ß-Estradiol and progesterone enhance biofilm development and persister cell formation in monospecies and microcosms biofilms derived from vulvovaginal candidiasis.

The present study aimed to: (1) evaluate the influence of the steroid hormones (SH) on biofilm development; (2) investigate the formation of persister cells (PC) in biofilms; and (3) investigate the influence of SH on PC formation. Biofilms were derived from vulvovaginal candidiasis (VVC) samples and evaluated by three models: microcosm biofilms grown in Vaginal Fluid Simulator Medium (MiB-VFSM); monospecies biofilms grown in VFSM (MoB-VFSM) and RPMI media (MoB-RPMI). SH altered cell counting and biomass of biofilms grown in VSFM; MoB-RPMI were negatively affected by SH. SH stimulated the formation of PC in MiB-VFSM but not MoB-VFSM; MoB-RPMI showed a lower number of PC in the presence of SH. The results showed that SH altered the dynamics of biofilm formation and development, depending on the study model. The data suggest the influence of hormones on the physiology of Candida biofilms and reinforce the importance of PC in the pathogenesis of VVC.

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.

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.

Yeasts from Scarlet ibises (Eudocimus ruber): A focus on monitoring the antifungal susceptibility of Candida famata and closely related species.

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garças Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species.

Aeromonas and Plesiomonas species from scarlet ibis (Eudocimus ruber) and their environment: monitoring antimicrobial susceptibility and virulence.

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.

Molecular methods for the diagnosis and characterization of Cryptococcus: a review.

Cryptococcosis is a fungal infection caused by yeasts of the genus Cryptococcus, with Cryptococcus neoformans and Cryptococcus gattii as the primary pathogenic species. This disease is a threat to immunocompromised patients, especially those who have AIDS. However, the disease has also been described in healthy individuals. The tests used to identify these microorganisms have limitations that make final diagnosis difficult. However, currently there are specific gene sequences that can be used to detect C. neoformans and C. gattii from clinical specimens and cultures. These sequences can be used for identification, typing, and the study of population genetics. Among the main identification techniques are hybridization, which was the pioneer in molecular identification and development of specific probes for pathogen detection; PCR and other PCR-based methods, particularly nested PCR and multiplex PCR; and sequencing of specific genomic regions that are amplified through PCR, which is especially useful for diagnosis of cryptococcosis caused by unconventional Cryptococcus sp. Concerning microorganism typing, the following techniques have shown the best ability to differentiate between fungal serotypes and molecular types: PCR fingerprinting, PCR-RFLP, AFLP, and MLST. Thus, the accumulation of data generated by molecular methods can have a positive impact on monitoring resistant strains and treating diseases.

Antifungal activity of different molecular weight chitosans against planktonic cells and biofilm of Sporothrix brasiliensis.

Sporotrichosis, caused by Sporothrix schenckii complex species, is the most prevalent subcutaneous mycosis in many areas of Latin America. Chitosan has been used as an antifungal agent; however the effects of the molecular weight (MW) of chitosan (i.e. high (HMW), medium (MMW) and low (LMW) molecular weight chitosan) on S. brasiliensis has not been well described, particularly on biofilms. Effects on the planktonic form activity of S. brasiliensis were quantified by broth microdilution, while anti-biofilm activity was quantified by measuring metabolic activity via XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide and biomass formation (crystal violet). The molecular weight of chitosan modulated its effect on the planktonic form of S. brasiliensis, presenting lower MIC values for LMW chitosan. With regards both the adhesive and mature phases of biofilm, the LMW chitosan reduced biomass and metabolic activity most effectively. This study confirms the effects of the molecular weight and deacetylation degree of chitosan on its antifungal properties for potentially pathogenic fungi.

Effect of the molecular weight of chitosan on its antifungal activity against Candida spp. in planktonic cells and biofilm.

Difficulties in the treatment of Candida spp. invasive infections are usually related to the formation of biofilms. The aim of this study was to determine the effects of molecular weight (MW) of chitosan (using high (HMW), medium (MMW) and low (LMW) molecular weight chitosan) on Candida albicans, Candida tropicalis and Candida parapsilosis sensu stricto. The deacetylation degree (DD) and molecular weight M were measured by potentiometric titration and viscosimetry, respectively. The planktonic shape activity was quantified by broth microdilution, and the activity against biofilm was quantified by metabolic activity through XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]- 2H-tetrazolium hydroxide and biomass formation (crystal violet). The influence of chitosan MW on the planktonic form of Candida spp. was strain dependent. Fungal growth decreased with increasing chitosan MW for C. tropicalis and C. parapsilosis, while chitosan MW did not modulate the effect for C. albicans. With regard to the formation of biofilms, in both the adhesion and mature phases, the biomass and metabolic activities of Candida spp. were reduced by about 70% and 80%, respectively for each phase.

A proposal for antifungal epidemiological cut-off values against Histoplasma capsulatum var. capsulatum based on the susceptibility of isolates from HIV-infected patients with disseminated histoplasmosis in Northeast Brazil.

Epidemiological cut-off values (ECVs) have been used as a tool to detect the acquisition of resistance mechanisms to antifungal drugs. In this context, the objective of this study was to determine the ECVs for classic antifungals against Histoplasma capsulatum var. capsulatum isolates from human immunodeficiency virus (HIV)-infected patients with a diagnosis of disseminated histoplasmosis. First, minimum inhibitory concentrations (MICs) for amphotericin B (AmB), itraconazole (ITR), fluconazole (FLU), voriconazole (VCZ) and caspofungin (CAS) were determined against 138 H. capsulatum isolates in the filamentous form by the broth microdilution method; antifungal ECVs were then calculated. MIC ranges were 0.0078-1 µg/mL for AmB, 0.0005-0.0625 µg/mL for ITR, 2 to ≥256 µg/mL for FLU, 0.0078-1 µg/mL for VCZ and ≤0.0156 to ≥32 µg/mL for CAS. The obtained ECVs were 0.5, 0.0313, 128, 0.5 and 16 µg/mL for AmB, ITR, FLU, VCZ and CAS, respectively. The percentage of wild-type isolates was 96.4% for AmB, 98.6% for ITR and 99.3% for FLU, VCZ and CAS. Although these results do not cover all phylogenetic species of H. capsulatum, they bring important information on strains from Brazil. In addition, the assessed isolates were from HIV-positive patients, which may not reflect the antifungal ECVs against isolates from immunocompetent individuals or from other sources. Finally, this study pioneers the initiative of establishing ECVs for five antifungal agents against H. capsulatum var. capsulatum, providing a criterion for the interpretation of susceptibility results as well as a monitoring strategy for the emergence of antifungal resistance.

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.