Mhii broth

The inclusion criteria for HiRC of K. pneumoniae as a MDR clone having caused at least four outbreaks and reported from ≥10 countries were used (7 (link), 19 (link)). The isolates used in the study were (i) sent to the CDC for reference antimicrobial susceptibility testing with limited clinical information on the specimen type (KP1–KP9) (21 (link)), (ii) isolated from expectorate, urine, perineal swab, blood culture, and wound secretions (KP10, KP11) (22 (link)), (iii) isolated from urine, sputum, wound, blood, catheter, and pus samples (KP12–KP20) (23 (link)), or (iv) isolated from urinary and lower respiratory tract samples (KP21–KP30) (24 (link)).
The bacteria were stored at −80°C and cultivated on cystine-lactose-electrolyte-deficient (CLED) or Mueller–Hinton II (MHII) (Sigma Aldrich) agar plates. Liquid cultures were done in MHII broth (Sigma Aldrich) or Luria–Bertani (LB) broth (Sigma Aldrich) unless stated otherwise. The isolates used in this study are presented in Table 1. Additional strains used for the construction of strains with fluorescent markers are listed in Table S2.

Bacterial growth was measured as optical density (OD₆₀₀) using the Bioscreen C MBR spectrophotometer (Oy Growth Curves Ab Ltd). One microliter overnight culture (~1 × 10⁹ CFU/mL in MHII broth, Sigma Aldrich) was added to 2 mL MHII media, whereafter 300 µL was transferred to 100-well “Honeycomb 2” plates (Oy Growth Curves Ab Ltd). Three biological replicates were included, as well as one technical replicate for each biological replicate. Bacterial growth was measured at 37°C with OD measurements every 4 min for 24 h. Plotting of bacterial growth curves and calculation of doubling time were performed using BAT 2.0 (Bioscreen Analysis Tool) (39 ). BAT 2.0 is a web browser version of an R-script (R Foundation for Statistical Computing) developed for analyzing growth curves from Bioscreen C MBR machines. For OD values between 0.02 and 0.09, bacterial growth was found to be exponential, and the doubling time (min) was calculated by log(2)/slope of the curve during the exponential growth phase.