Activity of biogenic silver nanoparticles in planktonic and biofilm-associated Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a gram-positive bacterium and is the etiologic agent of caseous lymphadenitis (CL) in small ruminants. This disease is characterized by the development of encapsulated granulomas in visceral and superficial lymph nodes, and its clinical treatment is refractory to antibiotic therapy. An important virulence factor of the Corynebacterium genus is the ability to produce biofilm; however, little is known about the characteristics of the biofilm produced by C. pseudotuberculosis and its resistance to antimicrobials. Silver nanoparticles (AgNPs) are considered as promising antimicrobial agents, and are known to have several advantages, such as a broad-spectrum activity, low resistance induction potential, and antibiofilm activity. Therefore, we evaluate herein the activity of AgNPs in C. pseudotuberculosis, through the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm activity, and visualization of AgNP-treated and AgNP-untreated biofilm through scanning electron microscopy. The AgNPs were able to completely inhibit bacterial growth and inactivate C. pseudotuberculosis at concentrations ranging from 0.08 to 0.312 mg/mL. The AgNPs reduced the formation of biofilm in reference strains and clinical isolates of C. pseudotuberculosis, with interference values greater than 80% at a concentration of 4 mg/mL, controlling the change between the planktonic and biofilm-associated forms, and preventing fixation and colonization. Scanning electron microscopy images showed a significant disruptive activity of AgNP on the consolidated biofilms. The results of this study demonstrate the potential of AgNPs as an effective therapeutic agent against CL.

In silico approaches for predicting natural compounds with therapeutic potential and vaccine candidates against Streptococcus equi.

Equine strangles is a prevalent disease that affects the upper respiratory in horses and is caused by the Gram-positive bacterium Streptococcus equi. In addition to strangles, other clinical conditions are caused by the two S. equi subspecies, equi and zooepidemicus, which present relevant zoonotic potential. Treatment of infections caused by S. equi has become challenging due to the worldwide spreading of infected horses and the unavailability of effective therapeutics and vaccines. Penicillin treatment is often recommended, but multidrug resistance issues arised. We explored the whole genome sequence of 18 S. equi isolates to identify candidate proteins to be targeted by natural drug-like compounds or explored as immunogens. We considered only proteins shared among the sequenced strains of subspecies equi and zooepidemicus, absent in the equine host and predicted to be essential and involved in virulence. Of these, 4 proteins with cytoplasmic subcellular location were selected for molecular docking with a library of 5008 compounds, while 6 proteins were proposed as prominent immunogens against S. equi due to their probabilities of behaving as adhesins. The molecular docking analyses revealed the best ten ligands for each of the 4 drug target candidates, and they were ranked according to their binding affinities and the number of hydrogen bonds for complex stability. Finally, the natural 5-ring compound C25H20F3N5O3 excelled in molecular dynamics simulations for the increased stability in the interaction with UDP-N-acetylenolpyruvoylglucosamine reductase (MurB). This research paves the way to developing new therapeutics to minimize the impacts caused by S. equi infections.Communicated by Ramaswamy H. Sarma.

Trypanosoma sp. infection in Boa constrictor snakes: morphological, hematological, clinical biochemistry, molecular, and phylogenetic characteristics.

There are few reports of Trypanosoma in snakes, as well as little information about its pathogenicity in these animals. Thus, the present study aimed to characterize Trypanosoma found in Boa constrictor snakes, to verify the influence of the parasitism on hematological and clinical biochemistry parameters, and to perform a phylogenetic study of the isolates. Blood samples from sixty-one boas were analyzed for the presence of trypanosomatids and by hematological and clinical biochemistry assays. The flagellates that were found in this analysis were used for cell culture, morphometry, and molecular analysis. Later, molecular typing phylogenetic studies were performed. Nine positive animals (14.75%) were identified by microscopy analysis. The hematological results showed that parasitized animals presented significantly lower levels of packed cell volume, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. In the leukogram, eosinophils and heterophils counts were higher in parasitized animals. Considering the molecular analyses, the isolates presented a higher identity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the 18S small subunit ribosomal RNA (SSU rRNA) gene fragments with Trypanosoma serpentis. The phylogenetic tree, using the GAPDH, clustered all isolates with T. serpentis and Trypanosoma cascavelli. This is the first description of T. serpentis parasitizing boas and of the clinical changes caused by trypanosomatid infection in snakes.

Detection of Hemopathogens in Chelonoidis carbonaria: Microscopic, Molecular, Hematological, and Clinical Biochemistry Aspects.

Background: The growing contact between men and wild animals, caused by the increase in the population in urban centers and the destruction of the habitat of these animals, has been leading to a greater circulation of pathogens between humans and wildlife. Chelonoidis carbonaria, a tortoise found throughout South America, is one of the animals most rescued from animal trafficking and illegal breeding. Considering this situation, this study aimed to verify the occurrence of hemoparasites in C. carbonaria. Materials and Methods: Blood samples from 73 C. carbonaria were collected from animals located in (1) a rural commercial breeding unit, (2) an urban zoo, and (3) a center of rescued animal screening. Genomic DNA was extracted from these animals and used in PCRs to detect specific genomic fragments of haemogregarines (i.e., Hepatozoon and Hemolivia), and members of the Anaplasmataceae Family (i.e., Ehrlichia sp. and Anaplasma sp.). Blood samples were screened for hemopathogens by direct microscopy and were used for hematological assays, and serum samples were analyzed to determine the concentration of serum components. Results: It was found that 34.2% of the tortoises presented Sauroplasma sp. in their blood samples; these animals showed clinical biochemistry changes that indicate altered liver function. Two zoo animals were positive for Ehrlichia sp. in PCR, and also presented clinical biochemistry and hematological changes. Conclusion: The present project is pioneer in the detection of Ehrlichia sp. in C. carbonaria, and was able to identify changes in clinical biochemistry that can be a result of the infection by hemopathogens in this species.

Exploring dysregulated immune response genes and endothelial dysfunction biomarkers as predictors of severe COVID-19.

Identifying individuals and factors associated with severe cases of COVID-19 is crucial as the pandemic continues to spread globally. Effective biomarkers for predicting severe cases are essential for optimizing clinical management, therapy, and preventing unfavorable outcomes. This exploratory observational study aimed to investigate the expression of dysregulated immune response genes (ARG1, NOS2, ITGA4, and SELPLG) in total leukocytes, plasmatic levels of P-selectin and PSGL-1, and their clinical associations in patients with mild and severe COVID-19. Data from 117 confirmed COVID-19 patients (severe = 58, mild = 59) were collected upon admission. Gene expression was measured using RT-qPCR, and plasma protein levels assessed with ELISA assay. The severe COVID-19 patient group had a higher median age of 62.0 (p = 0.0001), a higher proportion of black individuals (86.2%, p < 0.0001), and more males (65.5%, p = 0.007). The neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were significantly higher in the severe COVID-19 patient group (p < 0.0001), indicating ongoing systemic inflammation. Severe COVID-19 patients also exhibited increased expression of ARG1 (p < 0.05) and SELPLG (p < 0.0001) genes, as well as higher concentrations of soluble P-selectin (p < 0.005) and PSGL-1 (p < 0.05) proteins. Multivariate analysis revealed that NLR, PLR, the expression of SELPLG and sPSGL-1 were independent predictors of COVID-19 severity. In conclusion, this study suggests that biomarkers of endothelial dysfunction and dysregulated leukocyte responses are associated with COVID-19 severity, serving as promising predictive tools for optimizing clinical management and patient monitoring.

Comparative antimicrobial activity of four different endodontic sealers.

Dental cements are widely used in the clinical routine, specifically for root canal sealing. Within this context, it is expected that these materials present antimicrobial activity, since it would help in the prevention of apical and periapical infections. The present study aimed to comparatively verify the antimicrobial activity of four dental cements against microorganisms that are routinely isolated from endodontic infections. Reference strains of Enterococcus faecalis, Candida albicans and Escherichia coli were submitted to the agar diffusion test and to modified direct contact test using four different sealers: an eugenol zinc oxide compound, an epoxy resin associated to calcium hydroxide and bismuth, a mineral trioxide aggregate (MTA) and a bioceramics. Different E. coli, C. albicans and E. faecalis growth inhibition profiles were observed in the agar diffusion assay. In the direct contact test, the bioceramics presented a higher microbicide activity on all microorganisms tested herein. Dental cements have different antimicrobial activities, being that the bioceramics present the most consistent antimicrobial activity, and that the direct contact test presented more uniform results than the agar diffusion test. This study reveals the antimicrobial activities of different cements and allow dentists to decide which material to employ in their daily practice.

A serum NMR metabolomic analysis of the Corynebacterium pseudotuberculosis infection in goats.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants, is highly prevalent worldwide. Economic losses have already been associated with the disease, and little is known about the host-pathogen relationship associated with the disease. The present study aimed to perform a metabolomic study of the C. pseudotuberculosis infection in goats. Serum samples were collected from a herd of 173 goats. The animals were classified as controls (not infected), asymptomatic (seropositives but without detectable CLA clinical signs), and symptomatic (seropositive animals presenting CLA lesions), according to microbiological isolation and immunodiagnosis. The serum samples were analyzed using nuclear magnetic resonance (1H-NMR), nuclear Overhauser effect spectroscopy (NOESY), and Carr-Purcell-Meiboom-Gill (CPMG) sequences. The NMR data were analyzed using chemometrics, and principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed to discover specific biomarkers responsible for discrimination between the groups. A high dissemination of the infection by C. pseudotuberculosis was observed, being 74.57% asymptomatic and 11.56% symptomatic. In the evaluation of 62 serum samples by NMR, the techniques were satisfactory in the discrimination of the groups, being also complementary and mutually confirming, demonstrating possible biomarkers for the infection by the bacterium. Twenty metabolites of interest were identified by NOESY and 29 by CPMG, such as tryptophan, polyunsaturated fatty acids, formic acid, NAD+, and 3-hydroxybutyrate, opening promising possibilities for the use of these results in new therapeutic, immunodiagnosis, and immunoprophylactic tools, as well as for studies of the immune response against C. pseudotuberculosis. KEY POINTS: • Sixty-two samples from healthy, CLA asymptomatic, and symptomatic goats were screened • Twenty metabolites of interest were identified by NOESY and 29 by CPMG • 1H-NMR NOESY and CPMG were complementary and mutually confirming.

First description of Candida haemulonii infecting a snake Boa constrictor: Molecular, pathological and antifungal sensitivity characteristics.

Candida haemulonii is an emergent infectious pathogen that affects human presenting comorbidities and/or immunodepression. Little is known about other possible hosts. For the first time, this fungus was found causing a cutaneous infection in a snake, Boa constrictor, characterized by scale opacity and several ulcerative lesions. This C. haemulonii was isolated, identified using molecular techniques and a phylogenetic study, and had its growth totally inhibited by all the drugs tested; however, no fungicide effect was seen for fluconazole and itraconazole. The B. constrictor clinical signals subsided after a treatment using a biogenic silver nanoparticle-based ointment. These findings, along with the B. constrictor presence near human habitats, warn for the necessity of wildlife health monitoring for emergent and opportunistic diseases in peri-urban environments.

Photodynamic inactivation of different Candida species and inhibition of biofilm formation induced by water-soluble porphyrins.

BACKGROUND: Candida spp. is the main fungal genus related to infections in humans, and its treatment has become a challenge due to the production of biofilm and its resistance/multi-resistance profile to conventional antifungals. Antimicrobial photodynamic therapy stands out as a treatment characterized by a broad spectrum of antimicrobial action, being able to induce oxidative stress in pathogens, and porphyrins are photosensitizers with high selectivity to pathogens. Thus, this work aimed to analyze the photoinactivation of different species of Candida by two cationic (4-H2TMeP+ and 3-H2TMeP+) and one anionic (4-H2TPSP‒) porphyrins. MATERIALS AND METHODS: Microdilution assays were performed to determine the MIC100, with subsequent determination of MFC100. Determination of oxidative species was done through the use of scavengers, while biofilm morphological features were investigated using the atomic force microscopy. RESULTS: Cationic porphyrins were significantly efficient in inactivating Candida albicans and non-albicans species with 100% growth inhibition and fungicidal activity (MFC100/MIC100 ≤ 4.0). The cationic porphyrins were also able to interfere in Candida spp biofilm formation. The photo-oxidative mechanism activated by 3-H2TMeP+ in Candida spp. is concurrent with the production of singlet oxygen and oxygen radical species. In the AFM analysis, 3-H2TMeP+ was able to reduce yeast adhesion to the surface. CONCLUSIONS: Cationic porphyrins can photo-inactivate different species of Candida in both planktonic and biofilm-associated forms, and reduce the adhesion of these fungi to the surface.

AFFINITY OF BRAZILIAN WILD MAMMAL IMMUNOGLOBULINS TO BACTERIAL PROTEINS A AND G.

Staphylococcal A and streptococcal G proteins are widely used in immunoassays when specific immunological reagents are unavailable, such as for wild animals. The affinity of bacterial proteins A and G to the immunoglobulins of seven Brazilian mammals were tested, including black-tufted marmoset (Callithrix penicillata, n = 5), golden-bellied capuchin (Sapajus xanthosternos, n = 13), woolly mouse opossum (Micoureus demerarae, n = 6), long-nosed armadillo (Dasypus novemcinctus, n = 5), collared anteater (Tamandua tetradactyla, n = 5), ocelot (Leopardus pardalis, n = 6), and vampire bat (Desmodus rotundus, n = 5). Blood samples were collected from animals that were rescued in peri-urban rainforest fragments. Sera pools of each species were tested by ELISA to determine the intensity of each bacterial protein affinity to the immunoglobulins. When comparing the affinity to both proteins, immunoglobulins from D. rotundus, S. xanthosternos, and T. tetradactyla presented a higher affinity to protein G, whereas a higher affinity to protein A was found for immunoglobulins of C. penicillata and L. pardalis. The only species that presented a very low affinity to both bacterial proteins was M. demerarae. This study can be used as a reference for further studies on the development of sensitive and specific immunodiagnostic assays to be used for the monitoring of the health of these wild mammals.

Hematological and clinical biochemistry profiles in Canindé goats infected by Corynebacterium pseudotuberculosis and bred in a tropical semi-arid region.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in goats and sheep, is highly prevalent worldwide and is characterized by economic losses in small ruminant production. Currently available techniques for clinical and laboratory diagnosis of the disease lack market availability and/or sensitivity, and therefore, infected animals can remain in the herd, serving as a source of infection for other animals. The present study aimed to verify hematological and clinical biochemistry changes in goats naturally infected by C. pseudotuberculosis. One hundred seventy-three Canindé goats were included in this study, from which blood samples and caseous lesions were collected. The animals were classified as uninfected, asymptomatic, and symptomatic according to microbiological isolation and serological assays. A high dissemination of the infection was observed in the herd, with 86.13% of positive animals, being 74.57% asymptomatic and 11.56% symptomatic. In the hemogram and clinical biochemistry analyses, the only statistical difference found was a higher level of serum urea in asymptomatic individuals than in non-infected animals. In addition, this study points to the possibility of chronic CLA being potentially reflected in hepatic and renal biochemical markers.

Secretome from human adipose-derived mesenchymal stem cells promotes blood vessel formation and pericyte coverage in experimental skin repair.

Human adipose tissue-derived stem cells (hASC) secretome display various therapeutically relevant effects in regenerative medicine, such as induction of angiogenesis and tissue repair. The benefits of hASC secretome are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in host cells. However, the composition and the innate characteristics of hASC secretome can be highly variable depending on the culture conditions. Here, we evaluated the combined effect of serum-free media and hypoxia preconditioning on the hASCs secretome composition and biological effects on angiogenesis and wound healing. The hASCs were cultured in serum-free media under normoxic (NCM) or hypoxic (HCM) preconditioning. The proteomic profile showed that pro- and anti-antiangiogenic factors were detected in NCM and HCM secretomes. In vitro studies demonstrated that hASCs secretomes enhanced endothelial proliferation, survival, migration, in vitro tube formation, and in vivo Matrigel plug angiogenesis. In a full-thickness skin-wound mouse model, injection of either NCM or HCM significantly accelerated the wound healing. Finally, hASC secretomes were potent in increasing endothelial density and vascular coverage of resident pericytes expressing NG2 and nestin to the lesion site, potentially contributing to blood vessel maturation. Overall, our data suggest that serum-free media or hypoxic preconditioning enhances the vascular regenerative effects of hASC secretome in a preclinical wound healing model.

Immunoprophylactic properties of the Corynebacterium pseudotuberculosis-derived MBP:PLD:CP40 fusion protein.

Caseous lymphadenitis (CLA) is a disease that affects small ruminants, and the best way to prevent its spread on a herd is through immunoprophylaxis. Thus, we aimed to evaluate the MBP:PLD:CP40 fusion protein as a new CLA immunogen. The fusion protein was constructed by combining Corynebacterium pseudotuberculosis PLD and CP40 proteins with maltose-binding protein (MBP) as an intrinsic adjuvant. The antigenicity, allergenic potential, prediction of B epitopes, binding to MHC receptors, and docking on the Toll-Like 2 receptor were evaluated in silico. MBP:PLD:CP40 was expressed and purified. 40 BALB/c were divided into four groups (G1 - control, G2 - Saponin, G3 - MBP:PLD:CP40, and G4 - rPLD + rCP40). Total IgG, IgG1, and IgG2a were quantified, and the expressions of cytokines after splenocyte in vitro stimulation were assessed. Mice were challenged 42 days after the first immunization. The in silico analysis showed that MBP:PLD:CP40 has immunogenic potential, does not have allergic properties, and can dock on the TRL2 receptor. MBP:PLD:CP40 stimulated the production of IgG1 antibodies in a fivefold proportion to IgG2a, and TNF and IL-17 were significantly expressed in response to the antigenic stimuli. When rPLD and rCP40 were used together for immunization, they could induce IFN-γ and IL-12, but with no detectable antibody production. The G3 and G4 groups presented a survival of 57.14% and 42.86%, respectively, while the G1 and G2 mice were all dead 15 days after the challenge. MBP:PLD:CP40 partially protected the mice against C. pseudotuberculosis infection and can be considered a potential new CLA immunogen. KEY POINTS: • The fusion protein induced more IgG1 than IgG2a antibodies; • The fusion protein also induced the expression of the TNF and IL-17 cytokines; • Mice inoculated with MBP:PLD:CP40 presented a 57.14% survival.

P22 protein complex in the serodiagnosis of animal tuberculosis: Antigenic stability and cross-reactivity with Corynebacterium pseudotuberculosis infection.

The P22 ELISA was recently developed for the serodiagnosis of animal tuberculosis. Herein, the stability of the P22 antigen in different presentations and storage conditions, and the cross-reactivity with Corynebacterium pseudotuberculosis infection in small ruminants were evaluated. For the stability assay, serum samples from cows, sheep, goats, alpacas, badgers, and wild boar were used in the P22 ELISA. The cross-reactivity analysis used sera from sheep and goats with caseous lymphadenitis (CLA). Differences in the immune recognition of P22 were found when the antigen was stored at 40 °C, but without altering the negative or positive status of each sample. P22 ELISA presented 5.71 % cross-reactivity when CLA-positive sheep were evaluated, but no cross-reaction was observed among CLA-positive goat serum samples. This study showed that the P22 protein complex is stable under different formulations and temperatures, and that the assay presents a low cross-reactivity with CLA.

Mutational spectrum of breast cancer susceptibility genes among women ascertained in a cancer risk clinic in Northeast Brazil.

PURPOSE: There is a paucity of data on the spectrum and prevalence of pathogenic variants among women of African ancestry in the Northeast region of Brazil. METHODS: We performed BROCA panel sequencing to identify inherited loss-of-function variants in breast cancer susceptibility genes among 292 Brazilian women referred to a single institution cancer risk assessment program. RESULTS: The study included a convenient cohort of 173 women with invasive breast cancer (cases) and 119 women who were cancer-free at the time of ascertainment. The majority of the women self-reported as African-descended (67% for cases and 90.8% for unaffected volunteers). Thirty-seven pathogenic variants were found in 36 (20.8%) patients. While the spectrum of pathogenic variants was heterogeneous, the majority (70.3%) of the pathogenic variants were detected in high-risk genes BRCA1, BRCA2, PALB2, and TP53. Pathogenic variants were also found in the ATM, BARD1, BRIP1, FAM175A, FANCM, NBN, and SLX4 genes in 6.4% of the affected women. Four recurrent pathogenic variants were detected in 11 patients of African ancestry. Only one unaffected woman had a pathogenic variant in the RAD51C gene. Different risk assessment models examined performed well in predicting risk of carrying germline loss-of-function variants in BRCA1 and/or BRCA2 in breast cancer cases. CONCLUSION: The high prevalence and heterogenous spectrum of pathogenic variants identified among self-reported African descendants in Northeast Brazil is consistent with studies in other African ancestry populations with a high burden of aggressive young onset breast cancer. It underscores the need to integrate comprehensive cancer risk assessment and genomic testing in the management of newly diagnosed Black women with breast cancer across the African Diaspora, enabling improved cancer control in admixed underserved and understudied populations.

Chemokine production induced by Corynebacterium pseudotuberculosis in a murine model.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis. The main clinical sign of this disease is the development of granulomas, especially in small ruminants; however, the pathways that are involved in the formation and maintenance of these granulomas are unknown. Cytokines and chemokines are responsible for the migration of immune cells to specific sites and tissues; therefore, it is possible that chemokines participate in abscess formation. This study aimed to evaluate the induction of chemokine production by two C. pseudotuberculosis strains in a murine model. A highly pathogenic (VD57) and an attenuated (T1) strain of C. pseudotuberculosis, as well as somatic and secreted antigens derived from these strains, was used to stimulate murine splenocytes. Then, the concentrations of the chemokines CCL-2, CCL-3, CCL-4, and CCL-5 and the cytokines IL-1 and TNF were measured in the culture supernatants. The VD57 strain had a higher ability to stimulate the production of chemokines when compared to T1 strain, especially in the early stages of stimulation, which can have an impact on granuloma formation. The T1 lysate antigen was able to stimulate most of the chemokines studied herein when compared to the other antigenic fractions of both strains. These results indicate that C. pseudotuberculosis is a chemokine production inducer, and the bacterial strains differ in their induction pattern, a situation that can be related to the specific behavior of each strain.

Activity of Fusarium oxysporum-Based Silver Nanoparticles on Candida spp. Oral Isolates.

Candida spp. resistant to commercially available antifungals are often isolated from patients with oral candidiasis, a situation that points to the need for the development of new therapies. Thus, we evaluated the activity of Fusarium oxysporum-based silver nanoparticles (AgNPs) on Candida spp. isolated from denture stomatitis lesions. Candida isolates were molecularly identified and submitted to susceptibility assays using AgNPs and commercial fungicides. The interference on biofilm formation and the mechanisms of action of AgNPs on Candida spp. were also investigated. Scanning electron microscopy was used to evaluate the morphology of AgNP-treated Candida. Candida albicans was the most frequent species isolated from denture stomatitis cases. All Candida spp. were susceptible to AgNPs at low concentrations, except Candida parapsilosis. AgNPs caused surface damage, cell disruption, and biofilm formation inhibition. The ergosterol supplementation protected C. albicans against the AgNP action. AgNPs are effective against Candida spp. and can be faced as a promising new therapeutic agent against oral candidiasis.

Analysis of Gtpases Rab 5 and Rab 7 expression from macrophages infected with biofilm-producing and non biofilm-producing strains of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a facultative intracellular pathogen that uses various mechanisms to survive within macrophages. In phagocytosis, this survival can be attributed to the ability to inhibit phagosome-lysosome fusion. In this fusion, some proteins, including Rabs GTPases, are involved in the maturation process and are responsible for regulating membrane vesicle trafficking. Thus, to better understand these mechanisms, the capacity of biofilm-producing and non biofilm-producing strains of Corynebacterium pseudotuberculosis for modulating the expression of endosomal proteins GTPases Rab 5 and Rab 7 was evaluated in an in vitro study of infection of goat macrophages. Blood was collected from ten Canindé goats, infected with biofilm-producing and non biofilm-producing strains of C. pseudotuberculosis. Blood cells were separated in colloidal silica-polyvinylpyrrolidone gradients (GE Healthcare®). These cells were maintained at 37 °C, with 5% of CO2. After differentiation, macrophages were infected with the mentioned strains. The bacterial pellets were marked with Rab 5 and Rab 7 antibodies, and their expression was observed by flow cytometry. Both strains of C. pseudotuberculosis (biofilm-producing and non biofilm-producing) were observed to be capable of altering the expression of Rab proteins in macrophages cultivated in vitro. Macrophages from the animals infected with the biofilm-producing strain had an increase in the expression of Rab 5 protein, mainly when these macrophages were treated with the non biofilm-producing strain. The same mechanism was shown to function with Rab 7 protein, however at a lower intensity of expression when compared with Rab 5.

Activity of antifungal drugs and Brazilian red and green propolis extracted with different methodologies against oral isolates of Candida spp.

BACKGROUND: Oral candidiasis is an opportunistic disease caused by fungi of the Candida genus. The occurrence of Candida spp. resistance to the commercial antifungal drugs points to the search for alternative treatments. Propolis has been successfully used in the treatment of infectious diseases for centuries. It has been proposed that an ultrasound pretreatment in the propolis extraction protocol can enhance the concentrations of molecules with antimicrobial activities in the final extract. Thus, this study aimed to compare the antifungal activity against oral Candida spp. isolates of green and red propolis extracts submitted or not to an ultrasound pretreatment before the extraction procedure. METHODS: Candida spp. were isolated from denture stomatitis lesions and identified by sequencing. Oral Candida spp. isolates and reference strains were submitted to broth microdilution assays using commercial antifungals and Brazilian green and red propolis extracts submitted or not to an ultrasound pretreatment. Minimal Inhibitory Concentrations (MIC) and Minimal Fungicide Concentrations (MFC) were determined and biofilm formation interference was evaluated for resistant isolates. RESULTS: C. albicans, Candida tropicalis and Candida dubliniensis were isolated from denture stomatitis lesions. Growth inhibition was observed in all Candida isolates incubated with all green and red propolis extracts. At lower doses, red propolis extracts presented significant antifungal activity. The ultrasound pretreatment did not promote an increase in the antifungal activity of green or red propolis. Three isolates, which were highly resistant to fluconazole and itraconazole, were susceptible to low doses of red propolis extracts. These same three specimens had their biofilm formation inhibted by red propolis ethanolic extract. CONCLUSIONS: Thus, red propolis can be faced as a promising natural product to be used in the auxiliary antifungal therapy of denture stomatitis.

Activity of Ethanolic and Supercritical Propolis Extracts in Corynebacterium pseudotuberculosis and Its Associated Biofilm.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis in small ruminants, a chronic disease characterized by the development of granulomas in superficial and visceral lymph nodes as well as in several organs. An important characteristic of the infection with this bacterium is the formation of a biofilm and the absence of effective antibiotic therapy against the disease. From this scenario, the objective of this study was to evaluate the susceptibility of C. pseudotuberculosis to conventional antibiotics and to red, green, and brown propolis extracts obtained by the supercritical and ethanolic extraction methods as well as its activity in the bacterial biofilm. The results of the sensitivity test using antibiotics indicated a sensitivity of C. pseudotuberculosis strains to the antimicrobial agents. The ethanolic extract of green propolis and the supercritical red propolis extract showed the best antibacterial activities against planktonic C. pseudotuberculosis. A lower antimicrobial activity of the brown propolis extract was identified. Propolis extracts were effective in interfering with the formation of the C. pseudotuberculosis biofilm but had little activity on the consolidated biofilm. In conclusion, propolis extracts are more effective against C. pseudotuberculosis in the planktonic stage, being able to interfere with the formation of bacterial biofilm. However, the action of propolis extracts in a sessile and structured microbial biofilm is reduced.