Sheep blood

Clinical strains analysed in the study were isolated from different specimens: aspirates, wound swabs, blood, cerebrospinal fluid, sputum and peritoneal fluid. Samples from outpatients included two swabs, one taken from both anterior nares and one from the throat.
After the collection, all specimens were processed within 2h. S. aureus isolates were obtained by standard microbiological techniques. Specimens were cultured on Columbia agar with 5% sheep blood (bioMérieux, France), incubated for 24 hours aerobically at 37°C, and identified by the tube coagulase test with rabbit plasma (Torlak, Belgrade) and confirmed by MALDI-TOF MS (VITEK MS, bioMérieux) [5 (link), 6 (link)]. Methicillin resistance in S. aureus strains was determined by the disk diffusion method using cefoxitin disk in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendation (http://www.eucast.org).
MRSA isolates were stored in dextrose broth at -20°C, and re-cultivated on Columbia agar with 5% sheep blood (bioMérieux) for further examination.
The molecular characterisation of MRSA isolates was performed analysing 100 random MRSA clinical isolates, and all of 50 MRSA outpatient isolates.

The human blood was cultured by using a BacT/Alert3D continuous monitoring system (bioMérieux, Nürtingen, Germany). Aerobic and standard anaerobic culture bottles (bioMérieux, Germany) were inoculated with a 5 ml sample and were incubated at 37°C until a positive signal was detected. The bacterial isolate was cultured on Columbia agar with 5% sheep blood (bioMérieux, Germany).

Sterility was evaluated on samples of esophagi before and after ATB/ATM treatment and at the end of the whole process. Samples were incubated in Schaedler broth (Biomérieux SA, Craponne, France) for 10 days and then seeded into Chocolate agar + PolyViteX (Biomérieux SA) for aerobic culture, Columbia agar + 5% sheep blood (Biomérieux SA) for anaerobic culture, and Sabouraud chloramphenicol gentamicin agar (Bio‐Rad Inc, Hercules, USA) for fungal culture. The bacterial media were incubated at 37 °C for 8 days and the fungal medium at 30 °C for 11 days.

To determine the potential effects of culture medium on the test results, we performed the test with 20 colistin-susceptible isolates and 20 colistin-resistant isolates cultured overnight on different agar plates. The following culture media were tested: 1) nonselective culture medium such as Columbia agar + 5% sheep blood (bioMérieux, La-Balme-Les-Grottes, France); 2) chocolate agar + PolyVitex (bioMérieux); 3) nonselective chromogenic medium UriSelect 4 (Bio-Rad); 4) Eosin methylene blue agar (Sigma Aldrich); 5) Drigalski agar (Bio-Rad); 6) MacConkey agar (VWR BDH Prolabo, Leuven, Belgium); and 7) bromocresol purple (bioMérieux).

Bacteriological cultures were performed during the pre-experimental period to select the 88 goats enrolled in the experiment. Somatic cell counts were carried out both during the pre-experimental period and each week during the experimental periods (one per treatment). The procedure during the experiments included a 5 mL sampling of each mammary gland for somatic cell counts that was performed manually prior to machine milking. Later, forestripping and disinfection with 70% ethanol of each teat was performed. Then, another 5 mL of each mammary gland were taken for the bacteriological cultures. Somatic cell count samples were analyzed at the interprofessional dairy laboratory (LICOVAL, Valencia, Spain) using a Fossomatic 5000 device (Foss, Hillerød, Denmark). Bacteriological cultures were performed by seeding 20 μL of milk onto blood agar plates (5% sheep blood, Biomerieux, Lyon, France). The plates were incubated aerobically at 37 °C and examined at 24, 48, and 72 h. It was considered that glands in which milk had more than 5 colonies in the bacteriological cultures or more than 1,500,000 somatic cells/mL, suffered intramammary infection and these goats were not included in the experiment. Somatic cell count values of each gland during the experimental period were transformed into their logarithms in base 10 (log₁₀RCS).