Fusarium keratitis in a Brazilian tropical semi-arid area: Clinical-epidemiological features, molecular identification and antifungal susceptibility.

BACKGROUND: Fungal keratitis is a severe eye infection that can result in blindness and visual impairment, particularly in developing countries. Fusarium spp. are the primary causative agents of this condition. Diagnosis of Fusarium keratitis (FK) is challenging, and delayed treatment can lead to serious complications. However, there is limited epidemiological data on FK, especially in tropical areas. OBJECTIVES: This study aimed to describe the clinical, laboratorial and epidemiological characteristics of FK in a tropical semi-arid region of Brazil. PATIENTS/METHODS: Adult patients with laboratory-confirmed FK diagnosed between October 2019 and March 2022 were evaluated. Fusarium isolates were characterized at molecular level and evaluated regarding antifungal susceptibility. RESULTS: A total of 226 clinical samples from patients suspected of keratitis were evaluated; fungal growth was detected in 50 samples (22.12%); out of which 42 were suggestive of Fusarium spp. (84%). Molecular analysis of a randomly selected set of 27 isolates identified F. solani species complex (n = 14); F. fujikuroi sensu lato (n = 6) and F. dimerum sensu lato (n = 7); a total of 10 haplotypes were identified among the strains. All but one Fusarium strains were inhibited by amphotericin B, natamycin and fluconazole. Most patients were male (71.42%; 30 out of 42), aged from 27 to 73 years old. Trauma was the most important risk factor for FK (40.47%; 17 out of 42). Patients were treated with antifungals, corticoids and antibiotics; keratoplasty and eye enucleation were also performed. CONCLUSIONS: The study provided insights into the characteristics of FK in tropical regions and emphasized the importance of enhanced surveillance and management strategies.

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.

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.

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.

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.

Effects of lipopeptide biosurfactants on clinical strains of Malassezia furfur growth and biofilm formation.

Lipopeptide biosurfactants (LBs) are biological molecules with low toxicity that have aroused growing interest in the pharmaceutical industry. Their chemical structure confers antimicrobial and antibiofilm properties against different species. Despite their potential, few studies have demonstrated their capability against Malassezia spp., commensal yeasts which can cause dermatitis and serious infections. Thus, the aim of this study was to evaluate the antifungal activity of biosurfactants produced by new strains of Bacillus subtilis TIM10 and B. vallismortis TIM68 against M. furfur and their potential for removal and inhibition of yeast biofilms. Biosurfactants were classified as lipopeptides by FTIR, and their composition was characterized by ESI-Q-TOF/MS, showing ions for iturin, fengycin, and surfactin, with a greater abundance of surfactin. Through the broth microdilution method, both biosurfactants inhibited the growth of clinical M. furfur strains. Biosurfactant TIM10 showed greater capacity for growth inhibition, with no statistical difference compared to those obtained by the commercial antifungal fluconazole for M. furfur 153DR5 and 154DR8 strains. At minimal inhibitory concentrations (MIC-2), TIM10 and TIM68 were able to inhibit biofilm formation, especially TIM10, with an inhibition rate of approximately 90%. In addition, both biosurfactants were able to remove pre-formed biofilm. Both biosurfactants showed no toxicity against murine fibroblasts, even at concentrations above MIC-2. Our results show the effectiveness of LBs in controlling the growth and biofilm formation of M. furfur clinical strains and highlight the potential of these agents to compose new formulations for the treatment of these fungi.

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.

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.

Azole resistance in Candida from animals calls for the One Health approach to tackle the emergence of antimicrobial resistance.

This study initially aimed at investigating the occurrence of azole resistance among Candida spp. from animals and analyzing the involvement of efflux pumps in the resistance phenomenon. Then, the dynamics of antifungal resistance was assessed, by comparing the antifungal epidemiological cutoff values (ECVs) against C. albicans and C. tropicalis from humans and animals. Fifty azole-resistant isolates (24 C. albicans, 24 C. tropicalis; 2 C. parapsilosis sensu lato) were submitted to the efflux pump inhibition assay with promethazine and significant MIC reductions were observed for fluconazole (2 to 250-fold) and itraconazole (16 to 4000-fold). Then, the antifungal ECVs against C. albicans and C. tropicalis from human and animal isolates were compared. Fluconazole, itraconazole and voriconazole ECVs against human isolates were lower than those against animal isolates. Based on the antifungal ECVs against human isolates, only 33.73%, 50.39% and 63.53% of C. albicans and 52.23%, 61.85% and 55.17% of C. tropicalis from animals were classified as wild-type for fluconazole, itraconazole and voriconazole, respectively. Therefore, efflux-mediated mechanisms are involved in azole resistance among Candida spp. from animals and this phenomenon seems to emerge in animal-associated niches, pointing to the existence of environmental drivers of resistance and highlighting the importance of the One Health approach to control it.

Antifungal activity of promethazine and chlorpromazine against planktonic cells and biofilms of Cryptococcus neoformans/Cryptococcus gattii complex species.

Cryptococcus neoformans/Cryptococcus gattii are fungal pathogens that affect the central nervous system, mainly in immunocompromised individuals. Due to the limited pharmacological arsenal available for the treatment of cryptococcosis associated with cases of antifungal resistance of Cryptococcus spp. reported in some studies, the search for new compounds with antifungal potential becomes relevant. Thus, the objective of this study was to evaluate the inhibitory effect of phenothiazines (promethazine and chlorpromazine) on C. neoformans/C. gattii planktonic cells and biofilms. In vitro planktonic susceptibility testing was performed using the broth microdilution assay. The effect of phenothiazines was evaluated against biofilm formation and mature Cryptococcus biofilms. Biofilm morphology and ultrastructure were also evaluated by scanning electron microscopy. Promethazine and chlorpromazine showed antifungal activity against planktonic cells, with minimum inhibitory concentrations of 8-32 µg/ml and 4-16 µg/ml, respectively. As for biofilm formation, phenothiazines reduced biomass by 60% and metabolic activity by 90% at 64 µg/ml; while in mature biofilms, reductions of 85% and 90% in biomass and metabolic activity, respectively, were observed at 1024 µg/ml. Promethazine and chlorpromazine were also able to disrupt and fragment biofilms. In conclusion, promethazine and chlorpromazine have antifungal activity against planktonic cells and biofilms of Cryptococcus spp. These data show the potential of promethazine and chlorpromazine as antibiofilm drugs.

The yeast, the antifungal, and the wardrobe: a journey into antifungal resistance mechanisms of Candida tropicalis.

Candida tropicalis is a prominent non-Candida albicans Candida species involved in cases of candidemia, mainly causing infections in patients in intensive care units and (or) those presenting neutropenia. In recent years, several studies have reported an increase in the recovery rates of azole-resistant C. tropicalis isolates. Understanding C. tropicalis resistance is of great importance, since resistant strains are implicated in persistent or recurrent and breakthrough infections. In this review, we address the main mechanisms underlying C. tropicalis resistance to the major antifungal classes used to treat candidiasis. The main genetic basis involved in C. tropicalis antifungal resistance is discussed. A better understanding of the epidemiology of resistant strains and the mechanisms involved in C. tropicalis resistance can help improve diagnosis and assessment of the antifungal susceptibility of this Candida species to improve clinical management.

In vitro and ex vivo biofilms of dermatophytes: a new panorama for the study of antifungal drugs.

This study describes an ex vivo model that creates an environment for dermatophyte biofilm growth, with features that resemble those of in vivo conditions, designing a new panorama for the study of antifungal susceptibility. Regarding planktonic susceptibility, MIC ranges were 0.125-1 µg ml-1 for griseofulvin and 0.000097-0.25 µg ml-1 for itraconazole and terbinafine. sMIC50 ranges were 2->512 µg ml-1 for griseofulvin and 0.25->64 µg ml-1 for itraconazole and terbinafine. CLSM images demonstrated a reduction in the amount of cells within the biofilm, but hyphae and conidia were still observed and biofilm biomass was maintained. SEM analysis demonstrated a retraction in the biofilm matrix, but fungal structures and water channels were preserved. These results show that ex vivo biofilms are more tolerant to antifungal drugs than in vitro biofilms, suggesting that environmental and nutritional conditions created by this ex vivo model favor biofilm growth and robustness, and hence drug tolerance.

Mini-review: from in vitro to ex vivo studies: an overview of alternative methods for the study of medical biofilms.

Microbial biofilms are a natural adaptation of microorganisms, typically composed of multiple microbial species, exhibiting complex community organization and cooperation. Biofilm dynamics and their complex architecture are challenging for basic analyses, including the number of viable cells, biomass accumulation, biofilm morphology, among others. The methods used to study biofilms range from in vitro techniques to complex in vivo models. However, animal welfare has become a major concern, not only in society, but also in the academic and scientific field. Thus, the pursuit for alternatives to in vivo biofilm analyses presenting characteristics that mimic in vivo conditions has become essential. In this context, the present review proposes to provide an overview of strategies to study biofilms of medical interest, with emphasis on alternatives that approximate experimental conditions to host-associated environments, such as the use of medical devices as substrata for biofilm formation, microcosm and ex vivo models.