Antifungal resistance in cnlac1 possessing Cryptococcus neoformans isolated from domestic and feral pigeons in West Bengal, India

Pathogenic yeasts such as Cryptococcus neofor- mans and Candida spp. have emerged at the end of the 20^th century as important causes of human infections in immuno- compromised (AIDS), cancer and transplant patients (Bose et al., 2003). Cryptococcus neoformans is encapsulated hetero-basidiomycetous yeast which causes opportunistic infections and has a strong tropism for the central nervous system (Eastman et al., 2015). Important pathogenic species   are   Cryptococcus   neoformans - C. gattii  species  complex  which  includes  three varieties such as C. neoformans var. neoformans, C. neoformans var. grubii and C. gattii (C. bacillisporus) (Kumar et al., 2010). Cryptococcosis associated with HIV infection is mostly caused by C. neoformans var. neoformans irrespective of geographical location (Kwon-Chung and Bennett, 1992). Melanin forming ability associated with laccase enzyme (encoded by CNLAC1) in C. neoformans play an important role in virulence (Salas et al., 1996). C. neoformans var. neoformans and C. gattii are two major zoonotic pathogens transmitted by inhalation and cause pulmonary infections with probability of further dissemination into CNS producing meningitis (Kwon-Chung et al., 2014). C. neoformans sensu lato has a global distribution in avian excreta and contaminated soil, while C. gattii sensu lato is associated with soil and trees (Eucalyptus, almond, pottery) (Mitchel and Perfect, 1995). Presence of C. neoformans in the faeces of different avian species such as pigeon (Columba livia), budgerigars (Melopsittacus undulates), monk parakeet (Myiopsitta monachus), red-cowled cardinal (Paroaria dominicana), canary (Serinus canarius) is a saprobiotic phenomenon (Cafarchia et al., 2006; Samanta and Bandyo- padhyay, 2017). Avian faeces rich in creatine, urea, and uric acid and protected from sunlight and ultraviolet light, high flock density and poor sanitary conditions create a microenvironment for C. neoformans and can infect immunocompromised patients upon exposure to avian excreta throughout the world (Garcia-Hermoso et al., 1997; Kumlin et al.,1998; Lagrou et al., 2005). 
        
Major antifungals used for treatment of cryptococcosis are restricted to amphotericin B, flucytosine and fluconazole. Antifungal resistance is a current global concern like its counterpart. Few earlier reports regarding in-vitro resistance of C. neoformans to antifungal drugs are available (Archibald et al., 2004; Sar et al., 2004; Souza et al., 2005; Chowdhary et al., 2011), although there is scarcity of data regarding current antifungal resistance status of C. neoformans strains isolated from pigeon droppings. India has the second largest burden of cryptococcos is with a documented high prevalence (1.7-4.7%) in persons with AIDS (Kumarasamy et al., 2003; Vajpayee et al., 2003). Avian excreta especially of feral pigeon origin were earlier exploded as the source of C. neoformans in India (Sethi et al., 1966; Gugnani et al., 1976). The systemic approach for detection of C. neoformans occurrence with substantial numbers of samples incorporating both domestic and feral pigeons is still lacking.
        
In this scenario, the objective of the present study was to detect the occurrence of C. neoformans in domestic and feral pigeons’ excreta in and around Kolkata, a metropolitan city in India with considerable size of pigeon population, followed by study of the seasonal variation on the occurrence of the fungi and the detection of in-vitro antifungal resistance pattern of the pathogen against commonly used 8 antifungal agents.

Sample collection and transport
 
A total number of 917 weathered droppings of domestic and feral pigeons were collected with the help of sterile spatula from different pet bird shelters, buildings, congested slum areas and housing societies in and around Kolkata, West Bengal, India. The collected samples were placed into sterile plastic bags and transported to the laboratory aseptically. All the samples were processed within 6 to 8 hours of collection.
 
Isolation and identification of C. neoformans
 
The collected samples were serially diluted with sterile distilled water containing chloramphenicol (40mg/500ml). The diluted samples were inoculated into bird seed agar plates (HiMedia, India) and were incubated at 28°C. The characteristic brown coloured colonies were sub-cultured into Sabouraud’s dextrose agar (HiMedia, India) at 28°C for 2-7 days. The characteristic colonies were stained with indian ink for the detection of encapsulated budding yeast cells. Growth on corn meal tween 80 agar plates (2-3 days at 28°C; HiMedia, India), urease test, growth in presence of cycloheximide, growth in 1% peptone water, sugar fermentation test, assimilation test and growth in L-canavanine-glycine-bromothymol blue agar (HiMedia, India) were carried out for identification of the isolates (Carter and Wise, 2004).
 
Molecular detection of CNLAC1gene in C. neoformans isolates
 
Extractions of chromosomal DNAs of all positive C. neoformans isolates were performed using a commercial kit for yeast DNA extraction (Merck Biosciences, India) according to the protocol of the manufacturer. PCR was carried out with 25μl reaction volume containing 12.5μl of 2x Red Dye PCR Master Mix (Merck Biosciences, India), 2μl of isolated template DNA and 2μl of CNLAC1 primers (F-ACGGTGTCCCTGGTATAA; R-GCGTTGGACG ATT GAAAG). The amplification was carried out with 35 cycles consisting of 94°C for 1 minute, 57°C for 30 seconds and 72°C for 90 seconds (Chae et al., 2012).The amplified product was visualized by gel documentation system (UVP, UK) after electrophoresis in 1.2% (W/V) agarose (SRL, India) gel containing ethidium bromide (0.5µg / ml) (SRL, India).
 
Antifungal sensitivity testing
 
Antifungal sensitivity of the positive fungal isolates against ampho- tericin-B, fluconazole, itraconazole, ketoconazole, voriconazole, posaconazole and flucytosine were determined by EZY^TMMIC strips (HiMedia, India). MICs were determined following the recommendations proposed by the clinical labo -atory standards institute (CLSI) (CLSI 2008). The principal of 80% inhibition was used to visually select the end point. Break points used to interpret susceptibility results are described in Table 1 following the recommendations proposed by the clinical laboratory standards institute (CLSI 2008).
 

 
Statistical analysis
 
Occurrence of C. neoformans in different seasons was compared by chi-square test using SPSS software version 17.0 (SPSS Inc.).

Cryptococcus neoformans were isolated from 153 (16.68%) samples collected in this study. The isolates developed white mucoid colonies in Sabouraud’s dextrose agar and characteristic brown colonies (indicative of melanin production) in bird seed agar (BSA) (Fig 1). The present study determines a moderate occurrence (16.68%) of C. neoformans var. neoformans in domestic and feral pigeons in West Bengal, India which was also supported by Chae et al., (2012). In the soil contaminated with avian excreta, C. neoformans was detected to produce a mutual interaction with several organisms such as various bacteria, amoebas, mites, sowbugs and worms (Kwon-Chung et al., 2014). Overall the occurrence rate of C. neoformans in Indian pigeons detected in the present study (16.68%) is within the range (6-85%) reported earlier throughout the world (Yildiran et al., 1998). Higher prevalence of C. neoformans in pigeons was detected in few places such as Texas (30%) (Ramirez et al., 1976), Brazil (27.6%) (Costa et al., 2010) and Saudi Arabia (34%) (Abulreesh et al., 2015). Conductance of the study in a wide geographical area encompassing different agro-climatic zones might produce more accurate occurrence rate although the sample size in the present study was considerable.
 

        
All samples showed developed thick capsules when stained with indian ink (Fig 2). In corn meal agar, they produced characteristic white colonies with blastoconidia. All the isolates were characteristic urease (+ve), showed no growth in SDA with cycloheximide and chloramphenicol, and no growth or colour change in CGB agar. All the isolates were found to be positive for characteristic carbohydrate assimilation test with sugars like glucose, galactose, sucrose, cellobiose, trehalose, raffinose, L-arabinose and inositol. None of the isolates showed positive result in carbohydrate fermentation test with carbohydrates namely glucose, galactose, maltose, sucrose, dextrose, raffinose, trehalose, fructose and mannose. Therefore, all the isolates in this study showed typical cultural and biochemical characteristics as per Carter and Wise (2004). The weathered samples were selected for more probability to isolate C. neoformans as poor growth of other commensal organisms occurs in dried samples (Abulreesh et al., 2015). All the isolates produced specific amplified product for CNLAC1 outer gene in PCR (Fig 3). Laccase enzyme (CNLAC1) is a part of the copper-containing oxidase family which catalyzes the oxidation of catecholamines into quinines and polymerization into dark brown melanin-like compounds. The pigment protects the yeast from nitrogen and oxygen based oxidative stress (Wang and Casadevall, 1994). Development of a Mel- mutant with CNLAC1 deletion earlier confirmed the role of CNLAC1 in virulence of C. neoformans (Salas et al., 1996). All the C. neoformans isolates in the present study were found to possess CNLAC1 in PCR.  
 

 

        
The seasonal distribution of the isolates showed apparent higher occurrence in monsoon (19.12%) followed by winter (16.46%) and summer (15.36%) in the collected samples, though no statistically significant difference was observed (Table 2). Occurrence of C. neoformans var. neoformans was found to be apparently (not statistically significant) higher in monsoon (19.12%) which is corroborative with earlier study (Granados and Castaneda, 2005). Higher relative humidity during rainy season increased the probability of pulmonary infection caused by C. neoformans. The humidity was confirmed as an important factor to increase both desiccated Cryptococcus aerosol concentration and pulmonary retention in experimental mice (Springer et al., 2013).
 

        
Occurrence of C. neoformans var. neoformans in the environment such as soil contaminated with pigeon droppings makes the situation alarming in West Bengal (India) where estimated number of people living with HIV (PLHIV) is high (NACO 2016-17). The pigeons are not natural carriers of C. neoformans due to high body temperature (41°C-42°C) exceeding the optimum growth temperature of the yeast although the yeasts were isolated from crops, beaks, and feet of the pigeons which suggested the feed as a source of infection (Khan et al., 1978). The yeast concentration in the dried pigeon droppings remains high due to saprobiotic relationship and the exposure to dried excreta acts as additional predisposing factor for pulmonary infections and meningitis in PLHIV (Lagrou et al., 2005). Drying of avian excreta generated smaller cryptococcal cells and increased the probability of aerosolisation and inhalation into the susceptible host (Emmons, 1951). A recent analysis with 30 years data of the patients suffering with cryptococcal meningitis showed a positive correlation between occurrence of infection and pigeon population in Southern Brazil (Spina-Tensini et al., 2017).
        
All the samples were found to be resistant to caspofungin which is characteristic of C. neoformans. Resistance to amphotericin-B was highest among the antifungals tested (33.33%). It was followed by resistance to fluconazole (20.91%), flucytosine (11.11%), ketoconazole (8.49%) and itraconazole (3.26%). All the samples were found to be highly sensitive to voriconazole and posaconazole. MIC 90 values in case of ketoconazole, flucytosine, caspofungin, itraconazole, posaconazole and voriconazole are 0.125, 0.5, 16, 0.094, 0.064 and 0.19 µg/ml respectively. In case of fluconazole and amphotericin-B where higher resistance pattern is observed the MIC values obtained are 8 and 0.5 µg/ml. (Fig 4, Table 3). Resistance to amphotericin-B, fluconazole (16%) and itraconazole (7%) was observed earlier in clinical isolates of C. neoformans in India (Datta et al., 2003; Jain et al., 2005). Decreased susceptibility to fluconazole in C. neoformans serotype A isolated from pigeon excreta in Thailand was reported (Tangwattanachuleeporn et al., 2013). All the C. neoformans strains isolated from pigeon droppings in Cameroon showed resistance to fluconazole (MIC: 16 to > 256 μg/mL), ketoconazole (MIC:8-64 μg/mL) and amphotericin B (Dongmo et al., 2016). 
 

 

        
All the isolates in the present study were found to be highly sensitive to voriconazole and posaconazole. Several studies have confirmed potent antifungal activity of new triazoles (voriconazole and posaconazole) against C. neoformans isolated from clinical cases and environment throughout the world (Costa et al., 2010; Bii et al., 2007; Capoor et al., 2008; Matos et al., 2018). The antifungals can be used as alternative drug of choice for treatment of clinical cryptococcosis in future.
        
Therefore, it can be concluded that approx. 17% of the pigeon droppings were contaminated with Cryptococcus neoformans var. neoformans in and around Kolkata as reported in this study which can acts as a major etiology of human infections in immunocompromised, cancer and transplant patients. All the fungal isolates were having CNLAC1 outer gene as detected in PCR. In-vitro antifungal sensitivity testing revealed that these fungal isolates were considerably resistant to amphotericin-B, fluconazole, flucytosine etc. whereas voriconazole and posaconzole were sensitive against these. These findings are of great human health concern as Kolkata is a very densely populated city and the birds are in close proximity to the people.

The authors express their sincere thanks to the Honorable Vice Chancellor, WBUAFS for providing infrastructure and the Indian Council of Agricultural Research (ICAR) for the necessary funds, under the ‘Outreach Programme on Zoonotic Diseases’ at Department of Veterinary Public Health, WBUAFS.