Promethazine inhibits efflux, enhances antifungal susceptibility and disrupts biofilm structure and functioning in Trichosporon.

Trichosporon spp. are emerging opportunistic fungi associated with invasive infections, especially in patients with haematological malignancies. The present study investigated the in vitro inhibition of efflux pumps by promethazine (PMZ) as a strategy to control T. asahii and T. inkin. Planktonic cells were evaluated for antifungal susceptibility to PMZ, as well as inhibition of efflux. The effect of PMZ was also studied in Trichosporon biofilms. PMZ inhibited T. asahii and T. inkin planktonic cells at concentrations ranging from 32 to 256 µg ml-1. Subinhibitory concentrations of PMZ inhibited efflux activity in Trichosporon. Biofilms were completely eradicated by PMZ. PMZ potentiated the action of antifungals, affected the morphology, changed the amount of carbohydrates and proteins and reduced the amount of persister cells inside biofilms. The results showed indirect evidences of the occurrence of efflux pumps in Trichosporon and opens a perspective for the use of this target in the control of trichosporonosis.

Enterococcus faecalis and Candida albicans dual-species biofilm: establishment of an in vitro protocol and characterization.

Enterococcus faecalis is the most important agent of persistent apical periodontitis, and recently, Candida albicans has also been implicated in periapical infections. This study aimed to optimize an in vitro E. faecalis and C. albicans dual-species biofilm protocol for endodontic research. Different physicochemical conditions for biofilm formation were tested. Susceptibility assays to antimicrobials, biochemical composition and an ultra-morphological structure analyses were performed. Reproducible dual-species biofilms were established in BHI medium at 35 °C, for 48 h and in a microaerophilic atmosphere. An increase in biomass and chitin content was detected after vancomycin treatment. Structural analysis revealed that the dual-species biofilm was formed by both microorganisms adhered to the substrate. The proposed protocol could be useful for the study of interkingdom relationships and help to find new strategies against periapical infections.

Heterologous extracellular DNA facilitates the development of Trichosporon asahii and T. inkin biofilms and enhances their tolerance to antifungals.

Trichosporon asahii and T. inkin are emergent agents of deep-seated and disseminated infections in immunocompromised patients. The present study aimed to investigate the role of extracellular DNA (eDNA) and the enzyme deoxyribonuclease (DNase) on the structure of T. asahii and T. inkin biofilms, as well as to examine their effect on the susceptibility to antifungals. Biofilms reached maturity at 48 h; eDNA concentration in the supernatant increased over time (6 < 24 h < 48h). Exogenous eDNA increased biomass of Trichosporon biofilms at all stages of development, enhanced their tolerance to antifungals and improved their structural complexity. DNase reduced biomass, biovolume and thickness of Trichosporon biofilms, thereby rendering them more susceptibility to voriconazole. The results suggest the relevance of eDNA in the structure and antifungal susceptibility of Trichosporon biofilms and highlight the potential of DNase as adjuvant in biofilm control.

Vancomycin enhances growth and virulence of Trichosporon spp. planktonic cells and biofilms.

Invasive fungal infections (IFIs) are important worldwide health problem, affecting the growing population of immunocompromised patients. Although the majority of IFIs are caused by Candida spp., other fungal species have been increasingly recognized as relevant opportunistic pathogens. Trichosporon spp. are members of skin and gut human microbiota. Since 1980's, invasive trichosporonosis has been considered a significant cause of fungemia in patients with hematological malignancies. As prolonged antibiotic therapy is an important risk factor for IFIs, the present study investigated if vancomycin enhances growth and virulence of Trichosporon. Vancomycin was tested against T. inkin (n = 6) and T. asahii (n = 6) clinical strains. Planktonic cells were evaluated for their metabolic activity and virulence against Caenorhabditis elegans. Biofilms were evaluated for metabolic activity, biomass production, amphotericin B tolerance, induction of persister cells, and ultrastructure. Vancomycin stimulated planktonic growth of Trichosporon spp., increased tolerance to AMB, and potentiates virulence against C. elegans. Vancomycin stimulated growth (metabolic activity and biomass) of Trichosporon spp. biofilms during all stages of development. The antibiotic increased the number of persister cells inside Trichosporon biofilms. These cells showed higher tolerance to AMB than persister cells from VAN-free biofilms. Microscopic analysis showed that VAN increased production of extracellular matrix and cells in T. inkin and T. asahii biofilms. These results suggest that antibiotic exposure may have a direct impact on the pathophysiology of opportunistic trichosporonosis in patients at risk. LAY ABSTRACT: This study showed that the vancomycin stimulated Trichosporon growth, induced morphological and physiological changes on their biofilms, and also enhanced their in vivo virulence. Although speculative, the stimulatory effect of vancomycin on fungal cells should be considered in a clinical scenario.

Sodium butyrate inhibits planktonic cells and biofilms of Trichosporon spp.

Trichosporon spp. have been increasingly recognized as an important pathogen of invasive and disseminated infections in immunocompromised patients. These species are prone to form biofilms in medical devices such as catheters and prosthesis, which are associated with antifungal resistance and therapeutic failure. Therefore, new antifungals with a broader anti-biofilm activity need to be discovered. In the present study we evaluate the inhibitory potential of sodium butyrate (NaBut) - a histone deacetylase inhibitor that can alter chromatin conformation - against planktonic and sessile cells of T. asahii and T. inkin. Minimum inhibitory concentration (MIC) of NaBut against planktonic cells was evaluated by microdilution and morphological changes were analyzed by optical microscopy on malt agar supplemented with NaBut. Biofilms were evaluated during adhesion, development and after maturation for metabolic activity and biomass, as well as regarding ultrastructure by scanning electron microscopy and confocal laser scanning microscopy. NaBut inhibited the growth of planktonic cells by 50% at 60 mM or 120 mM (p < 0.05) and also reduced filamentation of Trichosporon spp. NaBut reduced adhesion of Trichosporon cells by 45% (10xMIC) on average (p < 0.05). During biofilm development, NatBut (10xMIC) reduced metabolic activity and biomass up to 63% and 81%, respectively (p < 0.05). Mature biofilms were affected by NaBut (10xMIC), showing reduction of metabolic activity and biomass of approximately 48% and 77%, respectively (p < 0.05). Ultrastructure analysis showed that NaBut (MIC and 10xMIC) was able to disassemble mature biofilms. The present study describes the antifungal and anti-biofilm potential of NaBut against these opportunist emerging fungi.

Farnesol inhibits planktonic cells and antifungal-tolerant biofilms of Trichosporon asahii and Trichosporon inkin.

Trichosporon species have been considered important agents of opportunistic systemic infections, mainly among immunocompromised patients. Infections by Trichosporon spp. are generally associated with biofilm formation in invasive medical devices. These communities are resistant to therapeutic antifungals, and therefore the search for anti-biofilm molecules is necessary. This study evaluated the inhibitory effect of farnesol against planktonic and sessile cells of clinical Trichosporon asahii (n = 3) andTrichosporon inkin (n = 7) strains. Biofilms were evaluated during adhesion, development stages and after maturation for metabolic activity, biomass and protease activity, as well as regarding morphology and ultrastructure by optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy. Farnesol inhibited Trichosporon planktonic growth by 80% at concentrations ranging from 600 to 1200 µM for T. asahii and from 75 to 600 µM for T. inkin. Farnesol was able to reduce cell adhesion by 80% at 300 µM for T. asahii and T. inkin at 600 µM, while biofilm development of both species was inhibited by 80% at concentration of 150 µM, altering their structure. After biofilm maturation, farnesol decreased T. asahii biofilm formation by 50% at 600 µM concentration and T. inkin formation at 300 µM. Farnesol inhibited gradual filamentation in a concentration range between 600 and 1200 µM. Farnesol caused reduction of filament structures of Trichosporon spp. at every stage of biofilm development analyzed. These data show the potential of farnesol as an anti-biofilm molecule.

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.

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.

Perfil epidemiológico de Staphylococcus spp. isolados de hemoculturas de pacientes internados em um hospital de atenção terciária da rede pública do Ceará / Epidemiological profile of Staphylococcus spp. isolated from hemocultures of interned patients in a tertiary attention hospital of the Ceará public network

Staphylococcus spp. vem ganhando destaque em infecções na corrente sanguínea (ICS), apresentando alta prevalência, multirresistência e considerável poder de letalidade. O presente estudo teve como objetivo analisar a prevalência e traçar o perfil de sensibilidade das espécies de Staphylococcus spp. isoladas de amostras de hemoculturas positivas obtidas de um hospital de atenção terciária da rede pública do Ceará, no período de janeiro de 2015 a dezembro de 2018. Dos 3292 exames de hemocultura realizados, apenas 15,88% tiveram resultado positivo, dos quais 24,85% eram cocos Gram positivos. S. aureus representou 1,53% das hemoculturas positivas com 50% das cepas resistentes à oxacilina. Os isolados de Staphylococcus sp. coagulase negativo obtiveram prevalência de 10,89%, representados por: S. epidermidis (n=23), S. haemolyticus (n=17), S. hominis (n=13), S. saprophyticus (n=2) e S. warneri (n=2). Verificou-se multirresistência em diversos isolados analisados, apresentando variações intra e interespécies. Portanto, nossos achados melhoram o entendimento da epidemiologia das ICS causadas por Staphylococcus spp., na instituição de estudo, bem como seu perfil de sensibilidade. A identificação precoce do agente infeccioso auxilia na escolha adequada do tratamento, aumentando as chances de cura e reduzindo o tempo de internação do paciente. (AU)

Staphylococcus spp. has been highlighted among bloodstream infections (BI), presenting high prevalence, multidrug resistance and considerable lethality. The present study aimed to analyze the prevalence and the susceptibility profile of Staphylococcus spp. isolated from positive blood cultures from a tertiary care public hospital of Ceará, from January 2015 to December 2018. Of the 3292 blood cultures performed during this period, only 15.88% were positive, of which 24.85% were Gram positive cocci. S. aureus represented 1.53% of positive blood cultures, of which 50% were oxacilin resistant. Isolates of coagulase-negative Staphylococcus spp. showed a prevalence of 10.89%, represented by: S. epidermidis (n = 23), S. haemolyticus (n = 17), S. hominis (n = 13), S. saprophyticus (n = 2) and S. warneri (n = 2). Multiresistance occurrence was verified in several of the analyzed isolates, presenting intra and inters species variations. Therefore, our findings improve the understanding of the epidemiology of BI caused by Staphylococcus spp. in the studied institution, as well as its susceptibility profile. Early identification of the infectious agent might aid in the appropriate choice of treatment, increasing the chance of cure and reducing the patient length stay in hospital. (AU)