Antibiotics Induced Membrane Damage in E. coli and K. pneumoniae

To study the damage of various antibiotics treatments on the bacterial cell membrane, E. coli and K. pneumoniae (OD₆₀₀ = 0.5) were inoculated into LB broth containing quercetin (½ MIC), colistin (½ MIC), and quercetin (½ MIC)+colistin (½ MIC) (Qu et al., 2019 (link)), followed by incubation in a shaker (180 rpm) at 37°C for 24 h. Then, these suspensions were centrifuged at 5,000 rpm for 5 min. The supernatant was collected and subjected to the alkaline phosphatase (ALP) activity test via a corresponding kit (Solarbio, Beijing). All experiments were performed in triplicate. In general, AKP/ALP uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (λ_max = 405 nm) when dephosphorylated by ALP.
Similarly, the β-galactosidase activity in the supernatant was tested via its corresponding kit (Solarbio, Beijing). β-galactosidase decomposes p-nitrobenzene-β-d-galactopyranoside into p-nitrophenol, and the activity of β-galactosidase is calculated by measuring its absorbance at 420 nm.