Antifungal Resistance Analysis of environmental isolates of Aspergillus in North india

triazoles are the major group of antifungals for treating Aspergillus infections. the morbidity and mortality associated with these infections is high and rate of treatment failure is more in patients infected with azole resistant Aspergillus. the azole resistant Aspergillus isolates have been recovered from both azole treated and azole naive patients. thus, there may be an environmental route of exposure to azole resistant Aspergillus. the present study was envisaged for the isolation and enumeration of environmental isolates of Aspergillus resistant to medically available antifungal azole drugs in North indian environment. A total of 25 soil samples were collected from North indian agricultural farms where azole pesticides were being used. the soil samples were screened for Aspergillus isolates by serial dilution pour plate method. Further, their drug susceptibility testing was performed using disc diffusion, estrip and micro-broth dilution method against medically available triazoles: itraconazole, ketoconazole, fluconazole and voriconazole. A total of 41 Aspergillus species were isolated from the soil samples. Based on conventional microscopic assay, 13 of them were identified as Aspergillus fumigatus, 9 as Aspergillus niger, 5 as Aspergillus terreus, 3 as Aspergillus nidulans, 1 as Aspergillus flavus and 9 as other Aspergillus species. Resistance for all tested antifungal drugs was detected in 7.3% Aspergillus isolates and 43.7% isolates were resistant to any of the tested azole drugs. the results demonstrated that Aspergillus isolates resistant to medical triazoles are present in the agricultural farms.


iNtRODUCtiON
Azoles are the common synthetic compounds that inhibit sterol biosynthesis. They are used as first choice of treatment against Aspergillus infections in humans. Besides, they are also used for preservation and plant protection (Horsfall, 1975;Hof, 2001;Russel, 2005; e Ribas et al., 2016). These have been widely used to treat human mycoses and plant pathogens effectively since 1970s and have similar mode of action in humans and plant fungal infections Cools et al., 2013). It has been reported that among 38 registered fungicides in India (Central Insecticide Board, Govt. of India), 14 are azole pesticides. Majority of these broad range azole fungicides used by farmers have negative impact on environment and human health (Berger et al., 2017). Large scale use of azole fungicide in agriculture is causing mutational resistance in Aspergillus species which are present in abundance in environment (Kumar et al., 2013). There have been various reports stating the emergence of cross-and multi-drug resistance of human pathogen Apergillus is related to the exposure of fungicides in the agro-ecosystem (Meneau and Sanglard, 2005; Bowyer and Denning, 2013; Lelievre et al., 2013). Azole pesticides have been used in agriculture during pre and post harvesting of crops to avoid the spoilage (Snelders et al., 2012). These pesticides persist in the soil for longer time, accumulate in plants and are transferred to humans through food web (Palazzini et al., 2018). These conditions enhance the development of azole resistant strains.
Azole drug resistance in patients suffering from Aspergillus infections has emerged as a global issue in recent years due to failure of drug efficacy, thereby increasing the mortality and morbidity rate. In India, 64%-71% of multi-triazole resistant strains have been isolated from in patients suffering from Aspergillus infections who were never been exposed to triazole treatments (Chang et al., 2016). Hence, an environmental origin for triazole resistant Aspergillus strains has been proposed. The probable reasons for the emergence of azole resistant strains in the environment include changes in drug target affinity (Perlin et al., 2015) and excessive use of azole pesticides in the agriculture Chowdhary et al., 2012). The Fungicide Resistance Action Committee (FRAC) fixes the baseline resistance level of fungi prior to commercialization of a fungicide whereas, the failures in disease control and detection of resistant isolates even below the baseline are the clear mark of the hazards of fungicide resistance development (Brent and Hollomon, 2007). The spores of the azole-pesticide resistant environmental strain of Aspergillus when inhaled by immuno-compromised individuals cause antifungal azole-resistant diseases, leading to treatment failure and deaths in patients (Pham et al., 2014).
The proposition of emergence of azole-resistant Aspergillus species due to azole fungicides in agri-ecosystem was initially suggested by studies conducted in the Netherland, where the triazole resistant Aspergillus was reported . All these epidemiological studies revealed that there is the link between triazole resistant clinical isolates and the widespread use of azole fungicides. Thus, continued surveillance of resistance in environmental Aspergillus strains is crucial. The present study was envisaged to study the prevalence of azole resistant Aspergillus species in agricultural fields of north western India.

screening and isolation of environmental A. fumigatus from soil samples
Soil samples were processed for isolation of Aspergillus colonies using serial dilution method (Chowdhary et al., 2012). Briefly, 1 g of soil was dissolved in 10 ml of 0.9% normal saline, vortexed and allowed to settle for 30 seconds. From this stock suspension, 10 -1 -10 -4 dilutions were prepared and 50µl of each dilution were spread on potato dextrose agar (PDA) plates supplemented with 0.5 mg/ml chloramphenicol. The plates were incubated for 5 days at 28°C. Each experiment was conducted in triplicates.
The macroscopic features like colony colour, texture were studied. The fungal colonies were identified by conventional microscopy and then by following mycological literature (de Hoog et al., 2000;Klich, 2002;McClenny, 2005). The filamentous colonies on PDA plates were teased apart and stained with lacto-phenol cotton blue and conidial heads, vesicle shape, seriation and conidia shape were investigated under light microscope at 40X magnification for identification of Aspergillus species (Nugent et al., 2006).

Procurement of azole-susceptible A. fumigatus, antifungal azole discs and e-strips
A. fumigatus (NAIMCC-F-02473) was procured from National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, India. The fungal strain was maintained by sub-culturing on PDA slants at 28 ± 2°C for 5 days to obtain conidial growth. The organism was maintained at 4 o C and sub cultured every 15 days.
Antifungal discs and e-strips of itraconazole, ketoconazole, fluconazole and voriconazole were procured from Himedia Chemicals.

Disc diffusion assay
Aspergillus conidia were harvested in sterile 0.85 % saline, conidial suspension was adjusted to 10 6 conidia per ml and 50µl of this suspension was inoculated on PDA plate. Discs containing the itraconazole (10µg/disc), ketoconazole (10 µg/disc), fluconazole (10µg/ disc) and voriconazole (1µg/disc) were impregnated to the surfaces of inoculated plates. Plates were incubated at 28°C for 5 days to allow fungal growth. Zone of inhibition diameters were measured in millimetres (Nweze et al., 2010). An azole-susceptible A. fumigatus (NAIMCC-F-02473) was used as control. All the experiments were run in triplicates.

e-strip test
Susceptibility to different azoles in Aspergillus isolates was determined by E-strip test as described by Pfaller et al., 2003 with minor modifications. Isolates of Aspergillus species were allowed to sporulate on PDA slants for 5 days. Conidial suspensions were prepared in 0.9% normal saline supplemented with 0.1% Tween-20. The concentration was adjusted to an optical density of 0.09-0.13 at 530 nm. The PDA culture plates were inoculated with 50µl of prepared conidial suspension. The E-strip of itraconazole (0.002-32µg/ml), ketoconazole (0.002-32µg/ml), fluconazole (0.016-256µg/ml) and voriconazole (0.002-32µg/ml) was placed across the central surface of the freshly inoculated PDA plate. An azole-susceptible control strain A. fumigatus (NAIMCC-F-02473) was used. The plates were incubated at 28°C and results were recorded for 5 days post inoculation.

Antifungal susceptibility testing (AFst)
Aspergillus isolates resistant to all tested azole drugs on agar plates were subjected to AFST using CLSI M38-A2 broth micro-dilution method (

ResUlts AND DisCUssiON
The emergence of drug-resistant strains has become a challenge for treating Aspergillus diseases especially among immune-compromised patients. The majority of cases of azole-resistant diseases are due to resistant Aspergillus originating from the environment (Verweij et al., 2016). In the present epidemiological study, we have studied the presence of azole resistant Aspergillus species in agricultural fields of North India. A total of 165 fungal isolates having different morphological characteristics were recovered from 25 soil samples collected from different sites. Conidial head and shape is important feature for fungal identification (Khalil and Hashem 2018). On the basis of microscopic and morphological examination, 24.8 % were Aspergillus isolates.

Disc diffusion method
Disc tests are inexpensive and easy to set up, and provide an ideal screening test

e-strip test
Agar based E-strip test is the modification of the CLSI reference method and is convenient and flexible (Posteraro and Sanguinetti, 2014). The strains AF-003, AF-006 and AF-008 were selected for E-strip test on the basis of disc diffusion assay, showing no inhibition zone for any of the tested azoles (Fig 2). E-strip test results were also consistent with the results of disc diffusion assay, where the drugs strips had no/negligible inhibitory effect on resistant isolates. According to Alastruey-Izquierdo et al., 2015, the E-strip test of itraconazole reveals a good correlation with the CLSI M38-AFST to detect Aspergillus resistance than other antifungal drugs.

Antifungal susceptibility testing (AFst)
At present, CLSI is one of the universally recognized standard methods to perform in vitro AFST for either yeasts or filamentous fungi (Sanguinetti and Posteraro, 2018) which apply broth micro-dilution method. The antifungal activity is expressed as MIC of antifungal drug indicating the minimal drug concentration that inhibits fungal growth. In the present study, resistant and susceptible isolates were distinguished using broth micro-dilution and were confirmed resistant on the basis of having MICs above ECVs and breakpoints. AF-003, AF-006, AF-008 had high MIC 100 in comparison to ECV against azole antifungals, where no hyphal growth was observed in 96 well micro-plate. No MIC 100 was observed for fluconazole in all the resistant isolates. Susceptible strain had MIC 100 < ECV against azole antifungals (Table 2).
Hence, among 41 environmental Aspergillus isolates from various parts of North India, 7.3% (n=3) isolates were resistant to all the tested azole drugs, 19.5% (n=8) were diazole resistant and 17.07% (n= 7) were resistant to itraconazole only. Multi-triazole resistance was only found in A. fumigatus which is similar to the results observed by Chowdhary

CONClUsiON
Environmental azole resistance Aspergillus strains are present in environment of North India. This indicates the possible exposure of the population to these resistant strains. The regular screening of environmental azole resistant Aspergillus strains is crucial to analyze development of resistance in human pathogenic fungi and developing mitigation strategies.