Production of hydrolytic enzymes by oral isolates of Eikenella corrodens.

Eikenella corrodens isolates from periodontally healthy subjects and adult periodontitis patients were compared for their ability to produce a range of potential virulence factors. All were positive for proline aminopeptidase, thiol-dependent haemolysin and esterase activities. Low or negative activities were found against casein, phospholipid, lipid, collagen, aminophosphate, phosphate under acid or alkaline conditions, and eleven other amino acid substrates tested. In oral infections, the haemolytic activity of E. corrodens could be amplified in the reduced environment of the periodontal pocket and damage host cells. Proline aminopeptidase may act against proline residues in collagen, immunoglobulins and complement proteins.

Streptococcus parasanguis sp. nov., an atypical viridans Streptococcus from human clinical specimens.

Molecular taxonomic studies were performed on ten strains of an unusual 'viridans streptococcus' that were originally isolated from human throats, blood and urine. On the basis of DNA-DNA hybridization studies the strains formed a single homology group distinct from all recognized species of oral and viridans streptococci. 16S ribosomal RNA reverse transcriptase sequence studies confirmed the genealogical distinctiveness of the human strains. The results of the present study clearly demonstrate that the human strains represent a new species of the viridans group for which the name Streptococcus parasanguis sp. nov. is proposed. The type strain is ATCC 15912.

A scheme for the identification of viridans streptococci.

A collection of strains representing all the currently recognised species of oral or viridans streptococci (Streptococcus sanguis, S. parasanguis, S. gordonii, S. oralis, S. mitis, S. salivarius, S. vestibularis, S. anginosus, S. constellatus, s. intermedius, S. mutans and S. sobrinus) were tested for the production of a range of glycosidase activities with 4-methylumbelliferyl-linked fluorogenic substrates, and for reactions in a range of conventional fermentation and hydrolytic tests. The resulting biochemical scheme, consisting of 14 tests, enabled the differentiation of all these species and distinguished three biotypes within S. sanguis. The scheme reported here represents an improvement over currently available schemes for the identification of viridans streptococci.

Preformed enzyme profiles of reference strains of gram-positive anaerobic cocci.

The preformed (constitutive) enzyme profiles of 30 type strains and reference strains of gram-positive anaerobic cocci were determined with two commercial systems, RapID ANA and a prototype system from API. Both systems identified Peptostreptococcus anaerobius, Ps. asaccharolyticus, Ps. indolicus, Ps. magnus and Ps. micros accurately, except for one strain of Ps. magnus misidentified as Ps. micros by the RapID ANA system. The indole-negative, butyrate-producing cocci (classified at present as Ps. prevotii and Ps. tetradius) produced several different, unique patterns with the prototype API system, but the results with RapID ANA were often misleading. Eight strains of Hare group cocci produced previously described profiles. Four strains of streptococci produced profiles easily distinguished from those of the gram-positive anaerobic cocci. We conclude that most gram-positive anaerobic cocci can be identified rapidly and reliably to the species level by their preformed enzyme profiles, providing that their underlying classification is sound. Problems were encountered with the butyrate-producing cocci, which appear to be a more heterogeneous group of organisms than is currently acknowledged; further taxonomic studies on these organisms are required.

Prevalence of helicobacter pylori at oral and gastrointestinal sites in children: evidence for possible oral-to-oral transmission.

Acquisition of Helicobacter pylori occurs mainly in childhood. However, the mode of transmission remains unclear. To help elucidate this, 100 children attending for upper gastrointestinal endoscopy were investigated for the presence of H. pylori at various sites. H. pylori was detected in antral gastric biopsies by the rapid urease test (13 patients), culture (13 patients), histology (15 patients) and PCR (20 patients). Gastric juice was positive for H. pylori in 3 patients by culture and 11 patients by PCR. The dental plaque from 68% of gastric biopsy-positive patients (as determined by culture or PCR) and 24% of gastric biopsy-negative patients was positive for H. pylori by PCR. The presence of H. pylori in dental plaque was significantly associated with the presence of this organism in the stomach. H. pylori was detected by PCR in the faeces of 25% of gastric biopsy-positive children sampled. H. pylori was not cultured on any occasion from the oral cavity or faeces. The evidence from this study suggests that oral-to-oral transmission may be a possible mode of spread of H. pylori in children.

A numerical taxonomic study of the "Streptococcus milleri" group based upon conventional phenotypic tests and pyrolysis mass spectrometry.

Clinical strains presumptively identified as Streptococcus milleri (60), and blind coded collection strains (21) were characterised in conventional tests and pyrolysis mass spectrometry. Comparison of the clusters found by these two approaches revealed five clearly distinct centres of variation. Three corresponded to the DNA homology groups suggested by Whiley and Hardie (1989) as representing the species S. anginosus, S. intermedius and S. constellatus; a fourth comprised three Lancefield group C beta-haemolytic strains; the fifth may represent a biotype of S. anginosus. The characteristics of the latter group are described.

Quantitative culture of Helicobacter pylori from gastric juice: the potential for transmission.

The transmission of Helicobacter pylori may occur by spread of organisms from gastric juice which has been introduced into the mouth by gastro-oesophageal reflux. The aim of this study was to quantify the load of H. pylori present in gastric juice available for transmission. Gastric antral biopsy and gastric juice samples were collected from 108 adult dyspeptic patients undergoing routine upper gastroscopy and the presence of H. pylori was determined. In all, 54 (50%) of 108 patients gave positive results in the gastric antral biopsy rapid urease test and for H. pylori histology. The gastric juice of 40 (37%) of patients gave positive results for the urease A gene by PCR assay; 34 (31%) of patients were positive by these three tests and H. pylori was cultured from the gastric juice of 13 (38%) of these patients. The median count of H. pylori in gastric juice was 1.75 x 10(1) cfu/ml. Viable organisms in gastric juice may lead to transmission of H. pylori when refluxed or vomited into the mouth.

Comparison of the effects of anaerobic and micro-aerophilic incubation on resistance of Helicobacter pylori to metronidazole.

To assess the influence of incubation conditions on the resistance of Helicobacter pylori this study compared the effect of micro-aerophilic and anaerobic incubation followed by micro-aerophilic incubation on the measurement of metronidazole resistance of 102 H. pylori isolates, by both disk diffusion and Epsilometer (E)-tests. Anaerobic incubation for 24 h before micro-aerophilic incubation for 48 h consistently increased metronidazole activity in both assay methods. Although statistically significant, this was microbiologically less significant, as only 4 of 102 isolates gave discrepant readings (all four were resistant in micro-aerophilic conditions but susceptible in anaerobic/micro-aerophilic conditions). In all four cases variation was by a few millimeters in zone size (i.e., all were close to the cut-off point). There was 100% agreement between disk diffusion and E-test results. Of 104 observations (52 duplicate assays: 13 strains, two atmospheric conditions, two methods of determining resistance) there was 100% intra-observer and inter-observer agreement with regard to susceptibility and resistance status for both E-test and disk diffusion methods. Anaerobic incubation followed by micro-aerophilic incubation had little effect on the estimation of prevalence of metronidazole resistance and seemed to add little, if any, significant advantage over micro-aerophilic incubation alone.

Conservation of the cytotoxin-associated (cagA) gene of Helicobacter pylori and investigation of association with vacuolating-cytotoxin activity and gastroduodenal disease.

Polymerase chain reaction (PCR) amplification and DNA hybridization analyses were used to test for the presence of the cytotoxin-associated (cagA) gene in 108 strains of Helicobacter pylori. Fifty-two geographically diverse strains of known vacuolating cytotoxin activity, and 56 recent UK clinical isolates from patients with duodenal ulceration (n = 28) and from healthy individuals who were endoscopically normal (n = 28) were studied. Overall, cagA was detected by PCR in 74 (69%) strains and DNA hybridization provided evidence of gene homologues in a further eight strains. For 96% of the cytotoxin-producing strains and 46% of the non-cytotoxin producing strains, there was a close association either with presence or absence of cagA. At the genomic level, Southern blot DNA hybridization showed that cagA was probably present in a single copy in most of the H. pylori tested, and that HaeIII restriction site variation within and around the gene provided additional markers of diversity for the species. As 40% of the cagA containing strains did not produce an active cytotoxin, and no significant association between cagA presence and DU-disease was observed, we concluded that the presence of the cagA gene in H. pylori could not be used as a single reliable predictor of higher risk patients.

Some serological cross-reactions between Streptococcus mutans, S sanguis, and other dental plaque streptococci.

From the preliminary observations described in this report, the following conclusions may be drawn. When attempting to produce diagnostic antiserums specific for S mutans, S mitior, or S sanguis, care must be taken to eliminate cross-reactions as a result of antibodies against extracellular glucans or dextrans. Other antigenic components, associated with the cell wall, may be common to different species. One soluble protein or polypeptide antigen normally present in S sanguis and S mitior may also be demonstrated in certain strains of S mutans. Antiserums prepared against S sanguis, S mitior, or S mutans should be carefully checked for interspecies cross-reactions before being used for identification purposes.

Physiological classification of oral viridans streptococci.

The human mouth harbors large numbers of a variety of greening streptococci. For many years the classification and nomenclature of these organisms have been confused, but recent studies have clarified the situation considerably. Five distinct species can be distinguished among the oral streptococci S mutans, S sanguis, S mitior, S milleri, and S salivarius, and these may be identified using a simple scheme consisting of seven physiological tests. Further subdivisions within these species may be achieved using serological methods. A small number of oral streptococci remain which cannot easily be identified with existing schemes. These may represent additional species that, as yet, have not been adequately described. There seems little justification for continuing the practive of calling all alpha-hemolytic streptococci simple "Streptococcus viridans".

The prevalence and significance of yeasts in persons wearing complete dentures with soft-lining materials.

Fifty-three persons wearing soft-lined mandibular dentures and heat-cured acrylic-resin maxillary dentures were studied, using imprint cultures, to determine the isolation frequency and density of colonization of denture and mucosal surfaces by yeasts. Yeasts were isolated from 35 (66%) of the persons studied. Nine species of Candida and one each of Trichosporon and Saccharomyces were identified. Candida albicans, occurring either alone or together with another strain, was identified in 66% of the isolates and was associated with a higher mean density/cm2 than that of other strains. An association between the method of denture cleaning, denture hygiene, and smoking habits and the isolation of yeasts was demonstrated, but a similar association could not be demonstrated with the sex of the person, denture-wearing habits, type and condition of the soft lining, or the clinical appearance of the mandibular denture-bearing mucosa. Although yeasts are more likely to colonize soft-lining materials than the fitting surface of conventional lower dentures, their presence did not significantly affect the soft-lining material. Further, the increased isolation of yeasts on the fitting surface of the soft-lined mandibular denture was not associated with an increased incidence of inflammatory changes in the mandibular denture-bearing mucosa.

Antigens from Actinomyces species and their value in identification.

Actinomyces have a complex antigenic structure that includes neutral cell wall carbohydrate and some polypeptide containing charged antigens. Wall carbohydrate can be prepared in a relatively pure form from cell walls or by DEAE Sephadex chromatography of acid extracts and supernatant culture fluid. Charged antigens are generally eluted in groups by batch-elution from columns. Cell wall carbohydrate can be responsible for species-specific reactions and cross-reactions, and strains may possess more than one cell wall carbohydrate determinant. The charged antigens also can be species specific, but they cause some cross-reactions, particularly those between the A israelii serotypes. Autoclave extracts of strains, tested against antiserums reacting with cell wall carbohydrate, may be valuable in routine identification of isolates.

A longitudinal epidemiological study on dental plaque and the development of dental caries--interim results after two years.

1. During the two-year period, caries developed at 20% of the target premolar sites. The attack rate for these surfaces was similar in the plaque panel and the other subjects in the study. 2. The microbial composition of plaque samples from caries-free sites and from carious sites before and after radiographic detection of lesions was broadly similar. 3. Numerical domination of particular sites by S mutans before detection of caries can occur, but has only been observed so far in 2 of 15 sites. 4. Pooled date from sites which have developed lesions indicate a rise in the isolation frequency and mean numbers of S. mutans after detection of caries. This trend was particularly obvious in the one subject who developed bilateral lesions by the second examination and in three of four sites where caries was detected at the fourth examination. Similar observations have been made with lactobacilli. 5. In two of 15 instances no isolations of S mutans were made from sites which developed caries. 6. To date, no single species appears to be uniquely associated with the onset of dental caries.

Taxonomic study of "tufted mitior" strains of streptococci (Streptococcus sanguinis biotype 11); recognition of a new genospecies.

The taxonomic position of tufted strains of streptococci, phenotypically resembling Streptococcus mitis and previously referred to as 'tufted mitior' was investigated. By 16S rRNA sequence analysis, it was clear that the "tufted mitior" strains belonged to the mitis group of species within the genus Streptococcus. It was confirmed that these strains were taxonomically independent at the species level, sharing less than 43%, DNA-DNA similarity with all established species of the mitis group. However biochemical test data obtained, using three commercial identification kits (Rapid ID32 Strep, STREPTOGRAM, and Biolog GP-plate) together with in-house biochemical tests employing 4-MUF-linked fluorogenic substrates did not reveal sufficient differential tests with which to identify the "tufted mitior" strains unequivocally. From these data, we conclude that these "tufted mitior" strains represent a new taxon within the mitis group of the genus Streptococcus, and propose that they should be considered as a genospecies until differential phenotypic characteristics are found for their identification.

Migration and Helicobacter pylori seroprevalence: Bangladeshi migrants in the U.K.

Helicobacter pylori, an infectious agent of worldwide public health importance, has higher seroprevalence in developing countries than in developed countries. We investigated whether Bangladeshi women, born in Bangladesh, have a greater H. pylori seroprevalence than Bangladeshi women born in the U.K. and, in addition, whether there is an association between H. pylori seropositivity and age of migration to the U.K. amongst Bangladeshi women. Women attending antenatal clinics at the Royal London Hospital were screened using ELISA for anti-H. pylori IgG. In Bangladeshi individuals born in the U.K., 13/16 (81%, 95% confidence interval (CI) 54%-96%) and, in Bangladeshi individuals born in Bangladesh 91/137 (66%, 95% CI 59%-74%) had antibodies to H. pylori. No significant association was found between H. pylori seropositivity and country of birth, or age at migration to the U.K. Public health strategies concerning H. pylori should consider migrant populations with high seroprevalence of H. pylori.

The evaluation of a new technique for anaerobic sampling of deep periodontal pockets.

A new technique for the anaerobic sampling of deep periodontal pockets has been developed and evaluated in vitro and in vivo. The mean percentage difference of total viable counts from pairs of samples from nine deep periodontal pockets was found to be 76.4%. This compared favorably with an established technique for which the equivalent figure was 147.4%. Evidence was obtained that the first sample taken with the new sampler depleted the site. When correction was made for that effect, the mean percentage difference was found to be 31.1%. That value was in good agreement with the variation obtained by taking repeat samples from centrifuged deposits of pure cultures of bacteria.

A comparison between commercial kits and conventional methods for enumeration of salivary mutans streptococci and lactobacilli.

Mutans streptococci (ms) and lactobacilli levels were determined by conventional and commercial dip-slide methods in three groups of young subjects, aged 5-6 years (93 subjects), 12-13 years (78 subjects) and 18-20 years (81 subjects). Using the same paraffin-stimulated saliva samples, ms and lactobacilli were estimated by conventional viable counts on modified mitis-salivarius agar (MSB) and Rogosa agar plates, and by inoculation of Dentocult SM and Dentocult LB dip-slides (Orion Diagnostica, Finland). The salivary ms and lactobacilli counts obtained from conventional agar plates were significantly correlated (P < 0.0001) with the dip-slide estimates of these organisms (Kendal Tau = 0.56, 0.71 respectively). Subjects in Group 2 showed the highest proportion (77 per cent) of individuals with salivary ms levels above 2.5 x 10(5) cfu/ml saliva; 99 per cent of that group had detectable levels of lactobacilli. Significantly different median salivary ms and lactobacilli levels were demonstrated between all groups except for lactobacilli levels between Groups 2 and 3. These dip-slide tests provided suitable and simple methods for screening salivary lactobacilli and ms levels which may have a useful role in the assessment of caries risk.

Oral microbiology: current concepts in the microbiology of dental caries and periodontal disease.

The microbial flora of the mouth is highly complex, containing a wide variety of bacterial species. The most common types of oral disease, dental caries and periodontal disease, are both related to dental plaque and seem to occur when the normal balance between the microorganisms and the host is disturbed in some way. Dental caries is usually associated with increased numbers of mutans streptococci and lactobacilli at the sites of disease; estimation of salivary levels of these organisms may be useful for assessing caries risk in patients and for monitoring their response to preventive measures. A large number of 'candidate pathogens' have been identified as potential aetiological agents in different types of periodontal disease, although the 'specific plaque hypothesis' may still be controversial. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, together with the poorly understood spirochaetes, have most frequently been reported as significant periodontopathogens and a number of possible virulence factors have been described. Application of modern molecular techniques to the study of the microbiology of oral diseases should allow rapid further progress to be made and will lead, hopefully, to improved methods of diagnosis, risk assessment and treatment.

Clinical and microbiological effects of topical minocycline in the treatment of elderly patients with periodontitis.

OBJECTIVE: To evaluate a 2% w/w minocycline gel (Dentomycin) in the treatment of older adults with chronic periodontitis. DESIGN: Vehicle-controlled, split-mouth SETTING: Dental Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry. SUBJECTS: 22 adults (mean age 60 years). INTERVENTIONS: At baseline, patients received scaling and root planing. Test and placebo were applied at contralateral disease sites surrounding 22 test and 22 control teeth at baseline, and at weeks 2 and 4. MAIN OUTCOME MEASURE: Probing pocket depth, bleeding on probing and supragingival plaque measurements, and microbiological sampling, at one pocket site per tooth were undertaken at baseline, and at weeks 2, 4, 6 and 16. RESULTS: Microbial counts of Porphyromonas gingivalis, Prevotella intermedia and Eikenella corrodens did not change significantly over 16 weeks. No significant reductions in bleeding on probing and supragingival plaque score occurred with either gel. A larger reduction in mean pocket depth, from baseline to week 16, was found at test (6.50 to 4.95 mm; 24% reduction; P < 0.01) than at control sites (6.41 to 5.53 mm; 14% reduction; P < 0.05). CONCLUSIONS: Minocycline gel administration is a useful adjunct to scaling and root planing in the treatment of older adults with periodontitis.