Biomedical subjects

P D Marsh

Publications and source records attributed to P D Marsh.

At least 19 recordsLinked to original sources

PubMed1995

Congo red binding by Porphyromonas gingivalis is mediated by a 66 kDa outer-membrane protein.

Congo red was bound from solution by strains of Porphyromonas gingivalis including W50, HG189, HG184, NCTC 11834, Bg 381, WPH35, the slower brown pigmenting colonial variant W50/BR1, and the avirulent mutant W50/BE1, and by Porphyromonas endodontalis HG370 and Porphyromonas asaccharolytica B537. SDS-PAGE of whole cells of all species examined displayed a 66 kDa Congo-red-binding component which was also detected in the outer membranes of P. gingivalis W50 grown in the chemostat under both haemin limitation and haemin excess, and which corresponded to a Coomassie-blue-stained band of the same mobility. Pretreatment of haemin-excess batch-grown cells of P. gingivalis W50 with polymyxin B, which binds to lipid A, did not inhibit binding, whilst binding was enhanced in the presence of 2 M ammonium sulphate, suggesting the involvement of non-specific hydrophobic interactions. Binding was also reduced by pretreatment with trypsin and papain, and by 8-anilino-1-naphthalenesulphonic acid, which binds to hydrophobic amino acids. The 66 kDa binding component was sensitive to proteinase K digestion, and loss of Congo red staining of this band correlated with the quantitative reduction in Congo red binding by whole cells. These data, and our previous work, show that Congo red and iron protoporphyrin IX (haemin) are bound to different outer-membrane components, and that Congo red binding may be of little value as a marker to detect virulent strains of P. gingivalis or those expressing haemin-binding proteins.

Bacterial Outer Membrane Proteins↗

PubMed1994

The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50.

Porphyromonas gingivalis strain W50 was grown under haemin-limitation and haemin-excess conditions in a chemostat at pH 7.5. The maximum specific growth rate (mumax) was determined at both haemin concentrations (mumax = 0.236 +/- 0.052 and 0.271 +/- 0.039 h-1, respectively). This enabled dilution rates to be adjusted so that the virulence and enzyme activity of haemin-limited and haemin-replete cells could be compared at identical relative growth rates (murel) of 0.25, 0.50 and 0.75 of their respective mumax. The data showed that the fastest growing cells were significantly more virulent than those grown more slowly, irrespective of haemin concentration. However, at each growth rate tested, cells grown under haemin-excess conditions were always more virulent than haemin-limited cells. Trypsin-like enzyme activity of whole cultures was also greater at each growth rate under haemin-excess conditions while, conversely, collagenolytic activity was generally higher in haemin-limited cultures. Thus, although growth rate had an effect on the virulence and enzyme activity of P. gingivalis, the availability of haemin for growth was the most significant factor.

Animals↗

PubMed1994

Metabolic cooperation in oral microbial communities during growth on mucin.

Hog gastric mucin has been used as a model glycoprotein to determine the role of particular glycosidases produced by different oral bacteria in the development of stable, diverse microbial communities. The patterns of glycosidase and protease activity were determined in pure cultures of ten representative species of oral bacteria using synthetic substrates. A five-member mixed culture was established in a chemostat, comprising species with minimal glycosidase and protease activity, in which hog gastric mucin was the major carbon and energy source. Introduction of additional species with novel enzyme activities (e.g. sialidase, alpha-fucosidase and endopeptidase) led to their establishment within the community to make communities with seven, eight, nine and ten members and resulted in an increase in the total viable counts of the microbial consortium. This increase in viable count was made up of the numbers of the newly added species as well as from a rise in the numbers of the existing community members. This result suggested that glycoprotein catabolism involved the synergistic and concerted action of several species with overlapping patterns of enzyme activity. Such metabolic cooperation results in the liberation of additional nutrients, and this may help to maintain the characteristic diversity of resident microbial communities found in many natural habitats.

Animals↗

PubMed1994

Effect of sugar alcohols on the composition and metabolism of a mixed culture of oral bacteria grown in a chemostat.

Xylitol and sorbitol are effective as non-cariogenic sugar substitutes. A number of studies suggest that xylitol may have an additional, caries-reducing effect. This study examines the effect of xylitol and sorbitol, when pulsed together with glucose, on the composition and metabolism of a mixed culture of oral bacteria grown in a chemostat. In this system, xylitol had the effect of reducing the rate and extent of acid production when pulsed with glucose, compared with glucose pulsed alone, enabling acid-sensitive species to persist in greater numbers. Furthermore, the enrichment of Streptococcus mutans, observed following glucose pulses in the absence of pH control, was prevented by xylitol. In contrast, sorbitol had the opposite effects and S. mutans increased in proportion. The study provides evidence for a mechanism by which xylitol could affect the ecology of dental plaque and thereby reduce the rate of dental caries.

Bacteria, Anaerobic↗

PubMed1994

Procedures for establishing efficacy of antimicrobial agents for chemotherapeutic caries prevention.

Chemotherapeutics are presently considered for use in caries-preventive programs. The laboratory and in vivo testing of these agents, to some extent, parallels the methodology developed for the evaluation of fluoride products. However, fluoride is primarily effective by interfering with the de- and remineralization balance between enamel and the oral fluids. Antimicrobial and chemotherapeutic agents interfere with the bacterial colonization, growth, and metabolism of dental plaque. The initial selection of promising agents is done, therefore, by determination of the minimum inhibitory concentration (MIC) against a wide range of relevant bacterial species, together with tests of the effects of sub-MIC levels on the expression of virulence factors. In the hierarchy of tests proposed in this paper, studies of bacterial adhesion and enamel caries models form the next phase in the evaluation of agents. Also, mixed-culture studies are recommended to determine how a treatment perturbs a stable microflora. A final stage before clinical testing might involve intra-oral studies on limited numbers of volunteers. These should be tests of intra-oral substantivity and activity of the agents in formulated products, and in situ models of enamel caries lesion formation and remineralization. The latter type of study seems particularly appropriate to ensure that new agents do not decrease the efficacy of the other active components (e.g., fluoride) in caries-preventive products.

Animals↗

PubMed1994

Flow rates of resting whole and stimulated parotid saliva in relation to age and gender.

Dry mouth is a common feature in the elderly, but it is not clear what proportion of incidences are related to functional disturbances and whether age per se and gender play a role. The aim of this study was to determine the effects of age and gender on salivary flow rates. The effect of age on unstimulated (resting) whole and stimulated parotid saliva flow rates was determined in 116 unmedicated, healthy individuals. The subjects were divided into four age groups: 20-39 years (group A), 40-59 years (group B), 60-79 years (group C), and 80 years and over (group D). A significant decrease in the secretion rates of unstimulated whole saliva in relation to age was observed in the study population (p < 0.001). However, the flow rates of stimulated parotid saliva were not significantly different in the four age groups. Females had significantly lower mean flow rates than males for both unstimulated (resting) whole saliva (p < 0.005) and stimulated parotid saliva (p < 0.05). In the study as a whole, significant negative correlations were found between either the DMF index (decayed, missing, and filled teeth) or the DMFS index (decayed, missing, and filled tooth surfaces) and the flow rates of unstimulated whole saliva (p < 0.02), but no relationship to stimulated parotid saliva flow rates was apparent. The results suggest that elderly subjects have no impairment in their ability to respond to sialogogues but that resting saliva rates are significantly lower than in younger individuals and may contribute to the increase in oral mucosal diseases seen in the elderly.

Adolescent↗

PubMed1994

Microbial ecology of dental plaque and its significance in health and disease.

Dental plaque forms naturally on teeth and is of benefit to the host by helping to prevent colonization by exogenous species. The bacterial composition of plaque remains relatively stable despite regular exposure to minor environmental perturbations. This stability (microbial homeostasis) is due in part to a dynamic balance of both synergistic and antagonistic microbial interactions. However, homeostasis can break down, leading to shifts in the balance of the microflora, thereby predisposing sites to disease. For example, the frequent exposure of plaque to low pH leads to inhibition of acid-sensitive species and the selection of organisms with an aciduric physiology, such as mutans streptococci and lactobacilli. Similarly, plaque accumulation around the gingival margin leads to an inflammatory host response and an increased flow of gingival crevicular fluid. The subgingival microflora shifts from being mainly Gram-positive to being comprised of increased levels of obligately anaerobic, asaccharolytic Gram-negative organisms. It is proposed that disease can be prevented or treated not only by targeting the putative pathogens but also by interfering with the processes that drive the breakdown in homeostasis. Thus, the rate of acid production following sugar intake could be reduced by fluoride, alternative sweeteners, and low concentrations of antimicrobial agents, while oxygenating or redox agents could raise the Eh of periodontal pockets and prevent the growth and metabolism of obligately anaerobic species. These views have been incorporated into a modified hypothesis (the "ecological plaque hypothesis") to explain the relationship between the plaque microflora and the host in health and disease, and to identify new strategies for disease prevention.

Bacteria, Anaerobic↗

PubMed1993

Haemin-binding proteins of Porphyromonas gingivalis W50 grown in a chemostat under haemin-limitation.

Porphyromonas gingivalis W50 was grown in a chemostat at pH 7.3 under haemin-limitation and haemin-excess at a constant mean doubling time of 6.9 h. Outer membranes (OM) were extracted from whole cells using EDTA and compared by SDS-PAGE. Haemin-limited cells expressed novel outer membrane proteins (OMPs) of mol. mass 115, 113 and 19 kDa when samples were solubilized at 100 degrees C. A 46 kDa OMP was observed in haemin-excess cells but not in those from haemin-limited conditions. Tetramethylbenzidine (TMBZ) staining of gels, after OM solubilization at 20 degrees C, was used to detect haemin-binding proteins (HBPs). HBPs were observed only in OM from haemin-limited cells. The major HBP (mol. mass 32.4 kDa) corresponded to a similar sized Kenacid-blue-stained protein which was not observed in haemin-excess-derived OM. Haemin-limited cells and OM displayed a ladder-like series of Kenacid-blue-stained proteins. Lighter TMBZ-stained proteins of mol. mass 51, 53, 56 and 60 kDa, with mobilities corresponding to those of silver-stained LPS components, were observed in haemin-limited OM. No soluble HBPs were detected extracellularly. The greater number of HBPs expressed by cells grown under haemin-limitation may reflect an additional cell surface receptor system for haemin acquisition under low environmental levels of this essential cofactor.

Bacterial Outer Membrane Proteins↗

PubMed1992

Amoxycillin-resistant oral streptococci and experimental infective endocarditis in the rabbit.

The ability of three amoxycillin-resistant strains of Streptococcus sanguis 254, 24 and 297 (MIC 40 mg/L) to cause infective endocarditis (IE) in the rabbit was investigated. These strains all produced infection in the rabbit, as did an antibiotic sensitive control strain NCTC 7864. Prophylactic amoxycillin (400 mg/kg body weight) administered one hour before bacterial challenge prevented 80% of the animals developing IE irrespective of the challenging strain. It is concluded that amoxycillin-resistant strains of S. sanguis can cause IE and that amoxycillin prophylaxis can still be effective against these bacteria.

Amoxicillin↗

PubMed1992

The influence of denture-wearing and age on the oral microflora.

The effects of denture-wearing and age on the prevalence of selected bacteria of dental significance and on the carriage of opportunistic pathogens in molar plaque and whole saliva were determined in 120 healthy subjects, 41 of whom wore partial dentures. The subjects were divided into four age groups: 20-39 years (group A), 40-59 years (group B), 60-79 years (group C), and greater than or equal to 80 years (group D). The proportions, mean log10 viable counts, and isolation frequency of yeasts and lactobacilli in saliva and plaque were consistently higher in partial-denture wearers. The proportions of staphylococci and mutans streptococci were also raised in denture wearers, but these differences did not reach statistical significance. When the data were analyzed for age effects, both yeasts and lactobacilli were found to be increased in saliva with age, but statistically significant differences were generally found only between denture wearers in group D and subjects in the control group A. The isolation frequency of yeasts from plaque was also significantly higher in denture wearers of the oldest age group (D) compared with those in group A. A. viscosus predominated over A. naeslundii in the older age groups, regardless of the presence of dentures. Enterobacteria were isolated occasionally but only from the saliva of denture wearers in group D. Spirochetes and black-pigmented anaerobes were generally found in lower numbers in denture wearers. Collectively, the data show that components of the oral microflora in adults can be independently influenced by both age and the wearing of partial dentures.

Actinomyces↗

PubMed1992

Microbiological aspects of the chemical control of plaque and gingivitis.

Antimicrobial agents, delivered either by mouthrinse or toothpaste, can be used to maintain plaque at levels compatible with oral health by (a) reducing existing plaque, (b) preventing the formation of new plaque, (c) selectively inhibiting those particular bacteria that are associated with disease, and (d) inhibiting the expression of virulence determinants. Although many antimicrobial agents would appear to be suitable for plaque control, few have been found to possess clinical efficacy. This is because of inherent problems in the mode of action of agents in the mouth, and with difficulties with their formulation into dental products. Currently formulated antimicrobial agents include metal salts (e.g., zinc, stannous, copper), phenols (triclosan), plant extracts (sanguinarine), enzymes (e.g., glucanase, amyloglucosidase/glucose oxidase), "essential oils" (e.g., thymol, menthol), and bisbiguanides (chlorhexidine). Although many of these agents exhibit a broad spectrum of antimicrobial activity in the laboratory, they may display valuable selective properties on plaque. The effect of an agent will be concentration-dependent. Initially, the inhibitor may be briefly at levels above its MIC, but thereafter, it will be desorbed off oral surfaces and operate at sub-lethal concentrations. At these latter levels, agents can be effective by inhibiting metabolism (e.g., acid production, protease activity), and slowing bacterial growth. Agents with complementary modes of action are being combined to increase their antibacterial effectiveness. The long-term use of dental products containing antimicrobial agents should not (a) disrupt the natural balance of the oral microflora, (b) lead to colonization by exogenous organisms, or (c) lead to the development of microbial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Infective Agents, Local↗

PubMed1991

Haemin-restriction influences haemin-binding, haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50.

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.

Animals↗

PubMed1991

The significance of maintaining the stability of the natural microflora of the mouth.

The mouth, like other areas of the digestive tract, possesses a natural microflora, the presence of which confers several beneficial properties to the host. However, in the absence of adequate oral hygiene, dental plaque can accumulate beyond levels compatible with oral health. This leads to shifts in the balance of the predominant bacteria away from those associated with health; such shifts can predispose a site to caries or periodontal diseases. Treatment should be designed, therefore, to control rather than to eliminate dental plaque. Possible strategies to maintain the stability and beneficial properties of the natural oral microflora include the stimulation of saliva flow for an extended period directly after a meal, the avoidance of fermentable carbohydrates during between-meal periods, and improvements to oral hygiene, for example, by using products containing antiplaque or antimicrobial agents.

Anti-Bacterial Agents↗

PubMed1991

Age-related microbiological changes in the salivary and plaque microflora of healthy adults.

The effect of age on quantitative or qualitative differences in selected bacteria of dental significance and on the carriage of opportunistic pathogens and transient oral species was determined in 79 healthy, non-denture wearing individuals divided into four age groups: 20-39 years (group A), 40-59 years (group B), 60-79 years (group C) and greater than or equal to 80 years (group D). Samples of dental plaque and whole saliva were cultured on appropriate selective and non-selective bacteriological media. The total numbers of viable bacteria in saliva, and the prevalence of mutans streptococci in plaque and saliva were similar in all age groups. Similarly, there was no correlation between the numbers of spirochaetes in plaque and age. In contrast, statistically significantly higher mean proportions (p = 0.004), mean log10 viable counts (p = 0.001) and isolation frequencies (p less than 0.01) of lactobacilli were found in the saliva of those aged greater than or equal to 70 years compared to subjects in group A. The isolation frequency (p less than 0.05) and proportions (p = 0.056) of staphylococci in saliva were also higher in those aged greater than or equal to 70 years. Yeasts were isolated most often and in higher numbers from saliva in those aged greater than or equal to 80 years and the proportion of yeasts was higher after 60 years of age, but these differences were not significant in comparison with results from individuals in group A.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinomyces↗

PubMed1991

T cell proliferative responses to molecular fractions of periodontopathic bacteria.

Soluble antigenic preparations of Veillonella parvula and Bacteroides gingivalis were separated by SDS-PAGE and used after electroblotting and solubilization for in vitro lymphocyte stimulation in 13 patients with severe periodontitis and 12 controls. The cellular responses of controls and patients to V. parvula antigens were represented by four main proliferation-inducing fractions with 74-66, 52-46, 22-19 and 12 kD mol. wt. These fractions induced slightly enhanced DNA synthesis in lymphocytes from eight patients who failed to respond to whole antigenic extract. Lymphocyte samples from Veillonella whole extract unresponsive patients were also examined for in vitro proliferation by B. gingivalis fractions. Almost all stimulatory activities could be classified into five regions of 84-74, 35-31, 28-25, 17-15 and 12 kD.

Adult↗

PubMed1991

Aspects of the growth and metabolism of Fusobacterium nucleatum ATCC 10953 in continuous culture.

Fusobacterium nucleatum ATCC 10953, a type strain of one of the newly proposed subspecies of this group of organisms, was grown anaerobically in continuous culture in a chemically defined medium. Its response to conditions of varying pH, nutritional environment, and imposed growth rate were then examined. The organism failed to grow at pH 7.8 but grew at pH 5.8, although the cell yield was greatly reduced. At pH 6.8 the cell yield was halved and less than 50% of available glucose was consumed. The optimum growth pH was around 7.4 when the culture appeared to be limited for both glucose and the amino acids glutamate, histidine and serine. Some intracellular polyglucose (IP) was produced and acetate, butyrate and ammonia were the major fermentation end-products, as they were under all growth conditions tested. Increasing the available glucose or amino acids did not alter cell numbers but the amount of IP was greatly increased. When glucose was omitted from the medium, the cell yield was halved and the culture then became limited for lysine as well as for glutamate, histidine and serine. Growth rate had little overall effect on the organism's physiology and the maximum growth rate at pH 7.4 was 0.20 h-1, a doubling time of 3.5 h. Glucose was thus channelled into stable IP synthesis only when the growth limitation imposed by lack of fermentable amino acids was relieved. The metabolism of IP and the ability to obtain carbon and energy from a variety of substrates may explain why F. nucleatum is one of the most commonly detected organisms in subgingival dental plaque.

Amino Acids↗

PubMed1991

The immunogenicity of outer membrane proteins of haemin-depleted Porphyromonas (Bacteroides) gingivalis W50 in periodontal disease.

The antigens from outer membrane protein extracts of Porphyromonas gingivalis (W50), grown under different haemin concentrations, were examined for binding with serum antibodies from patients with severe progressive periodontitis or from periodontally healthy control subjects. P. gingivalis was grown under haemin limitation (0.33 micrograms/ml) and haemin excess (2.5 micrograms/ml) conditions in a chemostat at a mean generation time of 6.9 h, at pH 7.5. Sarkosyl-insoluble fractions of outer membrane proteins from P. gingivalis were prepared, and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. The SDS-PAGE analysis of the outer membrane of haemin-limited P. gingivalis identified several new protein components, or changed expression of bands compared with cells grown under haemin excess. Immunoblot analysis showed IgG antibodies to 2 haemin deprivation-induced proteins in patients with severe progressive periodontitis, but not in the control sera. These results confirm the immunogenicity of some of the haemin-regulated outer membrane proteins of P. gingivalis in severe progressive periodontitis.

Adolescent↗

PubMed1991

Dentifrices containing new agents for the control of plaque and gingivitis: microbiological aspects.

Antimicrobial agents have been proposed as playing an important role in controlling plaque and gingivitis. Unfortunately, a large number of potential compounds are unsuitable for use in dentifrices because they lack "substantivity", produce undesirable side-effects, or are incompatible with toothpaste ingredients. New agents that have been successfully incorporated into dentifrices include plant extracts, phenolic compounds and metal salts. Several products are currently being based on the phenol, Triclosan. Triclosan has a broad spectrum of antimicrobial activity against yeasts and oral bacteria. To enhance its clinical efficacy, Triclosan has been combined either with a co-polymer or with another compatible antimicrobial agent, zinc citrate. The co-polymer acts to increase the oral retention of Triclosan, and has resulted in further reductions in salivary bacterial counts in vivo. Zinc salts also have antimicrobial activity, and at low concentrations, can inhibit glycolysis and bacterial proteases. In mixed culture chemostat studies, Triclosan selectively inhibited Gram-negative periodontopathic bacteria; additive effects were obtained when zinc citrate and Triclosan were combined. In an experimental human gingivitis study, a zinc citrate/Triclosan dentifrice reduced plaque accumulation and gingivitis compared to a placebo paste; the ratio of anaerobic/aerobic bacteria and the proportions of Actinomyces species in plaque were also reduced. The prolonged use of a zinc citrate/Triclosan dentifrice neither significantly altered the ecology of supragingival plaque nor led to the selection of Triclosan-resistant bacteria. The data suggest that dentifrices containing new antimicrobial agents could be of clinical relevance in the prevention and control of plaque and gingivitis.

Anti-Bacterial Agents↗