Selective growth inhibition of Porphyromonas gingivalis by bestatin

Abstract Recent work in our laboratory indicates that selected protease/peptidase inhibitors interfere with the growth of Porphyromonas gingivalis . The aim of the present study was to further investigate the inhibitory effect of bestatin on the growth of P. gingivalis . Complete growth inhibition of P. gingivalis (11 strains) was observed when bestatin was incorporated at 2.5 μg ml⁻¹ in a complex broth medium. Fifty percent inhibition was still obtained with bestatin at a final concentration of 0.5 μg ml⁻¹. The inhibitory effect of bestatin was highly specific as the growth of 20 different oral bacterial species, including Gram-positive and Gram-negative as well as saccharolytic and asacharolytic bacteria, was not affected even at bestatin concentrations up to 50 μg ml⁻¹. Bestatin did not significantly affect the viability of P. gingivalis indicating that it has a bacteriostatic rather than a bactericidal effect. Growth assays using other specific inhibitors suggested that the effect of bestatin on the growth of P. gingivalis was unlikely to be related to its aminopeptidase inhibitor activity. Cultivation of P. gingivalis with a subinhibitory concentration of bestatin did not modify the cell envelope protein profile, as determined by SDS-PAGE analysis, but significantly decreased the number of extracellular vesicles produced. The present study indicated that bestasin is a highly effective inhibitor of cell growth of P. gingivalis . Additional studies will indicate whether bestatin should be considered as a potential drug in the control of P. gingivalis , a suspected pathogen in adult chronic periodontitis.     

Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements

Abstract The nucleotide sequence of IS 1126 , the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis , has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transpsosase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposashad homology to putative transposases of IS 1106 and IS 1186 both of which belong to the IS 5 group within the IS₄ super-family of insertion elements. On the basis of this homology we propose that IS 1126 should also be included in the IS 5 group. Southern-blot analysis of a number of P. gingivalis strains using IS 1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS 1126 from other closely related Porphyromonas species. This should allow IS 1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis .     

Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis

Several molecular methods are currently available for identification and discrimination of bacterial strains within the same species, which vary in efficiency and required labour. Here we applied a novel method for fingerprinting genomes, called arbitrarily primed PCR (AP-PCR), to the delineation of strains within the species Porphyromonas gingivalis. Using a single primer on a set of nine strains, nine simple distinct banding patterns, indicative of genetic polymorphism, were observed. Common amplicons and amplicons shared by only some strains were also observed, the latter suggesting that AP-PCR can be used to generate polymorphic markers. Genomic fingerprinting obtained by AP-PCR was independent of the quality of DNA. Assays performed directly using whole cells as a source of DNA template indicated that AP-PCR from colony is a quick, simple and accurate procedure.