Primary versus post-treatment apical periodontitis: microbial composition, lipopolysaccharides and lipoteichoic acid levels, signs and symptoms.

OBJECTIVES: To compare the microbial load and composition and to determine the lipopolysaccharides (LPS) and lipoteichoic acid (LTA) concentrations found in primary apical periodontitis (PAP) and post-treatment apical periodontitis (PTAP), correlating these findings with clinical/tomographic features. MATERIAL AND METHODS: Sixty patients with PAP (31) and PTAP (29) were submitted to clinical and tomographic assessment. Samples were collected from each root canal using paper points for microbiological assessment (culture technique and Checkerboard DNA-DNA hybridization) and determination of LPS and LTA levels (limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively). Data were correlated with clinical/tomographic findings and statistically analyzed using the Mann-Whitney and Pearson correlation tests (α = 5%). RESULTS: A higher number of cultivable bacteria and LPS were found in PAP (p < 0.05). The median number of species per root canal found in PAP and PTAP was 9 and 22, respectively (p < 0.05). LPS was positively correlated with a larger periapical lesion volume (p < .05). LTA levels were similar in both infections and had no correlation with signs and symptoms. In PAP, gram-positive bacteria were correlated with spontaneous pain (p < .05) and exudate (p < .05). Tenderness to percussion and pain on palpation were correlated to the presence of both gram-positive and negative bacteria. In PTAP, a positive correlation was observed between both gram-positive and gram-negative bacteria with exudate and periapical lesion volume (p < .05). CONCLUSIONS: PAP had higher contents of microbial load and LPS compared with PTAP. However, PTAP presented a more diverse microbiota compared with PAP. Higher content of LPS was positively correlated with larger periapical bone destruction, whereas signs and symptoms with specific microorganisms. CLINICAL RELEVANCE: It was verified that PAP and PTAP are polymicrobial infections with predominance of gram-negative bacteria and a more diverse bacterial population found in PTAP. A wide interaction of specific microbial species resulted in different clinical features in both infections.

Titanium alloys: in vitro biological analyzes on biofilm formation, biocompatibility, cell differentiation to induce bone formation, and immunological response.

Biological effects of titanium (Ti) alloys were analyzed on biofilms of Candida albicans, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans, and Streptococcus sanguinis, as well as on osteoblast-like cells (MG63) and murine macrophages (RAW 264.7). Standard samples composed of aluminum and vanadium (Ti-6Al-4V), and sample containing niobium (Ti-35Nb) and zirconium (Ti-13Nb-13Zr) were analyzed. Monomicrobial biofilms were formed on the Ti alloys. MG63 cells were grown with the alloys and the biocompatibility (MTT), total protein (TP) level, alkaline phosphatase (ALP) activity, and mineralization nodules (MN) formation were verified. Levels of interleukins (IL-1ß and IL-17), tumor necrosis factor alpha (TNF-α), and oxide nitric (NO) were checked, from RAW 264.7 cells supernatants. Data were statically analyzed by one-way analysis of variance (ANOVA) and Tukey's test, or T-test (P ≤ 0.05). Concerning the biofilm formation, Ti-13Nb-13Zr alloy showed the best inhibitory effect on E. faecalis, P. aeruginosa, and S. aureus. And, it also acted similarly to the Ti-6Al-4V alloy on C. albicans and Streptococcus spp. Both alloys were biocompatible and similar to the Ti-6Al-4V alloy. Additionally, Ti-13Nb-13Zr alloy was more effective for cell differentiation, as observed in the assays of ALP and MN. Regarding the stimulation for release of IL-1ß and TNF-α, Ti-35Nb and Ti-13Nb-13Zr alloys inhibited similarly the synthesis of these molecules. However, both alloys stimulated the production of IL-17. Additionally, all Ti alloys showed the same effect for NO generation. Thus, Ti-13Nb-13Zr alloy was the most effective for inhibition of biofilm formation, cell differentiation, and stimulation for release of immune mediators.

Antimicrobial activity of noncytotoxic concentrations of Salvia officinalis extract against bacterial and fungal species from the oral cavity.

The use of medicinal plants can be an alternative method for the control of microorganisms responsible for human infections. This study evaluated the antimicrobial activity of Salvia officinalis Linnaeus (sage) extract on clinical samples isolated from the oral cavity and reference strains of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Candida albicans, Candida tropicalis, and Candida glabrata. In addition, testing assessed the cytotoxic effect of S officinalis on murine macrophages (RAW 264.7). Minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations of S officinalis extract were determined by broth microdilution method in 60 microbial samples. The cytotoxicity was checked by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The quantities of the proinflammatory cytokines interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) produced by RAW 264.7 were analyzed by an enzyme-linked immunosorbent assay. An S officinalis concentration of 50.0 mg/mL was effective against all microorganisms. Regarding cytotoxicity, the groups treated with 50.0-, 25.0-, and 12.5-mg/mL concentrations of S officinalis presented cell viability statistically similar to that of the control group, which was 100% viable. The production of IL-1ß and TNF-α was inhibited at a 50.0-mg/mL concentration of S officinalis. Thus, S officinalis extract presented antimicrobial activity on all isolates of Staphylococcus spp, S mutans, and Candida spp. No cytotoxic effect was observed, as demonstrated by the survival of RAW 264.7 and inhibition of IL-1ß and of TNF-α.

Immunomodulatory effects and anti-Candida activity of lactobacilli in macrophages and in invertebrate model of Galleria mellonella.

Due to the growing number of multi-resistant Candida spp., adjuvant treatments that may help combat these fungal pathogens are relevant and useful. This study evaluated the immunomodulation and anti-Candida activity of Lactobacillus rhamnosus (LR), Lactobacillus acidophilus and Lactobacillus paracasei suspensions, either single- or multiple-strain, in mouse macrophages (RAW 264.7) and Galleria mellonella (GM). Mouse macrophages were activated by different lactobacilli suspensions and challenged with C. albicans (CA). Tumor necrosis factor (TNF)-α, interleukin IL-1ß, IL-6 and IL-17 production and cell viability were investigated. LR was the best suspension for stimulating all evaluated cytokines and thus was used in subsequent in vivo assays. Two C. albicans clinical strains, CA21 and CA60, were then added to the GM assays to further confirm the results. LR suspension was injected into the larvae 24 h before challenging with CA. Survival curve, CFU per larva and hemocytes were counted. In the GM, the LR suspension increased the survival rate and hemocyte counts and decreased the CFU per larva counts for all groups. Lactobacilli suspensions presented strain-dependent immunomodulation; however, single suspensions showed better results. Anti-Candida activity was demonstrated by decreased Candida counts in the GM with the use of LR.

Biological activities of Rosmarinus officinalis L. (rosemary) extract as analyzed in microorganisms and cells.

R. officinalis L. is an aromatic plant commonly used as condiment and for medicinal purposes. Biological activities of its extract were evaluated in this study, as antimicrobial effect on mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory capacity, and genotoxicity. Monomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed of C. albicans with each bacterium were formed in microplates during 48 h and exposed for 5 min to R. officinalis L. extract (200 mg/mL). Its cytotoxic effect was examined on murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7), and cervical carcinoma cells (HeLa) after exposure to different concentrations of the extract, analyzed by MTT, neutral red (NR), and crystal violet (CV) assays. The anti-inflammatory activity was evaluated on RAW 264.7 non-stimulated or stimulated by lipopolysaccharide (LPS) from Escherichia coli and treated with different concentrations of the extract for 24 h. Interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) were quantified by ELISA. Genotoxicity was verified by the frequency of micronuclei (MN) at 1000 cells after exposure to concentrations of the extract for 24 h. Data were analyzed by T-Test or ANOVA and Tukey Test ( P ≤ 0.05). Thus, significant reductions in colony forming units per milliliter (CFU/mL) were observed in all biofilms. Regarding the cells, it was observed that concentrations ≤ 50 mg/mL provided cell viability of above 50%. Production of proinflammatory cytokines in the treated groups was similar or lower compared to the control group. The MN frequency in the groups exposed to extract was similar or less than the untreated group. It was shown that R. officinalis L. extract was effective on mono- and polymicrobial biofilms; it also provided cell viability of above 50% (at ≤ 50 mg/mL), showed anti-inflammatory effect, and was not genotoxic. Impact statement Rosmarinus officinalis L. extract effectively contributed to in vitro control of important species of microorganisms such as Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Pseudomonas aeruginosa in mono- and polymicrobial biofilms that are responsible for several infections in oral cavity as in other regions of the body. Furthermore, this extract promoted also cell viability above 50% at concentrations ≤ 50 mg/mL, excellent anti-inflammatory effect, showing inhibition or reduction of the synthesis of proinflammatory cytokines, being also non-genotoxic to cell lines studied. Thus, this extract may be a promising therapeutic agent that can be added in some medical and dental formulations such as toothpastes, mouthwashes, irrigating root canals, ointments, soaps, in order to control pathogenic microorganisms and biofilms, with anti-inflammatory effect and absence of cytotoxic and genotoxic.

Live and Heat-Killed Lactobacillus rhamnosus ATCC 7469 May Induce Modulatory Cytokines Profiles on Macrophages RAW 264.7.

This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1ß, IL-4, IL-6, IL-10, and IL-12) by mouse macrophages (RAW 264.7). Three microorganism preparations were used: live L. rhamnosus (LLR) suspension, heat-killed L. rhamnosus (HKLR) suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR) suspension, which were cultured with macrophages (37°C, 5% CO2) for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS) and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P < 0.05). SHKLR also significantly increased the production of TNF-α and IL-10 (P < 0.05) but not IL-6 (P > 0.05). All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1ß or significant levels of IL-4 and IL-12 (P > 0.05). In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses.

Control of microorganisms of oral health interest with Arctium lappa L. (burdock) extract non-cytotoxic to cell culture of macrophages (RAW 264.7).

OBJECTIVES: To evaluate the antimicrobial activity of Arctium lappa L. extract on Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Candida albicans, C. tropicalis and C. glabrata. In addition, the cytotoxicity of this extract was analyzed on macrophages (RAW 264.7). DESIGN: By broth microdilution method, different concentrations of the extract (250-0.4 mg/mL) were used in order to determine the minimum microbicidal concentration (MMC) in planktonic cultures and the most effective concentration was used on biofilms on discs made of acrylic resin. The cytotoxicity A. lappa L. extract MMC was evaluated on RAW 264.7 by MTT assay and the quantification of IL-1ß and TNF-α by ELISA. RESULTS: The most effective concentration was 250 mg/mL and also promoted significant reduction (log10) in the biofilms of S. aureus (0.438 ± 0.269), S. epidermidis (0.377 ± 0.298), S. mutans (0.244 ± 0.161) and C. albicans (0.746 ± 0.209). Cell viability was similar to 100%. The production of IL-1ß was similar to the control group (p>0.05) and there was inhibition of TNF-α (p<0.01). CONCLUSIONS: A. lappa L. extract was microbicidal for all the evaluated strains in planktonic cultures, microbiostatic for biofilms and not cytotoxic to the macrophages.