Isolates were cultured onto lysogeny broth (LB) plates and grown for 16 h at 37°C. To express resistance, samples were also cultured on LB with 2 μg/mL colistin sulfate plates. Isolates with MICs less than 2 μg/mL had reduced growth. Lipid A analysis was conducted using FLAT per the methods in Sorensen et al. (31 (link)). Briefly, a single colony was spotted on a MALDI plate, overlaid with 1 μL of buffer of 0.2 M anhydrous citric acid and 0.1 M trisodium citrate dihydrate, incubated at 110°C for 30 min, and rinsed with endotoxin-free water (31 (link)). Each isolate was spotted in triplicate. Subsequently, 1 μL of 10 mg/mL norharmane matrix suspended in 2:1 chloroform-methanol was spotted onto the extracted lipid sample on a MALDI plate. A Bruker Microflex LRF MALDI-TOF MS in the negative ion and reflectron mode was used to collect mass spectra. The MALDI-TOF MS comprises a 337 nm nitrogen laser. Analyses were conducted at 43% global intensity with approximately 300 laser shots for each spectrum acquisition. Spectra were recorded in triplicate. Electrospray tuning mix (Agilent, Palo Alto, CA) was used for mass calibration. Flex analysis (v3.4) software processed the mass spectra with smoothed and baseline corrections. Signal-to-noise ratios above three were considered optimal for inclusion of signature ions (52 (link)).
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