Mannitol salt agar

Two bacterial samples from the ventral conjunctival sac were taken from one eye of each of two healthy beagles using sterilized cotton swabs. The swab samples were suspended in 1 mL of the brain heart infusion broth (Eiken Chemical Co., Ltd., Tokyo, Japan) with 7.5% sodium chloride and were incubated for 48 h at 37 °C. After incubation, each bacterial suspension was inoculated into mannitol salt agar (Eiken Chemical Co., Ltd., Tokyo, Japan) with an egg yolk (MSEY) plate and were incubated for 48 h at 37 °C. Presumptive S. pseudintermedius colonies were confirmed with the coagulase test using rabbit plasma (Eiken Chemical Co., Ltd., Tokyo, Japan). Further confirmatory tests for S. pseudintermedius were performed on coagulase-positive isolates (multiplex polymerase chain reaction) [26 (link)]. Total DNA was extracted from one colony of each sample with MightyPrep for DNA analysis (Takara Bio Inc., Shiga, Japan), according to manufacturer’s instruction.

After AlEW and StAEW treatment (day 0), all samples were aseptically and immediately placed in a stomacher bag containing 90 mL of sterile PBS and homogenized for 2 min with iMIX (Interlab; Monti Rina, Roma). After homogenization, 0.1 mL aliquots of the samples were serially diluted in 0.9 mL of sterile PBS as needed, and 0.1 mL of the appropriate dilutions were spread‐plated onto each selective medium. Total viable counts were determined by plating appropriately diluted samples onto BHI agar. Xylose lysine deoxycholate (XLD; Merck, Darmstadt, Germany), XM‐G (Nissui pharmaceutical Co., Ltd., Tokyo, Japan) and Mannitol salt agar (Eiken Chemical, Tokyo, Japan) were used for Salmonella spp., E. coli, and S. aureus respectively. All inoculated agar plates were incubated at 37°C for 1–2 days, following which the CFU levels were enumerated. The samples (inoculated) treated with sterilized water were used as control throughout the experiment.