Molecular detection of Staphylococcus aureus enterotoxin genes isolated from mastitic milk and humans in El-Behira, Egypt

Background and Aim: Milk is a chief source of many nutrients. However, we must also bear in mind that it is a potential source for many cases of food poisoning. This study was conducted to investigate the prevalence of cow mastitis and evaluate the presence of enterotoxins and antibiotic resistance patterns in Staphylococcus aureus isolated from milk and contact humans in El-Behira Province, Egypt. Materials and Methods: A total of 680 milk samples from 170 cows and 86 human samples consisting of 43 hand swabs and 43 nasal swabs were analyzed. The milk samples were subjected to the California mastitis test. Results: The general occurrence was 23.1% (157/680) where 48 quarters had clinical mastitis and 109 had subclinical mastitis. Subsequently, S. aureus was isolated in Baird-Parker agar where typical and atypical colonies were selected and submitted to coagulase and complementary tests. Out of 48 samples of mastitic milk studied, 16 (33.3%) showed contamination by S. aureus whereas 109 samples of subclinical mastitis showed contamination in only 18 (16.5%). On the opposite hand, of the 86 human samples, 33 revealed S. aureus contamination, corresponding to 38.37% of the samples. Furthermore, multiplex polymerase chain reaction targeting nuc and the staphylococcal enterotoxin-encoding genes sea, seb, sec, sed, and see were performed after culture, revealing that 88.2% (30/34) of milk samples and 93.9% (31/33) of human samples were variably positive to those genes. Conclusion: The use of nuc gene based PCR is an accurate and rapid method for S aureus isolates detection. A high prevalence of multiple drug-resistant isolates of S. aureus recovered from both human and milk represents further evidence for possible veterinary hazards as well as public hazards, especially to those that consume milk from this region.


Introduction
Mastitis has a significant impact on dairy production, affecting animal health, welfare and milk quality, and causing major economic losses to the dairy industry [1]. Staphylococcus aureus is one of the major bacterial pathogens causing bovine mastitis [2,3]. Subclinical and clinical mammary gland infection can produce large numbers of S. aureus, which are believed to be extremely contagious, and are easily spread through milking [4,5].
S. aureus is incriminated in most food toxicity outbreaks. Its pathogenicity is due to the production of staphylococcal enterotoxins (SEs) [6]. Several varieties of SEs can be distinguished serologically. Most staphylococcal food intoxication outbreaks are associated with classical SE, sea-see; however, sea is the most widely implicated [7,8]. Several molecular techniques are used for the rapid, accurate detection of S. aureus isolates, including multiplex polymerase chain reaction (PCR) [9,10].
S. aureus exist in the throats, nasal passages, hair, and skin of nearly 50% of healthy individuals. Food handlers are, therefore, assumed to be a potential source of contamination for food, either directly or indirectly [11,12]. Food handlers act as vehicles for human infection and may, therefore, pose a public health threat [13,14]. The signs of staphylococcal food poisoning, such as abdominal cramps, nausea, vomiting, and diarrhea, occur 2-4 h following the ingestion of contaminated foods, and vary in severity depending on health status of the individual and the amount of SEs ingested [15,16].
The use of nuc gene-based PCR which encode thermonuclease is a very common use and can identify all isolates of S aureus from intramammary infection faster than conventional culturing methods. There is a clear need for strict hygienic measures, to reduce the risk of bacterial contamination [17][18][19].
The use of antimicrobial drugs in humans and animals is poorly controlled, leading to the development of drug resistance. Multidrug-resistant bacteria, including S. aureus, are, therefore, frequently isolated from both humans and animals [20,21].
This study was conducted to investigate the prevalence of mastitis and evaluate the presence of enterotoxins and antibiotic resistance patterns in S. aureus isolated from milk and contact humans in El-Behira Province, Egypt.

Ethical approval and informed consent
All procedures performed in this study, including collection of human and milk samples from animals were done in accordance with the Egyptian ethical standards of the National Research Committee. All human subjects gave their consent for the collection of samples, with the agreement that any identifying details of the individuals should not be published.

Study area and period
This study was conducted in five dairy farms in El-Behera Governorate, which is a Governorates of Egypt with the capital city of Damanhur, located in the West Delta. The study was conducted from January to July 2020.

Samples and sample preparation
Samples were collected at random from El-Behira Province. The samples were 680 milk samples from 170 cows, and 86 human samples, including 43 hand swabs and 43 nasal swabs, from apparently healthy individuals in contact with the cows. The samples were transferred immediately to the laboratory under full aseptic conditions and tested for mastitis using the California mastitis test (CMT) followed by bacteriological isolation and identification of S. aureus. The histories of the animals and the results of clinical examination for the presence of clinical or subclinical mastitis were recorded.

CMT
The CMT is a screening test for mastitis [22]. The CMT results can be 0 (negative, trace); 1 (weak positive); 2 (distinct positive); or 3 (strong positive). A test score of 0 was interpreted as a negative result, while scores of 1 or 2 were considered subclinical mastitis, and a score of 3 indicated clinical mastitis. Herds with 25% or more cows with a CMT score of >1 were defined as positive for mastitis. The 680 milk samples collected from 170 cows were tested using being the CMT and aseptically gathered into sterile plastic tubes (10-15 mL/sample). The positive samples were transported in an icebox to the laboratory.

Pre-enrichment
All samples were diluted with buffered peptone water at a proportion of 1:9.

Isolation and identification of S. aureus
All samples were cultured on mannitol salt agar (HiMedia, India) and then incubated at 37°C for 24 h. Colonies suspected to be positive were recultured on Baird-Parker medium (HiMedia ) with egg yolk tellurite emulsion (HiMedia) and incubated for 24-48 h at 37°C. For further identification of their cultural, morphological, staining, and other biochemical characteristics, one colony was selected and streaked on mannitol salt agar and incubated at 37°C for 24-48 h [23].

Morphological characteristics
The smear contained Gram-positive, spherical cells arranged in irregular clusters after staining with Gram stain.

Tube coagulase test
Five typical colonies were incubated in a tube holding brain heart infusion broth (HiMedia) at a temperature of 37°C for 24 h. Then, 0.3 mL was transported to a sterile tube containing 0.5 mL of rabbit plasma (HiMedia) and incubated at a temperature of 37°C for 6 h. The reaction was scored as positive if the resulting clot was immobile when the tube was tilted.

Catalase test
The presence of catalase was tested using 3% hydrogen peroxide (H 2 O 2 ). Liberation of oxygen bubbles was taken as indicating a positive reaction.
PCR of the nuc gene was used to confirm the classification of the cells as S. aureus. Entire isolates were then preserved in brain heart infusion broth with 15.0% glycerol at −80°C.

DNA extraction and purification
The chromosomal DNA of S. aureus was extracted by the boiling method as previously described Aldous et al. [24], with some alterations. Baird-Parker agar colonies were subcultured onto nutrient agar plates and incubated overnight 37°C. Pure colonies were then transferred to 5 mL of nutrient broth and incubated with aeration at 37°C, employing a shaker machine set at 120 rpm. One milliliter of each culture was then transferred into a 1.5 mL Eppendorf tube, centrifuged at 13,000 rpm for 10 min, and the cell pellets were collected. The cell pellets were cleaned with distilled water and recentrifuged. Then, 200 µL of deionized water was dissolved and mixed by manual shaking. Each Eppendorf tube was maintained at 100°C for 10 min followed by cold shock for 10 min on ice. The tubes were centrifuged again at 10,000 rpm for 10 min, and the supernatant (100-150 µL) was transferred into a fresh Eppendorf tube. The DNA purity and concentration were assessed using optical density with a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA) Available at www.onehealthjournal.org/Vol.7/No.1/9.pdf at various wavelengths [25] and stored at −80°C until further use.

Multiplex PCR conditions
PCR was used to detect the nuc gene and the SEs sea, seb, sec, sed, and see the specific primers described in Table-1. The PCR mixture contained 1.5 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton ® X-100, 200 mM of each deoxynucleotide triphosphate, 0.2 mM of the individual primers, and 0.625 U Taq polymerase (all the reagents from Promega Corp., Madison, Wisconsin, USA). The amplification was accomplished using an automatic thermocycler T-1 (Biometra). The positive control was S. aureus reference strain (ATCC 29213; sea, seb, sec, sed, and see).
The PCR cycles consisted of pre-heating at 95°C for 10 m, denaturation at 94°C for 5 m, annealing at 55°C for 30 s, and extension at 72°C for 1.5 min. The amplification was performed for 35 cycles with a final extension step at 72°C for 3.5 min.
The PCR products were analyzed using electrophoresis in 1.5% agarose gel containing 0.5 mg of ethidium bromide per mL. Gels were visualized and photographed with Image Master VDS (Pharmacia Biotech). The sizes of the amplification products were estimated by comparison with a 100 bp DNA step ladder (Promega Corp.).
The antibiogram pattern of the isolated S. aureus to some antimicrobial agents was constructed using the disk diffusion method, as described by Clinical and Laboratory Standards Institute [26].

Results and Discussion
S. aureus is a widespread member of the natural microflora of the human nasal passages and skin [27]. It is considered to be a possible pathogen, since it may deleteriously affect both human and animal health by causing severe necrotic lesions, abscesses, food poisoning, and bacteremia [28].
In this study, 680 milk samples from 170 cows were analyzed (Table-2). CMT was initially performed on all milk samples, and the overall cowlevel prevalence of mastitis was 39.4% (67/170). The overall occurrence in quarter of the udder was 23.1% (157/680), with 48 quarters having clinical mastitis and 109 having subclinical mastitis.
The results presented in Table-3 show that out of 48 samples of mastitic milk studied, 16 (33.3%) were positive for S. aureus, whereas 109 samples with subclinical mastitis had only 18 (16.5%) positive for S. aureus, producing an overall incidence of 21.6%. Our results are very similar to those of Kirkan et al. [29] who confirmed that S. aureus was the major pathogen causing bovine mastitis, being identified from 23.1% of the inspected farms, and from 28.3% of the milk samples studied in Turkey. Zeinhom et al. and Mansour et al. [30,31] reported that 12% and 16.3% of tested milk samples were S. aureus positive, respectively. Jahan et al. [32] reported that the prevalence of S. aureus was 25.53%, and Pexara et al. [33] reported that the overall occurrence of S. aureus in milk was 24.3%, and Ameen et al. [34] found a prevalence of 30%. Many studies have detected a higher occurrence of S. aureus such as 62.0% [35], 36.3% [36], and 37.32% [37]. The variation in frequencies of S. aureus in milk may be because of improper or suboptimal hygiene or poor farm management [38].
Of 86 human samples, 33 (38.37%) (44.2% of hand swabs and 32.6% of nasal swabs) were S. aureus positive (Table-4). This result agreed with those of Khalifa et al. [39] who found a prevalence of S. aureus of 43.33% from milkers' hands and 36.66% from their    noses. El-Shenawy et al. [40] recovered S. aureus from 45% of Egyptian dairy workers. The nasal prevalence S. aureus was 33% [41]. The results of this experiment are quite different from those reported by Rasha [42] who confirmed that the isolation rate from hand swabs was 60%. S. aureus exists in the environment, and humans can transfer it to food, serving as a source of contamination [12]. PCR assay is a rapid and extremely sensitive procedure, and is a very useful tool for the recognition of SE genes in clinical S. aureus isolates [43,44]. Multiplex PCR using nuc and SE-encoding genes was performed after culture (Table-5 [19,45]. Those authors discussed the role of the nuc gene as a potential gold standard marker gene in the detection of S. aureus. Another study was conducted in Egypt and confirmed the existence of the nuc gene in all S. aureus strains [45,46]. This contrasts with the results published by Abdeen et al. [47] who found that approximately 70% of S. aureus isolates carried the nuc gene.
The noses and hands of food handlers carry enterotoxigenic S. aureus and so are considered the chief cause of food contamination. The classical SE gene is distributed differently in different countries, due to differences in the habits of food consumption in each region. The PCR products of the recovered S. aureus confirmed a relationship between food contaminated with S. aureus and the handlers of food [39]. Table-6 shows the enterotoxin profile genes found in S. aureus isolates from bovine mastitis, in which sea was the most common enterotoxin, which agreed with previous findings [7,[48][49][50].
The results presented in Table-7 show that sea was the most widespread SE gene in the skin [40] and noses of Egyptian food handlers El-Shenawy et al. [51], either from carriers or patients [43]. Wongboot et al. [43] suggested that the differences in the geographic distribution of S. aureus SEs genes may be explained if SEs are located on mobile genetic elements that may be exchanged between bacteria of the same or different species.
Sec is the most widespread SE in Italian dairy products [52], while sed is the most predominant SE in dairy products [53]. Udo [54] reported that seb is the most detected SE on the hands of food handlers. Soriano et al. [55] reported that seb and sed were the predominant SEs in food handlers, while Collery et al. [56] reported that seb is the SE most shared among nasal isolates. Chang et al. and Gholamzad et al. [57,58] disagreed with our findings in that they reported that seb was the most common S. aureus enterotoxin gene.
The determination of combined SEs, such as sea-seb and seb-sed in both milk and human isolates, indicates the zoonotic importance of such toxins. This finding agrees with that of Hoque et al. [35] who detected sea+seb. Khalifa et al. [39] and Hasan and Hoshyar [50] reported a combined sea+sec SE.
Globally, the misuse and unsupervised use of antimicrobial agents in the treatment of animal and human infections has contributed to the emergence of antimicrobial resistance [20,37].
Our results concerning the sensitivity of S. aureus to different antibiotics show multiple drug resistance, providing further evidence of a potential public and veterinary health hazard (Table-8). Animals can also transmit resistant strains to humans [4,59,60].
We found high levels of resistance to gentamycin and ciprofloxacin in milk and human isolates ( Table-9 [30,35,61,62]. These authors also detected high resistance to gentamycin in animal PCR=Polymerase chain reaction isolates and to gentamycin and ciprofloxacin in human isolates. Higher sensitivity was recorded to gentamicin in human isolates in Iran [44], while, El Faramaway et al. [63] in Egypt found increased resistance in S. aureus isolates from clinical mastitis to penicillin and gentamicin. Pexara et al. [33] indicated that a high percentage of resistance to clindamycin was detected among S. aureus isolates.

Conclusion
The data presented here show that pathogenic S. aureus was isolated from milk and human samples at various numbers, probably because of poor hygiene. Multiplex PCR targeting the nuc gene and SEs can lead to rapid and accurate detection of S. aureus in milk. S. aureus carries multiple virulence genes which are of clinical importance in S. aureus. A high prevalence   Available at www.onehealthjournal.org/Vol.7/No.1/9.pdf of multiple drug-resistant S. aureus isolates provides further evidence for potential public and veterinary health hazards.