Microflex lt maldi tof instrument

The identity of all microbial species was verified using the Microflex LT MALDI-ToF instrument (Bruker Daltonics, Coventry, UK) as previously described^{23 (link)}. In brief, single colonies were placed onto a 24-spot steel plate and embedded in an acidic solution of α-cyano-4-hydroxycinnamic acid as a UV-adsorbent matrix. Samples were analysed after drying following the manufacturer’s instructions. Data were processed using the Bruker Biotyper software (version 3.0) and library (version 5.0) for taxonomic classification of the isolates. The identity of the 135 P. aeruginosa isolates were confirmed.

Bacteriological analyses were performed following the Laboratory Handbook on Bovine Mastitis of the National Mastitis Council [National Mastitis Council (NMC), 2017 ]. In brief, 10 μL of each milk sample was streaked out on sheep blood agar (BA) plates (Biomèrieux Suisse SA, Geneva, Switzerland) and bacterial colonies obtained after 24 and 48 h of aerobic incubation at 37°C were evaluated based on their morphology. Samples were considered “contaminated” and not included in the study, if more than 3 morphologically different bacterial colonies could be identified (Wyder et al., 2011 (link)). Representatives of each colony type were selected for bacterial identification using MALDI-TOF MS according to the protocols of the manufacturer (Bruker Daltonics GmbH, Bremen, Germany). Analysis was performed by a Microflex LT MALDI-TOF instrument using the MALDI Biotyper (MBT) Compass Library 7,311 (both Bruker Daltonics GmbH). Isolates with a score ≥ 2.2 were identified at the species level. All distinct isolates were conserved in skim milk at-20°C for subsequent analyses.