Columbia

Primary microbiological cultures of respiratory samples were isolated at four ICUs and normal wards of the University Hospital rechts der Isar, Technical University of Munich, Germany, and they were cultured on Columbia, Schaedler, and chocolate agar (Becton Dickinson, Sparks, MD, USA). Gram stain identification, species identification (Matrix Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry, Bruker Daltonics, Billerica, MA, USA), and automated antimicrobial susceptibility testing, in accordance with EUCAST breakpoints and terminology (VITEK^®, bioMerieux, Marcy l’Etoile, France), were performed for all pathogens. The capsule type for different Klebsiella spp. strains was determined in the phage laboratory using wzi PCR, as previously described (Figure S1) [26 (link)]. The three study runs (phases I–III) are depicted in Table 2. Bacteria were included, regardless of their antibiotic resistance patterns, as pipeline performance for phage delivery was not to be affected.

Briefly, tracheal aspirates and drainage fluids were streaked manually on Columbia (Becton, Dickinson, Franklin Lakes, NJ, USA), chocolate (bioMérieux SA, Marcy l’Etoile, France), MacConkey (bioMérieux SA, Marcy l’Etoile, France), Schaedler and kanamycin-vancomycin (Bi-plate, Becton, Dickinson, Franklin Lakes, NJ, USA) and chromogenic Candida agar (Becton, Dickinson, Franklin Lakes, NJ, USA), while wound swabs were inoculated semi-automated by PREVI Isola™ instrument on the same agar types. All plates were incubated at 37 °C in 5% CO₂ for 24 to 48 h, except the Schaedler-KV bi-plates, which were incubated at 37 °C in an anaerobic chamber (GasPak; Becton, Dickinson, Franklin Lakes, NJ, USA) for 48 h as described [110 (link)]. Bacterial and fungal colonies were identified by MALDI TOF mass spectrometry and automated antibiotic susceptibility testing (AST) was performed on VITEK II instruments (bioMérieux SA, Marcy l’Etoile, France).