The study was reviewed and approved by the Institutional Animal Care and Use Committee (JUST–ACUC) and the Institutional Review Board (IRB) of Jordan University of Science and Technology (approval number 16/4/12/782, dated 12 December 2022). The protocol fully complied with the ethical principles of the Declaration of Helsinki (2013 revision), the Belmont Report, and the International Ethical Guidelines for Health-related Research Involving Humans (CIOMS, 2016). All participants provided written informed consent after receiving a detailed participant information sheet that explained the study purpose, procedures, potential risks (including minor discomfort from nasal swabbing), benefits, voluntary nature of participation, right to withdraw at any time without consequence, and measures for data confidentiality and anonymity.

Nasal swab collection was performed by trained medical or laboratory personnel using a standardized, non-invasive protocol under strict biosafety conditions. Participant data were de-identified using unique study codes, stored in password-protected databases accessible only to the principal investigator and authorized study team members, and handled in accordance with Jordanian data protection regulations and international standards (e.g., GDPR principles where applicable). No personally identifiable information was included in the manuscript or supplementary materials. The study posed minimal risk to participants, and appropriate provisions were made for referral to medical care if any adverse events occurred during or after sampling. Approval was obtained prior to the commencement of participant recruitment, and the study was conducted in full compliance with all local, national, and institutional ethical and legal requirements.

A total of 200 human nasal swab samples were collected from dairy cattle farm environments and surrounding communities in northern Jordan (Irbid and Ramtha districts) between February and May 2023. These districts contain a high density of dairy cattle farms in northern Jordan, providing access to individuals with regular occupational exposure to dairy cattle. In addition, non-exposed participants were recruited from the same geographical area to minimize potential regional and environmental differences between the study groups.

This cross-sectional study employed convenience sampling. Participants were categorized into two groups based on occupational exposure to dairy cattle and classified as cattle-exposed (occupational contact with dairy cattle) and non-exposed (no livestock contact). Both groups were recruited from the same area. The cattle-exposed group (n = 100) included individuals with regular daily direct contact with dairy cattle, such as farm workers, milkers, veterinarians, feed handlers, and farm owners. These participants were involved in activities including milking, animal handling, veterinary procedures, and contact with raw milk or farm equipment. The non-exposed group (n = 100) consisted of individuals from the same geographical area with no occupational or regular contact with livestock, including university students, administrative staff, and community residents.

Nasal swabs were collected from both nostrils by trained personnel using sterile cotton swabs moistened with sterile saline, following a standardized protocol to ensure consistency across all participants. The swabs were immediately placed into sterile transport tubes containing nutrient broth, properly labeled, and transported in insulated containers at 4°C to the Microbiology Laboratory, Department of Basic Veterinary Medical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology (JUST), within 2–3 hours of collection.

Nasal swabs were processed as previously described by Gharaibeh et al. [12]. Standard selective and differential media (such as Mannitol Salt Agar and blood agar) were used, followed by phenotypic identification of S. aureus based on colony morphology, Gram staining, catalase and coagulase tests. Presumptive S. aureus isolates were identified using standard bacteriological methods, and MRSA was confirmed by PCR detection of the nuc and mecA genes.

All confirmed S. aureus isolates were screened for virulence-associated genes, enterotoxin genes, antimicrobial resistance (AMR) genes, and biofilm-related genes using ten multiplex PCR assays targeting biofilm genes, AMR determinants, hemolysins, and VGP1–VGP8, using primers and PCR amplification conditions as previously described [13–20].

Data were entered and managed using Microsoft Excel and analyzed with GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). Descriptive statistics were used to summarize the percentage of S. aureus, MRSA, and virulence- and AMR–associated genes, and results were expressed as frequencies and percentages. Comparisons between cattle-exposed and non-exposed groups were performed using the chi-square (χ²) test. A p-value of < 0.05 was considered statistically significant.

Among the 200 human nasal swab samples analyzed, S. aureus was detected in 42% (42/100) of cattle-exposed individuals and 40% (40/100) of non-exposed individuals. MRSA was confirmed by PCR detection of the species-specific nuc gene and the methicillin resistance gene mecA. Of the 82 S. aureus isolates recovered, 63 (76.8%) were identified as MRSA, including 32 isolates from cattle-exposed individuals and 31 from non-exposed individuals (Table 1).

The high prevalence of MRSA in both exposed and non-exposed groups suggests that methicillin resistance is not restricted to farm environments. The comparable MRSA rates observed between the two groups indicate a substantial contribution of CA-MRSA circulation, which has been increasingly reported in Jordan and neighboring countries [21, 22]. These findings support growing evidence that MRSA is now widely established in community settings beyond healthcare and agricultural environments [22, 23].

The detection of MRSA among cattle-exposed individuals supports the potential role of contact with livestock and their products in the distribution of MRSA strains. Close human–animal contact enhances dissemination through direct contact or via indirectly contaminated farm equipment and inadequate hygienic practices [23]. The adaptability of MRSA to multiple hosts, partly mediated by mobile genetic elements such as plasmids, further enhances its transmission potential [24]. Previous molecular studies have shown that certain S. aureus lineages, including ST8, circulate between humans and livestock, highlighting the potential bidirectional transmission [8]. The direct transmission of LA-MRSA to humans is less frequent than CA-MRSA; however, livestock-exposed workers may serve as a key reservoir or source for MRSA dissemination. The genetic relatedness between LA-MRSA and CA-MRSA strains further suggests the zoonotic potential of LA-MRSA [25, 26]. These findings confirm the necessity for continuous surveillance and characterization of MRSA in human populations with various levels of livestock exposure, including farmers, milkers, veterinarians, and individuals without direct livestock contact. Understanding the relative contributions of community and occupational transmission is critical for designing targeted prevention strategies and implementing integrated control measures under a One Health framework [26, 27].

All confirmed MRSA isolates from human nasal swabs were screened for key AMR genes, including blaZ, mecA, tetK, tetM, ermB, ermC, strB, msrA, and aph. The percentage and distribution of these genes differ among cattle-exposed and non-exposed individuals, reflecting variations in exposure and selective pressures across the groups (Figure 1).

The β-lactamase gene blaZ was the most prevalent AMR determinant detected, occurring in 100% of isolates from cattle-exposed humans and 83.9% of isolates from non-exposed individuals, with a significant difference between groups (p < 0.05). The ubiquitous presence of blaZ confirms the widespread resistance of S. aureus to penicillin and reflects the long-standing and often inappropriate use of β-lactam antibiotics in both human and veterinary medicine [28]. Tetracycline resistance genes (tetM and tetK) were frequently detected in isolates from both groups. Among cattle-exposed human isolates, tetM and tetK were identified in 40.4% and 91.5% of isolates, respectively, while corresponding frequencies among non-exposed human isolates were 67.7% (tetM) and 90.3% (tetK), with no statistically significant differences between groups. The high percentage of tetracycline resistance genes in both populations suggests shared or overlapping exposure pathways. This likely reflects the extensive use of tetracyclines for prophylactic and therapeutic purposes in livestock production, as well as their continued use in human medicine [29].

Macrolide resistance genes (ermB and ermC) were comparatively infrequent. ermB was detected in 2.1% of cattle-exposed human isolates and was absent in non-exposed human isolates. ermC was found in 4.3% and 12.9% of isolates, respectively, with a significant difference between groups (p < 0.05). In contrast, strB (conferring streptomycin resistance) and msrA (encoding a macrolide efflux pump) were highly prevalent, with 80.9% and 78.7% in cattle-exposed isolates and 54.8% and 90.3% in non-exposed isolates, with a significant difference between groups (p < 0.05). The widespread occurrence of strB and msrA suggests potential cross-resistance between aminoglycosides and macrolides, which may complicate treatment strategies in both human and veterinary settings [29, 30]. The aminoglycoside-modifying enzyme gene aph was not detected in cattle-exposed human isolates but was present in 3.2% of non-exposed human isolates, suggesting the potential for horizontal gene transfer and genetic exchange between strains from different sources [31].

Overall, the observed distribution of AMR genes of MRSA isolates indicates a high percentage of β-lactamase, tetracycline, streptomycin, and macrolide resistance among MRSA isolates from both groups. Similar AMR gene patterns in exposed and non-exposed humans suggest that MRSA resistance is not only linked to direct cattle contact. Instead, resistant strains likely circulate through multiple routes, including food chains, environment, healthcare settings, and human-to-human transmission [29]. This indicates widespread dissemination of AMR determinants beyond farms into the community. It reflects the interconnected role of humans, animals, and the environment in AMR spread. Therefore, a One Health antimicrobial stewardship approach is urgently needed in Jordan.

The pattern of AMR gene detection between cattle-exposed and non-exposed humans shows the interaction between livestock, humans, and the environment in shaping the AMR profiles of MRSA strains and raises the urgent need for an integrated AMR stewardship program under a One Health approach for MRSA control in Jordan.

A total of 63 (32 cattle-exposed and 31 non-exposed) MRSA isolates were screened for virulence-associated genes (VGP1–VGP8), hemolysins, immune evasion cluster genes, and biofilm-associated genes (Table 2).

Among enterotoxin genes grouped within VGP1, the sea gene was highly prevalent in both exposed (94%) and non-exposed isolates (89%), with no significant difference (p = 0.5). Other genes, including sec and sed, showed moderate to low percentages without statistical significance (p > 0.05), while seb and see were absent in both groups. The percentages of these genes did not significantly vary among cattle-exposed and non-exposed isolates, reflecting diverse virulence profiles and suggesting no potential effect of dairy cattle exposure on adaptation and transmission dynamics.

In VGP2, the majority of genes (seg, seh, sei, and sej) were widely distributed in both groups with no significant differences (p > 0.05). However, selp was significantly more prevalent in non-exposed isolates (97%) compared to exposed isolates (59%) (χ² = 12.1, p < 0.001). Within VGP3, genes (selm, selo, and tst) were common in both groups with no statistically significant differences. The VGP3 genes are key mediators of staphylococcal food poisoning; the high percentage of these virulence genes among both cattle-exposed and non-exposed isolates suggests that they are well established within human-associated MRSA populations, indicating that their distribution may persist independently of livestock exposure [32]. This conserved percentage of VGP 1, 2, and VGP3-associated enterotoxins among circulating human strains suggests that these virulence factors are likely maintained through community transmission, rather than being influenced by occupational exposure to dairy cattle. These findings contest with previous reports of the stability of enterotoxin gene clusters among human-adapted S. aureus lineages. Previous reports on genetic analyses also showed that enterotoxin gene clusters and individual enterotoxin genes (e.g., sea, seg, sei) are widely distributed across diverse S. aureus lineages and are conserved in human-associated strains, suggesting they are maintained through community transmission rather than solely by specific host exposures [33].

In contrast, VGP4 revealed a marked difference for selr, which was significantly more prevalent in exposed isolates (97%) than in non-exposed isolates (45%) (χ² = 19.5, p < 0.001). This finding suggests that occupational contact with dairy cattle may influence the acquisition or persistence of specific enterotoxin gene variants. Enterotoxin-like genes such as selr are frequently located on mobile genetic elements, including pathogenicity islands, which facilitates their dissemination among MRSA populations circulating at the human–animal interface [34]. Previous molecular studies have reported the presence of selr in S. aureus isolates from dairy cattle, raw milk, and dairy products, indicating that this gene may be enriched in livestock-associated or food-related strains [34]. The high occurrence of selr among cattle-exposed individuals supports the hypothesis that repeated contact with colonized animals or contaminated farm environments may promote zoonotic spillover or genetic exchange between livestock-associated and human-adapted S. aureus lineages [35]. Therefore, the significantly higher detection of selr in cattle-exposed isolates underscores its potential relevance as a marker of occupational exposure and highlights the importance of monitoring non-classical enterotoxin genes in One Health–based surveillance programs. Other VGP4 genes showed no variation between groups.

For VGP5, both seln and selu genes, part of the enterotoxin-like (SEl) family, were highly prevalent in the two groups without significant differences (p > 0.05), indicating these genes circulate in the community as well as in livestock settings [36]. This finding may enhance colonization, immune evasion, and zoonotic transmission, highlighting the need for continuous surveillance.

The VGP6 group encodes hemolysin cytotoxic proteins that lyse erythrocytes and immune cells, facilitating bacterial survival, tissue invasion, and pathogenesis [20, 37]. The results showed that these genes were broadly distributed among isolates. The hla and hld genes showed high percentages (>90%) in both groups with no significant differences. However, hlg was significantly more common in exposed isolates (100%) compared to non-exposed isolates (71%) (χ² = 10.6, p = 0.001). The hlb gene was more frequent in non-exposed isolates (55%) than in exposed isolates (31%) (χ² = 3.8, p = 0.05). No significant difference was observed for hlg2 (p = 0.09). The overall higher percentage of VGP6 in cattle-exposed isolates (p < 0.05) indicates that occupational exposure to livestock may contribute to colonization by more virulent MRSA strains, consistent with findings in other human and animal studies [38].

VGP7 comprises exfoliative toxins (eta, etb, and etd), which are proteases that cause loss of keratinocyte cohesion in the superficial epidermis and sometimes mucous membranes. Exfoliative toxins eta and etb mainly cause staphylococcal scalded skin syndrome (SSSS), while etd is linked to other skin infections [37]. Our results showed that the etd gene was detected in 50% of exposed isolates but was completely absent in non-exposed isolates, representing a highly significant association (χ² = 22.4, p < 0.001). In contrast, eta and etb were not detected in either group. The absence of eta and etb in both human groups suggests that SSSS-associated toxins are not circulating in these populations, which is expected since SSSS is mainly a pediatric disease and rarely linked to adults exposed to livestock [37, 38]. The detection of etd exclusively in individuals exposed to cattle indicates a potential zoonotic transmission of etd-producing MRSA from cattle to humans. The finding also highlights that occupational exposure to livestock can influence the carriage of specific virulence factors, suggesting that humans in close contact with cattle may serve as reservoirs or temporary carriers of etd-positive strains [38].

VGP8 included leukocidin genes. The lukED gene was highly prevalent in both exposed (97%) and non-exposed (94%) isolates with no significant difference (p = 0.6), whereas lukM was absent in both groups. The lukM gene, typically associated with ruminant-specific strains, was not detected in human samples, suggesting a host-adapted role in bovine infections [39].

Biofilm-associated genes (icaA and icaD) were detected in 100% of isolates across both cattle-exposed and non-exposed isolates, indicating universal biofilm-forming potential with no observable variation.

Among the immune evasion cluster genes, chp and scn encode the chemotaxis inhibitory protein of S. aureus (CHIPS) and the staphylokinase (SAK) enzyme and were highly prevalent (>90%) in both groups without significant differences. However, sak was significantly more common in non-exposed isolates (97%) compared to exposed isolates (81%) (χ² = 4.2, p = 0.04). None of the isolates carried the scn gene encoding the staphylococcal complement inhibitor protein (SCIN).

Overall, our findings show a broad distribution of virulence-, biofilm-, and immune evasion–associated genes among human MRSA isolates. Statistically significant differences between cattle-exposed and non-exposed groups were observed for selected enterotoxin gene profiles, hemolysin-associated gene clusters, the leukocidin gene lukM, and capsular polysaccharide genes (cap5 and cap8). These differences suggest that occupational exposure to cattle may be associated with variations in the virulent gene repertoire of human MRSA isolates. The detection of multiple virulence and enterotoxin genes among MRSA isolates indicates their potential to cause a wide range of clinical outcomes, from mild skin and soft tissue infections to more severe invasive diseases. The concurrent presence of virulence determinants and AMR genes may contribute to increased bacterial fitness and persistence, thereby complicating infection management in both human and animal hosts. Furthermore, the occurrence of shared virulence-associated genes among cattle-exposed and non-exposed human isolates supports the importance of reinforcing hygiene measures during milking, milk processing, and food handling. In addition, continuous molecular surveillance is warranted to better characterize transmission dynamics and to monitor the emergence of virulent MRSA lineages within a One Health framework.