Carlos Eduardo Cordeiro Teixeira

Minimum inhibitory concentrations of amphotericin B, azoles and caspofungin against Candida species are reduced by farnesol

The objective of this study was to evaluate the antifungal activity of farnesol and its interaction with traditional antifungals against drug-resistant strains of Candida species. To do so, we studied the minimum in vitro inhibitory concentration (MIC) of amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), caspofungin (CAS) and farnesol against 45 isolates of Candida spp., i.e., 24 C. albicans, 16 C. parapsilosis and 5 C. tropicalis through the use of the broth microdilution method. Then, the isolates were tested with the combination of farnesol plus drugs to which they were previously found to be resistant. Additionally, the strains were pre-incubated at sub-inhibitory farnesol concentrations and their antifungal susceptibilities were re-evaluated. We found the MIC values for farnesol varied from 4.68-150 µM for Candida spp., with 19 isolates having a MIC > 1 mg/l, 18 a MIC ≥ 64 mg/l, 35 having a MIC ≥ 1 mg/l and 6 isolates a MIC ≥ 2 mg/l or were resistant to AMB, FLC, ITC and CAS, respectively. Significant MIC reductions were observed when farnesol and antifungal drugs were combined (P < 0.05) and when Candida strains were incubated with farnesol (P < 0.05). We conclude that the in vitro effects of farnesol improved the activity of traditional antifungals to which the Candida spp. isolates were resistant. These results support further investigation of the role of farnesol in the balance of the sterol biosynthetic pathway and how it interferes with cell viability.

Yeasts from Macrobrachium amazonicum: a focus on antifungal susceptibility and virulence factors of Candida spp.

In the present study, it was sought to compare yeast microbiota of wild and captive Macrobrachium amazonicum and evaluate the antifungal susceptibility and production of virulence factors by the recovered isolates of Candida spp. Additionally, cultivation water was monitored for the presence of fungi. Overall, 26 yeast isolates belonging to three genera and seven species were obtained, out of which 24 were Candida spp., with Candida famata as the most prevalent species for both wild and captive prawns. From cultivation water, 28 isolates of filamentous fungi were obtained, with Penicillium spp., Cladosporium spp. and Aspergillus spp. as the most frequent genera. Eight out of 24 Candida spp. isolates were resistant to azole derivatives, out of which four were recovered from wild-harvested prawns. As for production of virulence factors, three (12.5%) and eight (33.3%) isolates presented phospholipase and protease activity, respectively. This is the first comparative study between wild and captive prawns and the first report on yeast microbiota of M. amazonicum. The most relevant finding was the high percentage of resistant Candida spp., including from wild individuals, which suggests the occurrence of an environmental imbalance in the area where these prawns were captured.

Yeast microbiota of raptors: a possible tool for environmental monitoring

Twenty-two raptors from a rehabilitation centre were evaluated for the presence of yeasts prior to returning them to the wild, and the recovered Candida isolates were tested for in vitro antifungal susceptibility and phospholipase production. Samples were collected from the crop/lower esophagus and cloaca. In vitro antifungal susceptibility and phospholipase production of 21 Candida strains were assessed through broth microdilution and growth on egg yolk agar respectively. Twenty-seven isolates, belonging to seven species, were recovered from 16 tested birds, with C. albicans and C. famata as the most prevalent species. Three out of 21 isolates (2 C. albicans and 1 C. tropicalis) were simultaneously resistant to fluconazole and itraconazole. As for phospholipase production, 8 (8/21) isolates (6 C. albicans, 1 C. famata and 1 C. parapsilosis) showed enzymatic activity. The most relevant finding in this study was the isolation of resistant Candida spp. from wild raptors that had never been submitted to antifungal therapy, which suggests exposure to environmental contaminants. Based on this, we propose the assessment of Candida spp. from the gastrointestinal tract of raptors as a tool for environmental monitoring.

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.

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.

Azole-resistant Candida albicans from a wild Brazilian porcupine (Coendou prehensilis): a sign of an environmental imbalance?

This study aimed at evaluating the in vitro antifungal susceptibility of Candida albicans isolates obtained during necropsy of a wild Brazilian porcupine and the mechanism of azole resistance. Initially, we investigated the in vitro susceptibility of the three isolates to amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole. Afterwards, three sub-inhibitory concentrations (47, 21 and 12 mg/l) of promethazine, an efflux pump inhibitor, were tested in combination with the antifungal drugs in order to evaluate the role of these pumps in the development of antifungal resistance. In addition, the three isolates were submitted to RAPD-PCR and M13-fingerprinting analyses. The minimum inhibitory concentrations (MICs) obtained with the isolates were 1, 0.03125, 250, 125, 8 and 250 mg/l for amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole, respectively, and the isolates were found to be resistant to all tested azoles. The addition of the three subinhibitory concentrations of promethazine resulted in statistically significant (P < 0.05) reductions in the MICs for all tested drugs, with decreases to azoles being statistically greater than those for amphotericin B and caspofungin (P < 0.05). The molecular analyses showed a genetic similarity among the three tested isolates, suggesting the occurrence of candidemia in the studied animal. These findings highlight the importance of monitoring antifungal susceptibility of Candida spp. from veterinary sources, especially as they may indicate the occurrence of primary azole resistance even in wild animals.

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.

Antifolates inhibit Cryptococcus biofilms and enhance susceptibility of planktonic cells to amphotericin B.

The Cryptococcus neoformans species complex contains the most important agents of fungal meningoencephalitis. Therapeutic choices are limited and issues related to toxicity and resistance to antifungals have been described. The present study evaluated the inhibitory effect of the antifolate combinations sulfamethoxazole–trimethoprim (SMX/TMP) and sulfadiazine–pyrimethamine (SDZ/PYR) against planktonic cells and biofilms of C. neoformans and C. gattii. The influence of the antifolate combinations on the amphotericin minimum inhibitory concentration (MIC) of planktonic cells was also investigated. In addition, the effect of these combinations on the cellular ergosterol content of planktonic cells was studied. Strains of C. neoformans (n = 15) and C. gattii (n = 15) obtained from environmental or clinical sources were evaluated by the broth microdilution method. SMX/TMP and SDZ/PYR showed antifungal activity against free living cells and sessile cells of Cryptococcus spp. Moreover, planktonic cells showed increased susceptibility to amphotericin B after pre-incubation with sub-inhibitory concentrations of SMX/TMP or SDZ/PYR. The drug combinations SMX/TMP and SDZ/PYR were able to prevent the biofilm formation and showed inhibitory effect against mature biofilms of both species. Additionally, the study showed that antifolate drugs reduced the ergosterol content in C. neoformans and C. gattii planktonic cells. Our results highlight the antifungal potential of antifolate drugs.

Exogenous tyrosol inhibits planktonic cells and biofilms of Candida species and enhances their susceptibility to antifungals.

Tyrosol is a quorum-sensing molecule of Candida albicans able to induce hyphal development in the early and intermediate stages of biofilm growth. In the present study, we evaluated the effect of high concentrations of exogenous tyrosol on planktonic cells and biofilms of C. albicans (n = 10) and C. tropicalis (n = 10), and investigated whether tyrosol could be synergic to antifungals that target cellular ergosterol. Antifungal susceptibility and drug interaction against planktonic cells were investigated by the broth microdilution method. Tyrosol was able to inhibit planktonic cells, with MIC values ranging from 2.5 to 5.0 mM for both species. Synergism was observed between tyrosol/amphotericin B (11/20 strains), tyrosol/itraconazole (18/20 strains) and tyrosol/fluconazole (18/20 strains). Exogenous tyrosol alone or combined with antifungals at both 10 × MIC and 50 × MIC were able to reduce biofilm of both Candida species. Mature biofilms were susceptible to tyrosol alone at 50 × MIC or combined with amphotericin at both 10 × MIC and 50 × MIC. On the other hand, tyrosol plus azoles at both 10 × MIC and 50 × MIC enhanced biofilm growth.

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.