TAxonomy of Candida Parapsilosis Complex Isolated from Neonates and the Role of HSP90 Inhibitors to Enhanced the Antifungal Activity of Micafungin.

Species from Candida parapsilosis complex are frequently found in neonatal candidemia. The antifungal agents to treat this infection are limited and the occurrence of low in vitro susceptibility to echinocandins such as micafungin has been observed. In this context, the chaperone Hsp90 could be a target to reduce resistance. Thus, the objective of this research was to identify isolates from the C. parapsilosis complex and verify the action of Hsp90 inhibitors associated with micafungin. The fungal identification was based on genetic sequencing and mass spectrometry. Minimal Inhibitory Concentrations were determined by broth microdilution method according to CLSI. The evaluation of the interaction between micafungin with Hsp90 inhibitors was realized using the checkerboard methodology. According to the polyphasic taxonomy C. parapsilosis sensu stricto was the most frequently identified, followed by C. orthopsilosis and C. metapsilosis, and one isolate of Lodderomyces elongisporus was identified by genetic sequencing. The Hsp90 inhibitor geladanamycin associated with micafungin showed a synergic effect in 31.25% of the isolates, a better result was observed with radicicol that shows synergic effect in 56.25% tested yeasts. The results obtained demonstrate that blocking Hsp90 could be effective to reduce antifungal resistance to echinocandins.

Levels of soluble TNF receptors (sTNFR1 and sTNFR2) increase with clinical worsening of patients and are related to COVID-19 mortality.

The present study aimed to inspect the serum levels of the soluble receptors, sTNFR1 and sTNFR2, in patients with COVID-19. The large production of inflammatory cytokines is an essential process in the pathogenesis of COVID-19. TNF is a multifaceted proinflammatory cytokine which has soluble and membrane receptors. Thus, knowing the role of these receptors will help better understand this disease's immunopathogenesis. We included 131 patients confirmed for SARS-CoV-2, separated into three groups: ward patients without O2 support, group A (n = 14); ward patients with O2 support, group B (n = 85), and patients in an intensive care unit (ICU), group C (n = 32), making up the receptors dosed by flow cytometry. The results showed that sTNFR1 and sTNFR2 are associated with disease severity, being higher in group C when compared to group A. As for the levels of receptors and their relationship with the degree of lung involvement, we found higher values of sTNFR1 in patients in group 1 (pulmonary involvement < 25%), suggesting that inflammatory processes related to TNF are not necessarily associated with the primary site of infection. When we analysed the patients who passed away compared to those who recovered, both receptors significantly increased the mortality numbers. These findings suggest a relevant influence of soluble receptors in the inflammatory processes involved in the pathogenesis of COVID-19. Wherefore, we suggest using these receptors as biomarkers of severity and mortality of the disease.

Efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates.

The aim of this study was to evaluate the efficacy and non-toxicity of ciclopirox olamine-loaded liposomes against Cryptococcus neoformans clinical isolates. Initially, 24-1 fractional experimental design was carried out to obtain an optimized formulation of liposomes containing CPO (CPO-LipoC), which were then used to prepare stealth liposomes (CPO-LipoS). Liposomal formulations were characterized by their mean size diameter, polydispersity index (PDI), and drug encapsulation efficiency (EE%). Immunosuppressed mice were exposed to CPO-LipoS at 0.5 mg/kg/day for 14 days to verify possible histopathological alterations in the liver and kidneys. Immunosuppressed mice infected with C. neoformans were treated with CPO-LipoS at 0.5 mg/kg/day for 14 days to quantify the fungal burden in spleen, liver, lungs, and brain. CPO-LipoS presented a mean size diameter, PDI, and EE% of 101.4 ± 0.7 nm, 0.307, and 96.4 ± 0.9%, respectively. CPO-LipoS was non-toxic for the liver and kidneys of immunosuppressed mice. At the survival curve, all infected animals submitted to treatment with CPO-LipoS survived until the end of the experiment. Treatment with CPO-LipoS reduced C. neoformans cells in the spleen (59.3 ± 3.4%), liver (75.0 ± 3.6%), lungs (75.7 ± 6.7%), and brain (54.2 ± 3.2%). CPO-LipoS exhibit antifungal activity against C. neoformans, and the encapsulation of CPO into stealth liposomes allows its use as a systemic drug for treating cryptococcosis.

Clinical and Epidemiological Characteristics of Neurocryptococcosis Associated with HIV in Northeastern Brazil.

Cryptococcal meningitis is a serious infection of the central nervous system that is predominant in developing countries, caused by fungi of the genus Cryptococcus, and which affects immunosuppressed patients, especially those with HIV. Here, we aim to diagnose and characterize the clinical-epidemiological profile of cryptococcosis in patients admitted to two tertiary public hospitals in northeastern Brazil. The study is divided into three moments: (1) the isolation of fungus and diagnosis from biological samples collected between 2017 and 2019, (2) a description of the clinical and epidemiological characteristics of the patients, and (3) the experimental tests related to an in vitro susceptibility antifungal profile. The species were identified by MALDI-TOF/MS. Among the 100 patients evaluated, 24 (24.5%) were diagnosed with cryptococcosis based on positive culture. Clinical-epidemiological analysis showed a slightly higher prevalence in men between 30 and 39 years. When comparing the date of HIV diagnosis and the development of cryptococcosis, it was observed that 50% received the diagnosis of infection by cryptococcosis after or equal to a period of 12 months from being diagnosed with HIV; the other 50% received it within the first 30 days of the HIV diagnosis. Neurocryptococcosis was the most prevalent clinical form, and, at the time of hospital admission, the most common clinical signs were high fever (75%), intense headache (62.50%), and neck stiffness (33.33%). The cerebrospinal fluid showed 100% sensitivity and positivity for direct examination by India ink, and fungal culture. The mortality rate in this study was 46% (11/24), a lower rate than in the other literature. An antifungigram showed that 20 (83.33%) isolates were susceptible to amphotericin B and 15 (62.5%) to fluconazole. Mass spectrometry identified 100% of the isolates as Cryptococcus neoformans. In Brazil, this infection is not mandatory notifiable. Therefore, although there is little information on the subject, it is obsolete and does not express the reality of the facts, mainly in the northeast region, where this information is insufficient. The data obtained in this research contribute to the epidemiological knowledge of this mycosis in Brazil and will serve as a basis for future globally comparative epidemiological studies.

Colonized patients by Candida auris: Third and largest outbreak in Brazil and impact of biofilm formation.

Objective: To describe the clinical-epidemiological features of patients colonized by Candida auris in the largest outbreak in Brazil and to show the biofilm formation capacity of yeast strains. Methods: Clinical yeasts suspected of C. auris isolated from urine and surveillance samples were seeded on chromogenic media at 30°C and Sabouraud agar at 42°C. matrix-assisted laser desorption/ionization-time of flight mass spectometry was used for reliable identification. After proteomic confirmation, the genomic approach and culture on Chromagar Candida Plus media were carried out. Biofilm formation was investigated based on metabolic activity, and the clinical-epidemiological profile of patients was described. Results: A total of 11 C. auris clinical yeasts from nine patients were identified between the end of December 2021 and March 2022. Two clinical yeasts were isolates from urine and nine clinical yeasts were isolates from axillary and inguinal surveillance swabs. No case is related to previous Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, all the yeasts showed a high ability of biofilm formation. Conclusion: C. auris requires great vigilance as its high capacity to colonize and form biofilms contributes to its dissemination. The rapid and precise identification of this species is essential for the management, control, and prevention of infections.

Virulence factors of Gram-negative bacteria from free-ranging Amazon river dolphins (Inia geoffrensis).

Freshwater cetaceans play a significant role as sentinel animals, providing important data on animal species and aquatic ecosystem health. They also may serve as potential reservoirs of emerging pathogens and host virulence genes in their microbiota. In this study, we evaluated virulence factors produced by Gram-negative bacteria recovered from individuals belonging to two populations of free-ranging Amazon river dolphins (Inia geoffrensis). A total of 132 isolates recovered from the oral cavity, blowhole, genital opening and rectum of 21 river dolphins, 13 from Negro River and 8 from Tapajós River, Brazil, were evaluated for the production of virulence factors, such as biofilms and exoproducts (proteases, hemolysins and siderophores), in planktonic and biofilm forms. In planktonic form, 81.1% (107/132) of the tested bacteria of free-ranging Amazon river dolphins were able to produce virulence factors, with 44/132 (33.4%), 65/132 (49,2%) and 54/132 (40,9%) positive for protease, hemolysin and siderophore production, respectively. Overall, 57/132 (43.2%) of the isolates produced biofilms and, under this form of growth, 66/132 (50%), 88/132 (66.7%) and 80/132 (60.6%) of the isolates were positive for protease, hemolysin and siderophore production. In general, the isolates showed a higher release of exoproducts in biofilm than in planktonic form (P < 0.001). The present findings show that Amazon river dolphins harbor potentially pathogenic bacteria in their microbiota, highlighting the importance of monitoring the micro-organisms from wild animals, as they may emerge as pathogens for humans and other animals.

Malassezia spp. and Candida spp. from patients with psoriasis exhibit reduced susceptibility to antifungals.

INTRODUCTION: Psoriasis is a chronic inflammatory disease that affects over 125 million people worldwide. Many studies have shown the importance of the microbiome for psoriasis exacerbation. AIM: Explore the fungal load and species composition of cultivable yeasts on the skin of psoriatic patients (PP) and healthy volunteers living in a tropical area and evaluate the susceptibility to antifungals. METHODOLOGY: A cross-sectional study with 61 participants (35 patients and 26 healthy controls) was performed during August 2018 and May 2019. Clinical data were collected from patient interviewing and/or medical records review. Samples were collected by swabbing in up to five anatomic sites. Suggestive yeast colonies were counted and further identified by phenotypical tests, PCR-REA, and/or MALDI-TOF. Susceptibility of Malassezia spp. and Candida spp. to azoles, terbinafine, and amphotericin B was evaluated by broth microdilution. RESULTS: Nearly 50% of the patients had moderate to severe psoriasis, and plaque-type psoriasis was the most common clinical form. Yeast colonies count was significantly more abundant among PP than healthy controls. Malassezia and Candida were the most abundant genus detected in all participants. Higher MIC values for ketoconazole and terbinafine were observed in Malassezia strains obtained from PP. Approximately 42% of Candida isolates from PP showed resistance to itraconazole in contrast to 12.5% of isolates from healthy controls. MIC values for fluconazole and amphotericin B were significantly different among Candida isolates from PP and healthy individuals. CONCLUSION: This study showed that Malassezia and Candida strains from PP presented higher MIC values to widespread antifungal drugs than healthy individuals.

Fungal esophagitis associated with tuberculous pericarditis in an human immunodeficiency virus-positive patient: a case report.

BACKGROUND: Opportunistic infections are frequent in people living with the human immunodeficiency virus who either do not have access to antiretroviral therapy (ART) or use it irregularly. Tuberculosis is the most frequent infectious disease in PLHIV and can predispose patients to severe fungal infections with dire consequences. CASE PRESENTATION: We describe the case of a 35-year-old Brazilian man living with human immunodeficiency virus (HIV) for 10 years. He reported no adherence to ART and a history of histoplasmosis with hospitalization for 1 month in a public hospital in Natal, Brazil. The diagnosis was disseminated Mycobacterium tuberculosis infection. He was transferred to the health service in Recife, Brazil, with a worsening condition characterized by daily fevers, dyspnea, pain in the upper and lower limbs, cough, dysphagia, and painful oral lesions suggestive of candidiasis. Lymphocytopenia and high viral loads were found. After screening for infections, the patient was diagnosed with tuberculous pericarditis and esophageal candidiasis caused by Candida tropicalis. The isolated yeasts were identified using the VITEK 2 automated system and matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry. Antifungal microdilution broth tests showed sensitivity to fluconazole, voriconazole, anidulafungin, caspofungin, micafungin, and amphotericin B, with resistance to fluconazole and voriconazole. The patient was treated with COXCIP-4 and amphotericin deoxycholate. At 12 days after admission, the patient developed sepsis of a pulmonary focus with worsening of his respiratory status. Combined therapy with meropenem, vancomycin, and itraconazole was started, with fever recurrence, and he changed to ART and tuberculostatic therapy. The patient remained clinically stable and was discharged with clinical improvement after 30 days of hospitalization. CONCLUSION: Fungal infections should be considered in patients with acquired immunodeficiency syndrome as they contribute to worsening health status. When mycoses are diagnosed early and treated with the appropriate drugs, favorable therapeutic outcomes can be achieved.

One Health Implications of Antimicrobial Resistance in Bacteria from Amazon River Dolphins.

Studies on the microbiota of freshwater cetaceans are scarce and may provide important data on animal and environmental health. This study aimed to evaluate the antimicrobial susceptibility of Gram-negative bacteria recovered from two populations of free-ranging Amazon river dolphins (Inia geoffrensis). Twenty-one animals were captured and released, 13 from Negro River and 8 from Tapajós River, Brazil. Swab samples were obtained from the oral cavity, blowhole, genital opening and rectum and were cultured on MacConkey agar. Isolates were biochemically identified, and antimicrobial susceptibility was assessed by disk diffusion method. Overall, 132 isolates were recovered, of which 71 were recovered from animals from Negro River and 61 from Tapajós River. The most commonly recovered bacterial species were Enterobacter cloacae, Morganella morganii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed (P < 0.001). The results indicate that free-ranging Amazon river dolphins host resistant bacteria, contributing for their maintenance in the environment. This study highlights the importance of the One Health approach to monitor the emergence of antimicrobial resistance. Summary Gram-negative bacteria recovered from 21 free-ranging Amazon river dolphins (Inia geoffrensis) from the Negro River and the Tapajós River populations were evaluated for their antimicrobial susceptibility. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed. Thus, free-ranging Amazon river dolphins, never treated with antimicrobials, host resistant bacteria, contributing for their maintenance in the environment and highlighting the importance of the One Health approach to monitor the emergence of antimicrobial resistance.

Trichosporon asahii and Trichosporon inkin Biofilms Produce Antifungal-Tolerant Persister Cells.

Persister cells are metabolically inactive dormant cells that lie within microbial biofilms. They are phenotypic variants highly tolerant to antimicrobials and, therefore, associated with recalcitrant infections. In the present study, we investigated if Trichosporon asahii and T. inkin are able to produce persister cells. Trichosporon spp. are ubiquitous fungi, commonly found as commensals of the human skin and gut microbiota, and have been increasingly reported as agents of fungemia in immunocompromised patients. Biofilms derived from clinical strains of T asahii (n=5) and T. inkin (n=7) were formed in flat-bottomed microtiter plates and incubated at 35°C for 48 h, treated with 100 µg/ml amphotericin B (AMB) and incubated at 35°C for additional 24 h. Biofilms were scraped from the wells and persister cells were assayed for susceptibility to AMB. Additionally, we investigated if these persister cells were able to generate new biofilms and studied their ultrastructure and AMB susceptibility. Persister cells were detected in both T asahii and T. inkin biofilms and showed tolerance to high doses of AMB (up to 256 times higher than the minimum inhibitory concentration). Persister cells were able to generate biofilms, however they presented reduced biomass and metabolic activity, and reduced tolerance to AMB, in comparison to biofilm growth control. The present study describes the occurrence of persister cells in Trichosporon spp. and suggests their role in the reduced AMB susceptibility of T. asahii and T. inkin biofilms.

Evaluation of cytotoxic, immunomodulatory effects, antimicrobial activities and phytochemical constituents from various extracts of Passiflora edulis F. flavicarpa (Passifloraceae).

The leaves of P. edulis were subjected to physicochemical analysis, such as ion content, extractives, and structural molecules. The hexanic, ethanolic and ethyl acetate extracts were submitted to phytochemical analyzes by GC-MS, HPLC-MS, and spectrophotometry. In addition, antioxidant (DPPH, ABTS and TAA methods) potential, antimicrobial (MIC method) action, cytotoxicity and immunostimulant activity (flow cytometry analysis) were performed. The extracts showed a moderate antioxidant capacity and revealed the presence of several metabolites, mainly phenols, such as caffeic acid, p-coumaric acid and luteolin. The ethyl acetate and ethanolic extracts showed antifungal activity. In addition, the extracts did not affect splenocytes viability at 12.5 µg/mL and promoted the production of IL-6, IL-10, IL-17 and TNF-α cytokines. P. edulis extracts showed antifungal and antioxidant activity and were able to induce immunostimulatory action in splenocyte cultures in vitro.

Lignin isolated from Caesalpinia pulcherrima leaves has antioxidant, antifungal and immunostimulatory activities.

Caesalpinia pulcherrima is a shrub with worldwide distribution used as an ornamental plant. In this study, we extracted a lignin from the C. pulcherrima leaves and investigated its biological functions. The lignin was characterized by FT-IR, UV-Vis, GPC, TGA and nuclear magnetic resonance (1H and 13C). The antioxidant activity was evaluated using phosphomolybdenum complexation methods (TAA), sequestration of DPPH and ABTS radicals, reducing power, formation of nitrite radical and iron chelating activity (Fe2 +). Antifungal activity was made using Candida spp., Aspergillus spp. and Cryptococcus neoformans strains. Cytotoxicity, oxidative stress, and cytokine production were performed using mouse splenocytes. The lignin showed maximal UV-Vis at ~280 nm, 22.27 L/g·cm of absorptivity and, 2,503 kDa of molecular weight. Phenolic compounds (41.33 ± 0.65 mg GAE/g) and indications of a guaiacyl-syringyl-hydroxyphenyl (GSH)-type composition were found. Antioxidant activities of lignin to TAA (40±1.2%) and to DPPH (16.9±0.2%) was high and showed antifungal potential, especially against Candida spp. (IC50 = 31.3 µg/mL) and C. neoformans (15.6 µg/mL). In mouse splenocytes, the lignin was not cytotoxic and stimulated the cell proliferation and cytokine release. These results indicate that C. pulcherrima lignin has the potential to be used as antifungal and immunostimulant compound.

Antimicrobial Resistance Profile and Biofilm Production of Microorganisms Isolated from Oropharynx of Rupornis magnirostris (Gmelin, 1788) and Caracara plancus (Miller, 1777).

The aim of this preliminary study was to identify microorganisms with antimicrobial resistance profile and biofilm producers in oropharynx of Rupornis magnirostris and Caracara plancus. Six R. magnirostris and six C. plancus maintained in Triage Center for Wild Animals (CETAS) facilities were studied. Coagulase-positive staphylococci (CoPS), enterobacteria, and yeasts were identified by the biochemical analysis or MALDI-TOF mass spectrometry. The resistance profile of the microorganisms was analyzed according to CLSI. The biofilm production was evaluated by Congo red and violet crystal staining methods. Among the 12 birds, 10 presented strains of CoPS and/or enterobacteria with resistance profile, such as methicillin-resistant CoPS (MR-CoPS), vancomycin-resistant CoPS (VR-CoPS), extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL), and Klebsiella pneumoniae carbapenemase- (KPC-) producing bacteria. Regards the fungal analysis, Candida spp., Cryptococcus spp., Rhodotorula mucilaginosa, R. glutinis, and Trichosporon coremiiforme were identified. All the Trichosporon coremiiforme strains were resistant to amphotericin B, as well as all the Rhodotorula mucilaginosa exhibited resistance to fluconazole. Related to the biofilm production, among the 8 CoPS, 27 enterobacteria, and 10 yeasts isolates, 3, 16, and 7 strains were biofilm producers, respectively. Thus, the presence of these microorganisms in birds of prey is worrisome, highlighting its possible influence in the spread of infections in urban centers.

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.

Incorporation of 2-amino-thiophene derivative in nanoparticles: enhancement of antifungal activity.

The objective of this study was to evaluate the effects of nanoparticles (nanospheres and nanocapsules) of the promising antifungal 2-amino-thiophene (6CN10) and 6CN10 complexed with 2-hydroxypropyl-ß-cyclodextrin (6CN10:HP-ß-CD) in vitro and compared with free drug against Candida and Cryptococcus, using a microdilution method to measure susceptibility. The Candida and Cryptococcus clinical strains were identified using phenotypic methods and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). To measure in vitro antifungal susceptibility, we used microdilution trials. Serial drug or nanoparticle dilutions were prepared according to the CLSI M27-A3 guidelines. Anti-biofilm activity was verified for Cryptococcus neoformans. All Candida isolates were sensitive to the free drug (MIC = 41.66-333.33 µg/mL) and were able to grow even at the higher concentration tested for all 6CN10 nanoparticles. However, the Cryptococcus neoformans strains presented MIC values of 0.32-83.33 µg/mL for 6CN10 nanoparticles, and MIC values of 0.1-0.2 µg/mL for 6CN10:HP-ß-CD nanoparticles, i.e., 3333 times more active than the free drug (MIC values 166.66-333.33 µg/mL), and presenting activity greater than that of the reference drug amphotericin B (MIC = 0.5-0.125 µg/mL). 6CN10:HP-ß-CD nanosphere also showed high anti-biofilm potential. The in vitro study showed that the nanoparticles allowed better drug efficiency against Cryptococcus than did the free drug. These results suggest that 6CN10-loaded nanoparticles may become a future alternative for cryptococcosis and candidiasis therapy. In vivo experiments are essential prior to clinical use.