Elx808 absorbance microplate reader

Growth of E. coli was determined in 200 μL of LB in a 96-well plate with shaking using an ELx808 absorbance microplate reader (Biotek). The specific growth rate, μ, was calculated as described (74 (link)). The ATP concentration was quantified using the BacTiter-Glo kit luminescence assay as per the manufacturer’s protocol (Promega). Biofilms were quantified as described (75 (link)).
For soft agar assays, five5 μL of culture was inoculated into the center of plates containing terrific broth (TB) (12 g/L tryptone, 10 g/L yeast extract, 0.4% glycerol [vol/vol]), 10% 1× phosphate-buffered saline [PBS]) with 0.3% agar (wt/vol). Plates were incubated for 15 h at 28°C.
For acid, alkaline, and osmotic stress tests, the pH of Luria Broth (LB) was adjusted to 4.5, 7.0, or 9.0 using HCl or NaOH or supplemented with 0.75 and 1 M for NaCl, 1.0 M and 1.5 M Sorbitol, and 0.5 M and 0.75 M KCl. Growth in stress conditions was determined as described above.
For MICs, cells were grown in LB to the log phase, diluted in 200 μL of fresh LB containing antibiotics at concentrations denoted in Fig. 4C, and shaken overnight using an ELx808 absorbance microplate reader (Biotek). Concentration (MIC) values were determined by the lowest antibiotic concentration added to the medium at which the growth rate was inhibited.

The enzymatic activity was measured in a 96-well plate using an ELx808 Absorbance Microplate Reader (Agilent, Santa Clara, CA, USA) at 405 nm according to [11 (link)]. The measurements were performed in the presence of 6 mM cysteine and 1 mM EDTA in 0.1 M acetate–phosphate–borate universal buffer (UB), pH 5.6 [40 ] using a selective substrate 0.5 mM Glp-Phe-Gln-pNA [41 (link)]. The reaction mixture was incubated at 37 °C. The activity was calculated in nmol/min from the initial rate of hydrolysis.

Antimicrobial susceptibility of all isolates after re-cultivation from cryo stocks was determined thrice by broth microdilution. Susceptibility tests were employed strictly according to the manufacturer’s instruction. From each isolate, a bacterial suspension in 0.9% saline solution was prepared. The suspension was adjusted to a McFarland value of between 0.48 and 0.52 using a DensiCHEK plus photometer (bioMerieux, Marcy-l’Etoile, France). For broth microdilution, Micronaut-S MDR MRGN-Screening MIC-Plates (Merlin, Bornheim, Germany) were utilized (tested antibiotics are listed in the legend of Table 2 in the “Results” section). Tests were performed with Mueller–Hinton broth (Merlin, Bornheim, Germany) and read with a BioTek ELx808 Absorbance Microplate Reader (now: Agilent Technologies Inc., Santa Clara, CA, USA). MICs were interpreted according to EUCAST 2022 v12 breakpoints (version 01.01.2022) for Enterobacterales and Pseudomonas, respectively.