Vetnuus | December 2025 11 3.2 Strengths. By documenting the presence of methicillin-resistant and multidrug-resistant S. pseudintermedius in dogs, this study highlights the zoonotic risks and reinforces the importance of prudent antimicrobial use within a One Health perspective. 4. Materials and Methods 4.1 Study Design. The study was conducted between 2022 and 2025 and involved the collection of samples from dogs diagnosed with otitis externa, as well as from clinically healthy dogs presented for routine procedures (such as sterilisation, vaccination, or deworming) at various veterinary clinics in Timisoara, Romania. 4.2 Sample Collection Samples were collected from 400 dogs presenting clinical signs of otitis externa and from 360 clinically healthy dogs. Bilateral ear swabs were taken using sterile cotton swabs, which were immediately placed in Amies (Oxoid, Basingstoke, UK) transport medium and stored at 4 ºC for a maximum of 24 h before processing. Samples were collected from 400 dogs presenting clinical signs of otitis externa and 360 clinically healthy dogs. Bilateral ear swabs were obtained using sterile cotton swabs, placed in Amies transport medium, and stored at 4 ºC for a maximum of 24 h before processing. Although samples were collected from both ears, only one S. pseudintermedius isolate per dog was included in the final analysis. This methodological choice was made to prevent pseudoreplication and overrepresentation of clonal isolates from the same individual, which could bias prevalence and antimicrobial resistance estimations. By focusing on a single representative isolate per host, the study ensured that epidemiological comparisons between diseased and healthy dogs reflected true host-level dynamics rather than intra-individual variability. In cases where multiple S. pseudintermedius isolates displaying identical antimicrobial resistance profiles were recovered from the same animal, only one representative isolate was retained to avoid data duplication and potential overestimation of strain prevalence. While this approach may have limited the detection of intra-host diversity and potential strain coexistence, it provided a more robust and accurate assessment of S. pseudintermedius occurrence and resistance patterns across the study population. This strategy aligns with standard epidemiological practices for prevalence and resistance studies, emphasising comparability between clinical and healthy cohorts. We acknowledge that phenotypic similarity does not necessarily imply clonal identity, as horizontal gene transfer or convergent evolution may result in similar resistance phenotypes among distinct strains. Therefore, future research incorporating molecular typing techniques, such as multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), or whole-genome sequencing (WGS), is warranted to explore intra-host diversity and confirm genetic relatedness among S. pseudintermedius isolates. 4.3 Bacterial Isolation and Species Identification Ear swab samples were cultured on two types of media: Columbia agar supplemented with 5% sheep blood ((Oxoid, Basingstoke, UK), a non-selective enriched medium that allows hemolysis evaluation, and Chapman agar (Mannitol Salt Agar; bioMérieux, Marcy-l’Étoile, France), a selective and differential medium for the isolation of halotolerant staphylococci and their differentiation based on mannitol fermentation. The plates were incubated aerobically at 35 ◦C for 18–24 h. Suspected colonies were selected based on characteristic morphology and subjected to Gram staining, catalase testing, and, when appropriate, coagulase testing. Suspect colonies were subcultured to obtain pure isolates, which were subsequently identified using MALDI-TOF mass spectrometry. Before antimicrobial susceptibility testing, presumptive S. pseudintermedius isolates were identified using MALDI-TOF MS (matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry; Bruker Daltonik, Bremen, Germany). Bacterial protein extracts were prepared following a standard ethanol/formic acid protocol. A volume of 1 µL from the prepared sample was placed on a MALDI target plate and subsequently layered with 1 µL of matrix solution composed of α-cyano-4-hydroxycinnamic acid (10 mg/ mL), prepared in 50% acetonitrile mixed with 2.5% trifluoroacetic acid. Spectra acquisition was performed using a Microflex™ mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), and data were processed via MALDI BioTyper™ 3.0 software (Bruker Daltonik GmbH, Bremen, Germany). Species determination was based on spectral comparison against the manufacturer’s database. According to the scoring criteria provided by Bruker, values ≥ 2.0 were accepted as reliable species-level identification, whereas scores ranging from 1.7 to 1.99 were interpreted as indicative of genus-level classification [39]. Quality control (QC) for MALDI-TOF identification was initially performed using Staphylococcus aureus ATCC 25923 as a general control strain, in accordance with manufacturer recommendations. To further ensure the accuracy of species identification, a representative subset of isolates was re-analysed using the wellcharacterised S. pseudintermedius DSM 21284 reference strain as a species-specific QC. The results confirmed the identity of all isolates originally included in the study, thereby reinforcing the robustness of the identification procedure. Moreover, the Bruker Biotyper database used in this study contained manufacturer-validated protein spectra for S. pseudintermedius, ensuring accurate and reliable identification. The Biotyper database used included manufacturer-validated protein spectra for S. pseudintermedius. 4.4 Antimicrobial Susceptibility Testing (AST) Antimicrobial susceptibility was assessed with the VITEK® 2 Compact system (bioMérieux, Marcy-l’Étoile, France), in line with the manufacturer’s recommendations. Initially, pure bacterial colonies were isolated, and suspensions were made in sterile saline, with turbidity adjusted to correspond to the 0.5 McFarland standard. To evaluate resistance, specific cards for Gram-positive strains (VITEK® AST-GP79; bioMérieux, Marcy-l’Étoile, France) were employed [40]. Antimicrobial susceptibility profiling was performed using 14 antimicrobial agents belonging to 11 different classes. The tested substances included: penicillin and oxacillin (β-lactams); gentamicin (aminoglycosides); tetracycline (tetracyclines); ciprofloxacin and moxifloxacin (fluoroquinolones); erythromycin (macrolides); clindamycin (lincosamides); linezolid (oxazolidinones); teicoplanin and vancomycin (glycopeptides); fusidic acid (fusidanes); tigecycline (glycylcyclines); and trimethoprim–sulfamethoxazole (sulfonamides + pyrimidines). Leading Article >>>12
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