Detection of 2-ethyl-1-hexanol and its modulating effect in biofilm of Fusarium oxysporum.

In immunocompetent individuals, Fusarium spp. stands out as the causative agent of onychomycosis, among the non-dermatophyte molds. Despite evidence indicating that Fusarium oxysporum organizes itself in the form of a biofilm causing onychomycosis, there is little literature on the etiopathogenesis of the biofilm on the nail, specifically the signaling molecules present, known as quorum sensing (QS). Thus, this study detected the presence of a molecule related to QS from the ex vivo biofilm of F. oxysporum on human nail and investigated its effect on preformed biofilm in vitro. The detection and physicochemical characterization of a QS molecule, from the extracellular matrix (ECM), was carried out by Fourier transform infrared (FTIR) spectroscopy with an attenuated total reflectance (ATR) accessory and by headspace gas chromatography coupled to mass spectrometry (GC-MS) analyses. Determination of viable cells, cell activity, total biomass, ECM components and scanning electron microscopy (SEM) were performed to evaluate the influence of the QS molecule on the in vitro biofilm of F. oxysporum. The beginning, in the ex vivo biofilm of F. oxysporum on human nails, the volatile organic compound 2-ethyl-1-hexanol (2EH) was detected as a component of QS. Thereafter in vitro analyses, synthetic 2EH was able to modulate the biofilm by stimulating its filament, increasing total biomass and ECM production in terms of total carbohydrates, but with a reduction in total proteins and nucleic acids. We thus evidence, for the first time, the presence of 2EH in the biofilm of F. oxysporum, developed on the human nail, and the in vitro action of this compound as a QS molecule.

Hospital Trichosporon asahii isolates with simple architecture biofilms and high resistance to antifungals routinely used in clinical practice.

Infections by Trichosporon spp. are increasing worldwide and its treatment remains a challenge. Colonization of medical devices has been considered as a predisposing factor for trichosporonosis, which is related to fungal biofilm production. Thus, this study aimed to evaluate the ability of six hospital T. asahii isolates to form biofilm on abiotic surface, as well as to investigate the impact of three classic antifungals on both planktonic and biofilm forms. The fungal identification was based on macro and micromorphological characteristics, biochemical tests and confirmation by mass spectrometry assisted by the flight time desorption/ionization matrix (MALDI-TOF MS). Antifungal susceptibility assay of planktonic cells showed inhibitory and fungicidal concentrations ranging from 2.5 to 10 µg/mL for voriconazole, 2 to 8 µg/mL for fluconazole, and 1 to 4 µg/mL for amphotericin B. All T. asahii strains were able to form biofilms on the polystyrene microplates surface within 24 h, showing a simple architecture when compared with Candida spp. biofilm. On the other hand, the same antifungals did not show action in neither the inhibition of biofilm formation nor on the formed biofilm. Concluding, the present study reinforced the relevance of the MALDI-TOF MS methodology for a safe identification of T. asahii. Classic antifungals were active on the planktonic form, but not on the biofilms. All isolates formed biofilms on the polystyrene microplates and showed a simple architecture.

Rhodotorula sp. and Trichosporon sp. are more Virulent After a Mixed Biofilm.

Rhodotorula spp. and Trichosporon spp. are opportunistic pathogens, and although an association between these two species in the same infection appears to be uncommon, it has been reported. This is the first study that aimed to evaluate the pathogenesis of a co-infection by R. mucilaginosa and T. asahii, using a new in vivo model, the Zophobas morio larvae. Suspensions from planktonic and biofilm-recovered cells were injected in the larvae as in monospecies as mixed (a ratio of 1:1 for both agents of a of 105 inoculum). Individual and mixed biofilms of R. mucilaginosa and T. asahii were produced for 24 and 48 h, and they were partially characterized by crystal violet and reduction of tetrazolium salt. When evaluating the impact of the planktonic suspension in vivo we verified that the fungi in monoculture were more able to kill the larvae than those from planktonic mixed suspension. On the other hand, regarding biofilm-recovered cells, there was an increase in the death of larvae infected for mixed suspensions. Moreover, the death rate was more pronounced when the larvae were infected with 48 h biofilm-recovered cells than the 24 h ones. T. asahii was the best producer of total biomass, mainly in 48 h. The metabolic activity for both yeasts organized in biofilm maintained the same pattern between 24 and 48 h. The present study proves a synergistic interaction between R. mucilaginosa and T. asahii after an experience in a mixed biofilm. Our results suggest that both species were benefited from this interaction, acquiring a greater potential for virulence after passing through the biofilm and this ability was acquired by the cells released from the biofilm.

Performance of Two Extracts Derived from Propolis on Mature Biofilm Produced by Candida albicans.

Species of the Candida genus represent the third most common cause of onychomycosis, the most frequent and difficult to treat nail infection. Onychomycosis has been attributed to fungi organized in biofilm and some natural products have proved promising for its treatment. This study aimed to evaluate the antibiofilm activity of propolis extract (PE) and its by-product (WPE) on 7-day preformed biofilms produced by Candida albicans in polystyrene microplates, as well as in an ex vivo model on human nail fragments. The cytotoxicity and permeation capacity were also assessed. Firstly, multiple parameters were evaluated over 7 days to elucidate the dynamics of biofilm formation by C. albicans. The cell viability and total biomass did not vary much from the beginning; however, days 3 and 4 were crucial in terms of metabolic activity, which was significantly increased, and the levels of extracellular matrix components, wherein proteins and nucleic acids experienced an increase, but polysaccharide levels dropped. Architecturally, one-day biofilm showed a monolayer of organized cells (blastoconidia, hyphae, and pseudohyphae), while in the seven-day biofilm there was a three-dimensional well-structured and complex biofilm. This yeast was also able to form a biofilm on both surfaces of the nail, without an additional nutritional source. Both extracts showed excellent antibiofilm activity against the 7-day preformed biofilm and were not toxic to Vero cells at concentrations compatible with the antifungal and antibiofilm activities. Both extracts permeated the experimentally infected nail, with WPE being more efficient. The results of this study, taken together, reinforce the potential of these natural products, containing propolis, as a safe option for the topical treatment of onychomycosis.

PanB over-representation as part of pyrazinamide action: a proteomic insight.

Background: Pyrazinamide (PZA) represents a milestone as a first-line antituberculosis drug due to its sterilizing activity against Mycobacterium tuberculosis. Materials & Methods: The protein changes induced by subinhibitory PZA exposure of M. tuberculosis in acidic pH were evaluated by a proteomic approach. Results: Among the 1059 M. tuberculosis proteins identified, the specific acidification in the culture medium induced the over-representation of MurF (Rv2157c), and its under-representation was induced by 12 h of PZA exposure. PanB (Rv2225) was over-represented at 24 h of PZA exposure. Conclusion: The authors highlight the over-representation of PanB in M. tuberculosis correlates of PZA action in acidic pH, reinforcing the role of the pantothenate pathway as a bacillus drug target to be explored.

Standardization of resazurin use in susceptibility testing of natural products against yeasts in planktonic cells and in biofilms formation

Natural products, such as the ethanolic propolis extract (PE), have been shown to be a safe and effective therapeutic alternative for the treatment of fungal skin and nail diseases. However, the presence of the resin and the physicochemical characteristics of the extract sometimes difficult the reading and determination of breakpoints of the in vitrotests, evidencing the need for alternatives that facilitate the reading. The present study aimed to standardize the use of resazurin in tests of susceptibility of PE with planktonic yeast cells and biofilm forms. The antifungal activity of PE was determined by minimum inhibitory concentration (MIC) and we observed that, for all Candida spp. tested, the most reproducible MIC results were obtained when resazurin was placed after 24 hours of incubation and remained more 24 hourswith yeasts plus PE. For encapsulated yeasts, there was no dye reduction and color transition. Resazurin was also used for the evaluation of minimal biofilm inhibitory concentration and minimal biofilm eradication concentration and it was metabolized and reproducedthe action of PE on Candida biofilms. In addition, microdilution checkerboard plates were made with the dye, which assisted reading the result of the interaction between PE and nystatin. We observed that the resin, the color and the turbidity of the PE slightly changed the color of the resazurin in high concentrations of the extract and did not impair the reading. Therefore, the resazurin standardization tests were proven to be efficient and grounds that it should be used as an auxiliary methodology for reading and interpretation of the susceptibility tests for non-encapsulated yeasts with natural products, which form turbidity or precipitation, such as propolis.

Microbiological and virulence aspects of Rhodotorula mucilaginosa.

We aimed to characterize microbiologically clinical isolates of R. mucilaginosa isolated from colonization of a patient with chronic renal disease (CKD), as well as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible infective agent. For this study, two isolates of R. mucilaginosa from oral colonization of a CKD patient were isolated, identified and characterized by classical (genotypic and phenotypic) methods. Susceptibility to conventional antifungals was evaluated, followed by biofilm production, measured by different techniques (total biomass, metabolic activity, colony forming units and extracellular matrix quantification). Finally, the pathogenicity of yeast was evaluated by infection of Tenebrio molitor larvae. All isolates were resistant to azole and sensitive to polyenes and they were able to adhere and form biofilm on the abiotic surface of polystyrene. In general, similar profiles among isolates were observed over the observed periods (2, 24, 48 and 72 hours). Regarding extracellular matrix components of biofilms at different maturation ages, R. mucilaginosa was able to produce eDNA, eRNA, proteins, and polysaccharides that varied according to time and the strain. The death curve in vivo model showed a large reduction in the survival percentage of the larvae was observed in the first 24 hours, with only 40 % survival at the end of the evaluation. We infer that colonization of chronic renal patients by R. mucilaginosa offers a high risk of serious infection. And also emphasize that the correct identification of yeast is the main means for an efficient treatment.

Promising Antituberculosis Activity of Piperine Combined with Antimicrobials: A Systematic Review.

Piperine, a bioactive compound from Piper nigrum and Piper longum, has shown promising activity as efflux pump (EP) inhibitor and as adjunct in treatment of tuberculosis (TB). The present systematic review investigated scientific studies of the activity of piperine against mycobacteria, with a focus on its mechanism of action, drug interactions, and antimycobacterial activity. A broad and rigorous literature search of three electronic databases (PubMed, Web of Knowledge, and LILACS) was performed according to the PRISMA statement. We considered studies that were published up to December 1, 2017. Google Scholar was also searched to increase the number of publications. We searched for articles using the search terms "piperine" and "Mycobacterium spp." The search yielded a total of 225 articles. After removing duplicate publications, 208 publications remained. Of these, we evaluated the full text of 13 articles. After applying the inclusion criteria, eight studies were included in the present systematic review. The results of the systematic review showed that piperine has promising anti-TB activity, mainly when combined with antimicrobials, and plays an important role as an EP inhibitor.

Intramacrophage Mycobacterium tuberculosis efflux pump gene regulation after rifampicin and verapamil exposure.

Objectives: Since resistance of Mycobacterium tuberculosis (Mtb) partially derives from efflux pumps (EPs) in the plasma membrane, the current study evaluates EPs in Mtb exposed to rifampicin in the presence of the EP inhibitor verapamil, within a macrophage environment. Methods: Human acute monocytic leukaemia cell line THP-1 was infected with Mtb H37Rv and exposed to rifampicin and verapamil alone and in combination for 24 and 72 h. After RNA extraction, quantitative PCR was carried out for 11 EP genes using SYBR green PCR master mix in the StepOne™ Real-Time PCR System. Results: After 24 h of exposure to rifampicin, Mtb H37Rv showed that 10 EP genes were up-regulated when compared with the control. The rifampicin/verapamil combination induced down-regulation of 54.5% (6/11) of the EP genes. At 72 h, rifampicin exposure induced up-regulation of 10 EP genes and rifampicin/verapamil induced down-regulation of 8 EP genes, which suggests effective EP-inhibitory activity of verapamil against Mtb H37Rv in an intramacrophage environment. Conclusions: The current study demonstrated that rifampicin/verapamil caused down-regulation of several EP genes in Mtb inside the macrophage environment. In vivo trials may show that rifampicin/verapamil therapy could be of value in enhancing anti-TB treatment.