Introduction

Reptiles are popular exotic pets and are well recognized, in western cultures, as a source of food, medicines, and leather1,2,3,4,5. The increase in mean global temperatures has resulted in overexpression of potentially pathogenic microbe lineages from reptiles, which ultimately could represent a risk as they could serve as reservoirs and spreaders of microorganism of zoonotic potential4,6. In human and veterinary medicine, reptiles have attracted the interest of the scientific community for their role as reservoirs of bacteria (e.g., Salmonella spp., Vibrio spp.), viruses (Arboviruses such as West Nile Virus, Chikungunya virus) and fungi (Candida spp.)5,6,7 and, intermediate hosts of parasites (e.g., Spirometra, Trichinella, Gnathostoma, Pentastomida1,3. Importantly, fungi are considered an emerging problem in human and veterinary medicine due to the increased number of immunocompromised hosts and therapies that affect host immune status8,9. Even if data on fungal infection in reptiles caused by filamentous fungi (i.e., Fusarium spp., Mucor spp., Trichoderma spp., Aspergillus spp., and Penicillium spp.) or yeasts (i.e., Candida spp., Trichosporon spp. and Geotrichum spp.) are seldomly reported10,11,12,13, studies on the mycobiota of these animals are scanty and mainly limited to lizards (i.e., Agama agama), wall geckos (i.e., Hemidactylus spp.), sea turtles (i.e., Chelonia mydas) and tortoises14,15,16,17,18. The occurrence of the isolated yeast species varies according to the origin of sampled animals and presents a direct correlation with that of yeasts causing fungal infections in humans. Overall, the above suggests that such animal species may be sentinel organisms for the emergence of zoonotic pathogens19,20. Additionally, the Candida spp. isolated from these animals show a decreased antifungal susceptibility profile against azoles and amphotericin B15,16,21. Since these yeast species may survive in the feces of reservoir animals for an appreciable number of years5,22, assessing the mycobiota of reptiles might provide information about the distribution of yeasts, even those with zoonotic potential5,20,22,23. This study aimed to investigate both the occurrence of yeasts from the cloacal swabs of snakes of different origins and the antifungal profile of the isolated strains.

Results

Snake species sampled

Number of snakes species sampled in both sites are reported in Table 1. Yeasts were isolated from 10% (18/180) of the snake cloacal samples with the highest occurrence in Group I (14.7%), compared to Group II (7.1%). The yeast population size in Group I (63.4 CFU/swab) was higher but not statistically significant from that recorded in Group II (59.6 CFU/swab; p = 0.94). A total of 72 yeast strains were isolated from the enrolled animals, with the highest frequency in Group I. Bitis arietans snakes harbored the highest percentage of strains (38.9%), followed by Elaphe quatuorlineata (22.2%; Table 2).

Table 1 Species and numbers (in bracket) of snakes sampled in Marrakech, Morocco (Group I) and Cocullo, Abruzzo, Italy (Group II).
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Table 2 Number and percentages in bracket of cloacal swabs positive for yeasts divided according to snake species and origin.
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Occurrence and molecular identification of isolated yeast strains

Nine yeasts species belonging to eight genera and five yeast species belonging to five genera were isolated from snakes from Group I and Group II, respectively (Table 3). Except for Rhodotorula mucilaginosa, no ITS nucleotide variations were detected among Candida species and non-Candida species and all representative sequence types were deposited in NCBI Sequence Read Archive under Accession Number (see Table 3). Two ITS sequence types were detected among R. mucilaginosa strains, one from Group I (accession number: PP264369) and one from Group II (accession number: PP001095). The yeasts species isolated from snakes from Group I were different from those isolated from Group II with exception of Rhodotorula mucilaginosa (Table 3). Since Debaryomyces hansenii, D. fabryi, and D. subglobosus cannot be distinguished from one another based on ITS sequences24, the isolated yeast strains were herein reported as Debaryomyces spp. Trichosporon asahii (22.2%) and Candida tropicalis (15.3%), were the most frequently isolated yeast spp. from snakes of Group I while Debaryomyces spp. (16.7%) and Metahyphopichia silvanorum (11.1%) were the most frequently isolated yeast spp. from snakes of Group II (Table 3).

Table 3 Yeast species with Gene bank Accession Number (in bracket) isolated from Group I (Marrakech, Morocco) and Group II (Cocullo, Abruzzo, Italy) snakes: sequence nucleotide identity with those present in Gene Bank is also reported.
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Antifungal susceptibility testing

The drug susceptibility profiles of isolated yeasts to azoles, Amphotericin B (AmB) and anidulafungin (ANI) are summarized in Table 4. The antifungal susceptibility varied according to the yeast spp. and within the yeast species according to the origin (Table 4). In particular, R. mucilaginosa from Group II showed higher MIC values for all the drugs tested than those registered for R. mucilaginosa from Group I. Comparatively, because of MIC values, Ketoconazole (KTZ) was the most active drug followed by Voriconazole (VOR) and Fluconazole (FLZ) for all yeasts species. The MIC mean values of all drugs tested for Candida tropicalis were statistically higher than those registered for other yeasts species in Group I (i.e., C. parapsilosis, D. catelunata, E. dermatidis, M. carribica, R. mucilaginosa, T. asahii and W. pararugosa; p = 0.0113; Table 4). Based on the clinical current CLSI breakpoints and ECV values, multiple azole, AmB and ANI resistance phenomena were detected among isolated yeasts (Table 5). Azole multidrug resistance phenomena were detected among isolates from Group I and R. mucilaginosa from Group II whereas AmB resistance were recorded among isolates (C. neoformans, D. hansenii and M. guilliermondii) from Group II (Table 5). ANI resistance was detected in all yeast spp. from Group I and II with exception of C. parapsilosis and C. tropicalis (Group I) and M. guilliermondii (Group II; Table 5).

Table 4 Minimum inhibitory concentration (MIC, µg /mL) data of fluconazole (FLZ), itraconazole (ITZ), ketoconazole (KTZ), posaconazole (POS), voriconazole (VOR), amphotericin (AmB) and anidulafungin (ANI) of isolated yeast species from snakes coming from Group I (Marrakech, Morocco) and Group II (Cocullo, Abruzzo, Italy).
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Table 5 Number and percentage of resistant yeast spp. to fluconazole (FLZ), itraconazole (ITZ), ketoconazole (KTZ), posaconazole (POS), voriconazole (VOR), amphotericin (AmB) and anidulafungin (ANI) isolated from snakes coming from Group I and II.
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Discussion

Data suggest that snakes may harbor pathogenetic yeasts, being potential reservoirs and spreaders of these organisms through their feces in the environment. In particular, in this study, different fungal species listed among WHO Critical and High Priority group of Fungal pathogens (i.e., C. neoformans, C. tropicalis and C. parapsilosis) were isolated from snakes according to their origin25. These yeasts in addition to other yeast species (i.e., D. catelunata, E. dermatidis, R. mucilaginosa, T. asahii, W. pararugosa and M. guilliermondii, M. caribbica), are considered a threat mainly for immunocompromised hosts26,27,28.

The isolation of these yeasts species from wild animals should be better assessed since humans and pet animals are more often in contact with reptiles and the environment where they thrive5,29. In addition, some of these reptiles are now kept as pets, source of food, medicines, and leather5, thus suggesting their potential pathogenic capabilities in spreading emerging yeasts in the environment.

Fungal species have been rarely isolated from snakes, though previous data suggest that yeasts may cause specific gastrointestinal infections in these reptiles11,30,31. However, the potential role of these animals in the transmission of yeasts to humans through bites and contaminated herpetological equipment31,32,33, emphasizes the zoonotic risk associated with wild reptiles, suggesting the need to perform studies on the mycobiota of such animals31. Indeed, wild animals may act as reservoirs of yeasts with different percentage of detection according to animal species, such as in bats (n = 7/57; 12.3%), cockatiel birds (n = 14/60; 23.3%), migratory birds (n = 66/421; 15.7%), lizards (n = 60/177; 33.9%) and wild boars (n = 62/124; 50%)5,19,24,34,35. Overall, the prevalence of yeasts reported above in different animal species are higher than those recorded in this study, suggesting that animal species as well as their lifestyle, might have a role in selecting the yeasts and the mycobiota composition. Also, the diversity of mycobiota identified in different snake groups tested, suggests that the environmental conditions where the snakes live, play a role in selecting the prevalent yeasts species36. In addition, the finding of a higher occurrence of yeasts in venomous snakes than in non-venomous ones, might be due to the antifungal and antibacterial activities of the venom in selecting pathogenic yeast populations that could be innocuous to these animals37,38,39. Indeed, these properties linked to the venoms could, to a certain extent, render venomous reptiles immune to high fungal and bacterial loads, such as in the case of the Komodo dragons40. The observation above is somehow in contrast with other studies showing that non-venomous snakes may harbor in the oral mucosa more fungi than venomous species41. Environmental conditions, husbandry and country of origin are clearly correlated with the fungal community composition of snakes, with animals kept in precarious husbandry conditions and overcrowded, and in highly urbanized areas (i.e., Marrakech, Morocco) being prone to have a higher fungal colonization. The lowest occurrence in Group II could be attributed to the management measures utilized in the last years to reduce the potential risk of disease and to ensure a better standard of handling and keeping wild ophidians on the occasion of the “Festa dei serpari”42. Noteworthy, the composition of yeast species in snakes reflects that in humans, in the same geographical areas43,44,45,46,47. For example, C. tropicalis and T. asahii were among the most frequently isolated yeasts from human infection in Morocco44,47, whereas M. guilliermondii and C. neoformans from humans in Italy43,45,46. Accordingly, the presence of R. mucilaginosa in animals from both sites might be due to the fact that this yeast is a ubiquitous saprophytic species that can be recovered from many environmental sources, including areas with unfavorable conditions48. Additionally, R. mucilaginosa was one of the most isolated species from human central venous catheter (CVC) mediated fungemia and skin ulcer infections in the countries where the study was performed48,49,50. Yeasts species herein isolated are also well recognized as causative agents of life-threatening infections characterized by high mortality in immune-compromised patients25,51,52,53,54,55,56, In addition, the finding that the occurrence of drug resistance phenomena varies according to the yeast species and their provenience suggests that this phenomenon might be associated with the quality of habitats/ environment from which animals came from. In particular, while azole resistance of some species of yeasts (e.g., C. tropicalis, E. dermatitis, and T. asahii) is a common phenomenon, having emerged worldwide due to the extensive use of conventional antifungal agents, the finding of azole resistance phenomena in R. mucilaginosa, mainly reported in European countries and Brazil26,57, is associated to environmental control through indiscriminate use of pesticides.

Indeed, the presence of azole drug-resistant pathogens in the cloaca of snakes suggests that these animal species may be useful as bio-indicator for the azole resistance phenomena in the environment and therefore, bio-indicators of environmental quality, considering that azole resistance is related to the use of antifungal compounds in agriculture58,59. Accordingly, the finding of AmB resistant strains only among isolates from Group II suggests an abuse of azole in the countries of origin60, being AmB resistance mainly linked to the prolonged use of azoles for the management of human and animal fungal infection61. The above hypothesis is confirmed by the fact that global consumption of triazoles and terbinafine is higher in high-income, than in middle or lower- income countries60.

Overall, results of this study suggest that snakes may harbor in their cloaca pathogenic yeasts which vary according to ophidian species and origin. Since the yeast species community from different groups of animals as well as their antifungal profile reflects the epidemiology of human yeast infections in the same geographical areas, snakes may be considered as sentinels for the emergence of zoonotic pathogenic micro-organisms and as bio-indicators of environmental quality. Based on that, future studies should investigate the genetic structure of isolated yeasts, to determine if these yeasts cross-border between animal, human and environment biospheres. Under the above circumstances snakes will be useful as sentinels to better manage the risks of emerging infectious diseases or the appearance of antifungal drug resistance phenomena as well as the antifungal drug prolonged use and abuse, under the context of One-Health.

Materials and methods

Ethics statement

The research adhered to relevant international, national, and institutional regulations regarding the handling of animals. The procedures for collecting samples from Morocco were approved by the Office National de Sècurité Sanitaire des Produits Alimentaires in the Kingdom of Morocco, under the authorization number 23355ONSSA/DIL/DPIV/2022. The protocols of snake sampling, handling, and capture by Serpari, in Italy, was allowed by the National authorizations (National law DPR 357/97) and approved by the Italian Ministry of Environment (n. 16271/2023 PNM and 79052/2023 PNM). The methods employed in this study were carried out in accordance with the regulations of the above listed authorizing bodies and with the recommendations in the ARRIVE guidelines.

Study areas, animal examination and sample collection

Between October 2022 and April 2023, a total of 180 snakes were enrolled in the study and divided into two groups. Group 1 included snakes (n = 68) kept around the Jeema-El-Fna square of Marrakech, Morocco, which were examined and screened under the frame of an epidemiological study of zoonotic pathogens associated to reptiles handled in the souks of Marrakech62 and, Group II, included snakes (n = 112) captured on the occasion of the “Festa dei serpari” an ancient ritual event which have remained unchanged for hundreds of years in Cocullo, Abruzzo, Italy (Fig. 1; Table 1). Snakes from Group II were screened and examined under the frame of an annual monitoring program performed by the local authorities and a scientific committee with authorization number 16,271/2023 PNM and 79,052/2023 PNM63. Animals were morphologically identified to species level using reference checklists62,63,64,65 and clinically examined. All snakes were apparently healthy. From each animal, cloacal swabs were collected and stored at 4 °C.

Fig. 1
figure 1

Map showing, Morocco (Pink) and Abruzzo, Italy (Light Orange) the sites where snakes cloacal samples were obtained. Map prepared using QGIS software.

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Fungal culture and identification procedures

Cloacal swabs were cultured in duplicates directly onto Sabouraud dextrose agar with chloramphenicol (0.5 g/l) (SDA, Liofilchem®, Abruzzo, Italy) incubated at 37 °C for 3 days and daily observed for growth. Cultures were considered “positive” when fungal yeast colonies were confirmed using the microscope after Gram staining. Colonies were counted and the yeast population size were expressed as colony forming units/swab (CFU/swab). Four pure colonies, for each positive sample were sub-cultured onto SDA slants for yeast identification. The strains were identified based on colonial morphology, microscopic and biochemical features and by Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS; Vitek MS -bioMérieux, France, knowledge Base V3.2)5,20.

Molecular identification of yeast species

The yeast strain identification was confirmed by sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region. Briefly, genomic DNA were isolated from each sample colonies using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany), adhering to manufacturer’s instructions. The nuclear ribosomal ITS region was amplified using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers5. The PCR program consisted of 5 min denaturing at 94 °C, 30 cycles of 30 s (s) at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, followed by 10 min at 72 °C for final extension. PCR products were analysed on 2% agarose gels stained with GelRed (VWR International PBI, Milano, Italy) and visualized on a Gel Logic 100 gel documentation system (Kodak, New York, USA). Subsequently, PCR products were enzymatically purified and sequenced in both directions using the above listed primers, employing the Big Dye Terminator v.3.1 chemistry in an automated sequencer (ABI-PRISM 377). Nucleotide sequences were edited, aligned, and analyzed using Bioedit sequence Alignment Editor 7.0.5.366, and compared with available sequences in the GenBank using Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Antifungal susceptibility testing

The sensitivity to antifungal drugs of the identified yeast strains were evaluated using broth microdilution (BMD) testing in accordance with the guidelines available in the Clinical laboratory standard institute (CLSI) document M27-A367. The following antifungal drugs: Amphotericin B (AmB), Ketoconazole (KTZ) and Itraconazole (ITZ) (Sigma-Aldrich Milan, Italy), Fluconazole (FLZ), Voriconazole (VOR) (Pfizer Pharmaceuticals; Groton, Connecticut, USA), Posaconazole (POS) (Schering-Plough Corporation, Kenilworth, NJ, USA), Anidulafungin (ANI), (Sigma-Aldrich, Milan, Italy) were tested. FLZ was dissolved in sterile water, whereas the remaining drugs were solubilized in dimethyl sulfoxide-DMSO (Sigma-Aldrich, Milan, Italy). The concentration of each antifungal drug ranged from 0.016 to 32 mg/L, except for FLZ (i.e., from 0.03 to 64 mg/L).

Visual reading of plates was performed after incubation at 35 °C for two-three days. All strains were tested in duplicates. For azoles, the minimum inhibitory concentration (MIC) endpoint was defined as the lowest concentration that produced a significant decrease (= 50%) in turbidity compared with that of drug-free control for the yeast isolated in this study68,69,70,71,72,73. For AmB, the MIC endpoint was defined as the minimum concentration at which no visible growth was detected. Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22,019 were used as quality control strains67. Yeasts strains were considered drug resistant using breakpoints or Epidemiological cut-off values (ECV) according to CLSI documents68,69,72. Yeasts with no available breakpoints or CLSI ECVs were classified resistant using C. albicans breakpoints and/or ECV according to those previously proposed70,71,74.

Statistical analysis

A two-way analysis of variance (ANOVA) was conducted to assess the effects of various categorical independent variables on a continuous dependent variable and where the homogeneity of variance assumption was violated or the observations were non-normally distributed, the Welch’s ANOVA was computed for any significant difference. The independent variables analysed were sampled location/region, snake species, antifungal drugs, and the yeast species from both groups. The dependent variable included CFU/swab, yeasts prevalence and MIC mean value. Where a significant difference of mean was computed, a Tukey’s Honestly Significant Difference (Turkey’s HSD) post-hoc analysis was performed to identify specific pairwise difference between group means. A critical p-value threshold of p ≤ 0.05 was applied to determine statistical significance.