Macconkey agar

Clinical specimens were cultured aseptically onto blood agar (Columbia agar with 5% Sheep blood, 254,005 BD), chocolate agar (GC II agar with IsoVitaleX, 254,060, BD or blood Agar No. 2 Base, 257,011, BD) and MacConkey agar (4,016,702, Biolife Italiana Srl) and incubated at 35–37 °C in aerobic conditions with 5–10% CO₂ for 24–48 h to address the isolation of respiratory bacterial pathogens.
Identification of isolates for respiratory pathogens was carried out by matrix assisted laser desorption ionization-time of flight (MALDI-TOF Biotyper System, Bruker Daltonics, Bremen, Germany) as previously described [13 (link)]. Actinobacillus pleuropneumoniae was also confirmed by PCR technique due to limitation of MALDI-TOF for the Actinobacillus genus [57 (link)]. Individual isolates were stored at -80 °C in brain heart infusion (CM1135, Oxoid) with 30% of glycerol (G9012, Sigma-Aldrich).

One hundred and two K. pneumoniae isolates with an MDR profile were provided by the microbiology laboratories of the Hospital de Braga and of the Centro Hospitalar De Trás Os-Montes E Alto Douro, E.P.E., both in northern Portugal. Isolates were collected over a period of ten years (2010–2020). This study was performed in line with the principles of the Declaration of Helsinki and the guidelines of Good Clinical Practice. Approval was granted by the Ethics Committee of Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal. The isolates provided were previously identified using VITEK^®2 Compact B System (bioMérieux, Marcy-l’Étoile, France). Even so, the bacterial isolates were cultured on MacConkey agar (BioLife, Milan, Italy) plates, and typical K. pneumoniae colonies were sub-cultured onto trypticase soy agar (TSA, Liofilchem, Roseto degli Abruzzi, Italy) for further analysis. Isolates were also preserved in 20% (v/v) glycerol (Riedel-de-Haën, Seelze, Germany) in tryptic soy broth (TSB, Liofilchem) and stored at −80 °C. The sample sources and isolation dates from all isolates were gathered together with the bacterial species information.

Clinical specimens were grown aseptically on Columbia blood agar base supplemented with 5% of defibrinated sheep blood (Oxoid), chocolate blood agar (GC II agar with IsoVitaleX, BD) and MacConkey agar (Biolife). All plates were incubated at 35–37 °C in aerobic conditions with 5–10% CO₂ for 24–48 h. Identification of isolates was carried out by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass espectrometry (Biotyper System, Bruker Daltonics, Bremen, Germany) as previously described [24 (link)].