Occurrence of Fungi on Duck Egg Shells and Drug Resistance Analysis of Aspergillus spp. Isolates

Abstract

Fungi are a typical part of the microbiome of poultry houses, but some of the genera can be pathogenic for poultry and humans. An investigation was conducted on 200 duck eggs from 10 flocks to determine total fungal contamination on the eggshells. The colony types were identified morphologically and microscopically, and a representative group was identified using PCR. The resistance profiles for all obtained Aspergillus isolates were conducted. The dominating genera on eggshells were Penicillium, Alternaria and Aspergillus and the number of fungal colonies ranged from 0 to 7100. Aspergillus fumigatus was cultured from 9.5% eggshells, and all isolates were obtained from three flocks. The minimum inhibitory concentration (MIC) values for A. fumigatus isolates ranged from 0.094–32 μg/mL for amphotericin B (MIC 50 1 mg/L and MIC 90 32 μg/mL), 0.125–32 μg/mL for caspofungin (MIC 50 0.38 μg/mL and MIC 90 32 μg/mL), 0.19–32 μg/mL for itraconazole (MIC 50 1.5 μg/mL and MIC 90 32 μg/mL), 0.047–12 μg/mL for posaconazole (MIC 50 0.5 μg/mL and MIC 90 8 μg/mL) and 0.023–32 μg/mL for voriconazole (MIC 50 0.19 μg/mL and MIC 90 32 μg/mL). A total of 73.7% of the isolates were resistant to posaconazole and 68.4% to itraconazole. Nearly half of the strains (47.4%) showed resistance to amphotericin B and 31.6% to voriconazole. Because of the lack of antifungals registered for poultry, hygiene and the regular disinfection of litter in particular are needed to prevent the contamination of the eggs by fungi for both animal and human health.

1. Introduction

Fungi are a typical part of the microbiome of poultry houses. They are present in the litter, feed and the organic dust emitted by farms. Some of the fungal genera, such as Aspergillus or Candida, may cause poultry and human infections [1,2]. Aspergillosis is one of the most common respiratory infections in poultry. Out of all animals, birds are the most susceptible group, due to their anatomic particularities such as the absence of an epiglottis and limited number of ciliated epithelial cells in the respiratory system, which allows the conidia of these fungi to directly reach the lower parts of the respiratory tract. The lack of diaphragm prevents the active expulsion of particles out of the lungs, and the lack of superficial macrophages and the presence of heterophils instead of neutrophils makes the poultry immune response less effective against aspergillosis [3,4].

The main route of infection in birds is inhalation or ingestion of conidia from the environment. Sick birds show respiratory symptoms such as dyspnea and an accelerated respiratory rate, but may also present suppressed growth and nervous symptoms [5,6,7]. The most susceptible are newly hatched and young birds in which infection is generally acute and the mortality rate is high (up to 90% of the flock) [6]. Early infection is possible, because Aspergillus species may penetrate eggshells and infect embryos. The eggshell porosity provides a means of entrance for microorganisms into the egg from the nesting material and environment. Fungi can proliferate due to the high air humidity and diffusion of vapor from the egg content while stored and incubated, and cracking up contaminated eggs in incubators generates spores that contaminate hatchery equipment, eggs and embryos [7,8,9]. The infected embryos’ mortality is mostly observed between the 15th and 18th day of incubation, which causes decreased hatchability even up to 30%. Potential embryo infection occurs when the dust contains more than eight hundred colonies per gram [9,10]. The eggshell infested by fungi can be a hazard for embryos and young birds, causing respiratory problems. Because aspergillosis can cause direct loss through higher mortality, impaired growth-feed conversion and immunosuppression, it is the major threat among mycotic diseases in birds. The lack of information about the occurrence of fungal genera on the eggshell of duck eggs and sparse data about the fungal contamination of eggshell of other poultry species resulted in a need to supplement the data.

To assess eggshell contamination and the potential risk to developing embryos and workers having contact with the eggs, we conducted studies to identify the species of fungi present on the eggshell surface acquired from duck reproductive flocks, with particular emphasis on Aspergillus contamination, and conducted a drug resistance analysis of isolated Aspergillus spp. isolates.

2. Materials and Methods

2.1. Egg Collection and Mycological Examination

Our research was conducted on 200 eggs collected from 10 Pekin duck flocks (coded as A–K) located in four provinces of Poland with the highest number of poultry farms. All flocks were kept on the straw, and the eggs were collected from the nests 2–4 h after laying. Protected against contamination, eggs were transported to the Department of Epizootiology with the Clinic of Birds and Exotic Animals, Wroclaw Environmental and Life Sciences.

The total fungal contamination of eggshells was examined according to Gentry and Quarles. The eggs were placed in sterile polyethylene bags with 10 mL of sterile NaCl added to each bag. The eggs were rubbed in the bag for 1 min to suspend the materials in the solution. Next, the eggs were allowed to stand in the bag for 5 min and were again rubbed for 1 min. After egg removal, 100 microliters were taken and inoculated on Potato Dextrose Agar (Graso, Starogard Gdanski, Poland) [11]. The cultures were incubated at 27 °C, and after 48 h, the total number of colonies was counted and calculated per egg and per group using measures of central tendency (mean ± standard deviation). In such a study, the detection limit of the method is 100 CFU.

The colony types of cultured fungi were identified morphologically and 10 isolates of each type were chosen for microscopic identification and recultured using Sabouraud Dextrose Agar (Graso, Starogard Gdanski, Poland). The cultures were incubated at 27 °C for 48 h up to 7 days and the distinguishing of species was achieved by assessing the conidial head and colony characteristics [12]. All colonies that looked like Aspergillus species (and were confirmed in microscopic examination) were recultured for future resistance examination (MIC tests).

2.2. PCR Amplification and Sequencing

For PCR amplification, 5 isolates of each fungus type and all Aspergillus spp. isolates were chosen. Genomic DNA was extracted using the Genomic Mini AX Yeast Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s instructions. PCR was performed in a 25 µL reaction mixture containing 50 ng of DNA in 2 µL, 0.25 µL of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primer at a concentration of 25 mM, 12.5 µL of PCR Mix Plus (A&A Biotechnology, Gdynia, Poland), and 10 µL DNAse- and RNAse-free water. Amplification of the ITS1—5.8rRNA—ITS2 fragment was performed using a Bio-Rad T100 PCR Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The reaction was carried out as follows: an initial denaturation step at 95 °C for 5 min, the reaction mixtures were subjected to 35 cycles of heat denaturation at 95 °C for 30 s, primer annealing at 55 °C for 1 min, DNA extension at 72 °C for 2 min and then a final extension step at 72 °C for 10 min.

Amplification products were electrophoresed in a 1.5% agarose gel stained with Sybr Green (Sigma-Aldrich, Poznan, Poland) and visualized under ultraviolet light. The size of the respective PCR products was determined using a molecular mass marker, DNA Marker 1 (A&A Biotechnology, Gdynia, Poland) [2].

The PCR products were excised from the gel and purified using a Gel Out Concentrator Kit (A&A Biotechnology, Gdynia, Poland) and sent for Sanger sequencing with the above-mentioned PCR primers (Genomed, Warsaw, Poland). The sequences were analyzed using online BLAST software of National Center for Biotechnology Information (NCBI) [13].

2.3. MALDI-TOF Mass Spectrometry

The Aspergillus strains were cultured on Sabouraud dextrose agar (Graso, Poland) and incubated at 37 °C for 24 h. The isolates were subjected to MALDI-TOF MS analysis using a Bruker MALDI Biotyper Sirius (Bruker Daltonics, Bremen, Germany). The sample preparation procedure was performed as described by the manufacturer as an extended direct transfer for each of the isolates. Mycelium was harvested from Sabouraud plates, smeared on the target plate, overlaid with 1 µL 70% formic acid and overlaid with 1 µL matrix solution in a second step. The sample overlaid with matrix was air-dried and then inserted into the MALDI-TOF MS.

The resulting spectra were assessed using the MBT HT Filamentous Fungi Module, which contains 225 species in the library. The score values were interpreted according to the manufacturer: identification scores of ≥2.0 were accepted for a reliable identification of the species level (high-confidence identification), scores of ≥1.70–1.99 indicated low-confidence identification and scores < 1.70 indicated no reliable identification.

2.4. Aspergillus fumigatus Antifungal Resistance Profile

The Aspergillus fumigatus resistance was determined using MIC tests strips impregnated with a predefined concentration gradient (0.002–32 μg/mL) of antifungals: amphotericin B (AMB), Caspofungin (CAS), itraconazole (ITC), posaconazole (POS) and voriconazole (VOR) (Liofilchem S.r.l., Roseto degli Abruzzi, Italy).

The inoculum suspensions of fungal isolates were prepared in 0.9% saline solution and adjusted to the turbidity of 0.5 McFarland standard according to the MIC test manufacturer’s instructions. This suspension was used directly to inoculate RPMI agar plates (Biomaxima S.A., Lublin, Poland) and the MIC test strip was placed on them after the surfaces of the RPMI agar plates were allowed to dry for 15 min. The plates were incubated at 35 °C for 48 h. MICs were read as the lowest drug concentrations at which the border of the ellipse touched the scale on the strip. A. fumigatus was classified following the breakpoints proposed by EUCAST table ver 10 ITZ and VRC ≤ 1 μg/mL (S) and >1 μg/mL (R); AMB ≤ 1 μg/mL (S) and >1 μg/mL (R); POS ≤ 0.125 μg/mL (S) and 0.25 > μg/mL (R) [14]; and CAS ≥ 0.5 μg/mL (R) [15].

3. Results

3.1. Egg Collection and Mycological Examination

Among 10 duck flocks, in 8 the presence of fungi on the eggshell was confirmed. The number of colonies varied between the eggs within the flock and between the flocks. In five flocks, eggs without fungal growth were observed. The number of colonies ranged from 0 to 7100 CFU per egg (

Table 1. The total fungal contamination of eggshells.

Flock Code	% of Eggs with Fungi Positive Growth	% of Eggs with Aspergillus spp. Growth	The Mean Colony Number per Group *	Colony Number Range (CFU) **
A	100	0	1.2 ± 1.0 × 102	1–34 × 102
B	90	0	0.8 ± 0.6 × 102	0–23 × 102
C	100	45	3.4 ± 2.0 × 102	5–71× 102
D	100	30	2.6 ± 1.7 × 102	12–62 × 102
E	0	0	-	0
F	0	0	-	0
G	80	0	0.4 ± 0.3 × 102	0–9 × 102
H	100	0	2.0 ± 1.0 × 102	6–38 × 102
I	60	0	2.0 ± 2.4 × 102	0–63 × 102
K	100	20	3.6 ± 1.6 × 102	12–68 × 102

* Mean plus/minus standard deviation and 95th percentile reference values. ** The detection limit of the method is 100CFU.

).

The dominating genera on the duck eggshells were Penicillium, Alternaria and Aspergillus (Figure 1a).

All flocks positive for fungi showed Penicillium growth, while 75% of the flocks tested positive for Alternaria and 37.5% of the flocks for Aspergillus [Table 1]. Nineteen isolates of Aspergillus obtained from three flocks were confirmed using colony and conidial head characteristics (Figure 1b). Those isolates were submitted for genetic testing to confirm the species affiliation of the strains.

3.2. PCR Amplification and Sequencing Results

All 19 Aspergillus isolates presented the ITS1—5.8rRNA—ITS2 fragment product size of about 600 bp, and the obtained sequences showed the homology to Aspergillus fumigatus GenBank sequences. Other Aspergillus species such as A. flavus and A. niger were not isolated. Among other fungal isolates, Penicillium chrysogenum, Penicillium griseofulvum, Alternaria alternata, Alternaria tenuissima and Lichtheimia ramosa were confirmed [

Table 2. The fungal species confirmed using PCR with homologous sequences from GenBank.

Fungus Species	Homologous Sequences
Aspergillus fumigatus	KY522968.1; MN58803; MN178807
Alternaria alternata	MN894079.1; KY099683.1
Alternaria tenuissima	MT078698.1; MW720805.1
Lichtheimia ramosa	FJ719386.1; MF919355.1
Penicillium chrysogenum	PP952058; MF363161.1
Penicillium griseofulvum	KJ467353.1; KU561903.1

].

3.3. MALDI-TOF Mass Spectrometry Results

The 19 isolates of Aspergillus were classified as Aspergillus fumigatus using the MALDI-TOF mass spectrometry method. All strains showed high confidence identification, with a score > 2.

3.4. Aspergillus fumigatus Strains Antifungal Resistance Profiles

The MIC values for A. fumigatus isolates ranged from 0.094–32 μg/mL for amphotericin B, 0.125–32 μg/mL for caspofungin, 0.19–32 μg/mL for itraconazole, 0.047–12 μg/mL for posaconazole and 0.023–32 μg/mL for voriconazole (Table 2).

According to EUCAST guidelines, an A. fumigatus isolate is considered resistant when the MIC exceeds 1 μg/mL for AMB, ITC and VOR or 0.25 μg/mL for POS and ≥0.5 μg/mL for CAS [15,16].

In accordance with the EUCAST guidelines on fungal drug resistance, high in vitro resistance to ITC (68.4%) and POS (73.7%) was observed. Among azoles, the lowest resistance to VOR (31.6%) was noted. Nearly half of the strains (47.4%) showed resistance to AMB. Thirteen isolates were resistant to more than one antifungal drug, and only two isolates were susceptible to all examined antifungal agents [

Table 3. Resistance of Aspergillus fumigatus isolates from eggshells to individual antifungals (MIC).

Isolate Number (n = 19)	AMB	CAS	ITC	POS	VOR
C1	3.0	0.25	3.0	0.38	0.19
C2	0.5	0.032	3.0	0.5	0.125
C3	4.0	0.38	3.0	1.5	>32
C4	4.0	0.38	1.5	0.75	0.094
C5	2.0	0.19	>32	8.0	>32
C6	0.094	0.125	0.19	0.047	0.064
C7	2.0	0.38	3.0	0.5	0.125
C8	2.0	0.25	12	8.0	>32
C9	0.75	0.38	2.0	0.38	0.19
D1	0.75	0.38	3.0	0.38	0.125
D2	0.094	>32	>32	12	>32
D3	0.50	>32	1.5	0.5	0.19
D4	0.75	0.25	0.19	0.19	0.023
D5	1.0	0.38	>32	8	>32
D6	>32	0.25	0.38	0.19	0.047
K1	>32	>32	1.5	2.0	>32
K2	0.75	0.38	0.19	0.5	0.19
K3	>32	0.25	0.38	0.19	0.047
K4	0.75	>32	0.19	0.19	0.5

AMB—amphotericin B, CAS—caspofungin, ITC—itraconazole, POS—posaconazole, VOR—voriconazole, bolded values—resistance according to EUCAST to: AMB, ITC, VOR > 1 μg/mL, POS > 0.25 μg/mL [15] and CAS ≥ 0.5 μg/mL [16].

].

The MIC 50 and MIC 90 values of AMB were 1 μg/mL and 32 μg/mL, respectively. The MIC 50 value for ITC was 1.5 μg/mL, and the MIC 90 value was 32 μg/mL. POS had MIC 50 and MIC 90 values of 0.5 μg/mL and 8 μg/mL, and VOR 0.19 μg/mL and 32 μg/mL, respectively [

Table 4. In vitro activity of 5 antifungal agents against Aspergillus fumigatus isolates (n = 19).

		MIC (μg/mL)
Antimicrobial Agents	MIC50	MIC90	Range	% Resistance
AMB	1	32	0.94–32	47.4
CAS	0.38	32	0.125–32	21.1
ITC	1.5	32	0,19–32	68.4
POS	0.5	8	0.047–12	73.7
VOR	0.19	32	0.023–32	31.6

AMB—amphotericin B, CAS—caspofungin, ITC—itraconazole, POS—posaconazole, VOR—voriconazole. Resistance to AMB, ITC, VOR > 1 μg/mL, POS > 0.25 μg/mL, CAS ≥ 0.5 μg/mL.

].

The A. fumigatus isolates showed a specific resistance profile to CAS. The MIC 50 and MIC 90 values of CAS were 0.38 μg/mL and 32 μg/mL, and those values group the strains into two: susceptible strains and highly resistant ones (without any ellipse around the MIC test strip).

4. Discussion

The results obtained from this study showed that eggshell samples from different reproductive duck flocks presented various levels of fungal contamination. The contamination scale did not depend on the different breeding systems, which was observed in hen flocks [8,16,17], because ducks’ parental flocks are kept in deep litter, and the eggs are laid in nests filled with straw. We suspect that various levels of eggshell contamination depended on the hygiene and the straw disinfection and its preservation on each farm.

The commonly cultured fungal genera in this study were Aspergillus, Alternaria and Penicillium. Polish research on table egg contamination showed the presence of fungi belonging to the Alternaria, Penicillium, Chaetomium and Aspergillus genera [9], which is consistent with our research. Not many data on the fungal contamination of duck eggs are available, but researchers from Egypt conducted a similar investigation, where Penicillium and Aspergillus were the most prevalent on the eggshells, followed by Cladosporium, Acremonium, Mucor and Fusarium [18,19]. Our research shows that Aspergillus contamination is not of a high level, and the dominating fungi were of other genera.

The litter could be the main source of fungal spores, which was confirmed by researchers studying different types of litter which were contaminated with fungal genera such as Aspergillus, Cladosporium, Penicillium, Stemphylium, Mucor, Rhizopus, Fusarium, etc. [20,21,22,23]. The investigations relating the type of litter with fungal contamination showed that the most contaminated type proved to be chopped straw, and flocks littered with sawdust and wood chips were the least contaminated by microscopic fungi [20]. The microbiologically safest litter seems to be rice husks [24], but this bedding is not used in most countries. In fresh litter material, Aspergillus fumigatus is the most often reported etiological agent of aspergillosis, and in ducks, infections with A. niger were also noted [10,24,25,26]. The prevalence of A. fumigatus on the duck eggs was 9.5%, and all isolates came from three flocks. The prevalence is low compared to other data from Egypt and Costa Rica (29–44%) [4,27], but consistent with research of fungal contaminations of poultry litter in Portugal, where Penicillium sp. was the most frequent genus noted (59.9%), followed by Alternaria sp. (17.8%), Cladosporium sp. (7.1%) and Aspergillus sp. (5.7%) [25]. Our research shows the low risk of contamination and aspergillosis development during the hatching process. Despite that, it is important to avoid the contamination of eggshells by spores and prevent embryo infections. Good litter management combined with assessment of its quality and the suitable collection and storage of eggs should be carried out. Control of the relative humidity in poultry houses and antifungal actions may be useful in order to control environmental contamination [5]. The spraying of antifungal agents like nystatin or copper sulfate [5,28] contributed to decreases in fungal poultry litter contamination.

Reports on the prevalence of aspergillosis in poultry and the resistance profiles of A. fumigatus isolates are scarce. In our study, antifungal resistance towards three classes of drugs was examined—polyenes (amphotericin B), azoles (itraconazole, posaconazole, voriconazole) and echinocandins (caspofungin).

The antifungal resistance profiles of the isolates were various. Only two isolates (10.5%) were susceptible to all of the tested antifungal agents. Most of the isolates were resistant to itraconazole, which is opposite to the results obtained in the same country in 2015–2016 by Nawrot et al. [29], and in France and China [30]. A. niger strains showed a similar resistance to itraconazole, isolated from poultry before 2012 in Poland [31], but all of those isolates were susceptible to voriconazole, and most (80%) were susceptible to amphotericin B. These observations are also consistent with our research, in which a group of A. fumigatus strains were susceptible to VOR (68.4%) and AMB (52.6%). The authors who investigated the drug resistance of A. fumigatus in geese [6] noted that resistance to AMB ranged from 90.6 to 70.6%, and full susceptibility was noted for VOR and enilconazole. The A. fumigatus isolated from Brazilian poultry also showed a high susceptibility to VOR (MIC range 0.5–32 μg/mL) and various resistance towards ITC (0.5–32.0 μg/mL) and AMB (0.5–8.0 μg/mL). The MIC values for CAS were 0.003–0.5 and were lower than observed in this investigation (0.19–32 μg/mL) [32].

The presence of Aspergillus in the poultry food, litter, eggs and in the poultry house and hatchery carries implications not only for animal health and welfare but also public health, especially the health of farmworkers [4,33,34]. Our research showed that most of the Aspergillus isolates (68.5%) were resistant to at least two antifungal agents. This is disturbing due to the possibility of human infection. Aspergillosis is a disease which can cause respiratory signs in children, immunocompromised patients and allergy sufferers if they are exposed to high concentrations or repeatedly exposed to Aspergillus [33,34]. In Europe, an antifungal resistance towards A. fumigatus isolates was observed. Amphotericin B resistance was observed in 2.6% and 10.8% of isolates in Denmark and Greece, respectively. The prevalence of triazole resistance towards A.fumigatus obtained from the included studies is 0.3% in Austria and 1% in Greece. The resistance in other countries was 1.2% in Switzerland, 2.1% in France, 3.9% in Portugal, 4.9% in Italy, 5.3% in Germany, 6.1% in Denmark, 7.4% in Spain, 8.3% in Belgium, 11% in the Netherlands and 13.2% in the United Kingdom [35].

In conclusion, the analysis of the fungal isolates obtained from the eggshells showed that the surface of the shell could be a potential source of fungal infection for the developing embryos. Among the detected species, Aspergillus fumigatus is the most common pathogen, which may cause aspergillosis. Because of the lack of registered antifungal drugs for poultry, hygiene and the regular disinfection of litter should be the main means of prevention, for both animal and human health.