Introduction. Diabetic foot infection (DFI) is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. DFIs are often complicated by ischaemia in the affected limb, the presence of polymicrobial biofilms and increasingly the occurrence of antibiotic resistant bacteria.Hypothesis/Gap statement. Antibiotic loaded beads could inhibit the growth of polymicrobial DFI communities with differing compositions in vitro.Aim. This study investigates the in vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure, Biocomposites Ltd. UK) against polymicrobial DFI communities and individual bacterial strains derived from DFIs.Methodology. Debrided tissue obtained from the base of infected diabetic foot ulcers was homogenised and spread over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates. Calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar and following incubation, zones of inhibition (ZOI) were measured. For individual bacterial strains isolated from the infected tissue, calcium sulfate beads containing vancomycin, gentamicin, flucloxacillin or rifampicin and beads containing a combination of vancomycin and gentamicin or flucloxacillin and rifampicin were tested for their ability to inhibit growth.Results. Calcium sulfate beads loaded with a combination of vancomycin and gentamicin were able to inhibit bacterial growth from all polymicrobial tissue homogenates tested, with ZOI diameters ranging from 15 to 40 mm. In the case of individual bacterial strains, beads containing combinations of vancomycin and gentamicin or flucloxacillin and rifampicin were able to produce ZOI with Gram-positive facultatitive anaerobic strains such as Staphylococcus aureus and Enterococcus faecalis, Gram-negative facultative anaerobic strains such as Pseudomonas aeruginosa and obligate anaerobic strains such as Finegoldia magna even where acquired resistance to one of the antibiotics in the combination was evidenced.Conclusion. The local use of calcium sulfate beads containing a combination of two antibiotics demonstrated high efficacy against polymicrobial DFI communities and individual DFI bacterial strains in in vitro zone of inhibition tests. These results show promise for clinical application, but further research and clinical studies are required.

Actinobacillus actinomycetemcomitans has been specifically implicated in the aetiology of one or more of the periodontal diseases, conditions in which inflammation of the gums is associated with destruction of the alveolar bone supporting the teeth. In these diseases there is loss of attachment of the gums (gingivae) to the teeth forming a periodontal pocket. The microenvironment of this pocket is extremely complex and it is likely that there will be substantial variation in the environmental conditions operating in this habitat. The aim of the current investigation was to study the effect of disease-relevant environmental factors on the production and release of secreted surface- associated proteins of A. actinomycetemcomitans. These secreted proteins contain many of the virulence determinants of this organism. A range of environmental conditions were investigated: growth in a CO(2)-enriched aerobic atmosphere vs anaerobic growth, presence of serum or blood, biofilm vs planktonic mode of growth and iron depletion. Differential expression of a number of the secreted surface-associated proteins was observed under different growth conditions and these included the glycolytic enzyme triose phosphate isomerase. An ability to adapt to prevailing environmental conditions may facilitate the survival of the organism in the changing microIenvironment of the periodontal pocket.

A phoA fusion library of Actinobacillus actinomycetemcomitans genomic DNA has been screened to identify genes encoding exported and secreted proteins. A total of 8,000 colonies were screened, and 80 positive colonies were detected. From these, 48 genes were identified with (i) more than half having homology to known or hypothetical Haemophilus influenzae genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. The proteins encoded by these genes may, by virtue of their presence on the cell surface, be novel virulence determinants.

There is evidence that the lipid A-associated proteins (LAPs) of enteric bacteria can induce the synthesis of interleukin 1 (IL-1) and therefore may be important virulence factors. Porphyromonas gingivalis is now recognized as a major pathogen in the chronic inflammatory periodontal diseases and it has previously been reported that a crude LAP fraction from this organism could induce IL-1 and interleukin 6 (IL-6) synthesis. In the present study the chemical and biological properties of the LAPs of this bacterium have been further characterized. Analysis by SDS-PAGE has shown that the LAPs comprise nine proteins of molecular masses 81, 68, 48, 47, 28, 25, 20, 17 and 16 kDa. These LAPs, at concentrations as low as 100 ng ml(-1), were shown to stimulate human gingival fibroblasts, human peripheral blood mononuclear cells and whole human blood to produce the pro-inflammatory cytokine IL-6. The cytokine-inducing activity of the LAPs was reduced after heat-inactivation and trypsinization, suggesting that the activity was not due to contaminating LPS. To establish which proteins in this mixture were the active cytokine inducers, the LAPs were separated by electrophoresis on polyacrylamide gels. The majority of the activity was associated with the 17 kDa LAP. N-terminal sequence analysis demonstrated that this protein was homologous to an internal region of a conserved adhesin domain contained within a family of P. gingivalis extracellular proteins including the RI protease, Lys-gingipain, porphypain and haemagglutinin A. In addition to a role in adherence, the adhesin domain(s) of these proteins may also have cytokine-inducing properties. Furthermore, it has also been shown that the previously observed degradation of cytokines by P. gingivalis may be attributable to the catalytic domain of the RI protease. Thus, different domains within the same molecule appear to have opposing actions on pro-inflammatory cytokine levels and the balance between these two activities may influence the cytokine status of the periodontium in patients with the common chronic inflammatory conditions known as the periodontal diseases.

The aim of this study was to determine whether biofilms of Porphyromonas gingivalis could proteolytically degrade the cytokines interleukin (IL)-1beta, IL-6, or IL-1 receptor antagonist (IL-1ra). Biofilms were grown on membrane filters on the surface of Wilkins-Chalgren blood agar. The biofilms were removed from the plates, and solutions containing 2.5 microg/ml of each cytokine were added. Following incubation for up to 4.0 h, supernatants from the biofilms were subjected to SDS-PAGE. The separated proteins were transferred by Western blotting to PVDF membranes and probed with peroxidase-conjugated antibodies recognizing both the intact cytokines and their degradation products. After 2 h, no intact IL-1beta, IL-6, or IL-1ra were detectable. Cytokine proteolysis also occurred in the presence of horse serum. These results demonstrate that biofilm-grown P. gingivalis can degrade both pro- and anti-inflammatory cytokines and so may be able to perturb cytokine networks in vivo by eliminating cytokines from the local environment.

Cytokines produced in response to plaque bacteria clearly play a key role in the periodontal diseases. However, we know very little about the interactions between cytokines and periodontopathogenic bacteria. The aims of this study were to determine whether the key pro-inflammatory cytokines interleukin-1 beta (IL-1 beta) and IL-6 could affect the growth of Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis and to determine whether these organisms could hydrolyse IL-1 beta, IL-6 or the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Culture medium containing up to 100 ng/ml of IL-1 beta or IL-6 was inoculated with A. actinomycetemcomitans (serotypes a, b and c) or P. gingivalis and growth was monitored by measuring changes in electrical conductivity every 3 min for up to 48 h. IL-1 beta, IL-6 or IL-1ra were added to culture supernatants and incubated for up to 24 h. Samples were taken at various times, analysed by SDS-PAGE and the separated proteins transferred by Western blotting to PVDF membranes and probed with anti-cytokine antibodies. None of the cytokines tested had any effect on the rate of growth or yield of A. actinomycetemcomitans or P. gingivalis. Supernatants from P. gingivalis cultures, but not those from A. actinomycetemcomitans, hydrolysed IL-1 beta, IL-6 and IL-1ra. The hydrolysate from the P. gingivalis supernatant-treated IL-1 beta was unable to stimulate the release of IL-6 from human gingival fibroblasts showing that it had lost biological activity. These results suggest that P. gingivalis can perturb the cytokine network, not only by stimulating the release of cytokines from host cells, but also by removing them from its local environment.

Biofilms of Streptococcus sanguis and planktonic cells of the organism were exposed to chlorhexidine gluconate and cetylpyridinium chloride, at concentrations used clinically, and survivors enumerated. S. sanguis exhibited a lower susceptibility to both antiseptics when it comprised a biofilm than when the organism was in the planktonic form. No viable bacteria were detectable after 5 min of exposure of planktonic cells to 0.2% (w/v) chlorhexidine gluconate or 0.05% (w/v) cetylpyridinium chloride, whereas viable bacterial survived in biofilms of S. sanguis even after exposure to these agents for 4 h. Older biofilms (7 days old) had similar susceptibilities to the antiseptics as younger biofilms (4 days old). Chlorhexidine achieved kills corresponding to approximately a 2 log10 reduction in the viable count of biofilms, containing approximately 10(7) colony-forming units after 5 min of incubation, whereas the corresponding kills achieved by cetylpyridinium chloride amounted to approximately a 1 log10 reduction. However, on a molar basis, cetylpyridinium chloride was the more effective of the two antiseptics. Minimum inhibitory concentration determinations showed chlorhexidine gluconate to be more effective against S. sanguis than cetylpyridinium chloride. The results of this study have revealed that the minimum inhibitory concentration is not a reliable predictor of the relative effectiveness of antimicrobial agents against S. sanguis biofilms.

A redox dye, methylene blue, was compared with subgingival root surface debridement and sterile water in the treatment of adult periodontitis. Plaque and gingival indices, bleeding on probing, and microbiological samples were obtained at baseline, and at 1, 4, 8 and 12 weeks following treatment. All subjects had matched pockets in each of the 4 quadrants, of 5 mm or more. One treatment consisted of 0.1% methylene blue gel irrigated professionally at 0, 1 and 4 weeks, and by subjects at days in between up to 4 weeks, at chosen sites within a randomly selected quadrant (split-mouth design). A 2nd treatment was sterile water irrigation as above. A 3rd quadrant received subgingival debridement, and sites in the 4th received methylene blue incorporated into a slow-release device of a biodegradable collagen alginate vicryl composite. All sites showed improvements in clinical and microbiological parameters. However, no statistically significant differences between treatment types were found for clinical measurements. Although plaque index tended to increase after week 1, gingival index was reduced, as was the papilla bleeding index. Probing depth reductions were approximately 1.2 mm for all treatments. Microbiological variables showed an increase in cocci and a decrease in motile organisms for all groups, the latter reaching statistical significance for subgingival debridement. The reductions in spirochaetes were significant for subgingival debridement and methylene blue by slow-release. Culture demonstrated an increase in the aerobe:anaerobe ratio for all groups, which was statistically significant initially (weeks 1 and 4) for subgingival debridement. Methylene blue was also effective statistically in improving this ratio, both by irrigation and slow-release (week 4). Methylene blue also significantly reduced the numbers of black-pigmented anaerobes during the trial period, both by irrigation and slow-release, which sterile water and subgingival debridement failed to do. No serious adverse experiences were seen, however, significantly greater morbidity was associated with subgingival debridement. These results clearly demonstrate that in altering the microflora to one that is more compatible with periodontal health, methylene blue treatment is comparable, or even better, than the currently standard treatment of subgingival debridement, and is better tolerated.