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

Abstract

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.

Differences in epidemiology of candidaemia in the Nordic countries – what is to blame?

“…By 2005, Denmark already used 100 tonnes of azole fungicides, nearly the same amount as Norway used of total fungicides in 2011 (http://www2.mst.dk). The agricultural azole tetraconazole was recently demonstrated to induce fluconazole cross resistance in C. parapsilosis in vitro when exposed to increasing concentrations over a 7 week period . It has also been reported that azole‐resistant Candida isolates have been found in lake water and in a porcupine in Brazil.…”

Azole resistance inCandida albicansfrom animals: Highlights on efflux pump activity and gene overexpression

“…albicans , emphasising the contribution of these pumps for the development of azole resistance. Moreover, this variability in RFU values is expected between isolates of the same species and even between clones of the same isolate, as demonstrated by Rocha et al., who reported that replicates of the same azole‐resistant isolate showed variable fluorescence values that were compatible with enhanced efflux activity. Even though no significant correlations between efflux activity and gene expression were found in this study, Bhattacharya et al.…”

“…Thus, we hypothesise that strains from veterinary sources may acquire azole resistance after exposure to environmental stressors . Even though non‐human Candida strains are not commonly submitted to selective pressures by azoles of clinical use, this pressure still occurs through the environmental exposure to this class of antifungals, especially in agricultural practices, and cross‐resistance between these drugs and those azoles used in medical and veterinary practice has been detected . Chowdhary et al., for instance reported the occurrence of azole‐resistant Aspergillus fumigatus infections in treatment‐naive patients.…”

“…This fluorescent dye is used as a tool for the evaluation of the efflux activity of these pumps. In this step, the efflux of rhodamine 6G was tested by the methodology described by Rocha et al., with modifications. Eleven randomly selected azole‐resistant C. albicans strains (numbers 3, 9, 10, 11, 18, 19, 21, 25, 27, 28 and 30) were grown in duplicate in 2 mL of YEPD broth, at 37°C, for 24 h. Afterwards, fungal inocula containing 2 × 10 7 cells mL −1 were prepared according to standard 2 on McFarland's scale, after prior validation by cell counting in Neubauer chamber and pour plate technique.…”

“…Wells containing only PBS were considered negative control for efflux activity, as they represent the basal fluorescence of each isolate. As previously reported by Rocha et al., after time 15 min, fluorescence reading stabilises tending to a plateau, so this time was used to compare the samples. For comparative purposes, two susceptible C. albicans strains from humans (C3 and C4) were added in this step of the research.…”