Occurrence of Cryptococcus neoformans and Other Yeast-like Fungi in Environmental Sources in Bonaire (Dutch Caribbean)

Abstract

Introduction: We report here the presence of Cryptococcus neoformans, and other potentially pathogenic yeasts and yeast-like fungi in environmental sources in Bonaire. Methods: Seventy environmental samples comprising 40 samples of old pigeon droppings, 18 of woody debris from hollows of living trees of Caesalpinia (‘Divi Divi’), Ziziphus jujuba (Red Indian date), Tamarindus indica (Tamarind), Terminalia catappa (Tropical almond), Azadirachta indica (Neem) and 3 of other unidentified species of trees, 3 of latex from a rubber tree and 6 of coral dust were processed for isolation of pathogenic Cryptococcus spp. and other potentially pathogenic yeasts and yeast-like fungi. A variety of mycological media were employed. Identification of the isolates was done with conventional techniques and species identification was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Results: Three of the 40 samples from old pigeon droppings yielded Cryptococcus neoformans, constituting the first record of environmental occurrence of this important pathogenic yeast in the Dutch Caribbean. Other potentially pathogenic yeasts and yeast-like fungi recovered from these environmental samples included 6 isolates each of Candida albicans, 8 of Candida parapsilosis, 4 each of Candida metapsilosis and Candida orthopsilosis, 2 each of Candida carpophila, Candida famata, Candida fabianii and Candida pelliculosa, 7 of Candida spp., 5 of Trichosporon spp. and 2 of Sporobolomyces spp. Conclusions: This study has demonstrated for the first time the occurrence of C. neoformans in a natural habitat in the Dutch Caribbean. The recovery of many species of potentially pathogenic yeast-like fungi and yeasts from environmental sources is remarkable.

Introduction

Members of the basidiomycetous fungal pathogen Cryptococcus genus are known to occur in a variety of ecological niches, such as soil, pigeon droppings, and tree hollows in many countries e.g., Argentina, Brazil, Colombia, India, Kenya, Korea, and South Africa [1,2]. There is lack of information on their ecology in the Caribbean region, especially in the Dutch Caribbean. So far there is only a solitary report about the recovery of C. gattii from woody debris, collected from inside trunk hollows of a living Divi-Divi tree (Caesalpinia coriaria) in Bonaire [3]. Occurrence of C. neoformans has so far not been reported from the Dutch Caribbean. This communication reports the isolation of Cryptococcus neoformans and other yeast-like fungi from environmental sources in Bonaire.

Methods

Sampling

Forty samples of desiccated pigeon droppings, 21 samples of woody debris from the hollows of different species of trees from several localities, viz. Ricon Village, Hato Village, Kralendijk, Klein Bonaire, and Marine Beach along Leeuwarden, three of latex from a rubber tree and 6 of dust deposit in the corals along the marine coast were collected.

About 5 grams of each sample were suspended in 10 mL of sterile saline and vortexed. One mL quantities of suspension of each sample were spread on Petri plates of tobacco agar and Bird Seed (Guizotia abyssinica) Agar (HiMedia Laboratories, Mumbai, India), and incubated at 22-28°C for up to 7 days. There was no facility in our laboratory to incubate the inoculated samples at 37°C. Tobacco agar was prepared as recommended by Tendolkar et al [4]. Fifty g of tobacco leaves purchased from a local market in Delhi, India were added to one liter of distilled water, boiled for 30 minutes, filtered through gauze and the volume was adjusted to one liter. To this were added 20 g agar, the medium was boiled until the agar dissolved, autoclaved at 121°C for 15 minutes and poured in plates. Chloramphenicol (0.05 mg/mL) and diphenyl (0.1%) were added to these media to cut down contamination due to bacteria and saprophytic molds respectively. We were able to see single brownish colonies suspected to be Cryptococcus, not overgrown by other molds in many of the inoculated plates. These and whitish mucoid colonies suggestive of Candida, and colonies suggestive of other yeasts were sub-cultured onto slants of Sabouraud dextrose agar supplemented with chloramphenicol (0.05 mg/mL) to get pure cultures. In some of the culture plates saprophytic molds overgrew on colonies of yeasts while in some other plates they appear as discrete colonies. These molds including species of Aspergillus, Penicillium, Fusarium, Cladosporium, Alternaria and Curvularia were similarly sub-cultured to obtain pure cultures.

Identification of isolates

The isolates suspected to be Cryptococcus neoformans were tentatively identified by study of their morphological characters, and standard biochemical tests, using VITEK2 and API 20C AUX (bioMérieux, Marcy l‘Étoile, France), with no change in color to blue on inoculating the plates of canavanine glycine bromothymol blue (CGB) medium (Hardy Diagnostics, Santa Maria, CA, USA). The identity of the isolates as C. neoformans was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) analysis [1]. Cryptococcal mating type and serotype were determined by qPCR using four PCRs that specifically amplify the STE20a and STE20α alleles that belong to serotype A and D as described by Arsenijevic et al [5]. The majority of Candida isolates were identified by MALDI-TOF at Post-graduate Institute of Medical Sciences & Research, Chandigarh, India.

Some of the isolates of saprophytic molds, which grew on culture plates inoculated with samples were identified as Aspergillus niger, A. fumigatus, A. flavus, A. candidus, Penicillium citrinum, Cladosporium cladosporioides, Alternaria alternata and Curvularia lunata. Since the aim of our study was to investigate natural occurrence of Cryptococcus and other potentially pathogenic yeasts, no attempt was made to identify other isolates of saprophytic molds.

Results

Cryptococcal isolates were identified as Cryptococcus neoformans (serotype A, mating-type α) and were recovered from three out of 40 samples of pigeon excreta examined from several locations in Bonaire. The positive samples originated from accumulated excreta under a pigeons’ roosting site on the top of a window air conditioner at the back of the Faculty lounge at a medical school in Bonaire. All the remaining 37 samples of pigeon droppings collected from several other locations in Bonaire were negative for C. neoformans. Several isolates of other yeast-like fungi, viz. Candida albicans, Candida parapsilosis, Candida orthopsilosis, Candida carpophila, Candida famata, Candida metapsilosis, Candida pelliculosa, Sporobolomyces and Trichosporon were recovered from different types of environmental samples examined (Table 1).

Table 1. Occurrence of C. neoformans and other yeasts in environmental sources in Bonaire (Dutch Caribbean) .

Table 1. Occurrence of C. neoformans and other yeasts in environmental sources in Bonaire (Dutch Caribbean) .

Discussion

There are only a few reports on the occurrence of pathogenic cryptococci in natural sources in the Caribbean region. Seventeen isolates of C. gattii were recovered from cacti and tree detritus in Puerto Rico; 16 of these belonged to VGII/AFLP6 genotype while one isolate was a VGIV/AFLP7 genotype [6]. Until now only two cases of infections due to C. gattii have been reported in Cuba: one from an HIV-negative patient, and the other one from a cheetah at the National Zoo in Havana [7]. Eighty-six C. neoformans var. grubii isolates were recovered from 222 pigeon houses across Cuba [7]; characterization of these isolates from pigeon excreta, and those from clinical samples by conventional, serological and molecular methods using high-resolution techniques (AFLP, sequencing D1/D2 and ITS1/5.8S/ITS2 fragments and conventional PCR using STE12alpha-and STE12a-specific primers) confirmed the dominance of C. neoformans var. grubii mating-type MATa [7]. Vanhove et al [8]. showed that C. neoformans can be divided into three major lineages, with the first two VNI and VNII being globally distributed, whilst the third one VNB seems to be restricted to Southern Africa.

There is a solitary report of recovery of C. deuterogattii from 10 samples of woody debris, collected from the inside trunk hollows of a living Divi-Divi tree (Caesalpinia coriaria) in Bonaire.3 Molecular typing of these 10 isolates showed that they all were of mating-type α and genotype AFLP6/VGII, being indistinguishable from each other.

Our study reports for the first time the occurrence of C. neoformans from an environmental source in the Dutch Caribbean. Several repeated attempts by the senior author to isolate Cryptococcus from woody debris in the hollows of Divi-Divi and other species of trees in several locations in Bonaire were unsuccessful. The current study and the previous report from Bonaire [3], which mentioned an earlier report in 1953 of isolation of C. gattii (isolate no CBS1930 in the culture collection) from a child in Aruba indicate possible sources of human cases of cryptococcosis caused by members of the C. neoformans/C. gattii species complexes in Bonaire and other islands in the Dutch Caribbean. It may be mentioned that common Eucalyptus trees, Eucalyptus camaldulensis and E. tereticornis, known to be frequently associated with C. neoformans and C. gattii [9,10] were not seen in the vegetation of Bonaire. A search of literature also did not reveal their presence in other islands in the Dutch Caribbean. The other type of Eucalyptus trees, viz. Rainbow Eucalyptus, Baby blue Eucalyptus, Eucalyptus pulverulenta (blue spiral Eucalyptus) are seen in Bonaire. These trees do not have hollows; thus, we did not sample any of them for Cryptococcus spp. and other yeast-like fungi. Failure to re-isolate C. gattii in our study may be due to lack of its persistent occurrence in Divi-Divi trees in Bonaire, thus emphasizing the need to investigate its presence in other natural sources in Bonaire and other countries in the Dutch Caribbean.

It is noteworthy that a variety of other yeast-like fungi including several species of Candida were recovered in our study. The importance of pigeon excreta in natural occurrence of C. neoformans is well known. Presence of other potentially pathogenic yeasts in pigeon excreta and other environmental sources is often not recognized. In a recent study from Gran Canaria Island [12], several species of Candida and other yeasts, viz. Candia albicans, C. guilliermondii, C. kefyr, C. pelliculosa, C. parapsilosis, C. rugosa, Trichosporon and T. asahii, T. mucoides were isolated from pigeon excreta. It is noteworthy that in our study isolates of C. albicans, C. parapsilosis, C. pelliculosa, C. orthopsilosis, C. carpophila, C. famata, and C. metapsilosis were recovered from samples of pigeon excreta, the association of the latter four species with pigeon excreta being demonstrated for the first time. C. parapsilosis is a human commensal [13] and is known to be associated with nosocomial outbreaks of candidemia [14,15]. This species is now recognized as an emerging major human pathogen that has drastically increased in importance and prevalence over the past two decades. Infections are particularly associated with solutions for hyperalimentation, prosthetic devices, indwelling catheters, and with the nosocomial spread through the hands of healthcare workers [16]. C. famata is echinocandin and azole resistant and has been described as a cause of several types of infections, including bloodstream catheter-related infections, peritonitis, acute zonal occult retinopathy and mediastinitis [17,18]. Among other species of Candida recovered in the study, C. orthopsilosis and C. metapsilosis have been shown to be virulent in reconstituted human tissue models [20]. A study from Brazil reported frequent occurrence of C. parapsilosis, occasional presence of C. famata, C. metapsilosis and C. orthopsilosis on the hands of healthcare workers in a neonatal intensive care unit [20]. C. parapsilosis has also been isolated from pine trees [21,22], and from soil [22]. Its isolation from the woody debris of the hollows of Divi-Divi trees and from dust in the coral, in our study is noteworthy. C. parapsilosis and other Candida species recovered here could have possibly transmitted from soil to old pigeon excreta, and woody debris of in the hollows of living trees.

Environmental occurrence of Histoplasma capsulatum in the environmental sources, and cases of human infection caused by this fungus in several countries in other parts of the Caribbean have been reviewed by Denning and Gugnani [23].

Conclusions

There is need for comprehensive investigation of the occurrence of C. neoformans and C. gattii species complexes, as well as that of other pathogenic fungi like Histoplasma in the environment and cases of human infections due to these fungi in the Dutch Caribbean.