Molecular Characterization of Multi-Drug Resistant Fungal Isolates Obtained from Raw Cow Milk in Bowen University, Nigeria

Cow milk is a highly nutritious food but many factors predispose it to microbial contamination. There is paucity of information on antifungal-resistant pathogens. Hence the study investigated the fungi from fresh cow milk samples and their resistance to some antifungals. Twenty-seven (27) fungi were isolated from forty-three (n=43) milk samples on PDA (Potato dextrose agar) media, re-cultured in glucose yeast extract broth, and incubated for 24 hours at 25°C. Taxonomic characterization on the isolates was done using photomicrography. Percentage occurrence of the isolates was determined. Molecular characterization was carried out on some isolates which were 100% resistant to antifungals; ketoconazole, amphotericin B, and clotrimazole using ITS1 and ITS4 primers. Isolate sequences were subjected to BLAST analysis and compared with representatives in GenBank. Kruskal-Wallis and Mann-Whitney Tests were used to analyze the data. Phylogenetic analysis and morphological characterization identified isolates as Rhodotorula paludigena, Candida sp and Candida tropicalis which had occurrence of 31.8%, 31.8% and 36.6% respectively. Level of resistance to ketoconazole (100%) and amphotericin B (92%) was significantly higher than clotrimazole (59%) (p< 0.05), while between ketoconazole and amphotericin B, there was no significant (p> 0.05) difference. Unprocessed raw milk is a potential source of drug-resistant pathogenic fungi. Pasteurization of raw milk is highly encouraged before consumption.


Introduction
Raw cow's milk is regarded as highly nutritious and is sometimes consumed raw in many countries as a normal cultural practice. Some of the benefits of consuming raw milk include improved respiratory allergies and atopic eczema, so its usage is rising in many nations [1,2]. But raw milk has proven to be an ideal media for microbial growth as it contains all the basic nutrients and conditions necessary for their survival, it is typically regarded as an optimal growth medium for microorganisms [3,4]. Many factors predispose raw milk to microbial contamination, such as animal welfare, which could impact the animal's physiological status, weather, and breeding conditions.
It was previously thought that milk contained in the mammary gland was sterile and free from microbial contamination and microbial isolates gotten from the mammary gland were contaminations from the external environment. But recent studies with the aid of more sophisticated molecular methods have suggested that the mammary gland of healthy individuals can be colonized by a wide variety of microorganisms [5]. Hence, microbial colonies isolated from the teat canal and teat surface inform the need for microbial analysis of raw milk [6]. Milk contamination is also influenced by poor hygienic practices, contaminated equipment, poor environmental conditions around the milking parlor, contaminated water supply, contaminated feed, and so on. [7,8].
Fungal contamination of milk is a growing concern in the agricultural sector of many sub-Saharan developing countries [9]. Akinyemi et al. (10) reported the contamination of raw cow milk sourced from local cows by multiple mycotoxins besides the regulated Aflatoxin M1. Adem et al. (11) also confirmed the presence of high quantities of disease-causing fungi and parasites in a large component of raw milk sourced from local animal breeders in the country. Scientific reports on multi-drug resistant bacteria are available in the country [12]. Thonda et al. (13) reported the prevalence of antibiotic resistant bacteria in raw cow milk samples in Osun state, Nigeria. But there is a dearth of reporting on multi-drug resistant fungi isolates in raw cow milk, which is often the most commonly available milk for consumption in many parts of the country. This research will provide information on the resistance pattern of microorganisms obtained from animal origin and the potential parameters that may be improved on in the adoption of relevant therapeutic decisions. Hence, this study aims to evaluate fungal communities associated with fresh cow milk samples collected from cows and used for public consumption in Southwest Nigeria and to study their antifungal resistance pattern.

Agar medium, Reagent and antifungals used
Potato dextrose agar (PDA) (Oxoid Limited, USA) was used for culturing the fungi, while the yeast isolates were sub-cultured in Yeast extract broth (Oxoid Limited, USA). Methylene blue (PS Pack Scientific Limited, UK) was used in staining the isolates for microscopic observation. The antifungal drugs; ketoconazole, amphotericin B and clotrimazole (Mast Group Limited, UK) were tested against the fungal isolates for sensitivity test.

Sample Collection
The study area was at Bowen University Iwo, Nigeria 7 o 38'N 4 o 11'E. Samples of raw milk were collected in sterile conical flasks from the Oluponna area, these were taken to the Microbiology laboratory under cold temperature of 4 0 C for analysis.

Microbiological analysis of the samples [14]
2.3.1. Culturing of fungi. The culture medium, Potato Dextrose agar (PDA) was prepared by dissolving 39 grammes of the powder in 1L of distilled water and was sterilized in the autoclave at 121 0 C for 15 minutes. Ten mL of streptomycin (10,000 I.U./mL) was added to the agar to prevent bacterial growth. The milk sample was agitated, serially diluted and 1mL of the sample of 10 4 dilution was inoculated into sterile Petri dishes. The sterile molten agar was poured inside the inoculated Petri dishes, swirled gently before allowing it to set. Culture plates were incubated at 25 o C for 72 hours [15]. Twenty-seven (27) yeasts were isolated from forty-three (n=43) milk samples cultured.

Yeast isolates in liquid medium.
The isolated yeasts were inoculated into sterile glucose yeast extract glucose broth containing 2% yeast extract and 20% glucose and 75 ug/ml Ampicillin in 100ml conical flasks plugged with cotton wool and incubated at 24 hours at 25 0 C before taken for molecular characterization

Identification and characterization of fungal isolates 2.4.1. Morphological Characterization.
Pure cultures of the isolates obtained by subculturing the mixed cultures on sterile PDA plates were morphologically identified using the cultural and microscopic features. The macroscopic morphological features include the colony shape, colour, texture of the colony. The microscopic characteristics of the fungi were viewed under the microscope by placing a drop of methylene blue (stain) on a clean slide and a small portion of the fungal growth on the stain using sterile inoculating loop. A cover slip was carefully placed on the preparation eliminating air bubbles. The slide was then mounted, viewed and the microphotographs taken using photomicrograph microscope at a magnification of 40 [16].

Antifungal Sensitivity Test (Kirby-Bauer disc diffusion technique) [17]
The yeast isolates were subjected to antifungal sensitivity test using three (3) antifungal drugs; ketoconazole (10µg), amphotericin B (20µg) and clotrimazole (10µg). This was carried out by swabbing the yeast growth on solidified plates of sterile PDA using sterile cotton swab. Sterile forceps was also used to pick and place the antifungal discs on the "seeded" agar plates. Inoculated plates were incubated at 25 0 C for 24 hours. Observed zones of inhibition were measured in millimeters (mm) using measuring ruler.

Molecular Characterization of the fungal isolates
Three (3) of the yeast isolates that were resistant to all the antifungal drugs were selected and sent to Macrogen Europe Laboratory, Netherlands for molecular characterization. (18). Fungal mycelia of about 100mg was macerated in an already sterilized laboratory mortal by adding 1ml of proteinase K (0.05mg/ml). DNA Extraction Buffer (DEB), was added to macerated fungal mycelia in 1.5ml Eppendorf tubes, and then 50µl of 20% Sodium Dodecyl Sulphate (SDS) was also added. The mixture was incubated at 65 o C for 30 minutes in a water bath. The tubes were cooled to room temperature. After reaching room temperature, 100µl of 7.5M Potassium acetate was introduced into the mixture and then the mixture was homogenized briefly. Centrifugation was carried out at a speed of 13000rpm for 10 minutes. The supernatants obtained were decanted into fresh autoclaved tubes, then, 2/3 volume of cold isopropanol was added. Incubation was done at -20 o C for 1 hour. The mixtures were further centrifuged at 13000rpm for 10 minutes, and then the supernatant portion were discarded. 500µl of cold 70% ethanol was subsequently added, and centrifugation was repeated for 5 minutes at 13000rpm. The supernatants were discarded with the DNA pellets intact, then the DNA pellets were dried at 37 o C for 30 minutes. 50µl of sterile distilled water was added to each DNA pellet to dissolve it. 2.6.3. Polymerase Chain Reaction (PCR Analysis. PCR cocktail comprising of 10µl of 5x GoTaq colourless reaction; 3µl of 25Mm MgCl2; 1µl of 10mM of dNTPs mix; 1µl of 10pmol each forward primer (ITS 1) -5' TCC GTA GGT GAA CCT GCG G 3' and reverse primer (ITS 4) -5' TCC TCC GCT TAT TGA TAT GC 3'; 0.24µl of 0.3units of Taq DNA polymerase (Promega, USA) made up to 42 µl with sterile distilled water and 8μl working DNA template. Amplification was done using 'PCR system thermal cycler' (Applied Biosystem Inc., USA) with PCR profile of an initial denaturation, 94°C for 5 minutes; 35 cycles of 94°C for 30 s, 55°C for 30s and 72°C for 1 minute 30 seconds; and a final extension at 72°C for 10 mins. The amplified DNA fragments of target sequences in PCR (PCR products) were purified with ethanol in order to remove the remnants of PCR reagents. The presence or absence of expected band size of amplified target ITS gene sequence was checked when fragment was run on a 2% agarose gel electrophoresis at a voltage of 100V for 50 minutes. It was viewed under UV light and picture taken.

Gel electrophoresis.
Products of PCR were viewed on a 2 % agarose gel containing maestro safe stain (New Biolab Group, USA) in 0.5x Tris-borate buffer (pH 8.0) using blue light transilluminator (New Biolab Group, USA). A molecular ladder marker (Jena Bioscience, midrange) was run simultaneously to determine the size of the amplicons.
2.6.5. Sequencing. Sequencing was done by Sanger sequencing method using AB1 3730XL sequencer and done by Macrogen Europe, Netherlands.

Phylogenetic analysis
To infer the evolutionary history of the fungal isolates the Neighbor-Joining (NJ) approach was used [19]. The bootstrap test (1000 repetitions) displayed the ideal tree, [20]. Kimura 2-parameter technique was used to calculate the evolutionary distances, these are measured in base substitutions per site [21]. The percentage of places where at least 1 unambiguous base is present in at least 1 sequence for each downstream clade were placed next to each internal node in the tree. There were 8 nucleotide sequences in this investigation with codon positions 1st+2nd+3rd+ Noncoding. All unclear places for each sequence pair was eliminated (pairwise deletion option). MEGA 11 software was used to conduct evolutionary analysis [22].

Statistical analysis
To compare the differences in the level of resistance of the isolates to the antifungals; ketoconazole, amphotericin B and clotrimazole, Kruskal-Wallis Test was used at p < 0.05 [23], while Mann-Whitney Test was applied for the pairwise comparison of the resistance exhibited against the isolates by the three antifungal drugs at p < 0.05 [24].

Results
On potato dextrose agar, Rhodotorula species appeared as coral red colonies, with smooth and glossy surfaces, while Candida appeared as cream colonies, having smooth, dull, wrinkled and tough surfaces. In the molecular characterization of the isolates, the gel result showed distinct band of the amplicons (Lanes 1, 2 and 3) which are within the expected base pair range (500-600bp) as represented in Plate 3. There was no negative result. Sequence analysis of ITS regions of the nuclear encoded rDNA of isolated fungal species showed significant alignments of 74 and 99.46% (similar percentage identity) with the complementary species on the database. The isolates were identified as Rhodotorula paludigena and Candida tropicalis. The phylogenetic relationship showed that the sequences of the yeast isolates show high similarity to others (Figure 3). The representation clustered with the ones retrieved from the GenBank database showing a maximum evolutionary relationship with their specific genera.
The antifungal assay carried out on the yeast isolates in this study revealed 92.6%, 100% and 59.3% resistance to antifungal drugs; ketoconazole, amphotericin B and clotrimazole respectively (Figure 2). These resulted in percentage susceptibility of 7.4, 0 and 40.7 for ketoconazole, amphotericin B and clotrimazole respectively. Table 1 is the summary of the comparison of resistance of the isolates against the tested antifungals. There is no significant difference between the resistance of the isolates against ketoconazole and amphotericin B (p>0.05), but there is significant difference between resistance against ketoconazole and amphotericin B; and clotrimazole (p<0.05). The pairwise comparison of the antifungal drugs using Mann-Whitney test is in Table 2. Comparing ketoconazole with amphotericin B, there was no significant difference in the resistance at p>0.05, whereas significant difference occurred between ketoconazole and clotrimazole, and also between amphotericin B and clotrimazole at p<0.05.

Discussion
The fungi isolated from the cow milk samples in this study were yeasts, which were Rhodotorula and Candida spp. According to Lavoie et al. [25], yeasts are usually more in cow raw milk, while in some ecosystems, moulds can be more. Their work revealed that 67% of the isolates were yeasts (37 species) while the remaining 33% were moulds. These yeasts have also been identified as part of the microbial community in Chinese milk fan; a cheesy fermented milk product produced from raw cow milk in China [26].
Garnier et al. [27] reported that the most frequently occurring moulds responsible for dairy products spoilage belong to the genera Penicillium and Mucor. Yeasts have the ability to grow in environments IOP Publishing doi:10.1088/1755-1315/1219/1/012001 9 with a wide pH range making them ideal spoilage organisms. Low pH dairy products such as yoghurt, fermented milk, cream cheese are easily spoilt by yeasts [28]. Yeasts, moulds and some bacteria can grow in milk at temperatures above 16 0 C. Fungal microbiota is mostly reported in fermented milk [29]. A few recent reports in different locations in Nigeria also documented the presence of fungi in raw milk, most of which produce mycotoxins [30,31]. It was confirmed in a study of milk's fungal microbiota that the variety and makeup of the microbiota can differ from one location to another. Milking, handling, storage, and other pre-processing operations can increase the population of fungi in raw milk [32,33,34]. They can also enter the milk through the cow, air, feedstuffs and the processor or personnel.
In this study, Candida tropicalis had a higher percentage of 70.4% while Rhodotorula paludigera had 29.6% occurrence. In a study carried out by De Casia Dos Santos and Marin [35], Candida had a high occurrence of more than 50% of the isolated yeast species. Some Candida spp. have been associated with bovine mastitis infection in cows [36]. Rhodotorula are recognized carotenogen and have industrial importance as they produce carotenoids [37]. Rhodotorula species have been discovered to be implicated in lung infections and otitis in cattle [38].
The molecular characterization of the isolates helped in the accurate identification of the yeasts. The morphological examination both at cultural and microscopic levels was authenticated by the molecular identification. According to Anderson and Parkin [39], ITS-rDNA is an important tool that is recommended for the identification of fungi from various sources. The phylogenetic results complemented the morphological characterization to identify isolates as yeasts; Rhodotorula paludigena and Candida tropicalis which are known to be pathogenic to man. Also, this study revealed that there is genetic relationship between Candida tropicalis BUSLCM6, Candida tropicalis BUSLCM6 isolated from raw cow milk in this study and Candida tropicalis LU 108 (MYV 113255) with 90% similarity; and also, Candida tropicalis LU 108 (MYV 113255) with 93% similarity. Rhodotorula paludigena BUSLCM6 isolated from the raw cow milk and the Rhodotorula paludigena strains that were isolated previously from other sources as obtained from the GenBank data [40] are also similar. Rhodotorula paludigena BUSLCM6 clade clustered with the identified Rhodotorula paludigena KV18 (ON411181) with a very high bootstrap value of 100% on the BLAST tool.
Statistically, the results of the resistance pattern of the isolated yeasts to the test antifungals showed that ketoconazole and amphotericin B were more effective in inhibitory action against the isolated yeasts than clotrimazole. This indicates that clotrimazole was the least effective drug against the isolated yeasts in this study. The multi-drug resistance to all the antifungals used in this study has many implications, for example, the development of the multidrug resistance could have been through imprudent and overuse of these antifungals in agriculture and veterinary sector [41]. Also, there is limited treatment option since only three major classes of antifungals being utilized in clinical practice; polyenes (including amphotericin B), triazoles (represented by ketoconazole and clotrimazole) and echinocandins (which was not readily available for this study) [42].
Even though there is scarcity of literature on resistance from fungal pathogens obtained from animal origin, yet previous exposure to antifungal drug in form of therapy, could be a possible reason for the development of resistant fungal strains [43]. The use of these drugs in livestock has been suggested to promote the spread of resistance in microorganisms associated within food products, and subsequently, represent an unacceptable risk to both the public health and the environment, in general [44].

Conclusion
The isolation of antifungal-resistant pathogens from the tested cow milk samples is an indication that fresh milk can be contaminated by fungi either from the environment or any other sources. Raw milk and milk products are potential sources of drug-resistant pathogenic fungi which can pose a serious public health risk. Prudent use of antifungals in agriculture and veterinary sectors is recommended. hence there is need to pasteurize raw milk before consumption.