Jamille Alencar Sales

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.

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 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).

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.

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.

Candida parapsilosis complex in veterinary practice: A historical overview, biology, virulence attributes and antifungal susceptibility traits

The Candida genus is composed by yeast that commensally live as part of human and animal microbiota. In the last years, C. parapsilosis complex, composed by the cryptic species C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis, has been frequently implicated in human nosocomial infections in Europe and Latin America. In veterinary medicine, C. parapsilosis sensu lato infections have been reported in different animal species. Several putative virulence factors have been associated with the pathogenicity of this species complex, including biofilm formation and the production of proteases, phospholipases, lipases and other hydrolytic enzymes. Additionally, these species have developed antifungal resistance, especially to azole derivatives and echinocandins. Thus, considering the pathogenic potential of the C. parapsilosis species complex, along with the emergence of antifungal resistant strains, this review was designed to approach historical and biological aspects, microbiological features, virulence factors and antifungal susceptibility traits of C. parapsilosis complex from animals.

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.

Antifungal susceptibility and virulence of Candida parapsilosis species complex: an overview of their pathogenic potential

Purpose. Antifungal resistance and several putative virulence factors have been associated with the pathogenicity of the Candida parapsilosis species complex. The objective of this study was to evaluate the antifungal susceptibility, the production of virulence factors and the pathogenicity of the C. parapsilosis complex. Methodology. Overall, 49 isolates of C. parapsilosis sensu stricto, 19 C. orthopsilosis and nine C. metapsilosis were used. The planktonic and biofilm susceptibility to fluconazole, itraconazole, voriconazole, amphotericin B and caspofungin was assessed using a broth microdilution assay. Finally, the production of biofilm and hydrolytic enzymes and the fungal pathogenicity against Caenorhabditis elegans were investigated. Results/Key findings. Overall, one C. orthopsilosis was resistant to caspofungin and susceptible‐dose‐dependent to itraconazole, the other two C. orthopsilosis were susceptible‐dose‐dependent to fluconazole and itraconazole, and one C. metapsilosis was susceptible‐dose‐dependent to azoles. A total of 67.5 % of the isolates were biofilm producers. Amphotericin B and caspofungin caused the greatest reduction in the metabolic activity and biomass of mature biofilms. Phospholipase and protease production was observed in 55.1 % of C. parapsilosis sensu stricto, 42.1 % of C. orthopsilosis and 33.3 % of C. metapsilosis isolates. Moreover, 57.9 % of C. orthopsilosis and 20.4 % of C. parapsilosis sensu stricto isolates were &bgr;‐haemolytic, and all C. metapsilosis were &agr;‐haemolytic. Finally, the C. parapsilosis complex caused high mortality of C. elegans after 96 h of exposure. Conclusion. These results reinforce the heterogeneity of these cryptic species for their antifungal susceptibility, virulence and pathogenic potential, emphasizing the relevance of monitoring these emerging pathogens.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.