Lactobacillus rhamnosus inhibits Candida albicans virulence factors in vitro and modulates immune system in Galleria mellonella.

AIM: The aim of this study was to evaluate the potential anti-Candida effects of Lactobacillus rhamnosus ATCC 9595 on Candida albicans ATCC 18804 using in vitro and in vivo models. METHODS AND RESULTS: The in vitro analysis evaluated the effects of L. rhamnosus on C. albicans's biofilm formation by CFU count and metabolic activity, filamentation capacity, and adhesion (ALS3 and HWP1) and transcriptional regulatory gene (BCR1 and CPH1) expression. The in vitro results showed that both the L. rhamnosus cells and supernatant reduced C. albicans biofilm formation, filamentation and gene expression. In the in vivo study, the treatment with L. rhamnosus supernatant increased 80% the survival of Galleria mellonella larvae infected with C. albicans. Furthermore, the supernatant of L. rhamnosus recruited haemocytes into the haemolymph (2·1-fold increase). CONCLUSIONS: Lactobacillus rhamnosus reduced the biofilm formation and filamentation of C. albicans in vitro by negatively regulating all studied C. albicans genes. Lactobacillus rhamnosus protected G. mellonella against experimental candidiasis in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first study to report the anti-Candida properties of L. rhamnosus ATCC 9595. The supernatant of this strain has immunomodulatory effects on the G. mellonella model and protects the larvae against pathogens.

Lactobacillus is able to alter the virulence and the sensitivity profile of Candida albicans.

AIMS: The study investigated whether the interaction with Lactobacillus rhamnosus (ATCC7469) interfere with the expression of virulence factors by Candida albicans (ATCC18804). METHODS AND RESULTS: These micro-organisms were grown in biofilms for 24, 48 and 72 h, Candida was isolated and the expression of the major virulence factors were investigated. The production of phospholipase, protease and haemolysin were observed in appropriate media; observation of germ tubes formation in serum; biofilm formation, after growth in microtitre plates and reading in spectrophotometer. Candida was also tested for antifungal sensitivity to amphotericin B, fluconazole and ketoconazole. The results were compared with the cells of Candida grown in the absence of lactobacilli (control group). Candida cells, which interacted with Lact. rhamnosus (test group), showed significantly lower proteinase and haemolysin activity, when compared with control group. The germ tube formation and biofilm formation capacity also decreased in tested groups, which demonstrated alterations in susceptibility to antifungal drugs. CONCLUSIONS: The results suggest that Lact. rhamnosus is able to influence the expression of virulence factors by C. albicans and can alter its antifungal sensitivity profile. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest reduction in the pathogenicity of Candida and improvement in candidiasis therapy and control.

Persea americana Glycolic Extract: In Vitro Study of Antimicrobial Activity against Candida albicans Biofilm and Cytotoxicity Evaluation.

This study evaluated the antifungal activity of Persea americana extract on Candida albicans biofilm and its cytotoxicity in macrophage culture (RAW 264.7). To determine the minimum inhibitory concentration (MIC), microdilution in broth (CLSI M27-S4 protocol) was performed. Thereafter, the concentrations of 12.5, 25, 50, 100, and 200 mg/mL (n = 10) with 5 min exposure were analyzed on mature biofilm in microplate wells for 48 h. Saline was used as control (n = 10). After treatment, biofilm cells were scraped off and dilutions were plated on Sabouraud dextrose agar. After incubation (37°C/48 h), the values of colony forming units per milliliter (CFU/mL) were converted to log10 and analyzed (ANOVA and Tukey test, 5%). The cytotoxicity of the P. americana extract was evaluated on macrophages by MTT assay. The MIC of the extract was 6.25 mg/mL and with 12.5 mg/mL there was elimination of 100% of planktonic cultures. Regarding the biofilms, a significant reduction (P < 0.001) of the biofilm at concentrations of 50 (0.580 ± 0.209 log10), 100 (0.998 ± 0.508 log10), and 200 mg/mL (1.093 ± 0.462 log10) was observed. The concentrations of 200 and 100 mg/mL were cytotoxic for macrophages, while the concentrations of 50, 25, and 12.5 mg/mL showed viability higher than 55%.

Oral microbial colonization in patients with systemic lupus erythematous: correlation with treatment and disease activity.

Treating patients with systemic lupus erythematosus (SLE) with steroids and immunosuppressive drugs may interfere in the presence of potentially opportunistic microorganisms in the oral cavity. The aim of this study was to evaluate the presence of Candida spp., Staphylococcus spp., Enterobacteria and Pseudomonas spp. in the oral cavity of SLE patients, compared with healthy controls. A group of 40 patients who had received therapy for at least 60 days was selected (19-53 years). For the control group, 40 healthy individuals matched for age, gender and use of partial prosthesis were selected. Oral rinse samples were collected and plated on specific culture media. After incubation, the number of colony forming units (CFU) was obtained and the isolates were identified at species level. Microbial counts were compared between SLE and control by analysis of variance (ANOVA) and Mann-Whitney (p < 0.05 significant). Microorganism counts in patients with and without immunosuppressive drugs, as well with active and inactive disease (according to SLEDAI score) were also compared. No significant differences in CFU/mL between SLE and control patients were observed (yeasts, p = 0.55; Staphylococci, p = 0.24; Enterobacteria/Pseudomonas spp., p = 0.26). No differences in microbial counts were observed regarding clinical parameters tested. The most frequent species isolated in the SLE group were Candida albicans, Staphylococcus epidermidis and Klebsiella oxytoca. In conclusion, no differences in frequency and microorganism levels were found between SLE patients and healthy individuals.

Photodynamic inactivation of biofilms formed by Candida spp., Trichosporon mucoides, and Kodamaea ohmeri by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H, 31H-phthalocyanine (ZnPc).

The biofilms formed by opportunistic yeasts serve as a persistent reservoir of infection and impair the treatment of fungal diseases. The aim of this study was to evaluate photodynamic inactivation (PDI) of biofilms formed by Candida spp. and the emerging pathogens Trichosporon mucoides and Kodamaea ohmeri by a cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine (ZnPc). Biofilms formed by yeasts after 48 h in the bottom of 96-well microtiter plates were treated with the photosensitizer (ZnPc) and a GaAlAs laser (26.3 J cm(-2)). The biofilm cells were scraped off the well wall, homogenized, and seeded onto Sabouraud dextrose agar plates that were then incubated at 37°C for 48 h. Efficient PDI of biofilms was verified by counting colony-forming units (CFU/ml), and the data were submitted to analysis of variance and the Tukey test (p < 0.05). All biofilms studied were susceptible to PDI with statistically significant differences. The strains of Candida genus were more resistant to PDI than emerging pathogens T. mucoides and K. ohmeri. A mean reduction of 0.45 log was achieved for Candida spp. biofilms, and a reduction of 0.85 and 0.84, were achieved for biofilms formed by T. mucoides and K. ohmeri, respectively. Therefore, PDI by treatment with nanostructured formulations cationic zinc 2,9,16,23- tetrakis (phenylthio)- 29H, 31H- phthalocyanine (ZnPc) and a laser reduced the number of cells in the biofilms formed by strains of C. albicans and non-Candida albicans as well the emerging pathogens T. mucoides and K. ohmeri.

Effectiveness of three instrumentation systems to remove Enterococcus faecalis from root canals.

AIM: To assess the effectiveness of three systems of mechanical preparation to reduce Enterococcus faecalis within root canals. METHODOLOGY: Twenty-four human single-rooted canine teeth were standardized to a length of 17 mm and the canal contents removed using a size 20 K-file, as the last apical file. After irrigation and sterilization, the canals were contaminated with E. faecalis and incubated for 21 days at 37 °C with 5% CO(2). Then, the teeth were divided into three groups for mechanical preparation with: ProTaper rotary system, ProTaper manual system and manual K-files. Samples of the root canal contents, before and after the debridement, were collected with sterile paper points for 1 min. Then, the samples were diluted and plated in Brain Heart Infusion (BHI) agar. The colony-forming units were counted and the percentage reduction calculated. The reduction and log CFU mL(-1) were compared between groups using Wilcoxon nonparametric test and two-way analysis of variance, respectively. RESULTS: There was a significant reduction in the number of CFU/mL (P = 0.000) before and after debridement for all the systems used. However, there was no significant difference between the systems. CONCLUSION: All the three instrumentation systems reduced E. faecalis counts to a similar degree.

Streptococcus mutans biofilm adhesion on composite resin surfaces after different finishing and polishing techniques.

This study evaluated Streptococcus mutans biofilm adhesion on the surface of three composite resins (nanofilled, Filtek Z350, 3M ESPE, Salt Lake City, UT, USA; nanohybrid, Vit-1-escence, Ultradent Products, South Jordan, UT, USA; and microhybrid, Esthet X, Dentsply, Milford, DE, USA) following different finishing and polishing techniques. Sixty standardized samples (6 × 3 mm) of each composite were produced and randomly divided into three finishing and polishing treatments (n=20): 1) control group: composite resin surface in contact with Mylar matrix strips with no finishing or polishing performed, 2) Sof-Lex aluminum oxide disc technique (3M ESPE, and 3) carbide bur finishing and Astrobrush polishing technique (Ultradent). Half the samples of each group were incubated in human saliva for 1 hour, and all the samples were subjected to S mutans (ATCC 35688) biofilm development. The mean log of CFU/mL present in the S mutans biofilm was calculated, and data were statistically analyzed by three-way analysis of variance and the Tukey test (p<0.05). Human saliva incubation promoted a significant increase of bacterial adherence on all three of the composites' surfaces, regardless of the polishing treatment performed (p<0.05). Of the three, the nanofilled composite (Filtek Z350) had the lowest bacterial adherence with each of the finishing and polishing techniques despite the presence or absence of human saliva (p<0.05). Mylar matrix strips (control group) promoted the lowest bacterial adhesion on the surface of the microhybrid and nanofilled composites in the absence of human saliva.

Prevalence and antifungal resistance profile of Candida spp. oral isolates from patients with type 1 and 2 diabetes mellitus.

OBJECTIVE: The goal of the study was to measure the prevalence of Candida spp. in the oral cavity of patients with diabetes types 1 and 2 when compared to healthy individuals and to study antifungal resistance profile of the isolates. DESIGN: There were 162 subjects in the study: diabetes type 1 (n=39); control group 1 (n=50): healthy individuals matched in gender, age, and oral conditions to diabetes type 1 patients; diabetes type 2 (n=37); control group 2 (n=36) who were matched to each patient of the diabetes type 2 group. Stimulated saliva was collected and isolates were identified with phenotypic tests. The presence of C. dubliniensis was determined by multiplex PCR. RESULTS: There were no statistically significant differences in Candida spp. frequency between the diabetes 1 group and its control (p=0.443) nor between the diabetes 2 group and its control (p=0.429). C. albicans was the most frequently isolated yeast in all groups. In the diabetes groups, C. stellatoidea, C. parapsilosis, C. tropicalis, C. lipolytica, C. glabrata, and C. krusei were also identified. Additionally, in control groups, C. kefyr was also detected. None of the isolates were resistant to amphotericin B and flucytosine. A low percentage of the isolates were resistant to ketoconazole. CONCLUSIONS: No differences were detected in colonization of Candida spp. oral isolates from type 1 and type 2 diabetes when compared to matched controls. The antifungal resistance of Candida spp. isolates for ketoconazole from type 1 diabetes patients was significantly higher than that of its matched control.

Antimicrobial photodynamic therapy: photodynamic antimicrobial effects of malachite green on Staphylococcus, enterobacteriaceae, and Candida.

OBJECTIVE: This study investigated in vitro the photodynamic antimicrobial effects of the photosensitizer malachite green on clinical strains of Staphylococcus, Enterobacteriaceae, and Candida. MATERIALS AND METHODS: Thirty-six microbial strains isolated from the oral cavity of patients undergoing prolonged antibiotic therapy, including 12 Staphylococcus, 12 Enterobacteriaceae, and 12 Candida strains, were studied. The number of cells of each microorganism was standardized to 10(6) cells/mL. Twenty-four assays were carried out for each strain according to the following experimental conditions: gallium-aluminum-arsenide laser and photosensitizer (n = 6, L+P+), laser and physiologic solution (n = 6, L+P-), photosensitizer (n = 6, L-P+), and physiologic solution (n = 6, L-P-). Next, cultures were prepared on brain-heart infusion agar for the growth of Staphylococcus and Enterobacteriaceae, and on Sabouraud dextrose agar for the growth of Candida, and incubated for 48 h at 37 degrees C. The results are reported as the number of colony-forming units (CFU/mL) and were analyzed with analysis of variance and the Tukey test. RESULTS: The Staphylococcus, enterobacterial, and Candida strains were sensitive to photodynamic therapy with malachite green (L+P+). A reduction of approximately 7 log(10) for Staphylococcus, 6 log(10) for enterobacteria, and 0.5 log(10) for the genus Candida. Significant statistical differences were observed between the L+P+ groups and the control groups (L-P-). CONCLUSION: The Staphylococcus, Enterobacteriaceae, and Candida strains studied were sensitive to photodynamic therapy with malachite green.

Frequency of Candida spp. in the oral cavity of Brazilian HIV-positive patients and correlation with CD4 cell counts and viral load.

The aim of this study was to evaluate the prevalence of Candida spp., and particularly C. dubliniensis, among oral isolates from Brazilian HIV-positive patients correlating these results with CD4 cell counts and viral load. Forty-five individuals (23 female and 22 male) diagnosed as HIV-positive by ELISA and Western-blot, under anti-retroviral therapy for at least 1 year and without oral candidosis signals were included in the study. The control group was constituted by 45 healthy individuals, matched to the test group in relation to age, gender, and oral conditions. Oral rinses were collected and the identification was performed by phenotypic tests. The existence of C. dubliniensis among the isolates was analyzed using a validated multiplex PCR assay. Candida spp. were detected at significantly higher number in the oral cavity of HIV-positive patients in relation to the controls (P = 0.0008). C. albicans was the most frequently isolated species in both groups. In the HIV group, C. glabrata, C. lipolytica, C. krusei, C. guilliermondii, and C. parapsilosis were also identified. In the control group, we additionally identified C. tropicalis and C. dubliniensis. Two isolates (1.9%, 2/108) from control individuals were identified as C. dubliniensis and this species was not verified in the HIV group. Candida spp. counts were statistically lower (P = 0.0230) in the oral cavity of patients with low viral load (<400 copies/mm(3)). Candida spp. counts did not differ statistically among groups with different levels of CD4 cells counts (P = 0.1068).

In vitro effects of calcium hydroxide and polymyxin B on endotoxins in root canals.

OBJECTIVES: To evaluate the effects of intracanal medicaments on endotoxins in root canals. METHODS: Seventy-five freshly extracted maxillary incisors were used in this study. The crowns of teeth were sectioned near the CEJ in order to standardize the root length to 14 mm. The root canals were instrumented to an apical size #50 file and irrigated with 1% sodium hypochlorite solution and sterilized with 60Co gamma irradiation. Standardized suspension containing Escherichia coli endotoxin was inoculated into the 60 root canals. The specimens were randomly assigned to 5 groups (n=15), according to the intracanal medicament used: (G1) calcium hydroxide; (G2) polymyxin B; (G3) combination neomycin-polymyxin B-hydrocortisone; (G4) positive control (no intracanal medicament); (G5) negative control (no endotoxin and no intracanal medicament). After 7 days, the detoxification of endotoxin was evaluated by Limulus lysate assay and antibody production in B-lymphocytes culture. RESULTS: Groups 1, 2 and 5 presented the best results by Limulus lysate and were significantly different to groups 3 and 4 (p<0.05). Stimulation of antibodies production in cell culture by groups 1 and 6 was smaller and statistically different than groups 2, 3, 4 and 5 (p<0.05). Groups 2 and 5 induced a small increase in the antibodies production in relation to the groups 1 and 6. Groups 3 and 4 induced a significant increase of antibodies production (p<0.05). CONCLUSIONS: The calcium hydroxide and polymyxin B intracanal medicaments detoxified endotoxin in root canals and altered the properties of LPS to stimulate the antibody production by B-lymphocytes. The combination neomycin-polymyxin B-hydrocortisone did not detoxified endotoxin.

In vitro evaluation of the effectiveness of irrigants and intracanal medicaments on microorganisms within root canals.

AIM: To evaluate in vitro the effectiveness of sodium hypochlorite (NaOCl), chlorhexidine (CHX) and five intracanal medicaments on microorganisms within root canals. METHODOLOGY: Ninety-six human single-rooted extracted teeth were used. After removing the crowns, canal preparation was completed and the external root surfaces were coated with epoxy resin. Following sterilization, the teeth were contaminated with Candida albicans and Enterococcus faecalis, and were incubated at 37 +/- 1 degrees C for 7 days. The teeth were divided according to the irrigant solution or intracanal medicament: group 1, sterile physiologic solution (SPS) and calcium hydroxide (Ca(OH)2) paste; group 2, SPS and camphorated paramonochlorophenol (CPMC); group 3, SPS and tricresol formalin; group 4, SPS and CaOH2 + CPMC paste; group 5, SPS and PMC furacin; group 6, 2.5% NaOCl without intracanal medication; group 7, 2.0% CHX without intracanal medication and group 8, SPS without intracanal medication (control group). Microbiological samples were collected with sterile paper points, and bacterial growth was determined. The data were submitted to the analysis of variance (anova, P = 0.05). RESULTS: For C. albicans, groups 3 and 8 were statistically less effective than groups 1, 2, 4 and 5 (Kruskal-Wallis (K-W) = 65.241; gl = 7; P = 0.001). For E. faecalis, groups 6 and 8 were statistically less effective than groups 1-4 and 7 (K-W = 61.048; gl = 7; P = 0.001). CONCLUSIONS: Ca(OH)2 + CPMC paste was the most effective intracanal medicament for the elimination of the two microorganisms; 2.0% CHX solution was more effective than 2.5% NaOCl against E. faecalis.

Caries risk tests and salivary levels of immunoglobulins to Streptococcus mutans and Candida albicans in mouthbreathing syndrome patients.

The aim of this study was to compare microbiological and salivary variables possibly related to caries risk in treated and untreated mouthbreathing syndrome (MBS) children and control children. Thirty control children, 30 mouthbreathers and 25 treated mouthbreathers were studied for the numbers of lactobacilli, mutans streptococci and yeasts in their saliva. Snyder's test, salivary flow and buffering capacity were also evaluated. Levels of immunoglobulins to Candida albicans and Streptococcus mutans in the saliva were quantified using ELISA. Considering the results obtained for the microbiological and salivary caries risk tests, no significant differences were observed among the proportions of patients with small/negative and high/moderate caries risk in the studied groups. The level of IgG to S. mutans was significantly higher in the treated MBS group in relation to MBS patients. On the other hand, the median anti-S. mutans IgM level was lower in the treated MBS patients than in the other groups. For the studied anti-Candida immunoglobulins, IgM level was significantly lower in the treated MBS group than in the other groups. No differences were observed for anti-S. mutans and anti-Candida IgA levels among the groups. The findings suggest that mouthbreathing cannot be considered a risk factor for dental caries.

Production of bacteriocin-like inhibitory substances (BLIS) by Streptococcus salivarius strains isolated from the tongue and throat of children with and without sore throat

"Streptococcus salivarius" strains, isolated from children with and without sore throat, were tested for bacteriocin production against "Streptococcus pyogenes. "S. salivarius" strains producing bacteriocin-like inhibitory substances (BLIS) against "S. pyogenes" were more frequently found in children without sore throat by the presence of BLIS-positive "S. salivarius" strains