Control of microbial contamination in dental unit water systems using tetra-sodium EDTA.

AIM: To examine the efficacy of tetra-sodium EDTA in controlling microbial contamination of dental unit water systems (DUWS). METHODS AND RESULTS: Ten dental units were treated once a week with either 4% or 8% tetra-sodium EDTA for four or two consecutive weeks, respectively. Before treatment, 43% and 60% of the water samples from the air/water triple syringe and high-speed hand-pieces, respectively, exceeded the American Dental Association (ADA) guidelines of 200 CFU ml(-1) water during a 6-week baseline period. After each weekend treatment, the levels of microbial contamination in all DUWS fell significantly (P < 0.001) to below the ADA guideline. By the end of the week, microbial counts in the outflowing water had returned to baseline levels indicating a transient effect of single doses of tetra-sodium EDTA, and the need for multiple applications. The biofilms were virtually eliminated after a single weekend treatment. CONCLUSIONS: Tetra-sodium EDTA is effective in controlling microbial contamination in DUWS. SIGNIFICANCE AND IMPACT OF THE STUDY: Inexpensive, effective and safe products for reducing the microbial load of water from DUWS are needed to meet ADA and other national guidelines. Tetra-sodium EDTA can significantly reduce microbial biofilms and bacterial counts in outflowing water, and is compatible for use in DUWS.

Inhibition of biofilms associated with dentures and toothbrushes by tetrasodium EDTA.

AIMS: We examined the efficacy of tetrasodium EDTA in eradicating biofilms derived from salivary inocula or pure cultures of Candida albicans on discs of polymethyl methacrylate (PMMA) denture base or on toothbrushes that had been used normally for 4-8 weeks. Its efficiency in virus neutralization was also determined. METHODS AND RESULTS: Overnight (16 h) treatment with 4% (w/v) tetrasodium EDTA solution reduced salivary and C. albicans biofilm viable counts by > or =99%. Biofilm removal was confirmed using confocal laser scanning microscopy. Presence/absence of sucrose during biofilm formation had no effect on killing efficacy. Prolonged treatment of PMMA with tetrasodium EDTA did not influence subsequent formation of C. albicans biofilms or affect surface roughness of the PMMA, but it reduced subsequent biofilm formation from a salivary inoculum. Infectivities of herpes simplex virus and polio virus suspensions were reduced by >99.99% by treatment for 1 and 2 h, respectively. CONCLUSIONS: Tetrasodium EDTA solution efficiently disinfected toothbrushes and PMMA discs, with the detachment of biofilms, and rapidly neutralized both nonenveloped and enveloped viruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Dentures and toothbrushes become contaminated by bacterial biofilms and by viruses. There is a need for disinfection methods that are rapidly effective, cost-effective, nontoxic and easily implemented. These studies indicate that tetrasodium EDTA solution has disinfection applications in the oral care field.

The oral microflora--friend or foe? Can we decide?

This paper discusses the factors that determine whether the oral microflora play a beneficial or detrimental role in the health of an individual during their life. The resident microflora of the mouth is diverse, and distinct from that found elsewhere in the body due to its unique biological and physical properties. This natural microflora is essential for the normal development of the physiology of the host, and contributes to the host defences by excluding exogenous micro-organisms. The oral microflora varies in composition on distinct surfaces (e.g. teeth, mucosa), and at sites on a specific surface (e.g. fissures, gingival crevice), demonstrating that subtle properties of a habitat influence the ability of individual species to colonise and dominate. The composition of these oral microbial communities remains relatively stable over time (microbial homeostasis). This stability does not indicate a passive relationship with the host, but reflects a dynamic balance among the component species. However, this stability can be perturbed by significant changes to the oral environment or in a person's life-style that occur during the life of an individual. Alterations in diet, medication, smoking, saliva flow, denture wearing, general health, etc, can lead to overgrowth by previously minor components of the oral microflora, which can predispose a site to disease. Likewise, the immune response can wane in old age, which may result in colonisation by exogenous and often pathogenic micro-organisms. Oral micro-organisms can also act as opportunistic pathogens, and cause serious disease elsewhere in the body. Therefore, active oral health care management is needed in order to maintain microbial homeostasis throughout life to ensure that we reap the benefits of our resident oral bacteria and not suffer from their mischief.

High power air-clad photonic crystal fibre laser.

An Ytterbium-doped photonic crystal fibre laser is demonstrated with a 100 microm2 core area and single transverse mode with an output efficiency of 30 %. Double-clad PCF laser structures are demonstrated with pump cladding NA greater than 0.8 and output power up to 3.9 W. Such lasers are potentially scalable to high power.

Temperature elevation regulates iron protoporphyrin IX and hemoglobin binding by Porphyromonas gingivalis.

Porphyromonas gingivalis, an obligate anerobe with a growth requirement for iron protoporphyrin IX (FePPIX), is exposed to increased temperatures in the inflamed periodontal pocket. In this study, P. gingivalis was grown in a chemostat at 37 degrees C (control), 39 degrees C, and 41 degrees C, and examined for hemagglutinating (HA) activity, hemoglobin binding and degrading activity, and iron protoporphyrin IX binding. HA activity decreased in cells as the growth temperature increased. Binding of mu-oxo bishaem (dimeric haem), and Fe(II)- and Fe(III)-monomeric forms was increased in 39 degrees C-grown cells but decreased in 41 degrees C-grown cells compared with controls. Cellular hemoglobin binding and degradation decreased with increased growth temperature. The decrease in cellular hemagglutination and hemoglobin degradation occurring with increased growth temperature would limit the potential overproduction of toxic monomeric haem molecules. The increased binding of mu-oxo bishaem and monomeric forms of FePPIX at 39 degrees C may reflect a defense strategy against reactive oxidants and a mechanism of dampening down the inflammatory response to maintain an ecological balance.

Identification of ragAB as a temperature-regulated operon of Porphyromonas gingivalis W50 using differential display of randomly primed RNA.

Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of P. gingivalis has the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island ragAB; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of P. gingivalis by changing its patterns of gene expression. In this study, RNA has been isolated from cells of P. gingivalis grown to steady state at temperatures of 37, 39, and 41 degrees C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the ragAB locus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of ragAB may be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.

Modulation of antibacterial peptide activity by products of Porphyromonas gingivalis and Prevotella spp.

This study investigated the ability of anaerobic periodontal bacteria to inactivate and resist killing by antimicrobial peptides through production of extracellular proteases. Antibacterial activities of peptides were assessed in a double-layer agarose diffusion assay, and MICs and MBCs were determined in broth microdilution assays. Culture supernates of Porphyromonas gingivalis and Prevotella spp. inactivated mastoparan, magainin II and cecropin B whilst Gram-positive oral supragingival bacteria had no effect. Inactivation was prevented by protease inhibitors and was unaffected by 45% human serum. Purified proteases from the periodontopathogen Porph. gingivalis inactivated peptides [cecropin B, brevinin, CAMEL (cecropin A 1-7 + melittin 2-9), mastoparan] as would be predicted from the amino acid sequences of the peptides and the known bond specificities of these Arg-x and Lys-x enzymes. MALDI-TOF MS revealed that inactivation of cecropin B by Porph. gingivalis protease was due to specific cleavage of the molecule. Inactivation of cecropin B by proteases took 10-15 min. Paradoxically, MICs of cecropin B against Porph. gingivalis and Prevotella intermedia were low, while Prevotella nigrescens was resistant, suggesting that production of proteases alone is insufficient to protect Porph. gingivalis and Prev. intermedia from the action of antimicrobial peptides. Thus, antimicrobial peptides could be developed as therapeutic agents targeted against specific periodontal pathogens.

Effect of temperature on growth, hemagglutination, and protease activity of Porphyromonas gingivalis.

Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation. Temperature is an environmental factor that can modulate gene expression. Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen, Porphyromonas gingivalis. P. gingivalis W50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41 degrees C; cultures were monitored for protease and hemagglutinin activity. P. gingivalis grew well at all three temperatures. An increase in growth temperature from 37 to 39 degrees C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P < 0.01). A further rise in growth temperature to 41 degrees C led to even greater reductions in arginine-specific (82%; P < 0.001) and lysine-specific (73%; P < 0. 01) activities. These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms. At 41 degrees C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains. The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41 degrees C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination. Thus, as the environmental temperature increased, P. gingivalis adopted a less aggressive phenotype, while retaining cell population levels. The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases.

Rapid and effective processing of blood specimens for diagnostic PCR using filter paper and Chelex-100.

AIM: To provide a more efficient method for isolating DNA from peripheral blood for use in diagnostic DNA mutation analysis. METHODS: The use of blood impregnated filter paper and Chelex-100 in DNA isolation was evaluated and compared with standard DNA isolation techniques. RESULTS: In polymerase chain reaction (PCR) based assays of five point mutations, identical results were obtained with DNA isolated routinely from peripheral blood and isolated using the filter paper and Chelex-100 method. CONCLUSION: In the clinical setting, this method provides a useful alternative to conventional DNA isolation. It is easily implemented and inexpensive, and provides sufficient, stable DNA for multiple assays. The potential for specimen contamination is reduced because most of the steps are performed in a single microcentrifuge tube. In addition, this method provides for easy storage and transport of samples from the point of acquisition.

Age-related changes in salivary antibodies to commensal oral and gut biota.

The prevalence of mucosally derived infections appears to increase with age, suggesting dysfunction at the mucosal surfaces. The present investigation was undertaken to examine any age-related changes in secretion rates and concentrations of secretory antibodies in whole and parotid saliva in a healthy adult population. A total of 116 subjects were subdivided into the following age groups: 20-39 years, 40-59 years, 60-79 years and 80 years and over. Specific immunoglobulin A (IgA), IgG and IgM antibodies in whole and parotid saliva to Streptococcus mutans (serotype c), Actinomyces viscosus NCTC 10951, and Escherichia coli NCTC 10418 were quantified by enzyme-linked immunosorbent assay. IgA antibodies to all three organisms examined increased with age in both whole and parotid saliva, whereas IgG antibody levels to S. mutans in whole saliva were significantly decreased with age. IgG antibodies to E. coli in parotid saliva were reduced in older age groups. IgM antibody levels to S. mutans were reduced with age in both secretions, whereas IgM antibodies to A. viscosus were greatest in the oldest age groups. No significant changes with age were observed in salivary IgM antibody levels to E. coli. No significant reduction in the secretion rates of IgA antibodies were observed in parotid or whole saliva, whereas IgG and IgM antibody secretion rates to all three microorganisms were reduced in most age groups in both whole and parotid saliva. The results of this investigation have demonstrated age-related changes with salivary antibodies, but, whereas salivary IgG and IgM antibodies showed decreases, salivary IgA levels generally increased with age. This suggests that the ability to form IgA antibody responses is not impaired with increased age, and that secretion rates and functional properties of antibodies may be as important as concentrations in protection against mucosal infective diseases.

A method for the quantitative site-specific study of the biochemistry within dental plaque biofilms formed in vivo.

The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ. A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conditions in vitro. Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources. Analytical data can be related to plaque volume rather than weight. Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.

Serum antibodies to commensal oral and gut bacteria vary with age.

This study examined the relationship between serum antibody levels to selected bacteria from the commensal oral and gut flora with increased age in a healthy adult population. A total of 116 healthy subjects were studied consisting of the following age groups: 20-39 years (group A), 40-59 years (group B), 60-79 years (group C) and 80+ years (group D). Only significantly lower mean IgM antibody levels to Streptococcus mutans strain Guy's serotype c were observed in older age groups (P < 0.001). With Actinomyces viscosus NCTC 10951 significantly reduced IgM levels (P < 0.02) and significantly elevated IgA levels were observed with increased age (P < 0.05). IgA and IgG antibodies to Escherichia coli NCTC 10418 were increased significantly in the older age groups (P < 0.001), whilst a trend toward lower levels of IgM antibodies was recorded with age. No changes in IgA antibodies to Streptococcus faecalis NCTC 775 were observed but the lowest level of IgM antibodies were detected in the oldest age group (P < 0.05). Mean specific activity was decreased with age with IgM antibodies to the oral bacteria and increased with age with IgG and IgA antibodies to E. coli. Overall, our results suggest a general reduction in serum IgM antibody responses. This impairment in the circulatory IgM immune response may contribute to the increased occurrence of infections in the elderly.

Age-related changes in immunoglobulin isotypes in whole and parotid saliva and serum in healthy individuals.

Mucosal infections account for the majority of infections seen in elderly people, but little is known of whether mucosal immunity decreases with age. The objective of this study was to investigate the effect of age on the levels of salivary and serum immunoglobulins and the salivary immunoglobulin secretion rates in a healthy adult population. Healthy subjects (116 total) were divided into the following age groups: 20-39; 40-59; 60-79 and >80 years. Unstimulated (resting) whole and stimulated parotid saliva and serum were collected from all participants. Salivary and serum immunoglobulins were quantified by enzyme-linked immunosorbent capture assays. The levels of serum immunoglobulin G (IgG) and IgM were significantly reduced in the oldest age group, whereas no significant reduction in the level of IgA with age was observed. The IgG and IgA levels in whole saliva increased significantly in the oldest age group D, but no changes were detected in IgM levels. No significant changes in any immunoglobulin levels with age were found in parotid saliva. However, significant reductions in the secretion rates of IgA and IgM, but not IgG, in whole saliva were detected in the oldest age group. No significant changes in the secretion rates in parotid saliva were found with age. Our results demonstrate a decline in immunoglobulin concentrations with increased age, which may contribute to the increased susceptibility of elderly individuals to infectious diseases.

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.

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.

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)

Biochemical and histological evidence that carcinoma of the prostate is associated with increased bone resorption.

We have investigated the hypothesis that carcinoma of the prostate with skeletal metastases is associated with increased bone resorption. In 54 affected patients a close correlation was observed between serum activity of alkaline phosphatase and urinary excretion of hydroxyproline (r = +0.818; P less than 0.001), comparable to that seen in Paget's disease of bone. The administration of synthetic salmon calcitonin (100 U subcutaneously) induced a significant fall in serum calcium and urinary excretion of hydroxyproline, proportional to the prevailing rate of bone turnover, as assessed by serum alkaline phosphatase or hydroxyprolinuria. Administration of the diphosphonate, etidronate, also decreased hydroxyprolinuria, suggesting that urinary hydroxyproline reflected increased rates of bone resorption in this disorder. Histology of bone in sites adjacent to and distant from skeletal metastases showed increased histological indices of bone resorption. These results suggest that the skeletal disease associated with prostatic carcinoma is characterized by generalized increases in bone resorption as well as focal increases in bone formation.

Skeletal effects of carcinoma of the breast and prostate.

Recent research has led to improved understanding of the pathology of skeletal metastases in carcinoma of the breast and prostate. Several humoral mechanisms have been identified which have both primary and secondary consequences on skeletal metabolism and probably depend on the complex interplay of a number of factors derived from tumour tissues. An improved understanding of these interactions may lead to new approaches in the management of these common disorders.