Antifungal Activity of Chitosan against Histoplasma capsulatum in Planktonic and Biofilm Forms: A Therapeutic Strategy in the Future?

Histoplasmosis is a respiratory disease caused by Histoplasma capsulatum, a dimorphic fungus, with high mortality and morbidity rates, especially in immunocompromised patients. Considering the small existing therapeutic arsenal, new treatment approaches are still required. Chitosan, a linear polysaccharide obtained from partial chitin deacetylation, has anti-inflammatory, antimicrobial, biocompatibility, biodegradability, and non-toxicity properties. Chitosan with different deacetylation degrees and molecular weights has been explored as a potential agent against fungal pathogens. In this study, the chitosan antifungal activity against H. capsulatum was evaluated using the broth microdilution assay, obtaining minimum inhibitory concentrations (MIC) ranging from 32 to 128 µg/mL in the filamentous phase and 8 to 64 µg/mL in the yeast phase. Chitosan combined with classical antifungal drugs showed a synergic effect, reducing chitosan's MICs by 32 times, demonstrating that there were no antagonistic interactions relating to any of the strains tested. A synergism between chitosan and amphotericin B or itraconazole was detected in the yeast-like form for all strains tested. For H. capsulatum biofilms, chitosan reduced biomass and metabolic activity by about 40% at 512 µg/mL. In conclusion, studying chitosan as a therapeutic strategy against Histoplasma capsulatum is promising, mainly considering its numerous possible applications, including its combination with other compounds.

Standardization of in vitro dual-species biofilms of Staphylococcus pseudintermedius and Malassezia pachydermatis: a strategy to establish an ex vivo biofilm model.

Ex vivo experiments have been performed aiming at mimicking in vivo environments. The main aim of this research was to standardize in vitro dual-species biofilm formation by Staphylococcus pseudintermedius and Malassezia pachydermatis as a strategy to establish an ex vivo biofilm model. Initially, the in vitro formation of biofilms in co-culture was established, using YPD medium, inoculum turbidity of 0.5 on the McFarland scale and maturation periods of 96 h for M. pachydermatis and 48 h for S. pseudintermedius. Subsequently, biofilms were formed on porcine skin using the same conditions, under which a greater number of cells/ml was observed in in vitro dual-species than in in vitro mono-species biofilms. Furthermore, ex vivo biofilm images demonstrated the formation of a highly structured biofilm with the presence of cocci and yeasts surrounded by the matrix. Thus, these conditions optimized the growth of both microorganisms within biofilms in vitro and ex vivo.

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.

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.

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.

Yeast microbiota of free-ranging amphibians and reptiles from Caatinga biome in Ceará State, Northeast Brazil: High pathogenic potential of Candida famata / Leveduras da microbiota de anfíbios e répteis do bioma Caatinga no Ceará, Nordeste do Brasil: alto potencial patogênico de Candida famata

ABSTRACT: Studies on the fungal microbiota of reptiles and amphibians are necessary to better understand of host-microbe interactions and the establishment of fungal disease in these animals. However, these studies are limited. The present researchidentified yeasts from free-ranging reptiles and amphibians from the Caatinga biome andevaluated the virulence factors production, the antifungal susceptibility in planktonic and biofilm growth and the pathogenicity of Candida famata isolates. Twenty-nine isolates of the genera Candida, Cryptococcus and Rhodotorula were identified by phenotypic and/or molecular methods and production of hydrolytic enzymes in vitro by these genera of fungi was evaluated. In addition, susceptibility of planktonic cells and biofilms to azoles and amphotericin B was evaluated. The pathogenicity of C. famata, the most prevalent yeast species isolated, was evaluated using Caenorhabditis elegans model. C. famata was the most prevalent yeast in amphibian and reptilian microbiota. Phospholipase and protease production was observed in 18/29 and 11/29 of the yeast isolates, respectively, while 100% formed biofilms. Itraconazole presented high minimal inhibitory concentrations against C. famata and C. tropicalis. Amphotericin B reduced the biomass and metabolic activity of biofilms. C. famata induced the mortality of C. elegans. In conclusion, reptiles and amphibians are colonized by yeasts capable of producing important virulence factors, especially by Candida spp. that present low susceptibility to azoles which may result from imbalances in ecosystem. Finally, C. famata isolated from these animals presented high pathogenicity, showing the importance of the study of reptile and amphibians fungal microbiota.

RESUMO: Estudos sobre a microbiota fúngica de répteis e anfíbios são necessários para melhor compreender as interações hospedeiro-microrganismo e o estabelecimento de doenças fúngicas nesses animais. No entanto, esses estudos são limitados. O objetivo da presente pesquisa foi identificar leveduras isoladas de répteis e anfíbios do bioma Caatinga e avaliar a produção de fatores de virulência, a sensibilidade a antifúngicos no crescimento planctônico e de biofilme e a patogenicidade de Candida famata. Vinte e nove isolados dos gêneros Candida, Cryptococcus e Rhodotorula foram identificados por métodos fenotípicos e/ou moleculares e a produção de enzimas hidrolíticas in vitro por esses gêneros de fungos foi avaliada. Além disso, foi avaliada a suscetibilidade de células planctônicas e biofilmes a azólicos e anfotericina B. A patogenicidade de C. famata, a espécie de levedura isolada mais prevalente, foi avaliada usando Caenorhabditis elegans. C. famata foi a levedura mais prevalente na microbiota de anfíbios e répteis. A produção de fosfolipase e protease foi observada em 18/29 e 11/29 dos isolados de levedura, respectivamente, enquanto 100% formaram biofilmes. O itraconazol apresentou altas concentrações inibitórias mínimas contra C. famata e C. tropicalis. A anfotericina B reduziu a biomassa e atividade metabólica dos biofilmes. C. famata induziu a mortalidade de C. elegans. Em conclusão, répteis e anfíbios são colonizados por leveduras capazes de produzir importantes fatores de virulência, especialmente por cepas de Candida spp. que apresentam baixa suscetibilidade a azólicos que podem resultar de desequilíbrio no ecossistema. Por fim, C. famata isolados desses animais apresentaram alta patogenicidade, mostrando a importância do estudo da microbiota fúngica de répteis e anfíbios.

Antifungal activity of deferiprone and EDTA against Sporothrix spp.: Effect on planktonic growth and biofilm formation.

The present study evaluated the antifungal activity of the chelators deferiprone (DFP) and ethylenediaminetetraacetic acid (EDTA) and their effect on biofilm formation of the S. schenckii complex. Eighteen strains of Sporothrix spp. (seven S. brasiliensis, three S. globosa, three S. mexicana and five Sporothrix schenckii sensu stricto) were used. Minimum inhibitory concentration (MIC) values for EDTA and DFP against filamentous forms of Sporothrix spp. ranged from 32 to 128 µg/ml. For antifungal drugs, MIC values ranged from 0.25 to 4 µg/ml for amphotericin B, from 0.25 to 4 µg/ml for itraconazole, and from 0.03 to 0.25 µg/ml for terbinafine. The chelators caused inhibition of Sporothrix spp. in yeast form at concentrations ranging from 16 to 64 µg/ml (for EDTA) and 8 to 32 µg/ml (for DFP). For antifungal drugs, MIC values observed against the yeast varied from 0.03 to 0.5 µg/ml for AMB, 0.03 to 1 µg/ml for ITC, and 0.03 to 0.13 µg/ml for TRB. Both DFP and EDTA presented synergistic interaction with antifungals against Sporothrix spp. in both filamentous and yeast form. Biofilms formed in the presence of the chelators (512 µg/ml) showed a reduction of 47% in biomass and 45% in metabolic activity. Our data reveal that DFP and EDTA reduced the growth of planktonic cells of Sporothrix spp., had synergistic interaction with antifungal drugs against this pathogen, and reduced biofilm formation of Sporothrix spp. LAY SUMMARY: Our data reveal that iron chelators deferiprone and ethylenediaminetetraacetic acid reduced the growth of planktonic cells of Sporothrix spp. as well as had synergistic interaction with antifungal drugs against this pathogen and reduced biofilm formation of Sporothrix spp.

Diclofenac exhibits synergism with azoles against planktonic cells and biofilms of Candida tropicalis.

This study aimed to evaluate the effect of diclofenac on minimum inhibitory concentrations of antifungals against planktonic cells and biofilms of Candida tropicalis. Susceptibility testing of planktonic cells was evaluated using the broth microdilution assay and checkerboard method. Biofilm formation by C. tropicalis in the presence of diclofenac, alone or in combination with antifungals, was also evaluated, and scanning electron microscope (SEM) and confocal microscope (CLSM) analyses were performed. Diclofenac showed an MIC of 1024 µg ml-1 against planktonic cells. The MICs of fluconazole and voriconazole against azole-resistant isolates were reduced 8- to 32-fold and 16- to 256-fold, respectively, when in combination with diclofenac. When in combination with fluconazole or voriconazole, diclofenac reduced the antifungal concentration necessary to inhibit C. tropicalis biofilm formation. In conclusion, diclofenac presents synergism with fluconazole and voriconazole against resistant C. tropicalis strains and improves the activity of these azole drugs against biofilm formation.

Ex vivo biofilm-forming ability of dermatophytes using dog and cat hair: an ethically viable approach for an infection model.

The aim of this study was to establish an ex vivo model for dermatophyte biofilm growth, using hair from dogs and cats. Strains of Microsporum canis, M. gypseum, Trichophyton mentagrophytes and T. tonsurans were assessed for in vitro and ex vivo biofilm production. All T. mentagrophytes and T. tonsurans isolates and 8/12 M. canis and 1/7 M. gypseum isolates formed biofilms in vitro, while all tested isolates presented biofilm growth on ex vivo models. T. mentagrophytes and M. canis formed more homogeneous and better-structured biofilms with greater biomass production on cat hair but T. tonsurans formed more biofilm on dog hair. Confocal and scanning electron microscopy demonstrated fungal hyphae colonizing and perforating the hair shaft, abundant fungal conidia, biofilm extracellular matrix and biofilm water channels. The present study demonstrated an ex vivo model for the performance of studies on biofilm formation by dermatophytes, using dog and cat hair.

Antifungal effects of the flavonoids kaempferol and quercetin: a possible alternative for the control of fungal biofilms.

This study aimed to determine the minimum inhibitory concentration (MIC) of kaempferol and quercetin against planktonic and biofilm forms of the Candida parapsilosis complex. Initially, nine C. parapsilosis sensu stricto, nine C. orthopsilosis and nine C. metapsilosis strains were used. Planktonic susceptibility to kaempferol and quercetin was assessed. Growing and mature biofilms were then exposed to the flavonoids at MIC or 10xMIC, respectively, and theywere also analyzed by confocal laser scanning microscopy. The MIC ranges were 32-128 µg ml-1 for kaempferol and 0.5-16 µg ml-1 for quercetin. Kaempferol and quercetin decreased (P < 0.05) the metabolic activity and biomass of growing biofilms of the C. parapsilosis complex. As for mature biofilms, the metabolic effects of the flavonoids varied, according to the cryptic species, but kaempferol caused an overall reduction in biofilm biomass. Microscopic analyses showed restructuring of biofilms after flavonoid exposure. These results highlight the potential use of these compounds as sustainable resources for the control of fungal biofilms.

Potassium iodide and miltefosine inhibit biofilms of Sporothrix schenckii species complex in yeast and filamentous forms.

This study aimed to evaluate the yeast biofilm growth kinetics and ultrastructure of Sporothrix schenckii complex and assess their mature biofilm susceptibility in filamentous and yeast forms to potassium iodide (KI) and miltefosine (MIL). Yeast biofilms were evaluated by crystal violet staining, XTT reduction assay and microscopic techniques. Susceptibility of planktonic and sessile cells was analyzed by broth microdilution. S. schenckii complex in yeast form produced biofilms, with an optimum maturation at 96 h, showing multilayered blastoconidia embedded in extracellular matrix. KI and MIL minimum inhibitory concentration (MIC) ranges against planktonic cells were 62,500-250,000 µg/ml and 0.125-4 µg/ml, respectively. KI and MIL reduced biofilm metabolic activity by 75.4% and 67.7% for filamentous form and 55.1% and 51.6% for yeast form, respectively. This study demonstrated that S. schenckii complex forms biofilms in vitro, and potassium iodide and miltefosine inhibit Sporothrix spp. biofilms in both filamentous and yeast forms.

Exposure of Candida parapsilosis complex to agricultural azoles: An overview of the role of environmental determinants for the development of resistance.

This work investigated the phenotypic behavior of Candida parapsilosis species complex in response to exposure to agricultural azoles and fluconazole. Three fluconazole-susceptible strains of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were used. Initial minimum inhibitory concentrations (iMICs) for agricultural and clinical azoles were determined by broth microdilution. Then, the strains were exposed to tebuconazole, tetraconazole and fluconazole for 15 days, at concentrations that were two-folded daily, starting at one-eighth the iMIC (iMIC/8) up to 64 times iMIC (64xiMIC). After 15-day-exposure, antifungal susceptibility, biofilm formation, CDR, MDR and ERG expression were evaluated. The three cryptic species developed tolerance to the antifungals they were exposed and presented reduction (P < 0.05) in fluconazole susceptibility. In addition, C. parapsilosis sensu stricto and C. metapsilosis also presented reduced susceptibility to voriconazole, after fluconazole exposure. Azole exposure decreased (P < 0.05) biofilm production by C. parapsilosis sensu stricto and C. orthopsilosis and increased (P < 0.05) the expression of ERG11 in all tested strains. The results show that exposure to agricultural azoles and fluconazole induces changes in the phenotypic behavior and gene expression by the three cryptic species of C. parapsilosis complex, highlighting the importance of environmental determinants for the development of antifungal resistance.

Malassezia pachydermatis from animals: Planktonic and biofilm antifungal susceptibility and its virulence arsenal.

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, biofilm-forming ability, protease, phospholipase, hemolysin and melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to>64 µg/mL for azole derivatives, 1 to >16 µg/mL for amphotericin B and 0.03 to 0.25 µg/mL for terbinafine. All strains were classified as strong biofilm producers, and ketoconazole, fluconazole and amphotericin B presented the best inhibitory effect against mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation in the presence of L-DOPA. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. This yeast species produces important virulence factors and presents high pathogenicity, corroborating its clinical importance.

In vitro activity of azole derivatives and griseofulvin against planktonic and biofilm growth of clinical isolates of dermatophytes.

As shown by recent research, most of the clinically relevant fungi, including dermatophytes, form biofilms in vitro and in vivo, which may exhibit antimicrobial tolerance that favour recurrent infections. The aim of this study was to determine the minimum inhibitory concentrations (MICs) of itraconazole (ITC), voriconazole (VCZ) and griseofulvin (GRI) against Trichophyton rubrum, Trichophyton tonsurans, Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum in planktonic and biofilm growth. For the planktonic form, susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI), document M38-A2, while biofilm susceptibility was evaluated using the XTT colorimetric essay. The planktonic growth of all strains was inhibited, with MIC values ranging from 0.00195 to 0.1225 µg/mL for VRC, 0.00195 to 0.25 µg/mL for ITC and <0.0039 to 4 µg/mL for GRI, while a 50-fold increase in the MIC was required to significantly reduce the metabolic activity (P < .05) of dermatophyte biofilms. In brief, the ability of dermatophytes to form biofilms may be a contributing factor for the recalcitrance of dermatophytoses or the dissemination of the disease.

Quantitative and structural analyses of the in vitro and ex vivo biofilm-forming ability of dermatophytes.

PURPOSE: The aim of this study was to evaluate the in vitro and ex vivo biofilm-forming ability of dermatophytes on a nail fragment. METHODOLOGY: Initially, four isolates of Trichophyton rubrum, six of Trichophyton tonsurans, three of Trichophyton mentagrophytes, ten of Microsporum canis and three of Microsporum gypseum were tested for production biomass by crystal violet assay. Then, one strain per species presenting the best biofilm production was chosen for further studies by optical microscopy (Congo red staining), confocal laser scanning (LIVE/DEAD staining) and scanning electron (secondary electron) microscopy. RESULTS: Biomass quantification by crystal violet assay, optical microscope images of Congo red staining, confocal microscope and scanning electron microscope images revealed that all species studied are able to form biofilms both in vitro and ex vivo, with variable density and architecture. M. gypseum, T. rubrum and T. tonsurans produced robust biofilms, with abundant matrix and biomass, while M. canis produced the weakest biofilms compared to other species. CONCLUSION: This study sheds light on biofilms of different dermatophyte species, which will contribute to a better understanding of the pathophysiology of dermatophytosis. Further studies of this type are necessary to investigate the processes involved in the formation and composition of dermatophyte biofilms.

Azole resistance in Candida albicans from animals: Highlights on efflux pump activity and gene overexpression.

This study investigated potential mechanisms of azole resistance among Candida albicans from animals, including efflux pump activity, ergosterol content and gene expression. For this purpose, 30 azole-resistant C. albicans strains from animals were tested for their antifungal susceptibility, according to document M27-A3, efflux pump activity by rhodamine 6G test, ergosterol content and expression of the genes CDR1, CDR2, MDR1, ERG11 by RT-qPCR. These strains were resistant to at least one azole derivative. Resistance to fluconazole and itraconazole was detected in 23 and 26 strains respectively. Rhodamine 6G tests showed increased activity of efflux pumps in the resistant strains, showing a possible resistance mechanism. There was no difference in ergosterol content between resistant and susceptible strains, even after fluconazole exposure. From 30 strains, 22 (73.3%) resistant animal strains overexpressed one or more genes. From this group, 40.9% (9/22) overexpressed CDR1, 18.2% (4/22) overexpressed CDR2, 59.1% (13/22) overexpressed MDR1 and 54.5% (12/22) overexpressed ERG11. Concerning gene expression, a positive correlation was observed only between CDR1 and CDR2. Thus, azole resistance in C. albicans strains from animals is a multifactorial process that involves increased efflux pump activity and the overexpression of different genes.

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.

Enterobacteria and Vibrio from Macrobrachium amazonicum prawn farming in Fortaleza, Ceará, Brazil.

OBJECTIVE: To investigate the isolation of enterobacteria associated with Macrobrachium amazonicum (M. amazonicum) farming and evaluate the in vitro antimicrobial susceptibility of Vibrio strains. METHODS: Strains were isolated from female M. amazonicum prawns and environmental and hatchery water. Biochemical assays were used to identify bacterial genera and those belonging to the genus Vibrio were submitted to further analyses for species identification, through Vitek 2 automated system and serotyping. Susceptibility test was performed according to Clinical Laboratory Standards Institute. RESULTS: The following genera of enterobacteria were recovered: Enterobacter (n = 11), Citrobacter (n = 10), Proteus (n = 2), Serratia (n = 2), Kluyvera (n = 2), Providencia (n = 2), Cedecea (n = 1), Escherichia (n = 1), Edwardsiella (n = 1) and Buttiauxella (n = 1). As for Vibrio, three species were identified: Vibrio cholerae non-O1/non-O139 (n = 4), Vibrio vulnificus (V. vulnificus) (n = 1) and Vibrio mimicus (n = 1). Vibrio spp. showed minimum inhibitory concentrations values within the susceptibility range established by Clinical Laboratory Standards Institute for almost all antibiotics, except for V. vulnificus, which presented intermediate profile to ampicillin. CONCLUSIONS: Enterobacteria do not seem to be the most important pathogens associated with M. amazonicum farming, whereas the recovery of Vibrio spp. from larviculture, with emphasis on Vibrio cholerae and V. vulnificus, deserves special attention due to their role as potentially zoonotic aquaculture-associated pathogens. Furthermore, the intermediate susceptibility of V. vulnificus to ampicillin reflects the importance of monitoring drug use in prawn farming.

Trends in antifungal susceptibility and virulence of Candida spp. from the nasolacrimal duct of horses.

This was a cross-sectional study to investigate the antifungal susceptibility and production of virulence factors in strains of Candida isolated from the outlet and the lumen of the nasolacrimal duct of horses in the state of Ceará, Brazil. The samples were obtained from 103 horses. Sterile cotton swabs were used to collect the material from the outlet of the nasolacrimal duct and urethral probes, for the instillation of 2 ml of saline solution, were used to collect samples from the lumen of the nasolacrimal duct. A total of 77 Candida isolates were obtained, with C. famata, C. tropicalis, C. guilliermondii, and C. parapsilosis sensu lato as the most prevalent species. One isolate (C. glabrata) was resistant to caspofungin. One isolate was resistant only to fluconazole (C. parapsilosis sensu lato), 11 were resistant only to itraconazole (7 C. tropicalis, 2 C. guilliermondii, 1 C. famata, 1 C. parapsilosis sensu lato), while eight C. tropicalis showed resistance to both azoles. Overall, 28 isolates produced phospholipases and 12 produced proteases. These results highlight the importance of investigating the antifungal susceptibility and virulence trends of Candida spp. from the microbiota of the nasolacrimal duct of horses.