Antifungal activity of sustainable histone deacetylase inhibitors against planktonic cells and biofilms of Candida spp. and Cryptococcusneoformans.

The limited therapeutic options for fungal infections and the increased incidence of fungal strains resistant to antifungal drugs, especially Candida spp., require the development of new antifungal drugs and strategies. Histone deacetylase inhibitors (HDACi), like vorinostat, have been studied in cancer treatment and have antifungal effects, acting alone or synergistically with classical antifungals. Here we investigated the antifungal activity of two novel sustainable HDACi (LDT compounds) based on vorinostat structure. Molecular docking simulation studies reveal that LDT compounds can bind to Class-I HDACs of Candida albicans, C. tropicalis, and Cryptococcus neoformans, which showed similar binding mode to vorinostat. LDT compounds showed moderate activity when tested alone against fungi but act synergistically with antifungal azoles against Candida spp. They reduced biofilm formation by more than 50% in C. albicans (4 µg/mL), with the main action in fungal filamentation. Cytotoxicity of the LDT compounds against RAW264.7 cells was evaluated and LDT536 demonstrated cytotoxicity only at the concentration of 200 µmol/L, while LDT537 showed IC50 values of 29.12 µmol/L. Our data indicated that these sustainable and inexpensive HDACi have potential antifungal and antibiofilm activities, with better results than vorinostat, although further studies are necessary to better understand the mechanism against fungal cells.

Fungal infections are neglected diseases that affect more than a billion people worldwide. Some histone deacetylase inhibitors can act against fungal cells. Our data reveal that HDACi LDT536 and LDT537 have potential antibiofilm and antifungal activities.

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.

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.

In vitro effect of the iron chelator deferiprone on the antimicrobial susceptibility and biofilms of Burkholderia pseudomallei.

This study evaluated the effect of the iron chelator deferiprone (DFP) on antimicrobial susceptibility and biofilm formation and maintenance by Burkholderia pseudomallei. Planktonic susceptibility to DFP alone and in combination with antibiotics was evaluated by broth microdilution and biofilm metabolic activity was determined with resazurin. DFP minimum inhibitory concentration (MIC) range was 4-64 µg/mL and in combination reduced the MIC for amoxicillin/clavulanate and meropenem. DFP reduced the biomass of biofilms by 21 and 12% at MIC and MIC/2, respectively. As for mature biofilms, DFP reduced the biomass by 47%, 59%, 52% and 30% at 512, 256, 128 and 64 µg/mL, respectively, but did not affect B. pseudomallei biofilm viability nor increased biofilm susceptibility to amoxicillin/clavulanate, meropenem and doxycycline. DFP inhibits planktonic growth and potentiates the effect of ß-lactams against B. pseudomallei in the planktonic state and reduces biofilm formation and the biomass of B. pseudomallei biofilms.

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.

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.

Ex vivo wound model on porcine skin for the evaluation of the antibiofilm activity of polyhexamethylene biguanide and ciprofloxacin.

This study aimed to standardize the use of an ex vivo wound model for the evaluation of compounds with antibiofilm activity. The in vitro susceptibility of Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 to ciprofloxacin and polyhexamethylene biguanide (PHMB) was evaluated in planktonic and biofilm growth. The effects of ciprofloxacin and PHMB on biofilms grown on porcine skin explants were evaluated by colony-forming unit (CFU) counting and confocal microscopy. Minimum inhibitory concentrations (MICs) against S. aureus and P. aeruginosa were, respectively, 0.5 and 0.25 µg mL-1 for ciprofloxacin, and 0.78 and 6.25 µg mL-1 for PHMB. Minimum biofilm eradication concentrations (MBECs) against S. aureus and P. aeruginosa were, respectively, 2 and 8 µg mL-1 for ciprofloxacin, and 12.5 and >25 µg mL-1 for PHMB. Ciprofloxacin reduced (P < 0.05) log CFU counts of the biofilms grown ex vivo by 3 and 0.96 for S. aureus and P. aeruginosa, respectively, at MBEC, and by 0.58 and 8.12 against S. aureus and P. aeruginosa, respectively, at 2xMBEC. PHMB (100 µg/mL) reduced (P < 0.05) log CFU counts by 0.52 for S. aureus and 0.68 log for P. aeruginosa, leading to an overall decrease (P < 0.05) in biofilm biomass. The proposed methodology to evaluate the susceptibility of biofilms grown ex vivo led to reproducible and reliable results.

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.

The herbicide paraquat alters growth and melanin production on the Cryptococcus neoformans/Cryptococcus gattii species complex.

Paraquat (1,10-dimethyl-4,4-bipyridinium dichloride; PQ) is a free-radical producing herbicide that affects cell membranes and can upset the environmental balance of microorganisms present in soil, such as Cryptococcus spp. This study aimed to evaluate the in vitro activity of PQ against Cryptococcus spp. in planktonic and biofilm forms, as well as the protective effect of antioxidant agents against the antifungal effect of PQ and the kinetics of melanin production in response to PQ. Susceptibility to PQ was evaluated by microdilution. Cryptococcus sp. strains exposed to PQ were grown in media with ascorbic acid (AA) and glutathione (GSH). Melanin production was assessed in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) + PQ. The minimum inhibitory concentration of PQ against Cryptococcus spp. ranged from 8 to 256 µg/mL. Furthermore, PQ reduced biofilm formation. AA and GSH restored the fungal growth of Cryptococcus spp. exposed to PQ. In addition, l-DOPA + PQ delayed melanin production by 24 and 48 h for C. deuterogattii and C. neoformans sensu lato, respectively, suggesting that PQ induces a fitness trade-off in melanin production. Taken together, our data suggest that the antifungal effect of PQ against Cryptococcus spp. possibly exerts selective pressures interfering with biofilm formation and melanin production by these yeasts.

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.

Inhibitory effect of proteinase K against dermatophyte biofilms: an alternative for increasing the antifungal effects of terbinafine and griseofulvin.

This study aimed to evaluate the effect of proteinase K on mature biofilms of dermatophytes, by assays of metabolic activity and biomass. In addition, the proteinase K-terbinafine and proteinase K-griseofulvin interactions against these biofilms were investigated by the checkerboard assay and scanning electron and confocal microscopy. The biofilms exposed to 32 µg ml-1 of proteinase K had lower metabolic activity and biomass, by 39% and 38%, respectively. Drug interactions were synergistic, with proteinase K reducing the minimum inhibitory concentration of antifungals against dermatophyte biofilms at a concentration of 32 µg ml-1 combined with 128-256 µg ml-1 of terbinafine and griseofulvin. Microscopic images showed a reduction in biofilms exposed to proteinase K, proteinase K-terbinafine and proteinase K-griseofulvin combinations. These findings demonstrate that proteinase K has activity against biofilms of dermatophytes, and the interactions of proteinase K with terbinafine and griseofulvin improve the activity of drugs against mature dermatophyte biofilms.

Collateral consequences of agricultural fungicides on pathogenic yeasts: A One Health perspective to tackle azole resistance.

Candida and Cryptococcus affect millions of people yearly, being responsible for a wide array of clinical presentations, including life-threatening diseases. Interestingly, most human pathogenic yeasts are not restricted to the clinical setting, as they are also ubiquitous in the environment. Recent studies raise concern regarding the potential impact of agricultural use of azoles on resistance to medical antifungals in yeasts, as previously outlined with Aspergillus fumigatus. Thus, we undertook a narrative review of the literature and provide lines of evidence suggesting that an alternative, environmental route of azole resistance, may develop in pathogenic yeasts, in addition to patient route. However, it warrants sound evidence to support that pathogenic yeasts cross border between plants, animals and humans and that environmental reservoirs may contribute to azole resistance in Candida or other yeasts for humans. As these possibilities could concern public health, we propose a road map for future studies under the One Health perspective.

Atypical chlamydoconidium-producing Trichophyton tonsurans strains from Ceará State, Northeast Brazil: investigation of taxonomy by phylogenetic analysis and biofilm susceptibility.

Chlamydoconidium-producing Trichophyton tonsurans strains isolated in Northeastern Brazil have morphological features different from the classic description of this dermatophyte species. This study investigated the phylogenetic relationship of chlamydoconidium-producing T. tonsurans strains isolated in Northeastern Brazil. Also, the effect of terbinafine and farnesol on mature biofilms of T. tonsurans strains was evaluated. The mass spectra of T. tonsurans strains were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The ITS and LSU loci regions of rDNA and the partial ß-tubulin gene were sequenced and the phylogenetic tree was analysed. The effects of terbinafine and farnesol on mature T. tonsurans biofilms were evaluated through the analysis of metabolic activity, quantification of biomass and observation by scanning electron microscopy. MALDI-TOF MS spectra of the chlamydoconidium-producing T. tonsurans strains differed from the spectrum of the control strain (ATCC 28942), presenting an intense ion peak at m/z 4155 Da. Phylogenetic tree analysis showed that the chlamydoconidium-producing strains isolated in Northeastern Brazil are allocated to a single cluster, differing from strains isolated from other countries. As for mature T. tonsurans biofilms, farnesol reduced biomass and metabolic activity by 64.4 and 65.9 %, respectively, while terbinafine reduced the biomass by 66.5 % and the metabolic activity by 69 %. Atypical morphological characteristics presented by chlamydoconidium-producing T. tonsurans strains result from phenotypic plasticity, possibly for adaptation to environmental stressors. Also, farnesol had inhibitory activity against T. tonsurans biofilms, demonstrating this substance can be explored for development of promising anti-biofilm drugs against dermatophytes.

Biofilm formation on cat claws by Sporothrix species: An ex vivo model.

This work aimed to evaluate the ability of Sporothrix species to attach and form biofilm on the surface of cat claws as an ex vivo model. A total of 14 strains (5 Sporothrix brasiliensis, 3 Sporothrix schenckii s. str., 3 Sporothrix globosa and 3 Sporothrix mexicana) were used. The biofilms were incubated for periods of 01, 03, 07, 10 and fifteenth 15 days. Their metabolic activities were evaluated by the XTT reduction assay and the morphology and structure were investigated by scanning electron microscopy (SEM). The analysis of the SEM images revealed that all the species can form biofilms on cat claws. The metabolic activity in the ex vivo biofilms was similar to that found in in vitro biofilms when incubated for the same period. This is the first report of an ex vivo biofilm model involving cat claws. The ability to form biofilms on cat claws can increase the viable period of the fungus and consequently the number of possibly infected animals and people.

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.

Azole-Resilient Biofilms and Non-wild Type C. albicans Among Candida Species Isolated from Agricultural Soils Cultivated with Azole Fungicides: an Environmental Issue?

This study aimed to identify Candida spp. from agricultural soils cultivated with azole fungicides and investigate their susceptibility to clinical (fluconazole, itraconazole, voriconazole, and amphotericin B) and agricultural (tetraconazole and tebuconazole) antifungals in planktonic form. Additionally, Candida biofilm-forming ability and biofilm susceptibility to agricultural antifungals and voriconazole were analyzed. Species identification was performed by phenotypic and molecular assays. The susceptibility of planktonic cells was evaluated by the broth microdilution method. The biofilm metabolic activity was evaluated by the XTT reduction assay. The recovered Candida spp. were identified as C. parapsilosis sensu stricto (n = 14), C. albicans (n = 5), C. tropicalis (n = 2), C. fermentati (n = 1), and C. metapsilosis (n = 2). Minimum inhibitory concentration ranges for clinical and agricultural antifungals were ≤ 0.03-4 µg/mL and 1-128 µg/mL, respectively. Two and one C. albicans strains were considered non-wild type for voriconazole and fluconazole, respectively. All strains were biofilm producers. The minimum biofilm inhibitory concentration ranges for tetraconazole and tebuconazole were 128-> 1024 µg/mL, while for voriconazole was 512-> 1024 µg/mL. In summary, this study shows that non-wild type and azole-resilient biofilm-producing Candida species colonize agricultural soils cultivated with azole fungicides.

Anthraquinones from Aloe spp. inhibit Cryptococcus neoformans sensu stricto: effects against growing and mature biofilms.

This study aimed to evaluate the in vitro effect of aloe emodin, barbaloin and chrysophanol on growing and mature biofilms of Cryptococcus neoformans sensu stricto. The compounds were added at the moment of inducing biofilm growth or after growth for 72 h to evaluate their effects on growing and mature biofilms, respectively. Then, biofilm biomass was evaluated by crystal violet staining and metabolic activity by the XTT reduction assay. Morphological alterations were also evaluated by laser scanning confocal microscopy. Aloe emodin and barbaloin affected growing biofilms and disrupted mature biofilms, reducing metabolic activity by > 60% and biomass by > 70%. Chrysophanol only inhibited mature biofilms, but to a lesser extent. In conclusion, anthraquinones, especially aloe emodin and barbaloin, show a relevant effect against growing and mature biofilms of C. neoformans sensu stricto.

Inhibitory effect of Brazilian red propolis on planktonic and biofilm forms of Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacillus which is the leading cause of health-care-associated infective diarrhea. The rising incidence of antibiotic resistance in pathogens such as C. difficile makes researches on alternative antibacterial products very important, especially those exploring natural products like propolis. Brazilian Red Propolis, found in the Northeast region of Brazil, is composed by products from regional plants that have the antimicrobial properties. This study aimed to evaluate the in vitro activity of Brazilian Red Propolis (BRP) against C. difficile strains in planktonic and biofilm forms. The susceptibility of four strains of C. difficile to BRP was analyzed by broth microdilution method and vancomycin was included as control drug. BRP-exposed C. difficile cells were evaluated by scanning electron microscopy (SEM). Then, the effects of BRP on growing and mature C. difficile biofilms were also evaluated. BRP minimum inhibitory concentration was 625 µg/mL against all tested strains, while vancomycin MIC range was 0.5-2 µg/mL. SEM showed the loss of homogeneity in bacterial cell wall and cell fragmentation, after BRP-exposure. BRP, at MIC, reduced (P < 0.05) the biomass, matrix proteins and matrix carbohydrates of growing biofilms, and, at 8xMIC, reduced (P < 0.05) the biomass and matrix proteins of mature biofilms. The present study demonstrated that BRP inhibits planktonic growth, damages cell wall, decreases biofilm growth and harms mature biofilms of C. difficile.

Cryptococcus neoformans/Cryptococcus gattii species complex melanized by epinephrine: Increased yeast survival after amphotericin B exposure.

Cryptococcus neoformans/Cryptococcus gattii complex species are etiological agents of cryptococcosis, a systemic mycosis that cause respiratory infection and meningoencephalitis. To establish the infection, these yeasts produce virulence factors, such as melanin, which contribute to pathogenicity and antifungal tolerance. The aim of this study was to investigate melanin production by the C. neoformans/C. gattii complex in the presence of different precursors of melanogenesis and evaluate the effect of melanization on the antifungal susceptibility of these species to fluconazole, flucytosine and amphotericin B. Epinephrine, norepinephrine, dopamine and caffeic acid were used as substrates for melanin production, and l-dopa was used as positive control. The susceptibility of melanized strains (n = 6), after exposure to epinephrine or l-dopa, was evaluated by broth microdilution assay, and non-melanized strains were used as control. The antifungal activity of amphotericin B against melanized strains was also investigated by time kill assay. All Cryptococcus spp. strains produced melanin after exposure to the tested substrates. After exposure to epinephrine, minimum inhibitory concentration (MIC) ranges were 1-8 µg/mL for fluconazole, 2-8 µg/mL for flucytosine and 0.125-1 µg/mL for amphotericin B, while, after exposure to l-dopa, MIC ranges were 2-8 µg/mL for fluconazole, 4-8 µg/mL for flucytosine, and 0.125-0.5 µg/mL for amphotericin B. Similar results were observed for non-melanized strains. The production of melanin after exposure to epinephrine was higher than that induced by l-dopa. Melanized cells of both species were more tolerant to amphotericin B than the non-melanized control, emphasizing the importance of melanin production for fungal virulence.

Antifungal effect of anthraquinones against Cryptococcus neoformans: detection of synergism with amphotericin B.

The emergence of tolerant Cryptococcus neoformans strains to antifungals has been described. It has directed researchers to screen for new antimicrobial compounds. In this context, several plant-derived compounds, such as anthraquinones (aloe emodin, barbaloin, and chrysophanol), have been investigated for their antimicrobial properties. This study aimed to evaluate the in vitro effect of aloe emodin, barbaloin and chrysophanol on C. neoformans in vitro growth. In addition, the interaction between these anthraquinones and amphotericin B and itraconazole was evaluated. Initially, the minimum inhibitory concentrations (MIC) of these compounds were determined against 17 strains of C. neoformans by the broth microdilution method and then pharmacological interaction assays were performed with 15 strains by the checkerboard method. Aloe emodin, barbaloin, and chrysophanol showed minimum inhibitory concentrations of 236.82-473.65 µM (64-128 µg/mL), 153-306 µM (64-128 µg/ml) and ≥1007 µM (≥256 µg/ml), respectively. Furthermore, aloe emodin (11/15), barbaloin (13/15), and chrysophanol (12/15) showed pharmacological synergism (FICI < 0.5) with amphotericin B at subinhibitory concentrations (MIC/4). The itraconazole-aloe emodin interaction was additive (1/15) (0.5 < FICI < 1.0). The itraconazole-barbaloin interaction were synergistic (2/15) and additive (5/15); whereas itraconazole-chrysophanol interactions were additive (2/15). Anthraquinones, especially aloe emodin and barbaloin, present in vitro antifungal activity against C. neoformans and potentiate the antifungal activity of amphotericin B.