Antibiofilm activity of promethazine, deferiprone, and Manuka honey in an ex vivo wound model.

This study evaluated the antibiofilm activity of promethazine, deferiprone, and Manuka honey against Staphylococcus aureus and Pseudomonas aeruginosa in vitro and ex vivo in a wound model on porcine skin. The minimum inhibitory concentrations (MICs) and the effects of the compounds on biofilms were evaluated. Then, counting colony-forming units (CFUs) and confocal microscopy were performed on biofilms cultivated on porcine skin for evaluation of the compounds. For promethazine, MICs ranging from 97.66 to 781.25 µg/ml and minimum biofilm eradication concentration (MBEC) values ranging from 195.31 to 1562.5 µg/ml were found. In addition to reducing the biomass of both species' biofilms. As for deferiprone, the MICs were 512 and >1024 µg/ml, the MBECs were ≥1024 µg/ml, and it reduced the biomass of biofilms. Manuka honey had MICs of 10%-40%, MBECs of 20 to >40% and reduced the biomass of S. aureus biofilms only. Concerning the analyses in the ex vivo model, the compounds reduced (P < .05) CFU counts for both bacterial species, altering the biofilm architecture. The action of the compounds on biofilms in in vitro and ex vivo tests raises the possibility of using them against biofilm-associated wounds. However, further studies are needed to characterize the mechanisms of action and their effectiveness on biofilms in vivo.

Effect of promethazine on biofilms of gram-positive cocci associated with infectious endocarditis.

This study evaluated the antimicrobial activity of promethazine against Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus mutans and its effect on the antimicrobial susceptibility of biofilms grown in vitro and ex vivo on porcine heart valves. Promethazine was evaluated alone and in combination with vancomycin and oxacillin against Staphylococcus spp. and vancomycin and ceftriaxone against S. mutans in planktonic form and biofilms grown in vitro and ex vivo. Promethazine minimum inhibitory concentration range was 24.4-95.31 µg/mL and minimum biofilm eradication concentration range was 781.25-3.125 µg/mL. Promethazine interacted synergistically with vancomycin, oxacillin and ceftriaxone against biofilms in vitro. Promethazine alone reduced (p < 0.05) the CFU-counts of biofilms grown on heart valves for Staphylococcus spp., but not for S. mutans, and increased (p < 0.05) the activity of vancomycin, oxacillin and ceftriaxone against biofilms of Gram-positive cocci grown ex vivo. These findings bring perspectives for repurposing promethazine as adjuvant in the treatment of infective endocarditis.

Reduced gut microbiota diversity in patients with congenital generalized lipodystrophy.

BACKGROUND: Previous studies suggest intestinal dysbiosis is associated with metabolic diseases. However, the causal relationship between them is not fully elucidated. Gut microbiota evaluation of patients with congenital generalized lipodystrophy (CGL), a disease characterized by the absence of subcutaneous adipose tissue, insulin resistance, and diabetes since the first years of life, could provide insights into these relationships. METHODS: A cross-sectional study was conducted with patients with CGL (n = 17) and healthy individuals (n = 17). The gut microbiome study was performed by sequencing the 16S rRNA gene through High-Throughput Sequencing (BiomeHub Biotechnologies, Brazil). RESULTS: The median age was 20.0 years old, and 64.7% were female. There was no difference between groups in pubertal stage, BMI, ethnicity, origin (rural or urban), delivery, breastfeeding, caloric intake, macronutrient, or fiber consumption. Lipodystrophic patients presented a lower alpha diversity (Richness index: 54.0 versus 67.5; p = 0.008). No differences were observed in the diversity parameters when analyzing the presence of diabetes, its complications, or the CGL subtype. CONCLUSION: In this study, we demonstrate for the first time a reduced gut microbiota diversity in individuals with CGL. Dysbiosis was present despite dietary treatment and was also observed in young patients. Our findings allow us to speculate that the loss of intestinal microbiota diversity may be due to metabolic abnormalities present since the first years of life in CGL. Longitudinal studies are needed to confirm these findings, clarifying the possible causal link between dysbiosis and insulin resistance in humans.

Meningitis caused by Capnocytophaga canimorsus in a COVID-19 patient: a rare complication of dog bites.

Capnocytophaga canimorsus is a gram-negative rod that is part of the commensal microbiota of dogs' and cats' mouths. In this case, we report an 85-year-old man with COVID-19 who had his right arm bitten by a dog. His symptoms were impaired consciousness, agitation and aggressive behavior. Physical examination revealed neck stiffness and Brudzinski's sign. The cerebrospinal fluid culture was compatible with Capnocytophaga canimorsus. He required intensive care and received a 14-day prescription of meropenem. After 40 days of hospitalization, the patient was fully recovered and was discharged. This case highlights the importance of physician and microbiologist be awareness of this disease, mainly in patients with neurological symptoms after a dog or cat bite.

Meningitis caused by Capnocytophaga canimorsus in a COVID-19 patient: a rare complication of dog bites

ABSTRACT Capnocytophaga canimorsus is a gram-negative rod that is part of the commensal microbiota of dogs' and cats' mouths. In this case, we report an 85-year-old man with COVID-19 who had his right arm bitten by a dog. His symptoms were impaired consciousness, agitation and aggressive behavior. Physical examination revealed neck stiffness and Brudzinski's sign. The cerebrospinal fluid culture was compatible with Capnocytophaga canimorsus. He required intensive care and received a 14-day prescription of meropenem. After 40 days of hospitalization, the patient was fully recovered and was discharged. This case highlights the importance of physician and microbiologist be awareness of this disease, mainly in patients with neurological symptoms after a dog or cat bite.

Proposal for a microcosm biofilm model for the study of vulvovaginal candidiasis.

This study proposes a microcosm biofilm (MiB) model for the study of vulvovaginal candidiasis (VVC). Different conditions that mimic the vaginal environment were tested for MiB formation. The best growth conditions were obtained with samples incubated in vaginal fluid simulator medium pH 4.5 at 35 °C under a microaerophilic atmosphere. MiBs were evaluated for growth kinetics, fluconazole susceptibility and morphology. Samples containing high numbers of bacteria were analyzed for metagenomics. At 48 h, MiBs presented a higher cell density (CFU ml-1), a higher biomass and tolerance to fluconazole than their corresponding monospecies biofilms. Morphological analysis of MiBs revealed blastoconidia preferentially adhered to epithelial cells. Abundant Lactobacillus spp. were detected in two clinical samples; their MiBs showed a lower biomass and a higher fluconazole susceptibility. The proposed model proved to be a useful tool for the study of the complex microbial relationship in the vaginal environment, and may help to find new strategies for VVC control.

An autochthonous case of paracoccidioidomycosis in a new area of Ceara State, Northeastern Brazil.

The largest endemic areas of paracoccidioidomycosis (PCM) in Brazil comprise the humid agricultural regions of the Southeast, South, and, recently, the Midwest and North regions. The Ceara State, located in the Brazilian Northeast region, presents semi-arid climate in most of its territory, characterized by high temperatures, scarce vegetation and low humidity. The objective of the present study was to describe a new autochthonous case of paracoccidioidomycosis from a distinct area of Ceara and review the characteristics of PCM occurrence in Northeastern Brazil. The patient was a 65-year-old male farmer who denied traveling outside the Ceara State or living in other locations. He was born and lived in the rural area known as Camara, bordering the municipalities of Itapaje and Itapipoca. Camara is one of the highest areas (around 720 m of altitude) of the Uruburetama mountains that exhibits tropical forests and is located in Northern Ceara, distant 139 km from the capital, Fortaleza. The patient sought for care, complaining of an oral lesion that appeared over the past three years. The hard palate lesion biopsy revealed multinucleated cells with cytoplasmic inclusions, compatible with PCM. After culture, P. brasiliensis was identified by polymerase chain reaction. Serological testing for PCM was reagent. The patient was treated with itraconazole for approximately 17 months, persisting free of symptoms after 15 months of follow-up. Regarding this new autochthonous case in the Ceara State, PCM should be considered in the differential diagnosis of patients with suggestive clinical manifestations, proceeding from the mountainous areas of Ceara.

Exposure of Candida parapsilosis complex to agricultural azoles: An overview of the role of environmental determinants for the development of resistance.

This work investigated the phenotypic behavior of Candida parapsilosis species complex in response to exposure to agricultural azoles and fluconazole. Three fluconazole-susceptible strains of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were used. Initial minimum inhibitory concentrations (iMICs) for agricultural and clinical azoles were determined by broth microdilution. Then, the strains were exposed to tebuconazole, tetraconazole and fluconazole for 15 days, at concentrations that were two-folded daily, starting at one-eighth the iMIC (iMIC/8) up to 64 times iMIC (64xiMIC). After 15-day-exposure, antifungal susceptibility, biofilm formation, CDR, MDR and ERG expression were evaluated. The three cryptic species developed tolerance to the antifungals they were exposed and presented reduction (P < 0.05) in fluconazole susceptibility. In addition, C. parapsilosis sensu stricto and C. metapsilosis also presented reduced susceptibility to voriconazole, after fluconazole exposure. Azole exposure decreased (P < 0.05) biofilm production by C. parapsilosis sensu stricto and C. orthopsilosis and increased (P < 0.05) the expression of ERG11 in all tested strains. The results show that exposure to agricultural azoles and fluconazole induces changes in the phenotypic behavior and gene expression by the three cryptic species of C. parapsilosis complex, highlighting the importance of environmental determinants for the development of antifungal resistance.

Quantitative and structural analyses of the in vitro and ex vivo biofilm-forming ability of dermatophytes.

PURPOSE: The aim of this study was to evaluate the in vitro and ex vivo biofilm-forming ability of dermatophytes on a nail fragment. METHODOLOGY: Initially, four isolates of Trichophyton rubrum, six of Trichophyton tonsurans, three of Trichophyton mentagrophytes, ten of Microsporum canis and three of Microsporum gypseum were tested for production biomass by crystal violet assay. Then, one strain per species presenting the best biofilm production was chosen for further studies by optical microscopy (Congo red staining), confocal laser scanning (LIVE/DEAD staining) and scanning electron (secondary electron) microscopy. RESULTS: Biomass quantification by crystal violet assay, optical microscope images of Congo red staining, confocal microscope and scanning electron microscope images revealed that all species studied are able to form biofilms both in vitro and ex vivo, with variable density and architecture. M. gypseum, T. rubrum and T. tonsurans produced robust biofilms, with abundant matrix and biomass, while M. canis produced the weakest biofilms compared to other species. CONCLUSION: This study sheds light on biofilms of different dermatophyte species, which will contribute to a better understanding of the pathophysiology of dermatophytosis. Further studies of this type are necessary to investigate the processes involved in the formation and composition of dermatophyte biofilms.

Azole resistance in Candida albicans from animals: Highlights on efflux pump activity and gene overexpression.

This study investigated potential mechanisms of azole resistance among Candida albicans from animals, including efflux pump activity, ergosterol content and gene expression. For this purpose, 30 azole-resistant C. albicans strains from animals were tested for their antifungal susceptibility, according to document M27-A3, efflux pump activity by rhodamine 6G test, ergosterol content and expression of the genes CDR1, CDR2, MDR1, ERG11 by RT-qPCR. These strains were resistant to at least one azole derivative. Resistance to fluconazole and itraconazole was detected in 23 and 26 strains respectively. Rhodamine 6G tests showed increased activity of efflux pumps in the resistant strains, showing a possible resistance mechanism. There was no difference in ergosterol content between resistant and susceptible strains, even after fluconazole exposure. From 30 strains, 22 (73.3%) resistant animal strains overexpressed one or more genes. From this group, 40.9% (9/22) overexpressed CDR1, 18.2% (4/22) overexpressed CDR2, 59.1% (13/22) overexpressed MDR1 and 54.5% (12/22) overexpressed ERG11. Concerning gene expression, a positive correlation was observed only between CDR1 and CDR2. Thus, azole resistance in C. albicans strains from animals is a multifactorial process that involves increased efflux pump activity and the overexpression of different genes.

RYP1 gene as a target for molecular diagnosis of histoplasmosis.

This study analyzed the RYP1 gene as a target for the molecular diagnosis of histoplasmosis. This assay detected fungal DNA in 13/13 blood samples from HIV/AIDS-patients with histoplasmosis. Therefore, the detection of RYP1 gene in whole blood sample is a quick and sensitive test to diagnose histoplasmosis.

Cross-resistance to fluconazole induced by exposure to the agricultural azole tetraconazole: an environmental resistance school?

This study aimed to investigate the influence of tetraconazole and malathion, both used in agricultural activities, on resistance to fluconazole, itraconazole and voriconazole in Candida parapsilosis ATCC 22019. The susceptibility to tetraconazole, malathion, fluconazole, itraconazole and voriconazole, through broth microdilution. Then, 12 independent replicates, were separated and exposed to four treatment groups, each one containing three replicates: G1: tetraconazole; G2: malathion; G3: fluconazole (positive control); G4: negative control. Replicates from G1, G2 and G3, were exposed to weekly increasing concentrations of tetraconazole, malathion and fluconazole, respectively, ranging from MIC/2 to 32 × MIC, throughout 7 weeks. The exposure to tetraconazole, but not malathion, decreased susceptibility to clinical azoles, especially fluconazole. The tetraconazole-induced fluconazole resistance is partially mediated by the increased activity of ATP-dependent efflux pumps, considering the increase in antifungal susceptibility after the addition of the efflux pump inhibitor, promethazine, and the increase in rhodamine 6G efflux and CDR gene expression in the G1 replicates. Moreover, MDR expression was only detected in G1 and G3 replicates, suggesting that MDR pumps are also involved in tetraconazole-induced fluconazole resistance. It is noteworthy that tetraconazole and fluconazole-treated replicates behaved similarly, therefore, resistance to azoles of clinical use may be a consequence of using azoles in farming activities.

Easy storage strategies for Sporothrix spp. strains.

The present study evaluated the maintenance of Sporothrix spp. (6 Sporothrix brasiliensis; 6 S. schenckii; 5 S. mexicana, and 3 S. globosa) in saline at 4°C, and in 10% glycerol plus either 10% lactose or 10% sucrose, at -20°C and -80°C. Viability was assessed after 3, 6, and 9 months of storage, through the recovery of strains on potato dextrose agar and analysis of macro- and micromorphological features. Conidium quantification was performed before and after storage, at 3, 6 and 9 months. 100% viability was observed, regardless of storage conditions or time period. Storage at 4°C and at -20°C did not alter the number of conidia, but lower conidium counts were observed at -80°C. This study shows that the combination of glycerol with lactose or sucrose is effective to maintain Sporothrix spp. at freezing temperatures.

Antifungal susceptibility and virulence attributes of animal-derived isolates of Candida parapsilosis complex.

This study aimed to identify strains of the Candida parapsilosis complex isolated from animals, as well as to assess their in vitro antifungal susceptibility profile and in vitro production of virulence attributes. We used 28 isolates of C. parapsilosis sensu lato recovered from clinically healthy animals. The strains were characterized phenotypically, followed by molecular identification of the species through PCR-restriction enzyme analysis. The susceptibility of the strains to amphotericin B, itraconazole, voriconazole, fluconazole and caspofungin was assessed through broth microdilution. Additionally, the ability of the strains to produce biofilm, phospholipases and proteases was analysed. Molecular analysis showed 13 C. parapsilosis sensu stricto, 10 Candida orthopsilosis and five Candida metapsilosis strains. In vitro resistance to fluconazole was observed in three strains of C. parapsilosis sensu stricto and two C. metapsilosis. All tested strains were able to form biofilms and 23/28 isolates presented protease production, whilst none was able to produce phospholipases. Our study showed that C. parapsilosis sensu stricto and C. orthopsilosis are the most common species of the C. parapsilosis species complex and that these cryptic species present no significant phenotypical differences.

Biochemical characterization of an in-house Coccidioides antigen: perspectives for the immunodiagnosis of coccidioidomycosis.

The objective of this study was to evaluate the reactivity of an in-house antigen, extracted from a strain of C. posadasii isolated in northeastern Brazil, by radial immunodiffusion and Western blotting, as well as to establish its biochemical characterization. The protein antigen was initially extracted with the use of solid ammonium sulfate and characterized by 1-D electrophoresis. Subsequently, it was tested by means of double radial immunodiffusion and Western blotting. A positive reaction was observed against the antigen by both immunodiagnostic techniques tested on sera from patients suffering from coccidioidomycosis. Besides this, two immunoreactive protein bands were observed and were revealed to be a ß-glucosidase and a glutamine synthetase after sequencing of the respective N-terminal regions. Our in-house Coccidioides antigen can be promising as a quick and low-cost diagnostic tool without the risk of direct manipulation of the microorganism.

Twelve years of coccidioidomycosis in Ceará State, Northeast Brazil: epidemiologic and diagnostic aspects.

Coccidioidomycosis is an endemic infection in the Americas caused by the dimorphic fungi Coccidioides immitis and Coccidioides posadasii. Although the disease occurs in Brazil in sporadic form, little information about these cases is available. In this study, we summarize the most important clinical, epidemiologic, and diagnostic features of coccidioidomycosis in Ceará State (Northeast Brazil) during the past 12 years. In this period, 19 cases of coccidioidomycosis were diagnosed. All the patients were young males and came from semiarid areas of the state. The majority of cases were associated to armadillo hunting, and pulmonary disease was the most common clinical presentation. In our laboratory, coccidioidomycosis was confirmed by culture, serology, and polymerase chain reaction tests, which together were very suitable for the diagnosis of this disease. Based on our local experience, we believe many cases of this disease are misdiagnosed or not diagnosed in our region. Therefore, some strategies for improvement of diagnosis should be encouraged by local authorities.

Epidemiologia e ecologia das dermatofitoses na cidade de Fortaleza: o Trichophyton tonsurans como importante patógeno emergente da Tinea capitis / Epidemiology and ecology or dematophytosis in Fortaleza city: Trichophyton tonsurans as an important emergent pathogen of Tinea capitis

As dermatofitoses säo os distúrbios infecciosos de pele mais comuns do mundo. Na presente pesquisa foram avaliados 2.297 pacientes com lesöes clínicas sugestivas de dermatofitoses das quais, 534 (23,2 por cento) mostraram-se positivas para dermatófitos. Destes, o T. rubrum foi a espécie mais prevalente (49,6 por cento; p