Glaucia Morgana de Melo Guedes

Cross-resistance to fluconazole induced by exposure to the agricultural azole tetraconazole: an environmental resistance school?

This study aimed to investigate the influence of tetraconazole and malathion, both used in agricultural activities, on resistance to fluconazole, itraconazole and voriconazole in Candida parapsilosis ATCC 22019. The susceptibility to tetraconazole, malathion, fluconazole, itraconazole and voriconazole, through broth microdilution. Then, 12 independent replicates, were separated and exposed to four treatment groups, each one containing three replicates: G1: tetraconazole; G2: malathion; G3: fluconazole (positive control); G4: negative control. Replicates from G1, G2 and G3, were exposed to weekly increasing concentrations of tetraconazole, malathion and fluconazole, respectively, ranging from MIC/2 to 32 × MIC, throughout 7 weeks. The exposure to tetraconazole, but not malathion, decreased susceptibility to clinical azoles, especially fluconazole. The tetraconazole‐induced fluconazole resistance is partially mediated by the increased activity of ATP‐dependent efflux pumps, considering the increase in antifungal susceptibility after the addition of the efflux pump inhibitor, promethazine, and the increase in rhodamine 6G efflux and CDR gene expression in the G1 replicates. Moreover, MDR expression was only detected in G1 and G3 replicates, suggesting that MDR pumps are also involved in tetraconazole‐induced fluconazole resistance. It is noteworthy that tetraconazole and fluconazole‐treated replicates behaved similarly, therefore, resistance to azoles of clinical use may be a consequence of using azoles in farming activities.

Terpinen-4-ol, tyrosol, and β-lapachone as potential antifungals against dimorphic fungi

This study aimed to evaluate the in vitro antifungal activity of terpinen-4-ol, tyrosol, and β-lapachone against strains of Coccidioides posadasii in filamentous phase (n = 22) and Histoplasma capsulatum in both filamentous (n = 40) and yeast phases (n = 13), using the broth dilution methods as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these compounds. The mechanisms of action of these compounds were also investigated by analyzing their effect on cell membrane permeability and ergosterol synthesis. The MIC and MFCf these compounds against C. posadasii, mycelial H. capsulatum, and yeast-like H. capsulatum, were in the following ranges: 350–5720 μg/mL, 20–2860 μg/mL, and 40–1420 μg/mL, respectively for terpinen-4-ol; 250–4000 μg/mL, 30–2000 μg/mL, and 10–1000 μg/mL, respectively, for tyrosol; and 0.48–7.8 μg/mL, 0.25–16 μg/mL, and 0.125–4 μg/mL, respectively for β-lapachone. These compounds showed a decrease in MIC when the samples were subjected to osmotic stress, suggesting that the compounds acted on the fungal membrane. All the compounds were able to reduce the ergosterol content of the fungal strains. Finally, tyrosol was able to cause a leakage of intracellular molecules.

Antiretroviral drugs saquinavir and ritonavir reduce inhibitory concentration values of itraconazole against Histoplasma capsulatum strains in vitro.

Recent studies have shown that some drugs that are not routinely used to treat fungal infections have antifungal activity, such as protease inhibitor antiretroviral drugs. This study investigated the in vitro susceptibility of Histoplasma capsulatum var. capsulatum to saquinavir and ritonavir, and its combination with the antifungal itraconazole. The susceptibility assay was performed according to Clinical and Laboratory Standards Institute guidelines. All strains were inhibited by the protease inhibitor antiretroviral drugs. Saquinavir showed minimum inhibitory concentrations ranging from 0.125 to 1μgmL(-1) for both phases, and ritonavir presented minimum inhibitory concentrations ranging from 0.0312 to 4μgmL(-1)and from 0.0625 to 1μgmL(-1) for filamentous and yeast phase, respectively. Concerning the antifungal itraconazole, the minimum inhibitory concentration values ranged from 0.0019 to 0.125μgmL(-1) and from 0.0039 to 0.0312μgmL(-1) for the filamentous and yeast phase, respectively. The combination of saquinavir or ritonavir with itraconazole was synergistic against H. capsulatum, with a significant reduction in the minimum inhibitory concentrations of both drugs against the strains (p<0.05). These data show an important in vitro synergy between protease inhibitors and itraconazole against the fungus H. capsulatum.

Yeasts from the microbiota of bats: a focus on the identification and antimicrobial susceptibility of cryptic species of Candida.

Bats harbour several pathogens that can be disseminated through their faeces, hence becoming important sources of environmental contamination once they are able to fly long distances (Botelho et al., 2012). Yeasts colonize the gastrointestinal tract of different animal species (Brilhante et al., 2013), but reports on the composition and antifungal susceptibility of the yeast microbiota of bats are scarce. Therefore, this study aimed at isolating yeasts from bats and their droppings, investigating the occurrence of the cryptic species Candida albicans–Candida dubliniensis, Candida parapsilosis complex, Candida famata complex and Candida guilliermondii complex and assessing the antifungal susceptibility of the recovered isolates. This project was approved by the Chico Mendes Institute of Biodiversity (licence 45268-1) and the Ethics Committee for the Use of Animals of the State University of Cear a (protocol 4797909/2014). Animals were captured in Fortaleza and Metropolitan Region, state of Cear a, Brazil, from January to April 2015, with mist or dip nets. Bat species were identified according to Reis et al. (2007).

Farnesol increases the susceptibility of Burkholderia pseudomallei biofilm to antimicrobials used to treat melioidosis.

The aim of this study was to analyse the in vitro activity of farnesol alone and combined with the antibacterial drugs amoxicillin, doxycycline, ceftazidime and sulfamethoxazole–trimethoprim against Burkholderia pseudomallei biofilms.

Virulence and antimicrobial susceptibility of clinical and environmental strains of Aeromonas spp. from northeastern Brazil.

The aims of the present study were to isolate and identify clinical and environmental strains of Aeromonas spp. by means of biochemical tests and the automated method VITEK 2 and to investigate the presence of the virulence genes cytotoxic enterotoxin (act), hemolysin (asa-1), and type III secretion system (ascV), and also the in vitro antimicrobial susceptibility of the strains. From the clinical isolates, 19 Aeromonas hydrophila, 3 Aeromonas veronii bv. sobria, and 1 Aeromonas caviae were identified, while from the environmental strains, 11 A. hydrophila, 22 A. veronii bv. sobria, 1 A. veronii bv. veronii, and 1 A. caviae were recovered. The gene act was detected in 69.5% of clinical isolates, asa-1 in 8.6%, and ascV in 34.7%. In the environmental strains, the detection rates were 51.4%, 45.7%, and 54.2% for the genes act, asa-1, and ascV, respectively. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was observed in 15 and 3 clinical strains, respectively, and resistance to ceftazidime, meropenem, imipenem, ciprofloxacin, and trimethoprim-sulfamethoxazole was observed in 1 strain for each drug. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was detected in 17 and 1 environmental strain, respectively. Higher resistance percentages were observed in clinical strains, but environmental strains also showed this phenomenon and presented a higher detection rate of virulence genes. Thus, it is important to monitor the antimicrobial susceptibility and pathogenic potential of the environmental isolates.

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.

Yeast microbiota of natural cavities of manatees (Trichechus inunguis and Trichechus manatus) in Brazil and its relevance for animal health and management in captivity.

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

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.

Effect of the molecular weight of chitosan on its antifungal activity against Candida spp. in planktonic cells and biofilm

Difficulties in the treatment of Candida spp. invasive infections are usually related to the formation of biofilms. The aim of this study was to determine the effects of molecular weight (MW) of chitosan (using high (HMW), medium (MMW) and low (LMW) molecular weight chitosan) on Candida albicans, Candida tropicalis and Candida parapsilosis sensu stricto. The deacetylation degree (DD) and molecular weight M were measured by potentiometric titration and viscosimetry, respectively. The planktonic shape activity was quantified by broth microdilution, and the activity against biofilm was quantified by metabolic activity through XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]- 2H-tetrazolium hydroxide and biomass formation (crystal violet). The influence of chitosan MW on the planktonic form of Candida spp. was strain dependent. Fungal growth decreased with increasing chitosan MW for C. tropicalis and C. parapsilosis, while chitosan MW did not modulate the effect for C. albicans. With regard to the formation of biofilms, in both the adhesion and mature phases, the biomass and metabolic activities of Candida spp. were reduced by about 70% and 80%, respectively for each phase.