Diversity, Virulence Factors, and Antifungal Susceptibility Patterns of Pathogenic and Opportunistic Yeast Species in Rock Pigeon (Columba livia) Fecal Droppings in Western Saudi Arabia.

Abstract Bird fecal matter is considered a potential source of pathogenic microbes such as yeast species that contaminate the environment. Therefore, it needs to be scrutinized to assess potential environmental health risks. The aim of this study was to investigate the diversity of the yeasts in pigeon fecal droppings, their antifungal susceptibility patterns, and virulence factors. We used culturing techniques to detect the yeasts in pigeon fecal droppings. The isolates were then characterized based on colony morphologies, microscopic examinations, and biochemical reactions. The molecular identification of all yeast isolates was performed by sequencing of the amplified ITS gene. Genes encoding virulence factors CAP1, CAP59, and PLB were also detected. Antifungal susceptibility patterns were examined by the disk diffusion method. A total of 46 yeast-like isolates were recovered, and they belonged to nine different genera, namely, Cryptococcus, Saccharomyces, Rhodotorula, Candida, Meyerozyma, Cyberlindnera, Rhodosporidium, Millerozyma, and Lodderomyces. The prevalence of two genera Cryptococcus and Rhodotorula was high. None of the yeast isolates exhibited any resistance to the antifungal drugs tested; however, all pathogenic Cryptococcus species were positive for virulence determinants like urease activity, growth at 37°C, melanin production, the PLB and CAP genes. This is the first report on the molecular diversity of yeast species, particularly, Cryptococcus species and their virulence attributes in pigeon fecal droppings in Saudi Arabia.


Introduction
Free-living wild birds are regarded as one of the indicators of a healthy environment. However, they also may be regarded as potential carriers of humanpatho genic viral, bacterial, fungal, and protozoan agents. Free-living rock pigeons (Columba livia), are found in large flocks within major cities around the world. They live in close proximity to humans, particularly in public parks, on rooftops, and sometimes close to catering establishments. Numerous reports highlight that pigeon fecal droppings in public areas are a source of bacterial infectious agents, such as Salmonella, Campylobacter, and E. coli O157, and that they may significantly affect public health (Abulreesh et al. 2007;Abulreesh 2014).
Carriage of pathogenic yeast in pigeon feces is a matter of growing interest and has been investigated worldwide, with much focus on the Cryptococcus species. Wu et al. (2012) reported the presence of eight different genera of yeast, such as Cryptococcus, Candida,

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and Rhodotorula, in pigeon fecal droppings in Beijing, China. They concluded that pigeon feces are a vector for medically important yeast species. Similarly, in Brazil, Costa et al. (2010) reported the predominance of Cry ptococcus, Candida, and Rhodotorula species in pigeon feces. Other reports from Egypt (Mahmoud 1999), the Canary Islands (Rosario et al. 2010), India (Xavier et al. 2013), and Sweden (Mattsson et al. 1999) highlighted the diversity of Cryptococcus species found in pigeon fecal droppings and the role of domestic pigeons as potential source of environmental pathogenic yeast. Soltani et al. (2013) even suggested a direct link between pathogenic yeast flora in pigeon feces and human infections.
The genus Cryptococcus is a basidiomycete yeast belonging to the Tremellales class. The genus Crypto coccus consists of two species complexes: C. neoformans species complex (comprise of two species: C. neofor mans and C. deneofirmans), and C. gattii species complex (comprises of five species: C. gattii; C. tetragattii; C. decagattii; C. deuterogattii and C. bacillisporus); these species are known for their potential clinical significance. C. neoformans species complex is responsible for cryptococcosis in humans, an infection that affects the lungs, the brain, and spinal cord (central nervous system) that usually affect HIV patients and other immunocompromised individuals, while C. gattii species complex causes serious infections and immunocompetent as well immunocompromised individuals. These species are commonly found in the excreta of wild birds, particularly pigeons, as well as soil, rotting vegetables, wood, and associated with certain species of plants. Cryptococcosis is not a contagious disease, however, it is acquired from environmental exposure to Cryptococcus species. Recent epidemiological data suggest that there are around 220 000 annual cases of cryptococcal meningitis in HIV patients worldwide. Therefore, cryptococcal meningitis may be the leading cause of death among HIV patients (Cogliati 2013;Hagen et al. 2015;Rajasingham et al. 2017;Esher et al. 2018;Magalhães Pinto et al. 2019).
Molecular identification tools such as real-time PCR, multiplex PCR, and RFLP PCR have been successful in detecting and identifying various yeast species in clinical and environmental samples. Furthermore, the identification of yeast-based on fungal ribosomal DNA (rDNA) has become popular as an accurate molecular tool. This tool allows for the detection of 18S and 26S subunits of rDNA that are separated by the internal transcribed spacers ITS1 and ITS2 (Pincus et al. 2007).
The city of Makkah is a major attraction for people around the world visiting for religious and spiritual purposes. Hence, the study of biological contamination of the environment by pigeons may be of great significance from a public health perspective. Very little information exists regarding the diversity and charac-terization of yeast species found in pigeon fecal droppings in Saudi Arabia, and particularly in Makkah city. The only available report describing the presence of C. neoformans in pigeon fecal droppings relied upon the phenotypical characterization of the isolates (Abulreesh et al. 2015). It did not involve the molecular identification of those isolates. The current study is, therefore, the first to report on the molecular diversity of yeast genera and their antifungal susceptibility patterns in pigeon feces within the city of Makkah, western Saudi Arabia. The paper also describes for the first time, the carriage of different genes encoding virulence factors among Cryptococcus and other yeast species found in pigeon feces in western Saudi Arabia.

Sampling.
A total of 100 samples of dried pigeon fecal droppings were collected from various locations within the city of Makkah, western Saudi Arabia, between May and November 2018. Each fecal sample was collected in a sterile universal bottle, protected from direct sunlight and transported to the laboratory on ice. All samples were processed within 6 h of collection.
Yeast isolation. Yeast species were isolated from pigeon fecal dropping following the methodology previously described by Abulreesh et al. (2015). First, 10 g of fecal droppings from each location was aseptically transferred to a flask containing 0.9 % saline solution with chloramphenicol (200 µg l -1 ). Then the mixture was shaken for 20 min and allowed to settle for 30 min. An aliquot of 0.5 ml of each supernatant was streaked onto Sabouraud dextrose agar (SDA) (Oxoid, Basingstoke, UK). Plates were incubated at 25°C and were examined daily for ten days to observe the growth of yeast. Individual colonies with the mucous appearance and yeast-like colonial morphology were selected and subcultured on SDA to obtain pure cultures.
Yeast identification. The selected colonies were microscopically examined for typical yeast cell morphology, pseudomycelium (specific for Candida spp.), and capsule in Indian ink preparations (in the case of Cryptococcus spp.). Biochemical identification for all yeast isolates included carbohydrate and nitrate assimilation (Teodoro et al. 2013), urease reaction on, urea agar base (Christensen's medium) (Oxoid, Basingstoke, UK) (Canteros et al. 1996), production of melanin on esculin agar (Oxoid), and ability to grow at 37°C on SDA (Abulreesh et al. 2015).
Molecular identification. All yeast isolates were identified at a molecular level by the detection of the internal transcribed spacer (ITS) regions. The ITS1-Diversity of yeasts in pigeon feces 4 495 5.8S-ITS2 fragment was amplified using universal primers ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) (White et al. 1990). Total DNA was extracted from colonies using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany), according to the manufacture's procedure. Amplification was performed in 50 µl reaction volume, containing 5.0 µl 10 × PCR Buffer, 4 µl dNTP, 0.5 µl rTaq, 1.0 µl ITS1 and ITS4, 3.0 µl genomic DNA, and 35.5 µl distilled water. The PCR protocol comprised of initial denaturation at 94°C for 5 min, followed by 35 cycles of 95°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. The PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN) and sequenced in both directions using the amplification primers on an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). BLASTN (http:// blast.ncbi.nlm.nih.gov) was employed to confirm the identified strains (Wu et al. 2012). Detection of virulence-encoding genes. The molecular detection of virulence-encoding genes, such as the capsular genes (CAP1 and CAP59) specific for Crypto coccus spp. and the phospholipase gene (PLB1) was performed on yeast isolates previously identified by ITS sequencing. CAP1 is a 700 bp long gene on chromosome IV and is a part of the MAT locus that encodes a capsule-synthesis associated protein. Primers used for CAP1 were, 5'-CGTTCGCGATAGAGAGAGGA-3' (forward) and 5'-CCTTACCTTCACAGTCGCCC-3' (reverse). CAP59 is a 502 bp long gene on chromosome I and encodes for a capsule-synthesis associated protein.
Antifungal susceptibility testing. To determine the antifungal susceptibility patterns, the agar disk diffusion method was employed as described by CLSI (2008). Four antifungal agents were used to examine the susceptibility patterns of the yeast species recovered from pigeon feces: Ciclopirox-Olamine (50 µg); Clotrimazole (10 µg); Nystatin (100 IU) and Fluconazole (25 µg), disks were purchased from Liofilchem Inc. (Waltham, USA). Fresh culture of each of the identified species was inoculated using swabs on the surface of Muller Hinton agar (Oxoid), plates supplemented with 2.0% glucose. Plates were incubated for 36 h at the optimum temperature for each fungal species. The sensitivity of the species against the antifungal compounds was determined by measuring the diameter of inhibition zones. A diameter of ≥ 15 mm for nystatin and ≥ 20 mm for the three antifungal agents was considered as susceptible. Susceptibility breakpoints of antifungal drugs used in our study are not species related (CLSI 2008). C. albicans ATCC 90028 was used as control.
Detection of virulence factors. The virulence factors were detected using (i) conventional methods, such as urease activity, and melanin production, and (ii) molecular methods probing for capsular (CAP1 and CAP59) and phospholipase (PLB1) genes.  Table II shows the presence and absence of the CAP1, CAP59 and PLB1 genes for all isolates. The CAP1gene was detected in six of 11 Cryptococcus neo formans isolates, while the CAP59 gene was detected in the other five isolates. Two isolates of C. gattii also possessed the CAP59 gene (Fig. 3). The PLB1 gene was detected in three Cryptococcus species, C. neofor mans; C. albidus, and C. gattii but not in C. liquefaciens. C. glabrata and S. cerevisiae were also positive for the PLB1 gene (Fig. 3).
Antifungal susceptibility patterns. None of the 46 yeast isolates showed resistance to any of the antifungal drugs, i.e., Ciclopirox-Olamine; Clotrimazole; Fluconazole and Nystatin used in this study (Table III).

Discussion
The presence of yeast species, especially the pathogenic ones, in pigeon fecal droppings and the role of pigeons in the dissemination and epidemiology of these pathogenic species have been investigated worldwide. However, in Saudi Arabia, there is a dearth of information regarding the environmental diversity of yeast,   (2015) were the first to report about C. neoformans in pigeon fecal droppings in Saudi Arabia. Their report was based on phenotypical identification of the presumptive isolates, and no molecular identification was performed to confirm the results. The aim of this work was to further investigate the diversity of Cryptococcus and other yeast species in pigeon excreta, in addition to their virulence factors and antifungal susceptibility patterns. Diversity of yeast species in pigeon feces. In this study, nine different genera of yeast were found in pigeon fecal droppings, in comparison to other studies conducted elsewhere, the current study has identified the most diverse yeast species ever reported. For example, Wu et al. (2012) reported six different yeast species in pigeon feces from various locations in Beijing, China. In our study, we reported C. neoformans, C. albidus, and C. gattii, with C. neoformans being the most prevalent than other species. These species have frequently been associated with pigeon fecal droppings in Brazil  (Mahmoud 1999;Mseddi et al. 2011;Abbass et al. 2017). One of our isolates is C. liquefaciens, which has not been reported in pigeon excreta to the best of our knowledge. We are the first to report its presence in pigeon excreta in the Middle East and Asia. In General, Cryptococcus spp. thrive in pigeon fecal droppings due to the high content of urea and organic matter (Costa et al. 2010;Abulreesh et al. 2015).
Similar to the previous studies, we found that S. cere visiae is less common than other saprophytic yeast species in pigeon feces (Wu et al. 2012;Rosario Medina et al. 2017). Although S. cerevisiae has no veterinary significance, its presence in bird excreta may be due to the physico-chemical nature of the droppings that provide a rich environment for yeasts to grow (Cafarchia et al. 2008).
Candida species, especially C. albicans have also been frequently found in pigeon excreta (Wu et al. 2012;Rosario Medina et al. 2017). However, C. albicans was not detected in our study, instead, C. glabrata, was the only Candida species encountered. Other Candidarelated genera, M. guilliermondii (formerly Candida guilliermondii), and Cyberlindnera fabianii (formerly Candida fabianii), were also present in this study, the former has been isolated in pigeon feces in Spain (Rosario Medina et al. 2017), while the latter has been very rarely detected in pigeon excreta.
We detected two other genera in our study, M. fari nosa (formerly Pichia farinosa), and Rhodosporidium paludigenum. Both have not been, detected in pigeon or other bird excreta worldwide. On the other hand, R. mucilaginosa and R. glutinis are very commonly found in pigeon fecal droppings (Wu et al. 2012;Marenzoni et al. 2016;Abbass et al. 2017). However, Lodderomyces elongisporus has seldom been reported in pigeon fecal droppings (Wu et al. 2012). It is worth noting that due to the method of sampling we adopted in the current study,  4 500 environmental contamination of fecal samples (e.g. from soil) cannot be ruled out and may play, in part, role in the diverse yeast genera reported in our study.
Virulence factors of yeast species isolated from pigeon excreta. Virulence factors play a vital role in pathogenesis. Various Cryptococcus species, like C. neo formans, C. gattii, and C. albidus possess an arsenal of such molecules. These factors enable them to successfully invade hosts, to resist defense mechanisms of their immune system and to cause infection, especially in the immunocompromised individuals. The prominent capsule of Cryptococcus species is an important virulence factor. All the Cryptococcus species isolated in this study displayed capsules in Indian ink preparations, as observed under the microscope. On a molecular level, the CAP1 gene was detected in 54.55% of C. neofor mans isolates, whereas, the CAP59 gene was detected in the rest of the isolates. The C. gattii exhibited only the CAP59 gene. The formation of the capsule by the Cryptococcus species was induced by many environmental conditions that include pH, CO 2 levels and iron deprivation (Alspaugh 2015). The polysaccharide capsule helps Cryptococcus species to proliferate within the phagocytic cells and to inhibit host any immune response. Once it has invaded the host cell, and colonized the vacuole, Cryptococcus will survive by the aid of the capsule and replicate despite the acidic nature of the vacuole (Srikanta et al. 2014). Both of the CAP1 and CAP59 genes are specific to C. neoformans and C. gattii, this explains the absence of these genes in other encapsulated isolates such as C. liquefaciens, C. glabrata, and Rhodotorula species.
Phospholipase is another virulence factor that helps the pathogenesis of Cryptococcus and other pathogenic yeast species. Phospholipase activity can alter the microenvironment of infection and can facilitate Cryptococcus species to survive better within the host cells (Alspaugh 2015). All Cryptococcus species detected in this study, except for C. liquefaciens, were positive for the PLB1 gene, it was also detected in C. glabrata. The presence of the PLB1 gene was also noted in S. cerevisiae isolates. The ability of phospholipase to hydrolyze phospholipids and to produce several bioactive compounds has given S. cerevisiae its industrial potential.
Melanin is a known protective determinant for Cry pto coccus against environmental stressors and, hence, considered a virulence factor (Alspaugh 2015). Similarly, urease activity has been associated with pathogenesis, in pathogenic yeasts such as the Crypto coccus species. The ammonia produced by urease activity damages the host cell endothelium; thus, the yeast to transmigrate toward the central nervous system (Feder et al. 2015). It is suggested that capsules, melanin production, and high-temperature growth (at 37°C) are key virulence determinants for pathogenic and oppor-tunistic yeasts (Boral et al. 2018). All these characteristics were observed for the isolates of Cryptococcus species in this study.
In general, several environmental yeast species possess the similar virulence factors as their pathogenic clinical counterparts (Magalhães Pinto et al. 2019). Our study reports this notion and highlights the presence of pathogenic yeast in the fecal droppings of free-living pigeons, suggesting the pathogenic potential of these environmental species of yeast.
Antifungal susceptibility. None of the 46 yeast isolates representing the nine different genera reported here, was resistant to the antifungal drugs tested. These results correlate with previously reported studies, particularly for Cryptococcus species, that it is rare for environmental (pigeon-derived) C. neoformans and C. gattii to exhibit resistance to antifungal drugs (Costa et al. 2010;Souza et al. 2010;Tangwattanachuleeporn et al. 2013;Teodoro et al. 2013). The clinical isolates of Cryptococcus species exhibit similar trends of low resistance to antifungal drugs (Souza et al. 2010;Govender et al. 2011). Environmental and clinical samples of C. glabrata have previously exhibited susceptibility to some of the antifungal drugs used in this study (Nenoff et al. 2011;Lotfalikhani et al. 2018;Miranda-Cadena et al. 2018). The lack of antifungal resistance was also observed in clinical R. mucilaginosa isolates (Razzaq Abed and Mohammed Hussein 2017). Resistance to fluconazole appears to be common in clinical isolates of R. mucilaginosa, this perhaps due to the fact that most of the patients are administered with fluconazole when fungemia is diagnosed (Wirth and Goldani 2012). The resistance mechanism of Rhodotorula to fluconazole is not known, thus, the observation of repeated resistance may suggest intrinsic resistance in some isolates (Duggal et al. 2011). So far, no available reports have described antifungal susceptibility patterns of environmental Rhodototrula species, therefore, whether environmental isolates exhibit similar resistance patterns, particularly to fluconazole, remains to be elucidated.
There is a lack of scientific evidence on the antifungal susceptibility patterns of both environmentally and clinically derived isolates of M. farinose, S. cerevi siae, R. glutinis, M. guilliermondii, C. fabianii, R. palu digenum, and L. elongisporus. Hence, we are unable to compare and discuss our results for these yeast species.
The lack of resistance observed in these environmental isolates of yeasts, particularly pathogenic species, may be explained by the role of various environmental factors that are not fully understood. Possibly there might be unique ecological niches in the environment where environmental yeast species can acquire drug resistance, or there might be a pattern of spread of drug resistance among environmental yeast species through geo-climatic factors, such as wind activity or Diversity of yeasts in pigeon feces 4 501 global warming that yet to be explored. Bird migration has been playing an important role in the spread of multidrug resistance in bacteria; it is not clear whether it plays a similar role in yeast drug resistance (Kontoyiannis 2017). Furthermore, it was hypothesized that environmental yeast could acquire resistance to antifungal drugs in the presence of industrial waste of pollutants that could promote altered expression of genes that may occasionally occur in pathways related to resistance (Milanezi et al. 2019). It is also possible that the disk diffusion method may have influenced the susceptibility results. It is highly likely that the use of dilution method or MIC test strips would provide more accurate susceptibility results in comparison to the disk diffusion method, i.e. some of the isolates might have exhibited resistance if tested by the dilution method or MIC strips.
Public health significance of this study. Earlier studies have implicated that free-living pigeons spread pathogens in the environment and have established a direct link between pigeon droppings and human infections (Haag-Wackernagel and Moch 2004). In this study, we observed three different species of pathogenic Cryptococcus in pigeon fecal droppings in western Saudi Arabia: C. neoformans, C. gattii, and C. albidus. Together they make up around 39% of all yeast species found in the excreta. Different Cryptococcus species have been reported to cause human infections, C. neoformans and C. gattii for cryptococcosis (Cogliati 2013), C. albidus for fungemia (Cleveland et al. 2013), and respiratory infections (Burnik et al. 2007). In Saudi Arabia, there is only one incidence of a clinical case involving C. neoformans. The pathogen caused abscess and osteomyelitis in an immunocompetent individual (Al-Tawfiq et al. 2007). However, this lack of reported incidents may not truly reflect low occurrence of clinical cases in the region. In contrast, we suggest that there may have been relatively few clinical investigations of medically important fungi.
Other yeast species found in pigeon feces in this study are also implicated in human infections. C. gla brata has been implicated in various diseases in humans including both superficial and systematic infections, such as brain abscess (Zhu et al. 2018), vertebral column (spondylodiscitis) infection (Gagliano et al. 2018), joint infection (Koutserimpas et al. 2018), and cutaneous granuloma (Fan et al. 2018).
R. mucilaginosa is an environmental yeast that has emerged as a causative agent of serious and even fatal opportunistic infections, including fungemia (Kitazawa et al. 2018) and meningitis (Miceli et al. 2011) particularly in immunocompromised patients, and immunocompetent individuals. Other species identified in our study that have been implicated in severe or fatal human infections include: C. fabianii (Hof et al. 2017), L. elongisporus (Hatanaka et al. 2016), M. guilliermondi (Cebeci et al. 2017), and the emerging invasive infections of S. cerevisiae (Popiel et al. 2015).
The most commonly transmitted pathogens via pigeons continue to be Chlamydophila psittaci and C. neoformans (Haag-Wackernagel and Moch 2004). Considering the relationship between environmental C. neoformans strains and human infection, Delgado et al. (2005) concluded that cryptococcosis could be acquired from the environmental strains in both urban and rural areas. Liaw et al. (2010) drew similar conclusions, finding strong similarities between clinical and environmental strains of C. neoformans and suggesting that patients might acquire yeast infection from the environment. Overall, the results of our study demonstrate that pigeon fecal droppings carried a number of pathogenic yeast species as well as emerging opportunistic yeast genera.
In Makkah city, western Saudi Arabia, massive numbers of pigeons inhabit the city. They flock in public parks, rooftops, and in close proximity to catering areas, making it almost unavoidable for humans to come in contact with their droppings. Pigeon excreta can be noted almost everywhere in the city. Additionally, dust from dried pigeon fecal droppings may contain different cryptococcal species and other opportunistic yeast. Despite the massive cleaning efforts of public spaces, dissemination of pathogenic yeast by air is inevitable, increasing the chances of acquiring infection through the respiratory system.
Risk assessment of environmental Cryptococcus species. C. neoformans and C. gattii are pathogenic yeasts that rarely cause infections in healthy individuals. However, immunocompromised patients, such as those who underwent organ transplant, those under medications that weaken the immune system (e.g. corticosteroid or rheumatic arthritis medications), or people with an advanced stage of HIV infection are at a high risk of getting C. neoformans infection. In addition, elderly individuals, over 50 years old with lung health issues may be at risk of C. gattii infection (Cogliati 2013). Cryptococcus infection is not contagious and there is low risk for healthy people to be infected when in contact with an infected individual (Delgado et al. 2005). However, Cryptococcus can infect healthy people when they inhale the dust containing the pathogens. This is common in the environment, in areas where pigeon fecal droppings exist in abundance. Every individual may inhale the yeast dust, yet they may not develop any symptoms immediately. Cryptococcus can stay hidden within the body and cause infection later, when the immune system is too weak to fight it (Esher et al. 2018). Currently, many countries around the world do not consider the detection of Cryptococcus in clinical routine work, particularly in meningitis cases. To mitigate the risk of Cryptococcus infections, it is necessary Abulreesh H.H. et al. 4 502 to perform extensive surveillance of the environmental distribution of the pathogens and laboratory detection of Cryptococcus infections in clinical specimens. Early detection of Cryptococcus infection in individuals may help to treat them promptly and to reduce the mortality rate of infected people.

Conclusions
Pigeon fecal droppings in western Saudi Arabia were found to harbor a wide range of pathogenic and opportunistic yeast species. Although none of them were resistant to the common antifungal drugs, all pathogenic species and some of the opportunistic species did carry different virulence factors. Our study confirms that pigeon fecal droppings provide a rich environment for the growth of saprophytic yeasts, particularly Cryptococ cus species. Additionally, we demonstrate that pigeons may act as reservoirs and carriers not only for pathogenic yeast (e.g. Cryptococcus and Candida), but also for opportunistic yeast species (e.g. R. mucilaginosa). This is the first report on the diversity and virulence factors of yeast species in pigeon fecal droppings in Saudi Arabia. Further investigations are required to understand the pathogenicity of these isolates in humans and animals using suitable experimental models.