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.

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.

Candida tropicalis isolates obtained from veterinary sources show resistance to azoles and produce virulence factors.

Candida tropicalis has been associated with invasive candidiasis, being the first or second most common non-Candida albicans Candida species isolated in humans with candidemia and candiduria, as well as being frequently isolated from healthy animals. This study aimed to characterize C. tropicalis isolates (n = 64) obtained from several animal species regarding antifungal susceptibility and production of virulence factors. The isolates were obtained from the microbiota of healthy animals (goats, n = 25; sheep, n = 6; psittacines, n = 14; rheas, n = 6; horses, n = 2; sirenians, n = 5; shrimp, n = 1), as well as from aquatic mammals found dead in the environment (cetaceans, n = 5). The isolates were subjected to in vitro susceptibility testing by broth microdilution according to the CLSI M27-A3 protocol against amphotericin B, caspofungin, itraconazole, and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases, as well as biofilm formation. Resistance to itraconazole (n = 29) and fluconazole (n = 30) was detected among isolates from every source; resistance to both azoles was detected in 24 isolates, but none of them were resistant to amphotericin B and caspofungin. Protease production was detected in the majority of the isolates (n = 59), but phospholipase was produced by only a few of them (n = 6). The isolates showed different patterns in biofilm production, being considered strong producers (n = 41), moderate producers (n = 11), weak producers (n = 9) or non-producers (n = 3). In summary, C. tropicalis isolated from animals showed high rate of resistance to azoles, expressed virulence factors and therefore may represent a potential threat to human and animal health.

Moringa oleifera inhibits growth of Candida spp. and Hortaea werneckii isolated from Macrobrachium amazonicum prawn farming with a wide margin of safety / Moringa oleifera inibe o crescimento de Candida spp. e Hortaea werneckii isoladas da criação de camarões Macrobrachium amazonicum com ampla margem de segurança

This study aimed to evaluate the antifungal activity of M. oleifera extracts against fungi isolated from farmed prawns and test the toxicity of the extracts on larvae of Macrobrachium amazonicum. The ethanol extracts of pods, seeds, leaves, stems and flowers and chloroform extract of flowers of M. oleifera were tested against 14 strains of Candida spp. and 10 strains of Hortaea werneckii isolated from farming water and the digestive tract of M. amazonicum. Antifungal activity was determined by microdilution, based on the M27-A3 and M38-A2 CLSI documents. Toxicity was evaluated by exposing larvae of M. amazonicum at concentrations between 10-1000mg mL-1, counting dead larvae (CL50) after 24 hours. The best results were verified with the chloroform extract of flowers, acting against all tested strains, with MICs ranging from 0.019 to 2.5 mg mL-1. Ethanol extracts of leaves, flowers and seeds acted against 22/24, 21/24 and 20/24 strains, respectively. The extract of pods was only effective against strains of Candida spp. (14/24) and extract of stem only against four strains of H. werneckii (4/24). Extracts of seeds, flowers (chloroform fraction), stems and leaves showed low or no toxicity, whereas extracts of pods and flowers (ethanol fraction) showed moderate toxicity. Thus, the antifungal activity of these extracts agaisnt Candida spp. and H. werneckii was observed, a wide margin of safety for larvae of M. amazonicum, demonstrating to be promising for the sustainable management of effluents from M. amazonicum farming.

Este estudo teve como objetivo avaliar a atividade antifúngica de extratos de M. oleifera frente a fungos isolados de camarões, cultivados em água doce, e testar a toxicidade dos extratos em larvas de Macrobrachium amazonicum. Os extratos etanólicos de vagens, sementes, folhas, caules e flores e o extrato clorofórmico de flores de M. oleifera foram testados contra 14 cepas de Candida spp. e 10 cepas de Hortaea werneckii isolados da água de cultivo e do trato digestório de M. amazonicum. A atividade antifúngica foi determinada por microdiluição, com base nos documentos M27-A3 e M38-A2 do CLSI. A toxicidade foi avaliada por exposição das larvas de M. amazonicum a concentrações entre 10-1000 mg mL-1 dos extratos, realizando contagem de larvas mortas (CL50), após 24 horas. Os melhores resultados foram verificados com o extrato clorofórmico de flores, agindo frente a todas as cepas testadas, com concentrações inibitórias mínimas variando entre 0,019-2,5 mg mL-1. O extrato etanólico de folhas, flores e sementes agiu ante 22/24, 21/24 e 20/24 cepas, respectivamente. O extrato de vagens foi eficaz contra cepas de Candida spp. (14/24) e o extrato de caule apenas contra quatro cepas de H. werneckii (4/24). Os extratos de sementes, flores (fração clorofórmica), caules e folhas apresentaram baixa ou nenhuma toxicidade, enquanto que extratos de vagens e flores (fração etanólica) apresentaram toxicidade moderada. Assim, observou-se atividade antifúngica dos extratos em Candida spp . e H. werneckii com uma ampla margem de segurança para as larvas de M. amazonicum, demonstrando ser promissor para o manejo sustentável dos efluentes do cultivo de M. amazonicum .

Macrobrachium amazonicum: an alternative for microbiological monitoring of aquatic environments in Brazil / Macrobrachium amazonicum uma alternativa para o monitoramento microbiológico de ambientes aquáticos no Brasil

This study aimed to evaluate the role of the Amazon River prawn, Macrobrachium amazonicum, as carrier of Candida spp., by analyzing the correlation between Candida spp. from these prawns and their environment (surface water and sediment), through M13-PCR fingerprinting and RAPD-PCR. For this purpose, 27 strains of Candida spp. were evaluated. These strains were recovered from the gastrointestinal tract of adult M. amazonicum (7/27) from Catú Lake, Ceará State, Brazil and from the aquatic environment (surface water and sediment) of this lake (20/27). Molecular comparison between the strains from prawns and the aquatic environment was conducted by M13-PCR fingerprinting and RAPD-PCR, utilizing the primers M13 and OPQ16, respectively. The molecular analysis revealed similarities between the band patterns of eight Candida isolates with the primer M13 and 11 isolates with the primer OPQ16, indicating that the same strains are present in the digestive tract of M. amazonicum and in the aquatic environment where these prawns inhabit. Therefore, these prawns can be used as sentinels for environmental monitoring through the recovery of Candida spp. from the aquatic environment in their gastrointestinal tract.

Este estudo teve como objetivo avaliar o papel do camarão Macrobrachium amazonicum como carreador de Candida spp. do ambiente aquático, por meio da análise da correlação entre Candida spp. isoladas desse camarão e do seu ambiente (água de superfície e sedimento) através das técnicas de M13-PCR fingerprinting e RAPD-PCR. Para tanto, 27 cepas de Candida spp. foram avaliadas. Essas cepas foram recuperadas a partir do trato gastrointestinal de M. amazonicum adultos (7/27), oriundos da lagoa do Catú, Ceará, Brasil, e do meio aquático (águas superficiais e sedimentos) desse lago (20/27). A comparação molecular entre as cepas de camarões e o ambiente aquático foi conduzida por M13-PCR fingerprinting e RAPD-PCR, utilizando os iniciadores M13 e OPQ16, respectivamente. A análise molecular revelou semelhanças entre os padrões de bandas de oito isolados de Candida com o iniciador M13 e 11 isolados com o primer OPQ16, indicando que elas estão presentes no trato digestivo de M. amazonicum e no ambiente aquático, onde esses camarões habitam. Portanto, essa espécie de camarão pode ser usada como sentinela para monitoramento ambiental através da recuperação de Candida spp. do ambiente aquático, a partir do seu trato gastrointestinal.

Antifungal susceptibility and virulence attributes of animal-derived isolates of Candida parapsilosis complex.

This study aimed to identify strains of the Candida parapsilosis complex isolated from animals, as well as to assess their in vitro antifungal susceptibility profile and in vitro production of virulence attributes. We used 28 isolates of C. parapsilosis sensu lato recovered from clinically healthy animals. The strains were characterized phenotypically, followed by molecular identification of the species through PCR-restriction enzyme analysis. The susceptibility of the strains to amphotericin B, itraconazole, voriconazole, fluconazole and caspofungin was assessed through broth microdilution. Additionally, the ability of the strains to produce biofilm, phospholipases and proteases was analysed. Molecular analysis showed 13 C. parapsilosis sensu stricto, 10 Candida orthopsilosis and five Candida metapsilosis strains. In vitro resistance to fluconazole was observed in three strains of C. parapsilosis sensu stricto and two C. metapsilosis. All tested strains were able to form biofilms and 23/28 isolates presented protease production, whilst none was able to produce phospholipases. Our study showed that C. parapsilosis sensu stricto and C. orthopsilosis are the most common species of the C. parapsilosis species complex and that these cryptic species present no significant phenotypical differences.

The calcineurin inhibitor cyclosporin A exhibits synergism with antifungals against Candida parapsilosis species complex.

Candida parapsilosis complex comprises three closely related species, C. parapsilosis sensu stricto, Candida metapsilosis and Candida orthopsilosis. In the last decade, antifungal resistance to azoles and caspofungin among C. parapsilosis sensu lato strains has been considered a matter of concern worldwide. In the present study, we evaluated the synergistic potential of antifungals and the calcineurin inhibitor cyclosporin A (Cys) against planktonic and biofilms of C. parapsilosis complex from clinical sources. Susceptibility assays with amphotericin, fluconazole, voriconazole, caspofungin and Cys were performed by microdilution in accordance with Clinical and Laboratory Standards Institute guidelines. Synergy testing against planktonic cells of C. parapsilosis sensu lato strains was assessed by the chequerboard method. Combinations formed by antifungals with Cys were evaluated against mature biofilms in microtitre plates. No differences in the antifungal susceptibility pattern among species were observed, but C. parapsilosis sensu stricto strains were more susceptible to Cys than C. orthopsilosis and C. metapsilosis. Synergism between antifungals and Cys was observed in C. parapsilosis sensu lato strains. Combinations formed by antifungals and Cys were able to prevent biofilm formation and showed an inhibitory effect against mature biofilms of C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis. These results strengthen the potential of calcineurin inhibition as a promising approach to enhance the efficiency of antifungal drugs.

Species of Candida as a component of the nasal microbiota of healthy horses.

Respiratory infections are a common problem among equines and occur with variable rates of morbidity and mortality. Although some fungal species are considered primary agents of respiratory tract infections in several mammals, their relevance in respiratory diseases of equines is frequently neglected. In the present study, we performed an active search for Candida spp. in the nasal cavity of horses. The presence of Candida spp. was investigated through the use of nasal swabs that were streaked on culture media. These yeasts were identified through physiological testing and their in vitro antifungal susceptibility were also characterized. The analysis of the material from the nasal cavity of 97 randomly chosen horses resulted in the isolation of Candida spp. from 35 animals (36.08%), out of which 18 (32.14%) were C. famata, 14 (25%) C. parapsilosis, 12 (21.42%) Meyerozyma guilliermondii (C. guilliermondii), 11 (19.64%) C. tropicalis and 1 (1.78%) Wickerhamomyces anomalus (C. pelliculosa). The minimum inhibitory concentration (MIC) values ranged from 0.03125-1 µg/ml for amphotericin B; and from 0.03125-> 16 µg/ml and 0.125 to > 64 µg/ml for itraconazole and fluconazole, respectively. Resistance to fluconazole and itraconazole was observed among C. tropicalis (n = 3) and C. guilliermondii (n = 1). The data show a predominance of non-C. albicans Candida species in the nasal microbiota of healthy equines, including antifungal resistant isolates, reiterating the importance of monitoring fungal pathogens in these animals.

Detection of Candida species resistant to azoles in the microbiota of rheas (Rhea americana): possible implications for human and animal health.

There is growing interest in breeding rheas (Rhea americana) in Brazil. However, there are no data on the yeast microbiota of the gastrointestinal tract of this avian species, and the phenotypic characteristics of these yeasts are not known. Therefore, the aim of this work was to isolate Candida species from the digestive tract of rheas and to evaluate the in vitro antifungal susceptibility and secretion of phospholipases of the recovered isolates. For this purpose, 58 rheas from breeding operations in the cities of Fortaleza and Mossoró, north-eastern Brazil, were used. Samples were gathered from the oropharynx and cloaca of the animals using sterile swabs. Stool samples were collected from their pens by scraping with a scalpel blade. For the primary isolation, the material was seeded onto 2 % Sabouraud dextrose agar supplemented with chloramphenicol (0.5 g l(-1)). The isolates were identified based on morphological and biochemical features. After identification, all the strains were submitted to antifungal susceptibility testing for amphotericin B, itraconazole and fluconazole. The phospholipase activity of the Candida species isolates was also tested by culturing on egg yolk agar. Candida species were isolated from at least one anatomical site in 36/58 birds (14/17 juveniles and 22/41 adults) and in 6/10 faecal samples. Mostly, only a single species was isolated from each collection site (36/56 positive sites), with up to three species being observed only in four cases (4/56). A total of 77 isolates were obtained, belonging to the species Candida parapsilosis sensu lato (19), Candida albicans (18), Candida tropicalis (13), Candida guilliermondii (12), Candida krusei (10) and Candida famata (5). C. albicans was more prevalent in the oropharynx of the juvenile rheas when compared with adult ones (P<0.001). All tested isolates were susceptible to amphotericin B, but 16 isolates were simultaneously resistant to the two azole derivatives (11/18 C. albicans, 1/10 C. krusei, 2/19 C. parapsilosis sensu lato and 2/13 C. tropicalis). C. albicans presented a particularly high resistance rate to fluconazole (15/18) and itraconazole (13/18). Finally, 23/77 strains secreted phospholipases. In summary, healthy rheas carry potentially pathogenic Candida species in their gastrointestinal tract, including azole-resistant strains that secrete phospholipases, and are prone to disseminating them in the environment. Thus, breeding and handling these animals may have some implications for human and animal health.

Farnesol inhibits in vitro growth of the Cryptococcus neoformans species complex with no significant changes in virulence-related exoenzymes.

Farnesol is a sesquiterpene alcohol that modulates cell-to-cell communication in Candida albicans. In recent years, several studies have shown that this molecule presents inhibitory effects against non-albicans Candida species, Paracoccidioides brasiliensis and bacteria. The present study aimed at determining the effect of farnesol on the growth of strains of the Cryptococcus neoformans species complex, through microdilution assays. In addition, the effect of farnesol on the synthesis of phospholipase and protease - important virulence-associated enzymes - by C. neoformans and Cryptococcus gattii was also investigated. A total of 36 strains were studied, out of which 20 were from veterinary sources, 8 were from human cases and 8 were from a reference collection. The minimum inhibitory concentrations (MICs) were determined in accordance with the M27-A3 protocol as described by the CLSI and farnesol was tested at a concentration range of 0.29-150 µM. Phospholipase and protease activities were evaluated through growth on egg yolk agar and spectrophotometry, respectively, after pre-incubating the strains at different farnesol concentrations (MIC/4, MIC/2 and MIC). It was observed that farnesol presents an inhibitory activity against C. neoformans and C. gattii (MIC range: 0.29-75.0 µM). Although farnesol did not significantly alter phospholipase activity, a tendency to decrease this activity was observed. Concerning protease, no statistically significant differences were observed when comparing the production before and after pre-incubation at different farnesol concentrations. Based on these findings, it can be concluded that farnesol has in vitro inhibitory activity against C. neoformans and C. gattii, but has little impact on the production of the analyzed virulence factors.

Extratos de Moringa oleifera e Vernonia sp. sobre Candida albicans e Microsporum canis isolados de cães e gatos e análise da toxicidade em Artemia sp. / Moringa oleifera and Vernonia sp. extracts against Candida albicans and Microsporum canis isolates from dogs and cats and analysis of toxicity to Artemia sp.

O aumento da incidência das infecções fúngicas, bem como o registro crescente de resistência e falha terapêutica, têm impulsionado a realização de estudos de prospecção de fitoquímicos com propriedades antifúngicas. Diante do exposto, o presente estudo investigou o potencial antifúngico de extratos de Baccharis ligustrina, B. schultzii, Croton jacobinensis, Licania rigida, Moringa oleifera, Vernonia sp. e V. brasiliana, e de óleos essenciais de Lippia alba (Quimiotipos 1, 2, 3 e 4) e Ocimum gratissimum. Inicialmente, foi realizada uma avaliação qualitativa da atividade antifúngica de cada amostra por meio do método de difusão em ágar, frente a cepas de Candida albicans e Microsporum canis, mostrando que apenas os extratos de M. oleifera (MLF-C) e Vernonia sp. (TVS-H) apresentaram atividade frente a C. albicans e M. canis, com halos de inibição =10mm. Também foram determinadas a concentração inibitória mínima (CIM), frente a 12 cepas de C. albicans e M. canis, e a toxicidade aguda de MLF-C e TVS-H, através de protocolos do Clinical and Laboratory Standards Institute (CLSI) e ensaio com Artemia sp., respectivamente. A CIM (80 por cento) de MLF-C e TVS-H variou de 0,156 a 2,5mg mL-1 frente C. albicans e de 0,039 a 1,25 e 0,039 a 0,625mg mL-1 para M. canis, respectivamente. A CIM (100 por cento) de MLF-C e TVS-H variou de 0,625 a 2,5mg mL-1 frente C. albicans é de 0,039 a 2,5 e 0,078 a 1,25mg mL-1 para M. canis, respectivamente. As doses letais (DL50) para o MLF-C e TVS-H foram de 201,09 e 204,17mg mL-1, respectivamente, sendo, portanto, demonstrada a baixa toxicidade desses extratos. Os extratos de M. oileifera e Vernonia sp. apresentaram atividade antifúngica frente cepas de C. albicans e M. canis, abrindo a perspectiva de estudos para caracterização dos seus componentes bioativos.

The increase in the incidence of fungal infections and the frequent report of resistance and therapeutic failure has promoted the performance of phytochemical screening for compounds with antifungal properties. Based on this, the present study investigated the antifungal potential of extracts of Baccharis ligustrina, B. schultzii, Croton jacobinensis, Licania rigida, Moringa oleifera, Vernonia sp. and V. brasiliana and of essential oils of Lippia alba (Chemotypes 1, 2, 3 and 4) and Ocimum gratissimum. Initially, a qualitative evaluation of the antifungal activity of each vegetal sample against strains of Candida albicans and Microsporum canis, through the agar diffusion method, was performed. Extracts of M. oleifera (MLF-C) and Vernonia sp. (TVS-H) presented activity against C. albicans and M. canis with inhibition halos =10mm. Then, minimum inhibitory concentrations (MICs) for MLF-C and TVS-H against 12 strains of C. albicans and M. canis were determined through the methodology established by the Clinical and Laboratory Standards Institute (CLSI), and acute toxicity tests against Artemia sp. were performed for both extracts. MICs (80 percent) for MLF-C and TVS-H varied from 0.156 to 2.5mg mL-1 against C. albicans and from 0.039 to 1.25mg mL-1 and 0.039 to 0.625mg mL-1 against M. canis, respectively. MICs (100 percent) for MLF-C and TVS-H varied from 0.625 to 2.5mg mL-1 for C. albicans and from 0.039 to 2.5mg mL-1 and 0.078 to 1.25mg mL-1 against M. canis, respectively. Lethal doses (DL50) of MLF-C and TVS-H were 201.09 and 204.17mg mL-1, respectively, being, therefore, demonstrated the low toxicity of these extracts. M. oleifera and Vernonia sp. extracts presented in vitro antifungal activity against C. albicans and M. canis, creating perspectives for the development of studies on the characterizations of their bioactive components.