Research and Publication Ethics: Ethical review and approval were waived for this study in accordance with national regulations and institutional requirements. The Animal Experiments Local Ethics Committee of Harran University (Meeting No. 2023/005, Decision No. 01-10, document date and number 15.09.2023–260686) concluded that diagnostic skin swab sampling in dogs is not subject to formal ethical approval.
Animals and Sample Collection: This study used clinical samples from dogs with cutaneous lesions presented to Harran University Animal Hospital, Şanlıurfa, Türkiye, between 2022 and 2025. Skin swabs were collected from 130 dogs with suspected bacterial infection using sterile swabs, transported under cold-chain conditions, and cultured on the day of sampling.
Bacterial Culture and Identification: Samples were streaked onto blood agar with 5% defibrinated sheep blood and mannitol salt agar (MSA; Oxoid, Basingstoke, UK) and incubated aerobically at 37 °C for 24-48 h. Colonies with staphylococcal morphology and growth on MSA that were Gram-positive and catalase-positive were regarded as Staphylococcus spp. Preliminary identification of S. aureus relied on colony morphology, mannitol fermentation on MSA, and a latex agglutination test (Phadebact Staph Aureus Test, Bactus AB, Sweden) with S. aureus ATCC 25923 as the positive control.
Molecular Assays: Genomic DNA was extracted from phenotypically identified Staphylococcus isolates using a classical phenol–chloroform method, and the purified DNA was resuspended in nuclease-free water and stored at −20°C until PCR analysis. Species-level confirmation of S. aureus was performed targeting the 23S rRNA gene using the primer pair Sau-327/Sau-1645 as previously described (Table 1) 17. Amplifications were carried out in 25 µL reaction mixtures containing standard PCR buffer, MgCl₂, dNTPs, Taq DNA polymerase, primers and 2 µL of template DNA, under the following cycling conditions: initial denaturation at 94°C for 2 min; 35 cycles, each composed of 94°C for 45 s, 64°C for 1 min and 72°C for 2 min; and a final extension at 72°C for 10 min.
The presence of the biofilm-associated genes icaA and icaD was assessed using gene-specific primers (Table 1) 18 with the same reaction mixture and a cycling profile of 95°C for 5 min; 33 cycles of 95°C for 30 s, 62°C for 30 s and 72°C for 30 s; and a final extension at 72°C for 5 min. PCR products were separated on 2% agarose gels alongside a FastRuler Middle Range DNA Ladder (Thermo Scientific, USA) and visualised under UV illumination. Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 35984 were used as positive controls for the 23S rRNA and icaA/icaD assays, respectively.
Phenotypic Assessment of Biofilm Formation: Biofilm formation was evaluated using a 96-well microtiter plate crystal violet assay as described by Christensen et al., with minor modifications 19. Briefly, overnight cultures grown in Tryptic Soy Broth (TSB) were adjusted to a 0.5 McFarland standard, diluted 1:100 in TSB supplemented with 1% (w/v) glucose and dispensed (200 µL) in triplicate into sterile, flat-bottom polystyrene microtiter plates. Plates were incubated statically at 37°C for 24 h, then gently washed with phosphate-buffered saline to remove non-adherent cells, air-dried, stained with 0.1% crystal violet, and rinsed with distilled water. Bound stain was solubilised with 95% ethanol, and the optical density (OD) at 570 nm was measured in a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA). The mean OD of triplicate wells was used for each isolate. Biofilm production was interpreted using an optical-density cut-off (ODc), calculated as the mean OD of negative-control wells plus three standard deviations (ODc=mean OD_NC + 3 × SD_NC). Isolates with OD ≤ ODc were classified as non-biofilm producers, those with ODc < OD ≤ 2 × ODc as weak, 2 × ODc < OD ≤ 4 × ODc as moderate, and OD > 4 × ODc as strong biofilm producers. Staphylococcus aureus ATCC 35556 was included as a positive control, and wells containing sterile TSB (Oxoid) served as negative controls.
Antimicrobial Susceptibility Testing: Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method in accordance with CLSI guidelines 20. Standardised bacterial suspensions (0.5 McFarland) were spread onto Mueller-Hinton agar supplemented with 5% defibrinated sheep blood. Commercial disks (Oxoid, Basingstoke, UK) containing penicillin (2 U), amoxicillin–clavulanic acid (30 µg), erythromycin (15 µg), tetracycline (5 µg), clindamycin (2 µg), enrofloxacin (5 µg), and trimethoprim–sulfamethoxazole (25 µg) were applied. Plates were incubated aerobically at 37°C for 18-24 h, and inhibition zone diameters were measured and interpreted as susceptible, intermediate, or resistant according to CLSI breakpoints for S. aureus.
Statistical Analysis: Statistical analyses were performed using GraphPad Prism version 10.4.1 (GraphPad Software, San Diego, CA, USA). Associations between categorical variables were assessed using Fisher's exact test (two-tailed), with p<0.05 considered statistically significant. For antimicrobial susceptibility, isolates with intermediate susceptibility were grouped with resistant isolates as non-susceptible, and biofilm production was dichotomised as low (non/weak) versus high (moderate/strong) for comparative analyses.
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