Biotyper library v4

Upon clinical suspicion of systemic infection, at least two blood samples were injected into BD BACTEC™ Peds Plus TM/F culture vials (enriched Soybean Casein Digest Broth with CO₂) and upon arrival at the lab immediately processed in the BACTEC FX (Becton Dickinson, Heidelberg, Germany) blood culture system. Blood cultures were incubated for five days. On positivity, Gram-staining and sub-cultivation on agar plates were performed according to the standard techniques [21 ]. Positive samples were cultivated on Columbia Blood Broth, chocolate, MacConkey and Schaedler Anaerobic Agar culture media (all Becton Dickinson, Heidelberg, Germany) and incubated for 24 h at 37 °C in aerobic and 48 h in anaerobic conditions.
Species identification of positive cultures was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonik, Bremen, Germany) using the reference Biotyper library v4.1 (Bruker Daltonik, Bremen, Germany). Antimicrobial susceptibility testing was performed according to NCCLS/CLSI guidelines until 2011 [22 ]. In 2011, Austrian microbiological laboratories switched their methodology to EUCAST (breakpoint tables for interpretation of MICs and zone diameters—2011–2021, Versions 1.3 to 11.0). Strains were classified as susceptible or resistant according to the breakpoints applied in the year of their isolation.

Secondary bacterial infections were determined by standard routine protocol at our institute according to our SOPs. In short, tracheal fluid was cultured overnight on selective medium agar plates (Columbia II blood agar, cooked blood agar, MacConkey agar) at 37 °C (MacConkey agar) or 37 °C under 5% CO₂ atmosphere (both blood agars). Species identification was achieved using matrix-laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonik, Bremen, Germany) combined with the reference Biotyper library v4.1 (Bruker Daltonik).

Routine blood culture testing was performed on the wards by inoculating at least one set of BACTEC™ Plus Aerobic/F and BACTEC™ Lytic/10 Anaerobic/F culture bottles (both BD Diagnostics, Vienna, Austria) with the patient’s whole blood. Culture bottles were immediately transported to the routine microbiological laboratory and incubated at 37 °C using the BACTEC FX Packaged continuous BC monitoring system (BD Diagnostics) until growth or for 5 days post-inoculation. Positive BCs were further used for pathogen identification after cultivation on agar plates using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) based on the reference Biotyper library v4.1 (both Bruker Daltonik, Bremen, Germany) and antimicrobial susceptibility testing (AST). Disk diffusion was used for the latter and was performed in accordance with the current EUCAST guidelines at the time point of sampling [26 ].