1. Introduction
Staphylococcus aureus (
S. aureus) is an opportunistic pathogen that is considered to be one of the leading causes of mastitis in dairy cattle [
1,
2,
3,
4]. A significant proportion (49.7%) of subclinical bovine mastitis was caused by
Staphylococci, of which
S. aureus was the predominant species (64.95%) [
5]. Mammary gland infection with
Staphylococcus aureus remains a main worldwide problem to the dairy industry due to its contagiousness, pathogenicity, persistence in the cow environment, colonization of skin or mucosal epithelia, and the poor therapeutic efficacy of different applied antimicrobials [
6]. Globally, mastitis is one of the most common diseases affecting dairy herds, leading to significant economic losses due to the increased somatic cell count (SCC), physical changes in milk, decreased milk production, increased costs of veterinary drugs, and early culling of infected animals [
7]. Most
S. aureus strains (94%) are resistant to penicillin and its derivatives due to the production of the penicillinase enzyme [
8,
9]. Strains of
S. aureus that are resistant to methicillin are known as methicillin-resistant
Staphylococcus aureus (MRSA) [
10]. The latter could be determined phenotypically by antimicrobial susceptibility testing to cefoxitin and/or oxacillin and genetically by the detection of the
mecA gene using PCR. The
mecA gene mainly encodes for a modified penicillin-binding protein (PBP2a) that leads to antimicrobial resistance (AMR) [
11].
S. aureus possesses different virulence factors, such as extracellular toxins, of which the enterotoxins cause food poisoning and others are responsible for causing many clinical manifestations in humans and animals [
11]. Enterotoxins are thermostable and cause food poisoning in humans when contaminated foods are consumed [
11,
12].
S. aureus and its enterotoxins are the third highest cause of foodborne illnesses globally [
2,
13]. It can colonize and persist in different environments, inanimate objects, or media [
14]. Virulence factors that have an important role in pathogenicity are adhesins and surface proteins, such as proteins A,
Staphylococcal enterotoxins (SEs), and β-Hemolysin (Hlb) [
15]. The organism has numerous ways to overcome phagocytosis and invade the udder, causing chronic inflammation [
16].
The presence of
S. aureus in milk indicates low hygienic standards adopted during the milking process [
17]. Dairy farms are major reservoirs of
S. aureus; it can be transmitted from cattle, farm environment, and farm workers to bulk tank milk (BTM), causing a public health hazard [
2].
The resistance of
S. aureus to antimicrobial agents is a growing challenge and can hinder the treatment of infections [
1]. In Egypt, a high proportion of
S. aureus isolates from humans and bovines were resistant to antibiotics [
18]. Unfortunately, the use of antimicrobials in livestock production is poorly regulated in Egypt [
19]. This resistance might be due to the fact that antimicrobials such as tetracycline, quinolones, and beta lactams are still used as growth promotors and feed additives for animals [
20,
21]. However, Egypt has launched a five-year National Action Plan (NAP) on AMR (2017–2022), aiming for the consistent investigation of AMR and promoting the judicious use of antimicrobials for humans and animals [
21].
In Egypt, the prevalence and genotypes of circulating
S. aureus and other
Staphylococci among dairy cattle and buffaloes have been reported in several studies [
22,
23], yet scarce and very limited data about the occurrence of antimicrobial-resistant strains of
S. aureus in animals and their environment still exist. This study was carried out to detect the occurrence of antimicrobial-resistant
S. aureus strains isolated from cattle, buffalo, their environment, and milk and dairy products and investigate the extent of animal, ecological, and food contamination by MRSA or enterotoxigenic
S. aureus.
3. Discussion
S. aureus is one of the major causes of mastitis in dairy cattle, resulting in severe health issues and economic losses. Molecular and epidemiological studies of S. aureus from animals, environment, and dairy products in Egypt are scarce. To our knowledge, this is the first study to explore the ecology and the co-circulation of multidrug-resistant S. aureus in livestock, dairy products and the environment.
Our results showed a lower percentage of
S. aureus from cattle and buffalo than in a previous study in Egypt, in which
S. aureus was detected in 50% to 83% of samples from dairy cattle and buffalo commercial farms and smallholders [
24]. In another study, the organism was detected in 72.5% of animals, barn environments, milking equipment, bulk milk tank (BMT), and workers [
25]. In a third study, it was detected in 36.3% and 31% of milk samples collected from healthy cattle and buffalo, respectively [
26]. Our results were higher than El-Ashker et al., who detected
S. aureus in 5.6% of milk samples collected from both small dairy householders and well-organized farms [
22]. Moreover, the organism was detected in 21.1% of milk samples collected from 140 household dairy cattle and buffalo with mastitis [
27]. Variations in
S. aureus occurrence may be due to the differences in the type of samples, the health status of the animals, the level of hygiene and the season of sampling [
28]. A high prevalence of
S. aureus may be due to the ability of the organism to survive in the udder, and under certain circumstances, it becomes capable of inducing chronic and subclinical infections and acting as a source of infection for other healthy cattle [
29].
Our detection rate of
S. aureus from feces and udder swabs from cattle was lower than [
25], who detected it in 55.6% of samples. This may be due to the fact that samples in the latter study were collected from open yards with cow sheds, earth floor system and a parlor that depended on pipeline milking machines. The degree of udder skin contamination may be attributed to the persistent association between udder skin and intra-mammary infection (IMI) in dairy cows [
30], and there was a minor role of teat skin contamination in IMI of
S. aureus previously reported [
31]. This indicates that there is an association between teat skin colonization and IMI with
S. aureus. It was concluded that
S. aureus on teat skin might be a risk factor for IMI. Therefore, proper teat skin hygiene and sanitation are recommended before and after the milking process.
In this study, the level of contamination of buffalo milk contamination by
S. aureus was higher than in cattle milk; this was similar to a previous study conducted by Abo-Shama et al. [
32], in which the organism was detected in buffalo and cattle milk at 46% and 37%, respectively. However, our results were higher than other studies, in which 42%, 36%, and 31% of milk samples from animals with mastitis, cattle with sub-mastitis and buffalo were positive, respectively [
23,
26]. Our results were also different to studies conducted in Turkey (56%) [
33], China (46% and 43%) [
34,
35] and Ethiopia (15%) [
29]. These variations might be attributed to the geographical area, climate conditions, animal species, farm hygiene, the health status of the animals, milking methods and hygiene [
36].
Limited data are available on the prevalence of
S. aureus in animal environments worldwide [
2]. There is a need for further epidemiological and ecological studies to better explain the transmission of the pathogenic
S. aureus between livestock, the environment, food chains and humans [
37]. In this study, the level of
S. aureus detection from environmental samples was in agreement with [
28] and [
2,
38,
39], who concluded that farm environments act as a vehicle for
S. aureus transmission to the farm equipment, cattle, workers and BMT.
S. aureus was detected in Karish cheese at a lower rate than [
40], in which 73.3% of Karish cheese samples processed from raw milk were positive. However, it was higher than [
41] in which 5% and 13% of samples from supermarkets and street vendors were contaminated, respectively. The frequent isolation of
S. aureus from raw milk intended for cheese production without pasteurization, dairy processing equipment, and environments, as well as food handlers, indicated its introduction to the dairy product supply chain [
42]. The critical points of contamination of raw milk with
S. aureus are the milking process, manure, milking collection facilities and handling during transportation [
43]. Therefore, hygienic standards during the production, transportation, and sale of raw milk and dairy products should be carefully monitored.
Resistance to methicillin is attributed to the existence of the
mecA and/or
mecC gene(s) on the
S. aureus chromosomes. Methicillin is stable in the presence of
β-lactamase enzymes and is effective in the treatment of
S. aureus infections, but not MRSA [
44]. The percentage of
mecA was lower than in several studies conducted in Egypt [
11,
22,
23,
26,
45]. This may be due to the differences in the health status of animals. The detection of MRSA was higher than [
2]. In our study, few isolates were resistant to cefoxitin and tested negative for
mecA. This was in agreement with [
46] and may be attributed to the existence of the
mecC gene.
The results of enterotoxigenic
S. aureus strains were similar to [
26]. The percentages of detection of enterotoxin genes,
sea,
sec,
sed, and
seb were in agreement with [
26], who detected the
sea, and
sec genes in 27%, and 7% of samples, respectively, while the
seb and
sed genes were not detected. The gene coding for enterotoxin A,
sea, was the most frequently found, as in studies by [
11,
47]. This toxin is thermostable, not inactivated by pasteurization, and maintains some biological activity after 28 min at 121 °C [
48]. The majority of
S. aureus infections and outbreaks, attributed to the consumption of contaminated milk or milk powder, were associated with
sea, sed,
sec and
seb toxins [
48,
49]. High percentages of
sea,
sec,
sed and
seb enterotoxin genes were detected in raw and pasteurized milk [
47]. Also, a higher detection rate of
S. aureus enterotoxins from animals, food and humans was reported by [
50]. The sources of enterotoxin and
mecA genes in the tested
S. aureus strains were animal, environment, milk and Karish cheese. These pose a potential risk of transmission of antimicrobial resistance to human consumers.
The percentage of MDR
S. aureus in this study was similar to a previous study in Egypt [
23], where 83.3% of
S. aureus isolates were MDR, with a high resistance against ampicillin (95.2%), penicillin (83.3%) and lower resistance against gentamicin (23.8%), amikacin (16.7%) and ciprofloxacin (14.3%) [
23]. MDR of our
S. aureus strains was high compared to other research studies worldwide. Other studies from Egypt reported a lower MDR percentage than the present study, in which 52.4% [
27] and 38.2% [
11] of
S. aureus strains were MDR
S. aureus strains. The phenotypic MDR phenomenon is mainly attributed to the frequent use of antimicrobials as well as the encoding of some antibiotic-resistant genes. The result of antibiogram agreed with [
51], who found that
S. aureus isolated from milk were sensitive to ciprofloxacin. In contrast, all
S. aureus isolates were susceptible to sulphamethazole—trimethoprim, erythromycin, gentamicin, ciprofloxacin, followed by penicillin (88.89%) and tetracycline (61.11%) [
29].
S. aureus strains from milk samples were found to be resistant to penicillin, tetracycline and cefoxitin, with a prevalence rate of 64.3%, 59.5%, and 35.7%, respectively.
The presence of high percentages of MAR
S. aureus from dairy cattle has been reported worldwide, 100% MAR from Egypt [
11], 98.3%, 50%, and 62% from Ethiopia, Italy, and South Africa, respectively [
52]. The difference in the MAR index may be attributable to the difference in the prevalence of antimicrobial-resistant genes that are responsible for MDR [
53]. The current farming practices make farm animals vulnerable to the development and acquisition of new resistance mechanisms that can propagate into the community and cause a significant risk to the human population [
54].
In conclusion, S. aureus is an important veterinary and public health issue, particularly for the small-scale dairy herds in Egypt. Further investigations into the prevalence, circulation and AMR of S. aureus and other pathogens with public health importance are required. Strict regulations for antimicrobial use in livestock production are highly recommended.