Staphylase test

TMC was quantified using GTK-M agar plates (MILCOM a.s., Tábor, Czech Republic) by culturing at 30 °C for 72 h under the aerobic conditions, according to ČSN EN ISO 4833-1 [14 ], which is the Czech equivalent of ISO 4833-1.
Identification of bacteria and their count: pathogenic bacteria in milk were determined by the Veterinary Laboratory Vedia s.r.o. (Strakonice, Czech Republic). The presence of coliform bacteria, staphylococci, streptococci, and corynebacteria was monitored. Basic cultivation of bacteria occurred on the Columbia blood agar (Oxoid, UK). ENDO agar (Oxoid, UK) was used to cultivate coliform bacteria. Enterococcus Selective Agar-BAA (Oxoid, UK) was used to distinguish streptococci and enterococci, and Edwards agar (Oxoid, UK) was used for streptococcal culture, and for staphylococci, Baird-Parker agar (Oxoid, UK) and Staphylococcus agar (Sigma-Aldrich, USA) was applied. A coagulase test was used to distinguish between coagulase-negative and -positive staphylococci (Staphylase Test, Oxoid, UK).
When necessary, a microflex LT MALDI TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with a microSCOUT ion source and a TOF flight time analyzer (Bruker Daltonics, Bremen, Germany) in conjunction with the MALDI Biotyper software system (Bruker Daltonics, Bremen, Germany) was used to identify bacterial species.

In total, 125 NAS isolates were collected from sheep milk samples in different provinces of Sardinia (Italy) over a period of 9 months (April–December 2017). The isolates belonged to a bank of NAS used for the preparation of inactivated autogenous vaccines, according to the Italian Ministerial Decree no. 287/1994. Basic identification of staphylococci was determined by colony morphology, Gram-stain, catalase and coagulase tests, clumping factor production (Staphylase Test, Oxoid, UK), and growth on mannitol salt agar (Becton Dickinson, Quebec, CDN).

Nasal samples were collected from nares of patients by a dry sterile swab. Each swab was rotated five times inside the nares and used for microbial isolation or for metagenomic analysis. The swabs were placed into the agar gel transport medium (Sterile transport swab, Oxoid) and sent to the microbiology laboratory for bacterial culture, isolation and recognition. Each swab was independently streaked on a set of culture media plates: blood agar, chocolate agar supplemented with or not with Bacitracin, Mannitol Salt Agar, MacConkey 3, Bile-esculin agar, Cetrimide, and.
Chromogenic Candida agar plates (all from Oxoid) and incubated for 24–48 h with or not 5% CO₂ at 25 and 37 °C. Sabouraud dextrose agar plates were incubated up to 7 days. The number of bacteria present on swabs was quantified by counting the colonies grown on plates, and annotated as low (< 10³ CFU/plate), medium (10³–10⁴ CFU/plate) and heavy (> 10⁴–10⁵ CFU/plate). Staphylococcus aureus strains were identified by the presence of β-hemolysis on blood agar and by coagulase-positive reaction (Staphylase test, Oxoid) and finally by MALDITOF. All the bacteria isolated, were identified by MALDI-TOF, Matrix Assisted Laser Desorption Ionization Time-of-Flight.