1. Introduction
The use of antimicrobial agents in animals for therapeutic or prophylactic purposes, as well as for animal growth promotion, significantly contributes to the development of antimicrobial resistance (AMR), a growing public health threat [
1]. Since 2006, the European Union has banned the use of antimicrobial agents as animal growth promoters, as did some other countries; nevertheless, this usage practice is still authorized in about 25% of countries at a global level [
2]. Many of the antimicrobials administered to food animals belong to the same family group as those used in human medicine, including penicillins, tetracyclines, cephalosporins, and fluoroquinolones [
3]. The increase in antimicrobial usage is correlated with the emergence of AMR in livestock animals [
4]. Therefore, farm animals are a significant source of multidrug-resistant bacteria and antimicrobial-resistant determinants [
5]. These bacteria include many zoonotic organisms that are frequently resistant to antibiotics, such as
Salmonella,
Escherichia coli, and
Staphylococcus aureus, among others [
6,
7,
8].
In humans and many animal species,
Staphylococcus aureus is considered to be a major opportunistic pathogen [
9]. It can cause a large array of infections, ranging in severity from superficial skin infections to more severe diseases, such as endocarditis, toxic shock syndrome, septicemia, and necrotizing pneumonia, among others [
10].
S.
aureus also causes a variety of infections with considerable economic impacts in livestock animals, including cows, sheep, goats, poultry, and rabbits [
11]. The most common disease in ruminants is mastitis, which is an inflammation of the udder tissue leading to abnormalities in milk production [
12].
S.
aureus colonizes its hosts without impacting their health, as is the case for any type of commensal bacterium [
13]. It has been reported to colonize the nares of 30% of humans and practically all domesticated farm animals, including pigs, cattle, poultry, as well as companion animals like cats, dogs, and horses. It has also been found in wild animals [
14].
The most challenging characteristic of
S.
aureus that has become a global health concern is its capacity to acquire resistance against several antibiotic molecules, including methicillin [
15]. Methicillin resistance is conferred by the
mecA gene, which encodes a modified penicillin-binding protein, PBP2a (or PBP2′), with a low affinity for most β-lactam antibiotics [
16,
17,
18]. The
mecA gene is part of a large mobile genetic element named staphylococcal cassette chromosome
mec (SCC
mec) [
19]. In the past, methicillin-resistant
S.
aureus (MRSA) has been associated with infections in health-care settings, and these strains have been named hospital-acquired MRSA (HA-MRSA). However, MRSA infections have also been reported outside hospitals in healthy people with no prior exposure to these hospital structures (CA-MRSA) [
20]. Recently, livestock-associated MRSA (LA-MRSA), mainly the complex clonal CC398, has been implicated in community infections [
21]. LA-MRSA strains differ from HA-MRSA and CA-MRSA in their genomic traits [
22]. As reported, livestock can be considered sources of MRSA, which can be transmitted to humans in close contact with farm animals [
19], such as farm workers and their family members, veterinarians, and veterinary students [
23]. Moreover, the handling or consumption of foods of animal origin contaminated with MRSA, including milk and meat, can also be involved in MRSA transmission through the food production chain [
24].
The increased number of reports describing community infections and the emergence of new highly virulent clones highlight the crucial importance of identifying potential reservoirs for newly emergent strains in humans [
11]. The aims of this study were to determine the occurrence of
S.
aureus in the nasal swabs of healthy dairy goats in two regions of Algeria (Tizi Ouzou and Bouira) and to characterize the recovered isolates both phenotypically and genotypically.
4. Discussion
Staphylococcus aureus is one of the main carriers of new and re-emerging antibiotic resistance determinants that represent a health risks for humans and animals [
15]. It is a common commensal bacterium both in humans and animals. Livestock animals represent a major source for antimicrobial-resistant bacteria, the transmission of which can occur either through contact with colonized animals and/or through the consumption of their products, such as meat, milk, and eggs [
13]. In this context, the aims of this study were to determine the prevalence of
S.
aureus in the nasal swabs of healthy dairy goats collected in various areas of Tizi Ouzou and Bouira (Algeria) and to investigate the phenotypic and genotypic characteristics of the isolated strains.
In this study, a low prevalence of
S.
aureus was observed among the nasal samples of healthy dairy goats, with different rates depending to the sampling regions. These results are in accordance with those of previous studies conducted in Algeria [
34], Tunisia [
35] and Saudi Arabia [
36], with rate values of 11.9%, 10.2%, and 19.2%, respectively. However, higher frequencies were reported in Denmark [
37], China [
38], and Korea [
39], with rates of 64%, 43.24%, and 82%, respectively. As is known, many factors could have an influence on the prevalence of
S.
aureus, including livestock density, isolation methods, breeding practices, and geographical conditions [
15,
40].
The results of this study showed that isolated
S.
aureus carried staphylococcal enterotoxin genes. Our results agree with those of other authors who have shown the presence of staphylococcal enterotoxin genes in
S.
aureus of goat origin [
35,
38,
39]. In this study, the most frequent enterotoxin genes carried by the
S.
aureus isolates were
sec and
sea, which corroborate the results obtained by Gharsa et al. [
35], who found a high prevalence of
sec and
sea among
S.
aureus isolates of goat origin. As reported by Normano et al. [
41], staphylococcal enterotoxin C has been implicated in high number of staphylococcal food poisoning instances associated with the consumption of dairy products. Fifteen isolates harbored the
tst gene encoding for the toxic shock syndrome toxin, which is consistent with findings among
S.
aureus of goat origin [
35,
42,
43]. None of
S.
aureus isolates carried the Panton–Valentine toxin (
lukF/S-PV) gene, an important virulence factor associated with pathogenicity. This contrasted with other studies, where the
pvl gene was observed both in goat nasal carriages and goat milk [
35,
38,
43,
44]. As reported by Abdullahi et al. [
15],
S.
aureus isolates of animal origin harbored several virulence factors, including
luk-S/
F-PV,
tst,
eta,
etb, and the enterotoxin genes, which could have an impact on public health, mainly if these isolates are implicated in human or animal infections.
Among our
S. aureus isolates, we found nine distinct
spa-types and we also detected one new
spa-type (t21230), suggesting that information about the population structure of
S.
aureus of goat origin is still limited, despite several studies having been conducted in this field [
35,
37,
39,
45]. Four STs were identified, including ST700, ST6, ST5, and ST88, which were assigned to three clonal lineages, including CC5, CC130/CC700, and CC88. CC5 was the most predominant in our study, including 28 isolates (51.8%). These results do not agree with those found by Shittu et al. [
43] in Nigeria, Porrero et al. [
45] in Spain, Saei and Panahi. [
46] in Iran, and Gharsa et al. [
35] in Tunisia, in which CC133 and CC522 were the predominant clones among goat populations. As reported by Aires-de-Sousa. [
47], CC5 seems to be predominant among poultry, in which it is frequently implicated in disease. However, the host jumps lead to specific lineages spreading and adapting within new animal hosts [
48]. ST700 associated with
spa-type t1773 was the second most prevalent genetic lineage in our study (42.6%). As a single locus variant of ST130 (
tsi allele different between them), the ST700 lineage is part of CC130 [
49]; however, due to their distinct epidemiology and their independent evolution, ST700 and some of its single-locus variants may be considered a separate lineage (CC700) [
50]; for this reason we included ST700 associated with both CCs. This ST700 lineage has previously been detected in ovine mastitis cases in Italy [
51], nasal carriages in healthy goats and sheep in Tunisia [
35,
52], zoo animals in Germany [
53], and abscesses of the submandibular lymph nodes of adult chamois in the Italian Alps [
54]. The CC130 clonal complex has been associated in other studies with MRSA through the
mecC mechanism in isolates of various hosts, including livestock, wildlife, companion animals, and humans, as well as environmental samples (wastewater and river water) [
55]. Three isolates were assigned in our study to CC88 and were associated with the
spa-type t2649. This lineage was also obtained from the nasal carriages of inpatients and hospital staff in Ghana [
56].
In the present study, approximately half of the
S.
aureus isolates exhibited resistance to penicillin; although this rate is high, in general, it is lower than the values detected in human clinical isolates (>80%) [
57]. Nevertheless, the rate detected in our study is in agreement with previous findings of other authors in animal isolates [
34,
36,
43,
46]. As reported by González-Candelas et al. [
58], the use of antibiotics in human and veterinary medicine, agriculture farming, and other areas can promote the selection and emergence of antibiotic-resistant organisms. The collection of
S. aureus isolates showed low resistance rates to tetracycline, erythromycin, gentamicin, sulfamethoxazole/trimethoprim, chloramphenicol, and ofloxacin. The same results were obtained in previous studies [
34,
35,
43,
46]. The use of phenicols in the veterinary sector (as in the case of florfenicol) may promote the emergence of resistance to chloramphenicol; this group of antibiotics (phenicols) could coselect for resistance to different classes of antibiotics (including linezolid) [
15].
It is necessary to conduct routine surveillance on MRSA clones of animal origin to gain a better understanding of the transmission routes of new lineages and for implementing appropriate preventive and control measures. Only two isolates (3.2%) were identified as MRSA, representing only 0.4% of the goats tested. A low prevalence was observed in other studies conducted in Saudi Arabia [
36], Spain [
59], Korea [
39], and Nigeria [
43], with values of 0.8%, 15.8%, 1.2%, and 4.4%, respectively. The detection of MRSA among our
S.
aureus isolates highlights the public health risks associated with the consumption of contaminated milk and the spread of potential zoonotic lineages between animals and humans, even though the prevalence of MRSA in our study was low. Published data report the zoonotic transmission of
S.
aureus between livestock and humans, especially people who work with farm animals [
60,
61]. Moreover, veterinarians and veterinary students were the most exposed to certain staphylococci predominantly found in farm animals [
23]. Their transmission may occur through direct contact with colonized animals and through the handling and consumption of contaminated food of animal origin [
13]. In our study, the two MRSA isolates were resistant to antimicrobial agents other than β-lactams, including tetracycline, macrolides (erythromycin), aminoglycosides (gentamicin), and chloramphenicol, indicating a multidrug-resistant phenotype, as in other studies [
36,
39,
43,
59]. None of the MRSA isolates harbored genes encoding Panton–Valentine leucocidin (
lukF/
S-PV), although these genes have been reported in the nasal carriages of goats [
43]. Similar to our results, previous studies have reported the ability of MRSA isolates of goat origin to carry staphylococcal enterotoxin genes [
39]. With regard to genetic typing, the two MRSA isolates recovered in this study belonged to the same CC (CC5) and ST (ST5), but they were ascribed to two different
spa-types: t450 and t688. Our results are in accordance with those of Titouche et al. [
62], who isolated
spa-types t450 and t688 (ST5) from raw and acidified milk (rayeb), respectively. Since ST5 has been observed in humans as well as in many domesticated animals, it can currently be considered an animal-adapted clone [
63]. However, the globalization of the broiler poultry sector was subsequently responsible for the dissemination of
S.
aureus CC5 [
64].
Bacterial cells have a tendency to adhere to solid surfaces and accumulate in multi-layered cell clusters called biofilms, with their microbial physiology being distinct from the planktonic state [
65]. This also applies to
S.
aureus, which has the ability to form a biofilm, as part of its normal life cycle [
66]. Their capacity to form a biofilm allows microorganisms to survive in hostile environments and to resist conventional treatments [
67]. However, few data were available concerning the biofilm formation ability of isolates of animal origin, and most of them were focused on bovine mastitis [
68]. As reported by Pedersen et al. [
69], the role of biofilms in bovine mastitis is still unclear, and more in vivo studies are required to gain a better understanding of the actual role of biofilm formation in the pathogenesis of bovine mastitis. In this study, we used two techniques to evaluate the capacity of recovered isolates to produce biofilms in vitro. Among all the recovered isolates, 27 (43.5%) were found to be biofilm producers using the CRA method. Our results show a greater difference with those of Lira et al. [
70], who reported a rate of 28% in a CRA test. Although the CRA test is not considered the most sensitive for determining biofilm formation, this simple qualitative phenotypic test is used for its acceptable sensitivity and specificity [
71,
72]. However, multiple factors, such as glucose and sodium chloride, among others, affect the slime production of
Staphylococcus spp. [
73]. The MPA test revealed that all isolates showed an ability to produce biofilms, which is similar to the results obtained by Silva et al. [
68] in
S.
aureus isolates from different animal species, including pets, livestock, and wild animals. Biofilms that are produced on food contact surfaces in the food industry are of great interest in food hygiene because they can harbor pathogenic and spoilage bacteria and cause contamination during post-processing, leading to a decrease in the shelf life of products and the transmissions of diseases [
74].