Data indicated that wild boar harbour, in their faeces many pathogenetic yeasts thus, suggesting their potential role in the spreading these organisms in the environment. The high density of yeasts in the faeces (i.e., CFU≥103) of a large population of wild boar (i.e., 50%) might indicate that the yeasts have also a role in causing infection in these animals. However, the absence of any signs of localized or systemic infections at the necropsy, confirm that these animals play a role in spreading pathogenic microorganisms in the environment. A similar picture was observed in others wild and domestic animals (i.e., rodents, cats), that are often asymptomatic carriers of pathogenic yeasts to humans [27]. In addition, the large range of wild boar movements (i.e., up to 16 Km) [28], as well as the changes of human habitation to suburban areas, the increased deforestation may increase the dissemination of fungal organisms of zoonotic concern [6, 29]. Similar prevalence of yeasts occurrence was previously observed in swine (44.2%), in cattle (46.8%), and in horses (56%) but it is lower than in wild birds (90%), thus confirming that host environment, as well as lifestyle-related factors of a host might play a primary role in shaping the fungal community [30]. In addition, the age of animals could be a factor affecting the diversity in the yeast species being lower in young animals compared to adults in which fungal flora showed the highest variety of yeast species. These finding might be due to the microbial community of adult animal is gradually formed based on exposure factors to different ecological and environmental conditions and immune system response [31].
In this study, Candida spp. was the most frequent isolated yeast genus with a mean population size ≥ 104 CFU/gram of faeces. The occurrence of these fungi in the faeces of examined animals could be explained by the fact that most of these yeast species are commensal organisms of intestinal and cloacal tracts of animals, whereas the high population density could be the result of compromised status of hosts or environmental contamination and consumption of contaminated food. On the other hand, these findings also indicate that wild boars harbour many important infectious agents that could be transmissible to domestic pigs and other animal species, including humans [6, 32]. Overall, all the isolated Candida species are causative agents of candidiasis or candidemia with clinical manifestations in humans (e.g., fungaemia, endophthalmitis, arthritis and endocarditis) which may ultimately cause high mortality and life-threatening infections in immune-compromised patients [33]. C. albicans is the most common agent of life-threatening human candidemia and candidiasis [34] and it has also been recovered from both wild and domestic animals, such as birds and reptiles [35]. Candida krusei and C. parapsilosis were retrieved in wild birds, cockatiels, dogs, cows and pigs faecal samples [10] and were considered the most common non-albicans Candida species causing severe infection in immune-compromised human patients [36]. In addition, C. krusei strains were characterized by a high mortality rate (40-58%) and poor response to standard antifungal therapies [37].
C. slooffiae was never reported as causative agent of severe infection in animals and humans but it has been frequently found in different parts of the gastrointestinal tract of apparently healthy pigs and therefore considered as a natural inhabitant of the porcine intestinal environment [38]. As far as C. guilliermondii and C. lusitaniae, they are rare human fungal pathogens, causing fungemia not exceeding 5% of nosocomial systemic infections worldwide. However, in certain geographical areas such as Brazil, India and Italy, over 10% of all the candidemia cases were caused by these species [39, 40]. All isolates molecularly identified to species level in this study showed very low intraspecific and high interspecific variability. An increasing number of studies reported that ITS-based identification system was accurate and applicable, even to strains with atypical morphological features [41]. Here, C. guilliermondii, C. fermentati, as well as C. parapsilosis and C. metapsilosis, were molecularly differentiated, confirming the utility of ITS sequence polymorphism analysis. Sequence analysis of the data revealed the circulation of four different STs for C. albicans, and one ST for others Candida spp. (C. parapsilosis, C. krusei, C. guilliermondii, C. fermentati, C. slooffiae, Clusitaniae, P.fermentans). These findings confirmed previous results suggesting that the genetic variability observed in ITS region, was more likely to be found in Candida species primarily involved as commensal organisms while species predominantly associated with an exogenously origin, such as, C. guilliermondii, C. fermentati, C. slooffiae, C. lusitaniae and C. lambica showed low intraspecific variability [2, 3]. The ML tree clearly indicates that all strains of each Candida species from wild boars were phylogenetically close to the same species recovered from immune-compromised patients suffering for a variety of lesions (e.g., oropharyngeal candidiasis, onychomycosis, persistent candidemia, candiduria, atopic dermatitis and vulvovaginal candidiasis). The phylogenetic analysis also showed that strains of C. albicans, C. parapsilosis, C. metapsilosis, and C. guilliermondii from wild boar clustered with those from domestic animals (i.e., sheep, cat and dog), from insect (i.e., Anopheles darling) and from environmental sources (i.e., tree, herb, egg, orange, water, soil) from Italy, as well as from other geographical locations (i.e., France, Poland, Hungary, Brazil, Argentina, Iran) [42]. However, MLST analysis should be done in the feature in order to confirm the molecular epidemiology of the fungal diseases caused by this yeast species. Overall, all these findings might suggest a clonal origin of these strains and their ability to circulate amongst different hosts and environment thus reinforcing the hypothesis that pathogens may be transmitted from wild boar (e.g., emerging zoonoses) to environment and to humans. Nowadays, wild boar populations are widespread, with considerably large home ranges, overlapping their habitat with livestock and humans and serving as an interface between human-influenced settings and natural areas [43].
As far the antifungal susceptibility profile of these yeasts the finding of high susceptibility for POS, VOR and ISA for all Candida spp. confirms the results of previous studies and suggests the usefulness of these drugs for treating tested these infections [44, 45]. However, a low susceptibility for azoles and echinocandins were detected among non-albicans Candida spp. strains. The high MICs for fluconazole in C. krusei and C. guilliermondii are a constant finding and may reflect an intrinsic resistance or acquisition of resistance followed to drugs exposure [46]. On the contrary, the high FLZ MIC values registered for C. lambica and C. sloffiae are of interest. In particular, these yeasts species are mainly isolated from environment or as commensal organism of porcine gut [47]. Usually, a very low MIC values for FLZ were registered in yeasts collected from their natural habitat whereas high FLZ MIC values were reported in isolates from human infections thus suggesting an acquired resistance phenomena during therapy. Consequently, the isolation of low FLZ susceptible yeast in faeces from wild board might be due the acquisition of these yeasts from anthropized environments or for the acquisition of azole resistance from environment azole exposition. However due to the small number of studies on antifungal susceptibility of these yeast species no any straightforward conclusions can be herein allowed. Accordingly, the poor susceptibility to echinocandins in C. guilliermondii and C. fermentati due to naturally occurring polymorphisms in the FKS1 hotspot regions [48], are usually registered in clinical isolates but the therapeutic level of drugs seems to be enough to successfully treat their infections [49].
In addition, the finding of the low level of azole or echinocandin resistance phenomena in the many Candida spp. herein registered is in accordance with the fact that azoles and echinocandins resistance is uncommon (< 1%) for Candida spp causing invasive fungal infections (IFIs) in patients from Southern Italy hospitals [50, 51]. All these findings might suggest that wild boards have no previous history of exposure to drugs or antifungal residues in the environment [52]. However, since azole fungicides are frequently used to treat fungal infections in agriculture, the acquisition of resistant phenomena is becoming a worldwide treat in the recent [53] and could at some point affect wild animals. Thus, the resistance rate registered from yeasts, isolated from wild boar, associated to the fact that wild boar is unlikely of being treated with antifungal drugs, might also suggest the importance of these animal species as bio-indicator of a good environmental quality.
In conclusion, this study showed for the first time the important role of wild boars in dissemination of pathogenic fungi in the environment, highlighting their potential role in zoonotic transmission of these microorganisms to immune-compromised human/animal hosts. The absence of resistance phenomena in the C. albicans and Candida non-albicans spp. strains from wild boar might reflect environmental free from residues of azoles antifungals pollution or chemical. Due to the ability of wild board to passively carry yeasts without affecting their susceptibility, these animals should be proposed as bio indicators of environment quality and/or as sentinel animal species for revelling the emergence of azole resistance phenomena. Further studies are needed in order to better understand the life cycle of pathogens and to disclose the evolution of resistance during transmission of yeast from wildlife animal to livestock/environment or human and vice versa.