Lauren Christine Gursky

Relationships between subgingival microbiota and GCF biomarkers in generalized aggressive periodontitis.

AIM To examine relationships between subgingival biofilm composition and levels of gingival crevicular fluid (GCF) cytokines in periodontal health and generalized aggressive periodontitis (GAP). MATERIALS AND METHODS Periodontal parameters were measured in 25 periodontally healthy and 31 GAP subjects. Subgingival plaque and GCF samples were obtained from 14 sites from each subject. Forty subgingival taxa were quantified using checkerboard DNA-DNA hybridization and the concentrations of eight GCF cytokines were measured using Luminex. Cluster analysis was used to define sites with similar subgingival microbiotas in each clinical group. Significance of differences in clinical, microbiological and immunological parameters among clusters was determined using the Kruskal-Wallis test. RESULTS GAP subjects had statistically significantly higher GCF levels of interleukin-1beta (IL-1beta) (p<0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF) (p<0.01) and IL-1beta/IL-10 ratio (p<0.001) and higher proportions of Red and Orange complex species than periodontally healthy subjects. There were no statistically significant differences in the mean proportion of cytokines among clusters in the periodontally healthy subjects, while the ratio IL-1beta/IL-10 (p<0.05) differed significantly among clusters in the aggressive periodontitis group. CONCLUSIONS Different subgingival biofilm profiles are associated with distinct patterns of GCF cytokine expression. Aggressive periodontitis subjects were characterized by a higher IL-1beta/IL-10 ratio than periodontally healthy subjects, suggesting an imbalance between pro- and anti-inflammatory cytokines in aggressive periodontitis.

Effects of periodontal therapy on GCF cytokines in generalized aggressive periodontitis subjects

AIM To examine changes in levels of gingival crevicular fluid (GCF) cytokines, after periodontal therapy of generalized aggressive periodontitis (GAgP). MATERIALS AND METHODS Twenty-five periodontally healthy and 24 GAgP subjects had periodontal clinical parameters measured and gingival crevicular fluid (GCF) samples collected from up to 14 sites/subject. GCF samples were analysed using multiplex bead immunoassay for: GM-CSF, IFN-γ, IL-10, IL-1β, IL-2, IL-6 and TNF-α. Aggressive periodontitis subjects were randomly assigned to either scaling and root planing (SRP) alone or SRP plus systemic amoxicillin (500 mg) and metronidazole (400 mg) 3 times a day for 14 days. Clinical parameters and GCF cytokines were re-measured 6 months after treatment. Differences over time were analysed using the Wilcoxon test and between groups using the Mann-Whitney test. RESULTS Significant reductions in GCF GM-CSF, IL-1β and the ratio IL-1β/IL-10 and increases in GCF IL-6 were detected after therapy. The mean change in GCF cytokines did not differ significantly between groups. CONCLUSIONS Periodontal therapy improved GCF cytokine profiles by lowering IL-1β and increasing IL-10 levels. The reduction in GCF GM-CSF after therapy implicates this cytokine in the pathogenesis of GAgP. There was no difference between therapies in changes of GCF cytokines.

Clinical and microbiological benefits of strict supragingival plaque control as part of the active phase of periodontal therapy

AIM To compare the clinical and microbiological effects of scaling and root planing (SRP) alone or combined with mechanical [professional plaque control (PPC)] or chemical [chlorhexidine rinsing (CHX)] control of supragingival plaque in the treatment of chronic periodontitis. MATERIAL AND METHODS Sixty subjects were randomly assigned to receive SRP alone or combined with PPC (twice a week) or with CHX rinsing (twice a day). The adjunctive treatments began with SRP and were continued for 42 days. Clinical and microbiological examinations were performed at baseline, 2 and 6 months post-therapy. Subgingival plaque samples were analysed for 38 bacterial species by checkerboard DNA-DNA hybridization. RESULTS The two test treatments were more effective in improving probing depth and clinical attachment level (CAL) than SRP alone, even in intermediate and deep sites. CAL gain was better maintained in the CHX group. The most beneficial microbiological changes were observed in CHX-treated subjects, who showed a significant reduction in the proportions of red and orange complexes, as well as an increase in the proportions of the host-compatible bacterial species. CONCLUSION Strict plaque control performed during and after SRP improves periodontal treatment outcomes. The greatest microbiological and clinical benefits were observed with the use of CHX rinsing.

Proposal of a low-cost protocol for colorimetric semi-quantification of secretory phospholipase by Candida albicans grown in planktonic and biofilm phases

Biofilms are aggregates of microorganisms living in multilayered structures inside polymeric matrices onto surfaces. These biofilms may subvert the physiological properties of adjacent tissues causing morphofunctional failure. Many studies have shown that the expression of virulence attributes is maximized when microbes form such communities. This study evaluated the differential phospholipasic activity of Candida albicans SC5314 grown in planktonic phase and in biofilm. We propose two distinct protocols for the colorimetric evaluation of phosphatidylcholine hydrolysis in neutral and acidic conditions. The results showed that both protocols are suitable for the proposed intention and that 72 h-old planktonic cultures of C. albicans SC5314 secrete higher quantities of neutral (6.42-fold) and acidic (3.85-fold) phospholipases than biofilms.

Enhancement of Secretory Aspartyl Protease production in biofilms of Candida albicans exposed to sub‐inhibitory concentrations of fluconazole

The production of Secretory Aspartyl Proteases (Sap) is an important virulence factor of Candida albicans. Many studies have shown that a challenge with sub‐inhibitory concentrations of antifungals lead species of Candida to the secretion of higher concentrations of Sap. Nevertheless, published studies only reported the secretion of such enzymes by cells growing in planktonic phase, with few mention of biofilms. The present study evaluated the alterations in the secretion of Sap by C. albicans grown in biofilms and exposed to sub‐inhibitory concentrations of fluconazole. The MICs for fluconazole of seven clinical strains were determined for planktonic cells. Biofilm and planktonic cells were grown in the presence of ½ MIC, ¼ MIC, and no medication (control). The relative metabolic activity, indirectly related to cell loads, were estimated by the absorbance of reduced XTT and the Sap activity was evaluated by bovine albumin test. It was observed that 72 h‐old biofilms under the influence of ½ MIC had fewer cells than ¼ MIC and control. The production of Sap was inversely proportional to the cell content, with higher secretion in ½ MIC, followed by ¼ MIC and control. Biofilms of C. albicans challenged by sub‐MICs of fluconazole tend to secrete higher quantities of Sap.

The role of candidal histolytic enzymes on denture-induced stomatitis in patients living in retirement homes

MATERIAL AND METHODS Fifty nine elders wearing complete dentures and living in retirement homes in Curitiba (southern Brazil), were divided into two groups: group #1, 26 patients with denture-induced stomatitis and group #2, 33 patients without denture-induced stomatitis. The two groups were evaluated in relation to the degree of denture-induced stomatitis, salivary fungal loads, and secretion of some histolytic enzymes. RESULTS Patients from group #1 showed higher degrees of colonisation by Candida albicans (p = 0.031). Candida krusei, Candida tropicalis, and Candida parapsilosis were also isolated, but there were no differences between the groups (p > 0.05). Secretory aspartyl protease (Sap) and chondroitinase did not show significant differences among the isolated Candida spp. in the two groups. Phospholipase secretion rates were higher among the strains of C. albicans from group #2 (p = 0.036). The same behaviour was not detected for non-albicans Candida species. CONCLUSIONS The results could infer that differences in the secretion rates of candidal histolytic enzymes should not be imputed as imperative for the progress of denture-induced stomatitis.

Non-steroidal anti-inflammatory drugs may modulate the protease activity of candida albicans

The phenotypic pressure exerted by non-steroidal anti-inflammatory drugs (NSAIDs) on autochthonous and pathogenic microbiota remains sparsely known. In this study, we investigated if some NSAIDs increment or diminish the secretion of aspartyl-proteases (Sap) by Candida albicans grown under different phenotypes and oxygen availability using a set of SAP knock-out mutants and other set for genes (EFG1 and CPH1) that codify transcription factors involved in filamentation and protease secretion. Pre-conditioned cells were grown under planktonic and biofilm phenotypes, in normoxia and anoxia, in the presence of plasma concentrations of acetylsalicylic acid, diclofenac, indomethacin, nimesulide, piroxicam, ibuprofen, and acetaminophen. For diclofenac, indomethacin, nimesulide, and piroxicam the secretion rates of Sap by SAP1-6, EFG1, and CPH1 mutants were similar or, even, inferior to parental wild-type strain. This suggests that neither Sap 1-6 isoenzymes nor Efg1/Cph1 pathways may be entirely responsible for protease release when exposed to these NSAIDs. Ibuprofen and acetaminophen enhanced Sap secretion rates in three environmental conditions (normoxic biofilm, normoxic planktonic and anoxic planktonic). In other hand, aspirin seems to reduce the Sap-related pathogenic behavior of candidal biofilms. Modulation of Sap activity may occur according to candidal phenotypic state, oxygen availability, and type of NSAID to which the cells are exposed.

Time-related Increase of Staphylococci, Enterobacteriaceae and Yeasts in the Oral Cavities of Comatose Patients

BACKGROUND/PURPOSE The composition of oral microbiota in comatose patients remains uncertain. Some pulmonary pathogens may be found in dental biofilms or as part of the saliva microbiota. It is supposed that some pneumopathogenic microorganisms may overgrow in the mouths of comatose patients and spread to their lungs. METHODS The oral colonization dynamics of staphylococci, Enterobacteriaceae and yeasts in nine comatose patients (group 1), and in 12 conscious patients that brushed their teeth at least twice a day (group 2) was evaluated. Both groups were followed up for 7 days after hospitalization. Daily samples of saliva were obtained, dispersed and plated on selective culture media and colony forming units of each microbial group were obtained. RESULTS For patients in group 1, the counts of total viable bacteria, staphylococci, Enterobacteriaceae and yeasts progressively increased in a time-dependant manner. For the conscious patients of group 2, there was no increase. CONCLUSION It would appear that concomitant consciousness and brushing teeth are determinants in controlling the selected pneumopathogen counts in resting saliva. The increase in microbial counts in comatose patients is understandable because these microorganisms could spread to the lungs.

Screening of reducing agents for anaerobic growth of Candida albicans SC5314

This study aimed to evaluate the influence of different redox potentials (Eh) on cell growth, whole-cell protein profile and cell surface hydrophobicity (CSH) of Candida albicans SC5314. The yeast was grown in YNB broth enriched with reducing (158mM sodium sulfite, 4mM sodium sulfite, 2.5mM sodium metabisulfite, 1.3mM 2-mercaptoethanol, 5.5mM thioglycolic acid, and 3.2mM l-cysteine hydrochloride) and oxidizing agents (15mM ammonium persulfate and 80mM potassium ferricyanide) and incubated in normoxic and anoxic atmospheres at 37°C, for 48h. Pre- and post-incubation Eh values were determined and cytoplasm proteins were extracted. Proteins were parted by SDS-PAGE and their profiles were compared. 3.2mM l-cysteine and 1.3mM 2-mercaptoethanol promoted and maintained negative Eh values during incubation. No differences were detected among SDS-PAGE profiles. CSH differences only were observed with 4mM sodium sulfite and 3.2mM l-cysteine. Results showed that 3.2mM l-cysteine is a reducing agent that allows maintenance of negative Eh in both anoxic and normoxic conditions and it seems not to interfere in the global expression of plasmatic proteins.