Background/Objectives: Staphylococcus aureus is a major pathogen of concern in raw milk due to its potential to cause foodborne illness and its increasing antimicrobial resistance (AMR). In Romania, data on the occurrence and resistance patterns of S. aureus in raw sheep milk from traditional farming systems remain limited. This study investigated the presence and antimicrobial resistance of S. aureus in 106 raw sheep milk samples collected from traditional farms in the Banat region of western Romania. Methods: Coagulase-positive staphylococci (CPS) were enumerated using ISO 6888-1:2021 protocols. Isolates were identified at the species level using the Vitek 2 system and molecularly confirmed via PCR targeting the 16S rDNA and nuc genes. Methicillin resistance was assessed by detecting the mecA gene. Antimicrobial susceptibility was determined using the Vitek 2 AST-GP79 card. Results: CPS were detected in 69 samples, with S. aureus confirmed in 34.9%. The mecA gene was identified in 13.5% of S. aureus isolates, indicating the presence of methicillin-resistant S. aureus (MRSA). Resistance to at least two antimicrobials was observed in 97.3% of isolates, and 33 strains (89.2%) met the criteria for multidrug resistance (MDR). The most frequent MDR phenotype involved resistance to lincomycin, macrolides, β-lactams, tetracyclines, and aminoglycosides. Conclusions: The high prevalence of S. aureus, including MRSA and MDR strains, in raw sheep milk from traditional farms represents a potential public health risk, particularly in regions where unpasteurized dairy consumption persists. These findings underscore the need for enhanced hygiene practices, prudent antimicrobial use, and AMR monitoring in small-scale dairy systems.

This study investigated the bacteriological quality of raw ovine milk produced by farms located in the Banat region of Romania. Additionally, the pathogenic potential and antimicrobial resistance of the isolated Escherichia coli strains were evaluated. A total of 95.8% (69/72) of the screened bulk tank milk samples, collected at the farm level immediately after milking, demonstrated appropriate total aerobic mesophilic bacteria (TMB) counts, varying from 3.32 to 6.09 log10 CFU/mL. However, 4.2% (3/72) of the samples were above the regulatory limit of 6.18 log10 CFU/mL. E. coli was identified in 66.6% of the examined samples, and from the total number (n = 48) of isolates, 18.8% harbored the stx2 gene, highlighting pathogenic potential. Antimicrobial susceptibility testing with the Vitek2 system of the isolated E. coli strains revealed resistance against ampicillin (45.8%), gentamicin (20.8%), ticarcillin–clavulanic acid (18.8%), cephalexin (18.8%), amoxicillin–clavulanic acid (8.3%), and trimethoprim–sulfamethoxazole (2.1%). Additionally, 64.6% of the strains showed intermediate resistance against amoxicillin–clavulanic acid, while no resistance was recorded against imipenem. Five (18.5%) strains were multidrug-resistant. This study’s results underline hygienic sanitary deficiencies during the milking phase and indicate that raw ovine milk can be contaminated with pathogenic and multidrug-resistant E. coli strains, highlighting a potential risk to public health. Further studies are encouraged to better understand the risk posed to the consumer via the consumption of ovine milk and derived products.

Subclinical mastitis is a major but often overlooked constraint to dairy productivity, causing economic losses through reduced milk yield and quality. In Romania, comprehensive data on the bacterial etiology and antimicrobial resistance (AMR) of subclinical mastitis are limited. This study aimed to characterize the etiological agents and antimicrobial susceptibility profiles of major bacterial pathogens isolated from subclinical mastitis cases in dairy cows from Western Romania. Between 2021 and 2022, milk samples were collected from 117 lactating cows diagnosed with subclinical mastitis on three dairy farms. Bacterial isolation and differentiation were performed on ChromID^® CPS^® Elite Agar, and isolates were confirmed by standard biochemical tests. Antimicrobial susceptibility testing of Staphylococcus aureus and Escherichia coli isolates was conducted using the automated Vitek^®2 system, interpreted according to CLSI veterinary standards. Multidrug resistance (MDR) was defined as resistance to at least one agent in three or more antimicrobial classes. Bacterial growth occurred in 51 of 117 samples (43.6%). S. aureus subsp. aureus predominated (28.2%), followed by E. coli (4.3%), Klebsiella pneumoniae subsp. pneumoniae (2.3%), and Streptococcus uberis (2.3%). Mixed infections occurred in 6.0% of positive samples. Among S. aureus, the highest resistance rates were to fosfomycin (58.3%), penicillin (44.4%), clindamycin (44.4%), and tetracycline (41.7%), with 47.2% MDR isolates. E. coli showed resistance to amoxicillin/clavulanic acid (88.9%), ampicillin (55.6%), and cefotaxime (55.6%), with 66.6% MDR. S. aureus remains the leading cause of subclinical mastitis in Western Romania. The high MDR rates highlight the need for targeted antimicrobial stewardship, culture-based therapy, and continuous AMR monitoring under the “One Health” framework.