In particular Candida tropicalis, Candida parapsilosis and C. albicans have been recovered from trees [12-14] as well as woodland are considered a privilegiate site for Saccharomyces spp. [15]. Furthermore, wooded and green areas are visited by large numbers of people and their pets, and in the last years reports of mycoses caused by yeasts, mostly referring to fungi belonging to Candida genus, are more frequently encountered than in the past [2,3,16,17]. For these reasons, considered also the increased occurrence of immunocompromised patients, the knowledge of the ecology of these fungal species may be of interest. To the best of our knowledge, there are no reports about the presence of yeast species, in particular Candida spp., associated with trees in Italy. Therefore, aim of the present study was to investigate the occurrence of potentially pathogenic yeasts on decaying vegetal material from trees in 2 city parks in Pisa, Italy, evaluating also the drug sensitivity pattern of Candida spp. isolated versus some among the most common used antifungals in human medicine.

Materials and Methods

Specimens were drawn from trees growing in 2 green recreational areas, within the urban area of Pisa (43°42′30″ N10° - 24′12″ E), Central Italy, visited by both people and pet dogs. hirtyone trees were sampled. In detail 6 Pinus pinea, 4 Tilia platyphyllos, 1 Platanus acerifolia, 1 Fagus sylvatica, 1 Chicas revoluta and 1 Taxus baccata from site 1, and 4 P. pinea, 2 Salix babylonica, 1 P. acerifolia, 3 Quercus ilex, 1 Chamaciparis lawsoniana, 1 Morus alba, 1 Laurus nobilis, 1 Nerium oleander, 1 Carpinus betulus and 1 Gleditsia triachantos from site 2, respectively, were selected. Only decaying parts of trees were selected and for each tree roots, tree hollows and leaves were sampled by swabbing technique, as reported by Maganti et al. [12]. For each sampled site 2 swabs were drawn, obtaining 186 total specimens. All samples were immediately transferred to the laboratory and therefore seeded onto Malt Extract Agar (MEA) supplemented with 0.1% byphenil. Subsequently, they were incubated at both 25°C and 37°C, until a noticeable fungal growth was ascertained. The latter temperature was selected in order to evaluate the thermotolerance of isolates. Yeast identification was achieved by microscopic analysis, as well as auxanographic profile using ID32C galleries (BioMérieux, Marcy l’Etoile, France). All strains presumptively referring to Candida genus were subsequently subjected to PCR analysis, performed by amplification and partial sequencing of the D1/D2 domain of 26S rRNA encoding gene, as described by Lopandic et al. [18], as confirmatory identification test. DNA partial sequences obtained were then compared with those present in the NCBI database, by matching the BLAST program from GenBank. Sequences with a 98 to 100% identity with GenBank sequences were retained as significant for the molecular identification of yeasts. Candida spp. sensitivity to most frequently employed antimycotic drugs (amphotericin B, fluconazole, voriconazole, caspofungin) was evaluated by Etest (BioMérieux, Marcy l’Etoile, France). Minimal inhibition concentrations (MIC) breakpoints, when available, were calculated following the CLSI recommendations for Candida spp. [19]. For amphotericin B this value is not available, so the antifungal clinical breakpoint provided by EUCAST was considered [20].

Results

Sixteen trees out of 31 (51.6%) scored positive for yeasts in at least one sampled site. Twenty-six fungal isolates were cultured and identified as Candida spp., Debaryomyces polymorphus, Rhodotorula sp. and Pichia etchelsii. In particular, thermotolerant Candida isolates were obtained for Candida famata (6/6), Candida guilliermondii (4/4), Candida membranaefaciens (2/3) and Candida parapsilosis (1/1). Eight P. pinea out of 10 scored positive, with Candida spp. as the most prevalent genus, recovered from at least 1 site. Candida spp. were isolated from 1 T. platyphyllos, 2 S. babylonica, 1 C. lawsoniana and 1 L. nobilis. Different fungal species from the same tree were detected in 3 P. pinea, 1 T. platyphyllos and 1 P. acerifolia. PCR analysis confirmed in all cases the presumptive Candida species identification based on phenotypic features, showing a high similarity (>99%) with the deposit species sequences described in the GenBank database determined by BLAST. Cryptococcus neoformans/gattii was never been isolated during the study. All Candida isolates were sensitive to amphotericin B, 2 C. famata were resistant to all azoles assayed, while fluconazole was effective versus all C. guillermondii isolates. Interestingly, C. famata was the sole yeast species fully sensitive to caspofungin. More detailed data about the source of yeasts and drug sensitivity pattern are reported.

Discussion

The fungal species isolated are in agreement with the findings of Maganti et al. [12], who report the occurrence of Candida, Rhodotorula and Pichia Genera from tree hollows in urban areas. A limitation of this study is the low number of sampled trees, indeed. Unfortunately, it was not possible to overpass this problem, since the study design was focused on decaying trees only, that were present in low numbers in sampled areas. Among Candida spp., C. parapsilosis is an important etiological agent of invasive candidiasis [21], representing the second or third most common species isolated in human candidemia worldwide. The isolate obtained in our study showed resistance to fluconazole and caspofungin. These findings agree with literature data, and the fluconazole resistance of some strains within this yeast species has been related to an overexpression of MRR1 [22]. C. famata was the most prevalent recovered yeast species and it is considered as a rare, but emergent [23] cause of candidiasis, with a prevalence ranging from 0.2% to 2% [24]. These yeasts have been reported as agents associated to urinary tract infection in pets, characterized by similar antimycotic drugs sensitivity patterns [25]. In the present study, 5 out of 6 isolates were resistant to fluconazole, while the other one had a dose dependent sensitivity. Moreover, 2 isolates resulted resistant to voriconazole. This finding would appear of interest, showing that isolates characterized by high rates of resistance would be widely spread in urban environment. C. guilliermondii has been reported as a cause of candidemia in neoplastic patients [26], being involved in up to 2% of human cases [27], and in a joint infection in a dog [16]. Three out of 4 strains scored resistant to caspofungin, while the other one showed intermediate sensitivity, in agreement with Marcos-Zambrano et al. [27]. C. membranaefaciens is a yeast infrequently reported in intravenous catheter-associated invasive infections [28,29], whose sensitivity to fluconazole is a matter of debate [28]. The isolates obtained in our work seem to confirm the resistance to fluconazole. The intermediate sensitivity of all the three isolates to caspofungin appears to be an interesting finding. The results obtained from this study would indicate that decaying vegetal material from trees occurring in investigated recreational green areas, seems to harbor potentially pathogen yeasts, characterized by low sensitivity against some currently used antimycotic drugs.

Disclosure of Interest

The authors declare no conflict of interest.

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