M ller hinton broth

We used the following bacterial species: Streptococcus agalactiae ATCC 12403, Staphylococcus aureus MRSA ATCC 43300, Klebsiella pneumoniae Extended Spectrum β-Lactamase (ESBL) ATCC 700603, Acinetobacter baumanii ATCC19606, and Enterococcus faecium (VRE) DSM17050. Bacteria were cultured at 37 °C/5% CO₂ in liquid THY broth (Todd-Hewitt Broth, Oxoid, Hampshire, UK) supplemented with 0.5% yeast extract (BD, Miami, FA, USA). Pseudomonas aeruginosa PA14 (DSM 19882) was cultivated in liquid Müller–Hinton–Broth (Carl Roth, Karlsruhe, Germany) at 37 °C, under orbital shaking at 160 rpm.
In addition, the employed Mycobacterium tuberculosis ATCC 27294 (Mtb) was grown in 7H9-medium containing 7H9 BBL Middlebrook broth (BD), glycerol (Honeywell, Charlotte, NC, USA) OADC (Oelic Albumin Dextrose Catalase; BD), Tween 80 (Roth, Karlsruhe, Germany), and ddH₂O. The pH was adjusted between 7.2–7.4 and sterile filtration was performed with a 0.2-µm filter membrane (Thermo Scientific^TM Nalgene^TM Rapid-Flow^TM, Thermo Scientific, Bremen, Germany).

For antimicrobial testing, P. aeruginosa PAO1 [58 (link)] and P. aeruginosa PA14 [30 (link)] were grown in 5 mL Müller–Hinton broth (Carl Roth GmbH (Karlsruhe, Germany)) liquid cultures for 16 h at 180 rpm at 37 °C.

The determination of the MIC was carried out in aqueous solution (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4) as well as in 4% human serum albumin (HSA; Kedrion Biopharmaceuticals, Gallicano, Italy) based on the standardized method described in DIN 58940-8 [43 ]. Additionally, the MIC testing was also performed in aqueous solution containing 5 IU/mL heparin. For bacterial growth, a Müller-Hinton broth (Carl Roth, Karlsruhe, Germany) was used. The inoculum with 2 × 10⁵–8 × 10⁵ colony forming units (CFU) per mL was spiked with AMPs, PMB, LL-37, and commercially available antibiotics at concentrations from 0.0625 to 128 μg/mL. After overnight incubation at 37 °C, the MIC was determined by turbidity measurement at 620 nm using an Anthos ht3 plate reader (Biochrom, Cambridge, UK).