Rimondia S. Percival

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)

Temperature-Dependent Modulation of Porphyromonas gingivalis Lipid A Structure and Interaction with the Innate Host Defenses

ABSTRACT Lipid A structure is a critical determinant of the interaction between pathogens and the innate immune system. Previously, we demonstrated the presence of non- and monophosphorylated tetra-acylated lipid A structures in the outer membrane of Porphyromonas gingivalis, an agent of human periodontal disease. These modifications to lipid A structure lead to evasion and suppression of innate defenses mediated by Toll-like receptor 4 (TLR4) and cationic antimicrobial peptides. In this investigation, we examined the influence of growth temperature on P. gingivalis lipid A structure and recognition by TLR4 as an example of an environmental influence which is known to vary between healthy and diseased sites in the periodontium. We demonstrate that P. gingivalis grown at a normal body temperature produces mainly nonphosphorylated and monophosphorylated tetra-acylated lipid A structures, whereas bacteria grown at 39°C and 41°C intended to mimic increasing levels of inflammation, producing increasing proportions of monophosphorylated, penta-acylated lipid A. The temperature-dependent alteration in lipid A renders the bacterium significantly more potent for activating TLR4 and more susceptible to killing by β-defensins 2 and 3. This is the first report of a lipid A remodeling system linked to temperature shifts associated with a deregulated inflammatory response. Temperature elevation at sites of inflammation in the periodontium may be a significant environmental regulator of the lipid A modification systems of P. gingivalis, which will influence the interaction of this organism with the innate host defense.

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.

Identification of ragAB as a Temperature-Regulated Operon of Porphyromonas gingivalis W50 Using Differential Display of Randomly Primed RNA

ABSTRACT 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. gingivalisgrown to steady state at temperatures of 37, 39, and 41°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.

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.

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).

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°C (control), 39°C, and 41°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 μ-oxo bishaem (dimeric haem), and Fe(II)- and Fe(III)-monomeric forms was increased in 39°C-grown cells but decreased in 41°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 μ-oxo bishaem and monomeric forms of FePPIX at 39°C may reflect a defense strategy against reactive oxidants and a mechanism of dampening down the inflammatory response to maintain an ecological balance.