Effects of α and ß-adrenergic signaling on innate immunity and Porphyromonas gingivalis virulence in an invertebrate model.

To investigate the role of adrenergic signalling (AS) in the host immune response and Porphyromonas gingivalis virulence, we compared norepinephrine (NE) and isoproterenol (ISO) responses in Galleria mellonella. P. gingivalis infection was evaluated by survival; humoral immune responses (i.e. melanization and cecropin and gloverin mRNA expression); cellular immune responses (i.e. haemocyte count, nodulation by histology); and P. gingivalis recovery (CFU/mL). P. gingivalis was cultivated in the presence of ISO (PgISO) or NE and injected into the larvae for survival evaluation. Finally, we co-injected ISO and PgISO to evaluate the concomitant effects on the immune response and bacterial virulence. None of the ligands were toxic to the larvae; ISO increased haemocyte number, even after P. gingivalis infection, by mobilizing sessile haemocytes in a ß-adrenergic-specific manner, while NE showed the opposite effect. ISO treatment reduced larval mortality and the number of recovered bacteria, while NE increased mortality and showed no effect on bacterial recovery. ISO and NE had similar effects on melanization and decreased the expression of cecropin. Although co-cultivation with NE and ISO increased the gene expression of bacterial virulence factors in vitro, only the injection of PgISO increased larval death, which was partially reversed by circulating ISO. Therefore, α- and ß-adrenergic signalling had opposite effects after P. gingivalis infection. Ultimately, the catecholamine influence on the immune response overcame the effect of more virulent strains. The effect of AS directly on the pathogen found in vitro did not translate to the in vivo setting.

Cryptococcus spp. and Cryptococcosis: focusing on the infection in Brazil.

Cryptococcosis is a global fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii yeast complex. This infection is acquired by inhalation of propagules such as basidiospores or dry yeast, initially causing lung infections with the possibility of progressing to the meninges. This infection mainly affects immunocompromised HIV and transplant patients; however, immunocompetent patients can also be affected. This review proposes to evaluate cryptococcosis focusing on studies of this mycosis in Brazilian territory; moreover, recent advances in the understanding of its virulence mechanism, animal models in research are also assessed. For this, literature review as realized in PubMed, Scielo, and Brazilian legislation. In Brazil, cryptococcosis has been identified as one of the most lethal fungal infections among HIV patients and C. neoformans VNI and C. gattii VGII are the most prevalent genotypes. Moreover, different clinical settings published in Brazil were described. As in other countries, cryptococcosis is difficult to treat due to a limited therapeutic arsenal, which is highly toxic and costly. The presence of a polysaccharide capsule, thermo-tolerance, production of melanin, biofilm formation, mechanisms for iron use, and morphological alterations is an important virulence mechanism of these yeasts. The introduction of cryptococcosis as a compulsory notification disease could improve data regarding incidence and help in the management of these infections.

The Fagerström and AUDIT Tests as Probable Screening Tools in Oral Cancer and Their Correlation with CYP1A1, GSTM1, GSTP1, and GSTT1 Gene Expression.

BACKGROUND: Cancer is currently a major public health problem worldwide, with a marked increase of about 70% in the number of expected diagnosed cases over the next two decades. The amount of tobacco and alcohol consumed is calculated based on the subjective information provided by the user. Tobacco exposure can be assessed using the Fagerström Test for Cigarette Dependence (FTCD) and alcohol consumption by the Alcohol Use Disorder Identification Test (AUDIT). MATERIALS AND METHODS: Forty-eight subjects answered the Fagerström, and AUDIT tests and we studied them as likely screening tools for oral cancer and their correlation with the expression of CYP1A1, GSTM1, GSTP1, and GSTT1 genes by the RT-qPCR method. RESULTS: There were significant differences in the AUDIT score and CYP1A1 expression between cancer and control groups. Participants in advanced stages, whether due to tumor size or regional metastasis, showed significant differences in the duration of tobacco use, FTCD, AUDIT score, and CYP1A1 expression when compared to patients in early stages. Among subjects without cancer, we found a significant correlation between participant age and GSTP1 expression. Furthermore, the expression of GSTP1 was significantly correlated with the number of cigarettes smoked per day, duration of tobacco use, and FTCD. CONCLUSIONS: Questionnaires designed to evaluate the degree of tobacco and alcohol exposure and dependence combined with gene expression tests can be useful to assess the risk of developing oral cancer. Furthermore, raising the awareness of individuals regarding their degree of dependence and encouraging them to participate in cessation programs are important educational measures for the prevention of tobacco-related malignancies.

Natural latex serum: characterization and biocompatibility assessment using Galleria mellonella as an alternative in vivo model.

Natural latex serum (NLS) is one of the natural rubber latex fractions from Hevea brasiliensis tree, which is formed by centrifuged serum and is composed of proteins, acids, nucleotides, salts and carbohydrates. The proteins present in NLS have demonstrated several interesting biological properties, including angiogenic, healing, osteogenic, anti-inflammatory, antimicrobial, in addition to inducing neovascularization, bone formation and osseointegration. Thus, we proposed to characterize NLS by physicochemical techniques and to investigate the biocompatibility by toxicological assays and safety test in Galleria mellonella. Infrared spectrum showed vibrational bands characteristic of amide I, II and III that are linked to the protein content, which was confirmed by the High Performance Liquid Chromatography profile and by the Electrophoresis analysis. This material did not exhibit hemolytic (rate <0.5%) and cytotoxic effects (viability >70%) and was able to enhance the proliferation of fibroblasts (>600%) after 3 days. The pronounced proliferative effect observed in fibroblast cells can be explained by the presence of the fibroblast growth factor (FGF) like protein revealed by the Western blot test. Moreover, NLS did not provoke toxic effects (survival ∼ 80%) on the G. mellonella model, indicating that it is a biocompatible and safe material.

The Anti-Biofilm Efficacy of Caffeic Acid Phenethyl Ester (CAPE) In Vitro and a Murine Model of Oral Candidiasis.

Candida albicans is the main fungal species associated with the development of oral candidiasis. Currently, therapeutic options for these infections are limited by the adverse effects of antifungal drugs and by the emergence of drug resistant strains. Thus, the development of new antifungal agents is needed for the prevention and treatment of oral Candida infections. Caffeic acid phenethyl ester (CAPE) is a natural compound from propolis polyphenolic groups that exhibits many pharmacological properties. In this study, we investigated whether CAPE can have antifungal and immunomodulatory effects on oral candidiasis. Preliminary tests to assess the antifungal activity of CAPE were performed using the Minimum Inhibitory Concentration (MIC) assay that demonstrated inhibition in a range from 16 to 32 µg/mL, confirming its antifungal activity on several C. albicans strains isolated from the oral cavity. Subsequently, we analyzed Candida spp biofilms formed in vitro, in which CAPE treatment at 5 x MIC caused a reduction of 68.5% in the total biomass and ~2.60 Log in the viable cell count (CFU/mL) in relation to the untreated biofilm (p<0.0001). Next, RNA was extracted from untreated and CAPE-treated biofilms and analyzed by real-time qPCR. A series of genes analyzed (ALS1, ECE1, EPA1, HWP1, YWP1, BCR1, BGR1, CPH1, EFG1, NDT80, ROB1, TEC1, UME6, SAP2, SAP5, PBL2, and LIP9) were downregulated by CAPE compared to the untreated control group (p<0.0001). In in vivo studies using Galleria mellonella, the treatment with CAPE prolonged survival of larvae infected by C. albicans by 44.5% (p < 0.05) and accompanied by a 2.07-fold increase in the number of hemocytes. Flow cytometry revealed the most prominent increases were in types P2 and P3 hemocytes, granular cells, which phagocytize pathogens. In addition, CAPE treatment decreased the fungal load in the hemolymph and stimulated the expression of antifungal peptide genes such as galiomicin and gallerimycin. The antifungal and immunomodulatory activities observed in G. mellonella were extended to a murine model of oral candidiasis, in which CAPE decreased the levels of C. albicans colonization (~2 log CFU/mL) in relation to the untreated control group. In addition, CAPE treatment significantly reduced pseudomembranous lesions, invasion of hyphae on epithelium surfaces, tissue damage and inflammatory infiltrate (p < 0.05). CAPE was also able to increase the expression of ß-defensin 3 compared to the infected and untreated group by 3.91-fold (p < 0.0001). Taken together, these results show that CAPE has both antifungal and immunomodulatory effects, making it a promising natural antifungal agent for the treatment and prevention of candidiasis and shows impact to oral candidiasis.

Antimicrobial effects of photodynamic therapy with Fotoenticine on Streptococcus mutans isolated from dental caries.

Photodynamic therapy (PDT) is a promising strategy to control cariogenic pathogens, such as Streptococcus mutans. Seeking to reach the total bacterial elimination from dental surfaces, novel photosensitizers have been investigated, such as Fotoenticine (FTC) derived from chlorin e6. The objective of this study was to investigate the photodynamic effects of FTC against several clinical strains of S. mutans. Clinical isolates were obtained from patients with active carious lesions, identified by molecular analysis and subjected to PDT using laser irradiation (660 nm and 39.5 J/cm2) in planktonic and biofilm stages. We identified 11 S. mutans strains from cervical, occlusal and proximal caries. PDT mediated by FTC has totally eliminated the S. mutans cells in planktonic growth for all analyzed strains. In biofilms, PDT with FTC reached statistically significant reductions compared with the non-treated control group, at 5.4, 5.5 and 6.5 Log10 (CFU/mL), respectively, for the strains from proximal, occlusal and cervical caries. The scanning electron microscopy evaluations confirmed that PDT mediated by FTC was able to disaggregate and kill the S. mutans cells adhered to enamel surface, suggesting its potential to disinfect the dental tissues.

Probiotic Effects of Lactobacillus paracasei 28.4 to Inhibit Streptococcus mutans in a Gellan-Based Formulation.

Streptococcus mutans is considered to be a major bacterium involved in dental caries, and the control of virulence mechanisms is fundamental to prevent disease. Probiotics present a promising preventive method; however, the use of probiotics requires its incorporation into delivery materials to facilitate oral colonization. Thus, we performed a comprehensive study examining preventive effects of Lactobacillus paracasei 28.4-enriched gellan hydrogel materials to inhibit S. mutans in planktonic and biofilm states, addressing its influence in the production of extracellular polysaccharides (EPS) and altered gene expression of several cariogenic virulence factors. L. paracasei 28.4, a strain isolated from the oral cavity of a caries-free individual, was incorporated in three gellan hydrogels (0.5%, 0.75%, and 1% w/v). The pretreatment with probiotic-gellan formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the planktonic growth of S. mutans, independent of the gellan concentrations and pH variations. This pretreatment also had inhibitory activity against S. mutans biofilms, exhibiting a reduction of 0.57 to 1.54 log10 in CFU/mL (p < 0.0001) and a decrease of 68.8 to 71.3% in total biomass (p < 0.0001) compared with the control group. These inhibitory effects were associated with the decreased production of EPS by 80% (p < 0.0001) and the downregulation of luxS, brpA, gbpB, and gtfB genes. The gellan formulation containing L. paracasei 28.4 exhibited probiotic effects for preventing S. mutans growth, biofilm formation, and production of cariogenic factors to suggest possible use in tooth decay prevention.

Chitosan enhances the antimicrobial photodynamic inactivation mediated by Photoditazine® against Streptococcus mutans.

Chitosan (CS), a biopolymer with intrinsic antimicrobial activity, can increase antimicrobial photodynamic inactivation (aPDI). The aim of this study was to evaluate the capacity of CS to potentiate the efficacy of Photoditazine® (PDZ)-mediated aPDI of the cariogenic bacterium Streptococcus mutans. CS effectively augmented the effects of aPDI, with reductions of approximately 4.5 logs in both planktonic and biofilm states. The combined treatment was also capable of reducing the number of S. mutans cells and amount of extracellular matrix in biofilms formed on enamel surfaces, which were characterized using scanning electron microscopy analysis. Furthermore, CS increased the absorption of PDZ by S. mutans cells. The combination of CS with PDZ-mediated aPDI is hence a promising antimicrobial approach against S. mutans and may be useful to control dental caries.

Oral Colonization by Candida Species in Patients with Multiple Sclerosis.

INTRODUCTION: Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease that affects the central nervous system. Since immune system plays a key role in this disease, patients with MS can present higher risk of infections. PURPOSE: This study aimed to investigate the prevalence of Candida spp. in the oral cavity of MS patients in relation to a control group METHODS: In total, 100 individuals were selected: 55 diagnosed with MS and 45 healthy individuals (control group). Saliva samples were collected and seeded in culture media selecting for Candida. Following an incubation period of 48 h, colony-forming units (CFU mL-1) were counted and colonies were isolated for Candida species identification by multiplex PCR. The results were analysed by chi-squared and Mann-Whitney U statistical tests considering a significance level of 5%. RESULTS: Candida spp. were confirmed in the oral cavity of 50.09% patients in the MS group and 35.55% individuals in the control group. In individuals positive for the growth of Candida spp., the median values of Candida colonies were 220 CFU mL-1 for the MS group and 120 CFU mL-1 for the control group. However, no statistically significant differences were observed between groups for both prevalence and CFU mL-1 count. Of the Candida species identified, 73.91% were C. albicans, 21.73% C. glabrata, 2.17% C. tropicalis, and 2.17% C. krusei. CONCLUSIONS: The colonization of Candida spp. in the oral cavity of individuals with multiple sclerosis was higher than in the control group; however these findings were not proven to be statistically significant.

Streptococcus mutans Secreted Products Inhibit Candida albicans Induced Oral Candidiasis.

In the oral cavity, Candida species form mixed biofilms with Streptococcus mutans, a pathogenic bacterium that can secrete quorum sensing molecules with antifungal activity. In this study, we extracted and fractioned culture filtrate of S. mutans, seeking antifungal agents capable of inhibiting the biofilms, filamentation, and candidiasis by Candida albicans. Active S. mutans UA159 supernatant filtrate components were extracted via liquid-liquid partition and fractionated on a C-18 silica column to resolve S. mutans fraction 1 (SM-F1) and fraction 2 (SM-F2). We found anti-biofilm activity for both SM-F1 and SM-F2 in a dose dependent manner and fungal growth was reduced by 2.59 and 5.98 log for SM-F1 and SM-F2, respectively. The SM-F1 and SM-F2 fractions were also capable of reducing C. albicans filamentation, however statistically significant differences were only observed for the SM-F2 (p = 0.004). SM-F2 efficacy to inhibit C. albicans was confirmed by its capacity to downregulate filamentation genes CPH1, EFG1, HWP1, and UME6. Using Galleria mellonella as an invertebrate infection model, therapeutic treatment with SM-F2 prolonged larvae survival. Examination of the antifungal capacity was extended to a murine model of oral candidiasis that exhibited a reduction in C. albicans colonization (CFU/mL) in the oral cavity when treated with SM-F1 (2.46 log) and SM-F2 (2.34 log) compared to the control (3.25 log). Although both SM-F1 and SM-F2 fractions decreased candidiasis in mice, only SM-F2 exhibited significant quantitative differences compared to the non-treated group for macroscopic lesions, hyphae invasion, tissue lesions, and inflammatory infiltrate. Taken together, these results indicate that the SM-F2 fraction contains antifungal components, providing a promising resource in the discovery of new inhibitors for oral candidiasis.

The Postbiotic Activity of Lactobacillus paracasei 28.4 Against Candida auris.

Candida auris has emerged as a medically important pathogen with considerable resistance to antifungal agents. The ability to produce biofilms is an important pathogenicity feature of this species that aids escape of host immune responses and antimicrobial agents. The objective of this study was to verify antifungal action using in vitro and in vivo models of the Lactobacillus paracasei 28.4 probiotic cells and postbiotic activity of crude extract (LPCE) and fraction 1 (LPF1), derived from L. paracasei 28.4 supernatant. Both live cells and cells free supernatant of L. paracasei 28.4 inhibited C. auris suggesting probiotic and postbiotic effects. The minimum inhibitory concentration (MIC) for LPCE was 15 mg/mL and ranges from 3.75 to 7.5 mg/mL for LPF1. Killing kinetics determined that after 24 h treatment with LPCE or LPF1 there was a complete reduction of viable C. auris cells compared to fluconazole, which decreased the initial inoculum by 1-logCFU during the same time period. LPCE and LPF1 significantly reduced the biomass (p = 0.0001) and the metabolic activity (p = 0.0001) of C. auris biofilm. There was also a total reduction (~108 CFU/mL) in viability of persister C. auris cells after treatment with postbiotic elements (p < 0.0001). In an in vivo study, injection of LPCE and LPF1 into G. mellonella larvae infected with C. auris prolonged survival of these insects compared to a control group (p < 0.05) and elicited immune responses by increasing the number of circulating hemocytes and gene expression of antimicrobial peptide galiomicin. We concluded that the L. paracasei 28.4 cells and postbiotic elements (LPCE and LPF1) have antifungal activity against planktonic cells, biofilms, and persister cells of C. auris. Postbiotic supplementation derived from L. paracasei 28.4 protected G. mellonella infected with C. auris and enhanced its immune status indicating a dual function in modulating a host immune response.

Expression of DNA repair genes in oral squamous cell carcinoma using reverse transcription-quantitative polymerase chain reaction.

OBJECTIVE: The aim of this study was to evaluate the expression of DNA repair genes in cases of oral squamous cell carcinoma (OSCC). STUDY DESIGN: Expression of the MLH1, MSH2, MLH3, ATM, MRE11A, XRCC1, and PMS2 genes was evaluated by reverse transcription-quantitative polymerase chain reaction in the OSCC group (32 patients) and the control group (15 patients). The groups were compared by using the Mann-Whitney test, with Bonferroni correction. Associations between gene expression levels and clinical data were explored by using Pearson's and Spearman's correlation coefficients, with P value less than .05 indicating a significant difference. RESULTS: The MLH1, MSH2, MLH3, ATM, MRE11A, XRCC1, and PMS2 genes were downregulated in the OSCC group compared with the control group, with significant values for MLH1 (P < .0001); MSH2 (P = .038); MLH3 (P < .0001); ATM (P < .0001); MRE11A (P < .0001); XRCC1 (P = .0004); and PMS2 (P = .008). Analysis of the correlation between gene expression and clinical data only revealed a significant negative correlation between age and expression of the PMS2 gene. CONCLUSIONS: Expression of the DNA repair genes MLH1, MSH2, MLH3, ATM, MRE11 AMRE11A, XRCC1, and PMS2 was reduced in OSCC.

Candida Biofilms: An Update on Developmental Mechanisms and Therapeutic Challenges.

Fungi of the genus Candida are important etiological agents of superficial and life-threatening infections in individuals with a compromised immune system. One of the main characteristics of Candida is its ability to form highly drug tolerance biofilms in the human host. Biofilms are a dynamic community of multiple cell types whose formation over time is orchestrated by a network of transcription regulators. In this brief review, we provide an update of the processes involved in biofilm formation by Candida spp. (formation, treatment, and control), as well as the transcriptional circuitry that regulates its development and interactions with other microorganisms. Candida albicans is known to build mixed species biofilms with other Candida species and with various other bacterial species in different host niches. Taken together, these properties play a key role in Candida pathogenesis. In addition, this review gathers recent studies with new insights and perspectives for the treatment and control of Candida biofilms.

Development of Probiotic Formulations for Oral Candidiasis Prevention: Gellan Gum as a Carrier To Deliver Lactobacillus paracasei 28.4.

Probiotics might provide an alternative approach for the control of oral candidiasis. However, studies on the antifungal activity of probiotics in the oral cavity are based on the consumption of yogurt or other dietary products, and it is necessary to use appropriate biomaterials and specific strains to obtain probiotic formulations targeted for local oral administration. In this study, we impregnated gellan gum, a natural biopolymer used as a food additive, with a probiotic and investigated its antifungal activity against Candida albicansLactobacillus paracasei 28.4, a strain recently isolated from the oral cavity of a caries-free individual, was incorporated in several concentrations of gellan gum (0.6% to 1% [wt/vol]). All tested concentrations could incorporate L. paracasei cells while maintaining bacterial viability. Probiotic-gellan gum formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacterium-impregnated gellan gum was achieved when L. paracasei 28.4 was lyophilized. The probiotic-gellan gum formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the growth of C. albicans, with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic-gellan gum formulations also had inhibitory activity against Candida sp. biofilms by reducing the number of Candida sp. cells (P < 0.0001), decreasing the total biomass (P = 0.0003), and impairing hyphae formation (P = 0.0002), compared to the control group which received no treatment. Interestingly, a probiotic formulation of 1% (wt/vol) gellan gum provided an oral colonization of L. paracasei in mice with approximately 6 log CFU/ml after 10 days. This formulation inhibited C. albicans growth (P < 0.0001), prevented the development of candidiasis lesions (P = 0.0013), and suppressed inflammation (P = 0.0006) compared to the mice not treated in the microscopic analysis of the tongue dorsum. These results indicate that gellan gum is a promising biomaterial and can be used as a carrier system to promote oral colonization for probiotics that prevent oral candidiasis.

Role of epstein-barr virus in the severity of recurrent respiratory papillomatosis.

OBJECTIVE: The objective was to investigate the prevalence of the Epstein-Barr virus (EBV) and its association with human papilloma virus (HPV) detection, clinicopathological features, and the severity of recurrent respiratory papillomatosis (RRP). METHODS: Cases of juvenile recurrent respiratory papillomatosis (JRRP) (n = 36) and adult recurrent respiratory papillomatosis (ARRP) (n = 44) were collected retrospectively and subdivided into low- and high-risk severity groups based on the Derkay score. We performed HPV detection and genotyping using a reverse hybridization protocol and investigated the presence of EBV by polymerase chain reaction (PCR) and in situ hybridization. CD21 levels were accessed by immunohistochemistry. RESULTS: All samples were HPV-positive, including 49 cases of HPV 6, 26 cases of HPV 11, four cases of HPV 6 and 11 coinfections, and one case of HPV 16. EBV-DNA was detected in nine samples by PCR, although none of the cases were positive by means of in situ hybridization. CD21 immunoexpression was not statistically associated with any of the variables analyzed. HPV 6 detection was significantly higher in ARRP cases (P = 0.03), whereas HPV 11 was more prevalent in JRRP cases (P = 0.02) and was even more prevalent in JRRP cases of greater severity (Derkay laryngoscopic scale ≥20) (P = 0.04). CONCLUSION: The presence of EBV does not seem to play an important role in the progression/severity of RRP. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E611-E618, 2020.

Effects of surface pre-reacted glass-ionomer (S-PRG) eluate on Candida spp.: antifungal activity, anti-biofilm properties, and protective effects on Galleria mellonella against C. albicans infection.

Surface pre-reacted glass-ionomer (S-PRG) is a bioactive filler produced by PRG technology, which is applied to various dental materials. The inhibitory effects of S-PRG eluate against Candida, the most common fungal oral pathogen, were investigated. Minimum inhibitory concentrations (MIC) and anti-biofilm activities were tested against Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis. For the in vivo study, Galleria mellonella was used as a model to evaluate the effects of S-PRG on toxicity, hemocyte counts and candidiasis. The MIC of S-PRG ranged from 5 to 40% (v/v). S-PRG eluate exhibited anti-biofilm activity for all the Candida species tested. Furthermore, injection of S-PRG eluate into G. mellonella was not toxic to the larvae and protected G. mellonella against experimental candidiasis. In addition, S-PRG eluate inhibited biofilm formation by C. albicans, C. glabrata, C. krusei, and C. tropicalis and exerted protective effects on G. mellonella against experimental candidiasis in vivo.

Antimicrobial photodynamic therapy against clinical isolates of carbapenem-susceptible and carbapenem-resistant Acinetobacter baumannii.

Infections caused by Acinetobacter baumannii have become a challenge for healthcare professionals because of the rapid increase in Gram-negative bacteria resistant to carbapenem antibiotics. The objective of this study was to evaluate the effect of antimicrobial photodynamic therapy (aPDT) against different strains of A. baumannii isolated from patients with infectious process and hospitalized at the intensive care unit of the hospitals of São Jose dos Campos, São Paulo. These isolates were obtained from the Valeclin Clinical Analysis Laboratory (SP, Brazil) and were tested for susceptibility to the carbapenems imipenem and meropenem by determination of the minimal inhibitory concentration (MIC) using the broth microdilution method. The strains susceptible and resistant to these antibiotics were submitted to aPDT using methylene blue and a low-level laser with a wavelength of 660 nm and fluence of 39.5 J/cm2 (energy of 15 J and time of 428 s). The number of colony-forming units (CFU/mL) was analyzed by ANOVA and the Tukey test. The laboratory of origin of the clinical isolates identified 1.54% of 13,715 strains tested over a period of 8 months as A. baumannii. Among the A. baumannii isolates, 58% were resistant to carbapenems by the disk diffusion test. Susceptible isolates exhibited MIC of 0.5 to 1 µg/mL and resistant isolates of 64 to > 128 µg/mL. PDT reduced the number of A. baumannii cells for all isolates tested, with this reduction ranging from 63 to 88% for susceptible isolates and from 26 to 97% for resistant isolates. The percentage of viability was dependent on the strain analyzed. In conclusion, these data indicate that PDT could be an alternative strategy for the control of infections caused by carbapenem-resistant A. baumannii.

Thiazole derivatives act on virulence factors of Cryptococcus spp.

Cryptococcosis is an opportunistic or primary fungal infection considered to be the most prevalent fatal fungal disease worldwide. Owing to the limited number of available drugs, it is necessary to search for novel antifungal compounds. In the present work, we assessed the antifungal efficacy of three thiazole derivatives (1, 2, and 3). We conducted in vitro and in vivo assays to investigate their effects on important virulence factors, such as capsule and biofilm formation. In addition, the phagocytosis index of murine macrophages exposed to compounds 1, 2, and 3 and the in vivo efficacy of 1, 2, and 3 in Galleria mellonella infected with Cryptococcus spp. were evaluated. All compounds exhibited antifungal activity against biofilms and demonstrated a reduction in biofilm metabolic activity by 43-50% for C. gattii and 26-42% for C. neoformans. Thiazole compounds promoted significant changes in the capsule thickness of C. gattii compared to that of C. neoformans. Further examination of these compounds suggests that they can improve the phagocytosis process of peritoneal murine macrophages in vitro, causing an increase in the phagocytosis rate. Survival percentage was examined in the invertebrate model Galleria mellonella larvae, and only compound 3 could increase the survival at doses of 5 mg/kg after infection with C. gattii (P = .0001) and C. neoformans (P = .0007), similar to fluconazole at 10 mg/kg. The results demonstrated that thiazole compounds, mainly compound 3, have potential to be used for future studies in the search for new therapeutics for cryptococcosis.

Influence of Streptococcus mitis and Streptococcus sanguinis on virulence of Candida albicans: in vitro and in vivo studies.

The aim was to evaluate in vitro possible interactions, gene expression, and biofilm formation in species of Candida albicans, Streptococcus mitis, and Streptococcus sanguinis and their in vivo pathogenicity. The in vitro analysis evaluated the effects of S. mitis and S. sanguinis on C. albicans's biofilm formation by CFU count, filamentation capacity, and adhesion (ALS1, ALS3, HWP1) and transcriptional regulatory gene (BCR1, CPH1, EFG1) expression. In vivo studies evaluated the pathogenicity of the interaction of the microorganisms on Galleria mellonella, with analyses of the CFU per milliliter count and filamentation. In vitro results indicated that there was an observed decrease in CFU (79.4-71.5%) in multi-species biofilms. The interaction with S. mitis inhibited filamentation, which seems to increase its virulence factor with over-expression of genes ALS1, ALS3, and HWP1 as well the interaction with S. sanguinis as ALS3 and HWP1. S. mitis upregulated BRC1, CPH1, and EFG1. The histological images of in vivo study indicate an increase in the filamentation of C. albicans when in interaction with the other species. It was concluded that S. mitis interaction suggests increased virulence factors of C. albicans, with periods of lower virulence and proto-cooperation in the interaction with S. sanguinis.

Effects of Bacillus subtilis on Candida albicans: biofilm formation, filamentation and gene expression / Efeitos de Bacillus subtilis sobre Candida albicans: formação de biofilme, filamentação e expressão gênica

Objective: The aim of this study was evaluate the effect of Bacillus subtilis on Candida albicans biofilm formation and filamentation by evaluating the gene expression of ALS3, HWP1, BCR1, EFG1 and TEC1. Material and Methods: Mixed (C. albicans / B.subtilis) and monotypic biofilms were cultured in plates at 37°C for 48 h under shaking for counting viable cells (CFU / mL) and analysis of gene expression by real-time PCR. The C. albicans filamentation assay was performed in medium containing 10% fetal bovine serum at 37°C for 6 hours. Data was analysed by t-Student and Mann­ Whitney tests. Results: B. subtilis reduced the biofilm formation of C. albicans in 1 log when cultured in the same environment (p<0.0001). In addition, it significantly reduced the yeast - hypha transition affecting the morphology of C. albicans. Among all of the analyzed genes, the ALS3 and HWP1 genes were the most affected, achieving 111.1- and 333.3- fold decreases in the C. albicans biofilms associated with B. subtilis, respectively. Conclusion: B. subtilis reduced the biofilm formation and filamentation of C. albicans by negatively regulating the ALS3, HWP1, BCR1, EFG1 and TEC1 genes that are essential for the production of biofilm and hyphae. (AU)

Objetivo: O objetivo deste estudo foi avaliar o efeito de Bacillus subtilis sobre a formação de biofilme e filamentação de Candida albicans através da avaliação da expressão dos genes ALS3, HWP1, BCR1, EFG1 and TEC1. Material e métodos: Biofilmes monotípicos e mistos (C. albicans / B.subtilis) foram cultivados em placas a 37°C por 48 h sob agitação, para a contagem de células viáveis (UFC/mL) e para a análise da expressão gênica por PCR em tempo real. O ensaio de filamentação de C. albicans foi realizado em meio contendo 10% de soro fetal bovino a 37°C por 6 h. Os dados obtidos foram analisados por testes t-Student e Mann­Whitney. Resultados: B.subtilis reduziu em 1 log a formação de biofilme por C. albicans quando cultivados no mesmo ambiente (p<0.0001). Além disso, reduziu significantemente a transição de levedura para hifa, afetando assim, a morfologia de C. albicans. Em relação aos genes analisados, os genes ALS3 e HWP1 foram os mais regulados negativamente, com uma diminuição de 111,1 e 333,3 vezes, respectivamente, na sua expressão em biofilmes de C. albicans associados a B. subtilis. Conclusão: B. subtilis reduziu a filamentação e a formação de biofilme de C. albicans através da regulação negativa dos genes ALS3, HWP1, BCR1, EFG1 e TEC1, que são essenciais na produção de hifas e de biofilme. (AU)