Xylene

Heart tissues were fixed at room temperature in buffered formaldehyde solution (10% in PBS) (Sigma, St. Louis, MO, USA) for 24 h, dehydrated using a graded series of ethanol solutions (Sigma, St. Louis, MO, USA), embedded in Paraplast (Sherwood Medical, Mahwah, NJ, USA), and cut into 7-micrometer-thick sections with a pfm rotary 3004 microtome (Leica Microsystems SpA, Milan, Italy) [34 (link)]. Sections were deparaffinized with xylene (Bio-Optica, Milano, Italy) and stained with hematoxylin and eosin (Bio-Optica, Milano, Italy) [35 (link)], Furthermore, Masson’s trichrome staining was performed for the detection of collagen according to the manufacturer’s protocol (Bio-Optica, Milan, Italy), as reported previously [36 (link)]. Sections were evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) equipped with a motorized stage and associated with the Leica LAS X Navigator software (Leica Microsystems SpA, Milan, Italy) [37 (link)]. Histologic score was evaluated as described above [38 (link)]. Based on the score value, it is interpreted as:

Negative score (score 0 or 1), the absence of myocardial damage.

Positive score (score 2–7), the existence of damage-

Mild myocardial damage (score 2–3),

-

Moderate myocardial damage (score 4–5),

-

Extensive myocardial damage (score 6–7).

The hydrophobicity of the investigated bacteria was determined using an in vitro method to detect the bacterial adhesion to hydrocarbons [25 (link),26 (link)]. Canine and chicken E. faecalis and E. faecium were grown in BHI broth at +37 °C for 18 h. The bacterial cells harvested by centrifugation (5000× g, +4 °C, 15 min) were washed twice with PBS (pH7.0) and resuspended in the same solution to an optical density (OD600) of 0.552–0.606 (A0). In total, 1 mL of xylene (Bio-Optica, Milan, Italy) was added to 3 mL of cell suspension in a glass tube and vortexed for 2 min after 10 min of incubation at RT. After phase separation, the aqueous phase was removed after 2 h of incubation at RT, and the OD600 nm was determined (A1) and compared with the initial value. Due to the destructive effect of xylene on plastic cuvettes, both optical density measurements (A0 and A1) were performed using glass cuvettes. The percentage of hydrophobicity (%H) was calculated using the equation %H = [(A0 − A1)/A0] × 100, and was expressed as follows: 0–35%, low hydrophobicity; 36–70%, medium hydrophobicity; and 71–100%, high hydrophobicity [27 (link)]. All measurements were made in triplicate and the mean hydrophobicity percentage was reported.

After fixation, HS were dehydrated in an increasing scale of ethanol (70%, 95%, 100%, Bio-Optica, Milan, Italy), clarified in xylene (Bio-Optica, Milan, Italy) and embedded in paraffin (VWR International Srl, Milan, Italy) and 4 µm sections were cut. Standard stain protocol was used to evaluate morphology and GAGs deposition. Briefly, the slices were hydrated to water using xylene and a decreasing ethanol scale (100%, 95%, 70%, 50%). The sections were stained by 1 min incubation of hematoxylin (Bio-Optica, Milan, Italy), followed by 10 min of washing in tap water. Then, the sections were incubated for 5 min in a 0.05% solution of Fast Green (FCF, Sigma-Aldrich, St. Louis, MA, USA) in distilled water, and quickly rinsed in 1% acetic acid (Sigma-Aldrich, St. Louis, MA, USA) solution in distilled water. A 5 min counterstain was performed with a 0.1% Safranin-O solution (Sigma-Aldrich, St. Louis, MA, USA). Finally, slices were dehydrated in alcohol solution (95%, 100%) and clarified with xylene.