Brain heart infusion broth

All L. monocytogenes strains were cultured overnight in brain heart infusion broth (Eiken Chemical, Tokyo, Japan) at 37°C. The bacterial DNA was extracted using the phenol-chloroform and ethanol precipitation method [30 ]. One milliliter of enriched culture was centrifuged at 10,000 × g for 10 min, and bacterial cells were incubated in 567 μL of Tris-EDTA buffer containing lysozyme (2 mg/mL) for 1 h at 37°C. Cells were lysed by the addition of 30 μL of 10% (wt/vol) sodium dodecyl sulfate and 3 mL of 20 mg/mL proteinase K, with incubation for 1 h at 37°C. Next, 100 μL of 5 M NaCl was added, and DNA was extracted with chloroform–isoamyl alcohol (24:1) followed by phenol–chloroform–isoamyl alcohol (25:24:1). DNA was then precipitated with isopropanol, washed with 70% ethanol, and dried. Purified DNA was dissolved in Tris-EDTA buffer and used as the DNA template for whole genome shot gun sequencing and Sanger sequencing.

Bacteria were cultured in brain heart infusion broth (Eiken Chemical, Tokyo, Japan) supplemented with 0.3% yeast extract (Becton Dickinson, Sparks, MD, USA) (BHI-YE) at 37°C without agitation, as described previously [14 (link)].

C. muytjensii ATCC51329 and C. sakazakii ATCC29544 were cultured at 37 °C for 16 h using Brain Heart Infusion Broth (Eiken Chemical Co., Ltd., Tokyo, Japan). An aliquot of 5 ml of the culture was placed in a 15-ml Falcon tube (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and subjected to refrigerated centrifugation at 3,000 × g for 10 min at 4 °C. The supernatant was removed, and 5 ml of physiological saline was subsequently added to the pellet to prepare a stock live cell suspension of Cronobacter. The live cell suspension was appropriately diluted with physiological saline as in the test sample. Independently, 1 ml of the aforementioned stock of live cell suspension was placed in a 1.5-ml volume microtube (Eppendorf, Hamburg, Germany), and the tube was subsequently immersed in a boiling water bath for 50 sec and immediately quenched. To generate a stock of heat-killed (dead) cell suspension, we confirmed that the bacteria did not thereafter form any colonies on a standard plate count agar medium (SPC agar: Eiken). The viable cell count of C. muytjensii or C. sakazakii live cell suspension was conducted on SPC agar medium, and measurements of turbidity were simultaneously performed at a wavelength of 600 nm using a spectrophotometer U-2800 A (Hitachi, Tokyo, Japan) to determine the relationship between the live cell count and turbidity.

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.