both E. nodatum and S. exigua was significantly higher in deep than shallow pockets (Wilcoxon, p < 0.001). The level of E. nodatum, but not S. exigua, was higher in patients than matched controls (Mann-Whitney U, p < 0.03). Using an ordered logistic regression model, the probing depth of the sampled sites had the greatest influence on the level of both species and significant variations occurred between individuals. Bleeding also influenced the levels of both species, with supragingival plaque influencing S. exigua. CONCLUSION: Both E. nodatum and S. exigua were associated with clinical indicators of periodontal disease.

Adult↗
PubMed2004

Culture-independent identification of periodontitis-associated Porphyromonas and Tannerella populations by targeted molecular analysis.

Periodontitis is the commonest bacterial disease of humans and is the major cause of adult tooth loss. About half of the oral microflora is unculturable; and 16S rRNA PCR, cloning, and sequencing techniques have demonstrated the high level of species richness of the oral microflora. In the present study, a PCR primer set specific for the genera Porphyromonas and Tannerella was designed and used to analyze the bacterial populations in subgingival plaque samples from inflamed shallow and deep sites in subjects with periodontitis and shallow sites in age- and sex-matched controls. A total of 308 clones were sequenced and found to belong to one of six Porphyromonas or Tannerella species or phylotypes, one of which, Porphyromonas P3, was novel. Tannerella forsythensis was found in significantly higher proportions in patients than in controls. Porphyromonas catoniae and Tannerella phylotype BU063 appeared to be associated with shallow sites. Targeted culture-independent molecular ecology studies have a valuable role to play in the identification of bacterial targets for further investigations of the pathogenesis of bacterial infections.

Adult↗
PubMed2004

Molecular analysis of the microflora associated with dental caries.

Molecular techniques have revealed many novel, presumed unculturable, taxa in oral infections. The aim of this study was to characterize the bacterial community of the middle and advancing front of carious dental lesions by cultural and molecular analyses. Samples were collected with a hand excavator from five teeth with carious lesions involving dentine. Samples were cultured on blood agar and Rogosa agar incubated in air plus 5% CO(2) and on fastidious anaerobe agar anaerobically. DNA was also extracted directly from the samples and 16S rRNA genes were amplified by PCR with universal primers. PCR products were singularized by cloning, and the cloned inserts and cultured isolates were identified by 16S rRNA gene sequence analysis. We identified 95 taxa among the 496 isolates and 1,577 clones sequenced; 44 taxa were detected by the molecular method alone; 31 taxa were previously undescribed. Only three taxa, Streptococcus mutans, Rothia dentocariosa, and an unnamed Propionibacterium sp., were found in all five samples. The predominant taxa by anaerobic cultivation were the novel Propionibacterium sp. (18%), Olsenella profusa (14%), and Lactobacillus rhamnosus (8%). The predominant taxa in the molecular analysis were Streptococcus mutans (16%), Lactobacillus gasseri/johnsonii (13%), and Lactobacillus rhamnosus (8%). There was no significant difference between the compositions of the microflora in the middle and advancing front samples (P < 0.05, Wilcoxon matched pairs, signed ranks test). In conclusion, combined cultural and molecular analyses have shown that a diverse bacterial community is found in dentinal caries and that numerous novel taxa are present.

Adult↗
PubMed2003

Dialister invisus sp. nov., isolated from the human oral cavity.

Six strains of anaerobic, Gram-negative coccobacilli isolated from the root canals of patients with endodontic infections (five strains) and from a deep periodontal pocket (one strain) were subjected to a comprehensive range of phenotypic and genetic tests and were found to comprise a homogeneous group. Following 16S rRNA gene sequence analysis, they were found to be most closely related to Dialister pneumosintes, with 93 % sequence similarity between the two taxa. A novel species, Dialister invisus sp. nov., is proposed. Biochemically, the species is largely unreactive and is asaccharolytic, with only traces of acetate and propionate detected as metabolic end-products. The G+C content of the DNA of D. invisus strains is 45-46 mol%. The type strain is E7.25(T) (=CCUG 47026(T)=DSM 15470(T)).

Bacterial Proteins↗
PubMed2002

Adjunctive effects to non-surgical periodontal therapy of systemic metronidazole and amoxycillin alone and combined. A placebo controlled study.

BACKGROUND AND AIM: Several studies have reported adjunctive benefits to scaling and root planing (SRP) of systemic amoxycillin and metronidazole in the treatment of periodontal diseases. To date no comparisons have been made of these antimicrobials alone or in combination. The aim of this study was to compare the adjunctive benefits to SRP of amoxycillin and metronidazole alone and combined. METHODS: 66 subjects <46 years of age with advanced chronic periodontal disease participated in this randomised, double blind, 4 parallel treatment group designed study. All subjects received quadrant SRP and then were prescribed amoxycillin capsules (250 mg) and metronidazole tablets (200 mg) (AM) or lactate capsules and metronidazole (PM) or amoxycillin and calcium lactate tablets (AP) or lactate and calcium lactate (PP). All medication was 3 of each per day for 7 days. Subgingival plaque samples were obtained and probing depth (PD), loss of attachment (LOA), bleeding on probing (BOP), suppuration (SUPP) and plaque (DEP) were recorded pre-treatment, 1, 3 and 6 months post-treatment. RESULTS: Final group sizes were: AM=15, PM=16, AP=16 and PP=15. PD improved in all groups. Treatment effects were highly significantly different and always greatest in the AM and least in the PP groups. Benefits of PM and AP over PP were also noted. LOA improved in all groups and showed the same highly significant treatment differences, again favouring AM. BOP improved in all groups, particularly in AM compared to the other groups. SUPP improved in all groups and was virtually eradicated in AM with differences among treatments highly significant. DEP changed little in any group and there were no significant differences among groups. Microbiological data showed significant differences in favour of AM compared to PP and PM for total aerobes and anaerobes at 1 month. P. intermedia counts were always lower in active groups compared to PP and reached significance for AM and AP at 1 month and AM and PM at 3 months. CONCLUSION: The significant differences among treatment groups and the overall trend in the data, in line with other studies, support the considerable adjunctive benefits to SRP of amoxycillin and metronidazole combined in the treatment of advanced chronic periodontal disease.

Adult↗
PubMed2002

Molecular and cultural analysis of the microflora associated with endodontic infections.

Cultural studies have indicated that a subset of the oral microflora is responsible for endodontic infections. Approximately 50% of oral bacteria are unculturable, so it is likely that currently unknown bacteria are present in such infections. In this study, cultural and molecular analyses were performed on the microflora in aspirate samples collected from 5 infected root canals. 16S rDNA sequences from 261 isolates and 624 clones were identified by comparison with database sequences. Sixty-five taxa were identified, of which 26 were found by the molecular method alone. A mean of 20.2 taxa was found in each sample. A new species of Dialister was the only organism present in all 5 samples. Twenty-seven novel taxa were detected, 18 of which belonged to the phylum Firmicutes and 8 to Bacteroidetes. Culture-independent, molecular analysis has revealed a more diverse microflora associated with endodontic infections than that revealed by cultural methods alone.

Adult↗
PubMed2001

The clinical and microbiological effects of a novel acidified sodium chlorite mouthrinse on oral bacterial mucosal infections.

Acidified sodium chlorite mouthrinses have been shown to have equivalent anti-plaque activity to those containing chlorhexidine, the current 'gold standard'. In this study, sodium chlorite mouthrinses (ASC) acidified with either malic or gluconic acids were compared to each other and with a chlorhexidine rinse and sterile water for their effect on salivary bacterial counts. Sixteen subjects participated in the study, which had a cross-over Latin square design. In a second study, a sodium chlorite/gluconic acid rinse was compared with chlorhexidine for its clinical and microbiological effects in 36 patients with oral mucosal infections. The sodium chlorite rinses acidified with malic and gluconic acids and the chlorhexidine rinse caused significant reductions in salivary bacterial counts up to 7 h after a single rinse compared with water. There were no significant differences between the three active treatments. In the mucosal infection study, there was a significant reduction in erythema in the chlorhexidine group compared with the ASC group. Patients who received the ASC rinse reported significantly less discomfort following treatment than those receiving the chlorhexidine rinse. Staphylococcus aureus counts were significantly reduced in the group who received the sodium chlorite rinse. There were no other significant differences between the treatments. ASC appears to be an effective alternative to chlorhexidine mouthrinse.

Adult↗
PubMed2001

Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae: description of olsenella gen. nov., reclassification of Lactobacillus uli as Olsenella uli comb. nov. and description of Olsenella profusa sp. nov.

The diversity of organisms present in the subgingival pockets of patients with periodontitis and acute necrotizing ulcerative gingivitis (ANUG) were examined previously. The 16S rRNA genes of subgingival plaque bacteria were amplified using PCR with a universal forward primer and a spirochaete-selective reverse primer. The amplified DNA was cloned into Escherichia coli. In one subject with ANUG, 70 clones were sequenced. Seventy-five per cent of the clones were spirochaetal, as expected. Twelve of the remaining clones fell into two clusters that represent novel phylotypes in the family Coriobacteriaceae. The first novel phylotype was most closely related to Atopobium rimae (98% similarity). The phylotype probably represents a novel Atopobium species, but will not be named until cultivable strains are obtained. The second novel phylotype was only 91% similar to described Atopobium species and 84% similar to Coriobacterium glomerans. The 16S rRNA sequences of the type strain of Lactobacillus uli and a strain representing the Moores' Eubacterium group D52 were determined as part of on ongoing sequence analysis of oral bacteria. The sequence for L. uli was more than 99.8% similar to sequences for the second clone phylotype. It therefore appears that the second clone phylotype and L. uli represent the same species. The sequence for the Eubacterium D52 strain was 95.6% similar to that of L. uli. The G+C content of the DNA of L. uli and Eubacterium D52 is 63-64 mol %. These organisms are thus distinct from the neighbouring genus Atopobium, which has a DNA G+C content of 35-46 mol%. A new genus, Olsenella gen. nov., is proposed for these two species on the basis of phenotypic characteristics and 16S rRNA sequence analysis to include Olsenella uli comb. nov. and Olsenella profusa sp. nov.

Actinobacteria↗
PubMed2000

Bulleidia extructa gen. nov., sp. nov., isolated from the oral cavity.

Five strains of anaerobic non-sporing Gram-positive bacilli isolated from advanced periodontitis (four strains) and a dentoalveolar abscess (one strain) that did not correspond to existing species were subjected to phenotypic and genetic characterization. Following 16S rDNA sequence analysis, they were found to constitute a novel branch of the low G+C Gram-positive division of the phylogenetic tree related to Erysipelothrix rhusiopathiae and Holdemania filiformis. A new genus Bulleidia, and the species Bulleidia extructa, are proposed. Growth of B. extructa in broth media was poor but was enhanced by the addition of fructose, glucose or maltose together with Tween 80. Glucose and maltose were fermented and arginine was hydrolysed. Acetate, lactate and trace amounts of succinate were the end products of glucose fermentation. The G+C content of the DNA of the type strain is 38 mol%. The type strain of Bulleidia extructa is DSM 13220T.

Bacteria, Anaerobic↗
PubMed2000

Production of volatile sulphur compounds in diseased periodontal pockets is significantly increased in smokers.

OBJECTIVE: This study was undertaken in order to test the hypothesis that the consequences of tobacco smoking may include increased synthesis of toxic volatile sulphur compounds in diseased periodontal pockets. DESIGN: A cross-sectional, parallel study comparing groups of smokers and non-smokers with periodontitis and the level of volatile sulphur compounds in the gingival sulci of these subjects. PATIENTS AND METHODS: Levels of volatile sulphur compounds were measured in diseased periodontal sites of 12 smokers and 11 non-smokers using a portable sulphide monitor. Anaerobic and aerobic counts of the total cultivable subgingival microflora of both groups were also determined. RESULTS: The percentage of sites per subject with high levels of sulphides (> or = 10 units) detected in moderate (4-6 mm) and deep (> or = 7 mm) periodontal pockets was found to be significantly higher in smokers, compared to non-smokers (P = 0.040 and P = 0.005, respectively). No significant difference in the microbiological parameters tested were observed between the two groups. CONCLUSIONS: Increased production of volatile sulphur compounds may represent a further mechanism of increased susceptibility to periodontitis in smokers and also help to explain the reported association between smoking and halitosis.

Adult↗
PubMed1999

Phospholipid analogue distribution in Capnocytophaga.

Polar lipids of nineteen previously characterised culture collection strains of Capnocytophaga were analysed using fast atom bombardment mass spectrometry (FAB MS) in negative mode. All strains examined had a major peak at m/z 241, consistent with the expected presence of the pentadecanoate anion. The most intense higher mass anions, consistent with expected presence of phospholipid molecular species, were as follows: m/z 574, 588, 618 and 662 which are consistent with presence of PE(24:2), PE(25:2), PE(27:1) and PE(30:0) respectively. Other anions putatively identified as phospholipid anions were: m/z 572, 578, 592, 602, 604, 616 and 720 consistent with presence of PE(24:3), PE(24:0), PE(25:0), PE(26:2), PE(26:1), PE(27:2) and OH-PE(33:0). Capnocytophaga isolates share a distinctive phospholipid fingerprint which appears to lack the somewhat higher mass phospholipid analogues observed in related oral bacteria. Within the genus, the profiles obtained showed only quantitative differences which did not correlate with previous studies.

Capnocytophaga↗
PubMed1999

Diversity of oral asaccharolytic Eubacterium species in periodontitis--identification of novel phylotypes representing uncultivated taxa.

Oral asaccharolytic Eubacterium species are associated with periodontal disease and other oral infections. The aim of this study was to use a culture-independent molecular technique to determine the diversity of asaccharolytic Eubacterium species in subgingival plaque in periodontitis. An oligonucleotide PCR primer designed to amplify 16S rRNA genes of the oral asaccharolytic Eubacterium branch of the phylogenetic tree was constructed. This primer was used together with a universal primer in PCRs to amplify gene sequences directly from a single subgingival plaque sample. Fifty PCR products were purified by cloning, fully sequenced and subjected to molecular phylogenetic analysis. The sequences were assigned to four groups within a single lineage of the low G + C gram-positive bacteria. Group I (58% of the cloned sequences) was assigned to a branch that included Eubacterium nodatum, and Group II (22%) to a branch including Eubacterium infirmum. Group III (8%) was distinct from but related to E. infirmum at the species level, and Group IV (12%) was another novel taxon more distantly related to E. infirmum and E. nodatum.

Adult↗
PubMed1999

The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al. 1996) and Peptostreptococcus heliotrinreducens (Lanigan 1976) as Slackia exigua gen. nov., comb. nov. and Slackia heliotrinireducens gen. nov., comb. nov., and Eubacterium lentum (Prevot 1938) as Eggerthella lenta gen. nov., comb. nov.

16S rRNA gene sequences were determined for Eubacterium exiguum and Peptostreptococcus heliotrinreducens. These species were found to be closely related and, together with Eubacterium lentum, to constitute a branch of the Coriobacteriaceae. Two new genera are proposed on the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons: Slackia to include the bile-sensitive species Eubacterium exiguum and P. heliotrinreducens, and Eggerthella to include the bile-resistant Eubacterium lentum. It is proposed that Eubacterium exiguum and Peptostreptococcus heliotrinreducens are transferred to the genus Slackia gen. nov. as Slackia exigua gen. nov., comb. nov. (type strain ATCC 700122T) and Slackia heliotrinireducens gen. nov., comb. nov. (type strain NTCC 11029T), respectively, and Eubacterium lentum is transferred to the genus Eggerthella gen. nov. as Eggerthella lenta gen. nov., comb. nov. with Eggerthella lenta as the type species.

Base Composition↗
PubMed1999

Eubacterium minutum is an earlier synonym of Eubacterium tardum and has priority.

The recently proposed species Eubacterium minutum and Eubacterium tardum appeared to be similar from their published descriptions. The aim of this study was to perform phenotypic and genetic analyses of strains of both species to clarify their taxonomic position. The type strains of E. minutum and E. tardum exhibited identical biochemical and protein profiles and their 16S rRNA gene sequences displayed 99.9% similarity. The G + C content of the DNA of both strains was estimated at 45 mol%. It is concluded that E. minutum and E. tardum are synonyms; E. minutum has priority. An emended description of E. minutum is given.

Bacterial Typing Techniques↗
PubMed1999

Detection of unculturable bacteria in periodontal health and disease by PCR.

Recently developed molecular methods have made it possible to characterize mixed microflora in their entirety, including the substantial numbers of bacteria which do not grow on artificial culture media. In a previous study, molecular analysis of the microflora associated with acute oral infections resulted in the identification of three phylotypes, PUS3.42, PUS9.170, and PUS9.180, representing as-yet-uncultured organisms. The aim of this study was to design and validate specific PCR primers for these phylotypes and to determine their incidences in samples collected from healthy and diseased periodontal tissues. Two specific reverse primers were devised for each phylotype, and these were used in duplex PCRs with universal forward and reverse primers. All three phylotypes were detected in periodontal sites; PUS9.170, related to oral asaccharolytic Eubacterium spp., was significantly associated with disease. This study demonstrates the possibility of using unculturable, and therefore uncharacterized, organisms as markers of disease.

Adult↗
PubMed1998

The deconvolution of pyrolysis mass spectra using genetic programming: application to the identification of some Eubacterium species.

Pyrolysis mass spectrometry was used to produce complex biochemical fingerprints of Eubacterium exiguum, E. infirmum, E. tardum and E. timidum. To examine the relationship between these organisms the spectra were clustered by canonical variates analysis, and four clusters, one for each species, were observed. In an earlier study we trained artificial neural networks to identify these clinical isolates successfully; however, the information used by the neural network was not accessible from this so-called 'black box' technique. To allow the deconvolution of such complex spectra (in terms of which masses were important for discrimination) it was necessary to develop a system that itself produces 'rules' that are readily comprehensible. We here exploit the evolutionary computational technique of genetic programming; this rapidly and automatically produced simple mathematical functions that were also able to classify organisms to each of the four bacterial groups correctly and unambiguously. Since the rules used only a very limited set of masses, from a search space some 50 orders of magnitude greater than the dimensionality actually necessary, visual discrimination of the organisms on the basis of these spectral masses alone was also then possible.

Artificial Intelligence↗