Bactiter glotm microbial cell viability assay

CFS-treated pathogen viability was assessed with BacTiter-Glo^TM Microbial Cell Viability Assay (Promega Italia S.r.l., Milan, Italy). Pathogens were seeded at OD₆₀₀ = 0.01 (approximately 5 × 10⁶ CFU/mL) into a 96-well-plate, immediately treated with probiotic CFSs (50% v/v) and then incubated at 37 °C in static conditions. A plate for each pathogen and each time point of 24, 48, and 72 h was used. The viability assay was then performed following the manufacturer’s instructions and the luminescence was detected with a Spark microplate reader (Tecan Trading AG, Switzerland). A complex viability assay with pathogen co-culture was also optimized. Pathogens were plated altogether at the same OD₆₀₀ = 0.01 and allowed to adapt for 1 h at 37 °C before CFS treatment. Then, the assay was executed as described above. In all the experiments, TSB, iMRS, and iCysMRS were used as controls. Each experiment was done with five replicates and repeated three times independently.

The BacTiter-Glo^TM microbial cell viability assay (Promega) was used for quantitation of ATP content of bacteria cultures. Bacteria cultures were adjusted to A₆₀₀ 0.05 and dispensed into 96-well white plates, and drug compounds were subsequently added. Quantitation of ATP was conducted after 24 h of incubation at 37 °C.

For measuring the intracellular ATP levels, we used the BacTiter-Glo^TM Microbial Cell Viability Assay (Promega, G8230), according to the manufacturer’s instruction with slight modifications. Briefly, bacteria were grown overnight in N-minimal media containing 10 mM Mg²⁺. Then, 50 μl of the overnight culture was washed in N-minimal media without Mg²⁺ and grown for 5 h in 5 ml of N-minimal media containing 0.01 mM or 10 mM Mg²⁺. Cells were normalized by measuring OD₆₀₀ and resuspended in 1 ml of PBS (phosphate-buffered saline). Then, 80 μl of this cell suspension was dispensed into an opaque 96-well microplate (PerkinElmer), followed by the addition of 80 μl of BacTiter-Glo^TM Reagent. The contents were then mixed briefly by pipetting and incubated for 5 min. The luminescence of the samples was measured using Synergy H1 plate reader (BioTek).

To investigate the impact of the tat mutation and Tat inhibitor Bayer 11–7082 [48 (link)] on PQS signalling, transcriptional fusions between the promoter regions of pqsA, pqsR, rhlA, phzA1 and phzA2 and the luxCDABE operon were constructed using the miniCTX-lux plasmid as previously described [17 (link)]. In addition, a constitutively bioluminescent reporter using a miniCTX::tac-luxCDABE promoter fusion was constructed as a control for Bayer 11–7082. Bioluminescence as a function of bacterial growth was quantified in 96 well plates using a combined luminometer- spectrometer.
Semi-quantification of cellular ATP was carried out using the BacTiter-Glo^TM Microbial Cell Viability Assay (Promega). Briefly, the P. aeruginosa PA14, ΔtatABC mutant and ΔpetA mutant were grown in LB broth for 8 h, diluted 1000-fold with fresh media and mixed with equal volume of BacTiter-Glo^Tm reagent in a 96-well plate. After a 5 min incubation period, luminescence for each well was recorded in an automated plate reader.

A. veronii ACCC61732 cells (1 × 10⁵ CFU/mL mid-log phase in MHB) were exposed to various concentrationds (1/2×, 2×, 4×, 8×, 16×, and 32× MIC) of N6NH₂ (MIC 4 μg/mL), DN6NH₂ (MIC 4 μg/mL), N6PNH₂ (MIC 16 μg/mL), V112N6NH₂ (MIC 16 μg/mL), Guo-N6NH₂ (MIC 8 μg/mL), and CIP (MIC 0.125 μg/mL) for 60 min. ATP was measured using the BacTiter-GloTM Microbial Cell Viability Assay (Promega) following the manufacturer’s instructions. The assay was performed for three biological replicates at 37 °C, with luminescence recorded using a Tecan Infinite M1000 Pro plate reader. The fold reduction of ATP was calculated as: Fold reduction ATP = 1 − (L_treat − L_media/L_control − L_media), where L_treat is the luminescence of treated cells, L_media is the luminescence of MHB without cells, and L_control is the luminescence of untreated cells. Resultant graphs show mean (n = 3) and SEM for each data point, prepared in Prism 7 [46 (link)].

Adenosine triphosphate (ATP) measurements were performed using a BacTiter-Glo^TM Microbial Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, 100 µL of sample was mixed with an equal volume of the BacTiter-Glo^TM reagent in a 96-well white microtiter plate. Afterwards, the luminescence (in relative luminescence units—RLU) was recorded using a FLUOstar^® Omega microtiter plate reader (BMG Labtech, Ortenberg, Germany). Data were calculated as RLU/cm².

E. coli K12 MG1655 (ATCC 700926) cells (5 × 10⁵ CFU mL⁻¹ mid-log phase in CAMHB) were exposed to various MIC-folds (MIC and 1/4×, 1/2×, 2×, 4×, 8×, 16×, 32× MIC) of arenicin-3 (MIC 0.25 μg mL⁻¹), AA139 (MIC 0.125 μg mL⁻¹), piperacillin sodium salt (Sigma-Aldrich, Cat # P8396; MIC 2 μg mL⁻¹), and colistin sulfate (Sigma-Aldrich, Cat # C4461; MIC 0.03 μg mL⁻¹). ATP was measured using the BacTiter-Glo^TM Microbial Cell Viability Assay (Promega) following the manufacturer’s instructions. The assay was performed for three biological replicates at 37 °C, with luminescence recorded using a Tecan Infinite M1000 Pro plate reader. The fold reduction of ATP was calculated as: Fold reduction ATP = 1 − (L_treat − L_media/L_control − L_media); where L_treat is the luminescence of treated cells, L_media is the luminescence of CAMHB without cells, and L_control is the luminescence of untreated cells. Resultant graphs show mean (n = 7) and std error for each data point, prepared in Prism 8. DMSO was included as a control as piperacillin was solubilized in DMSO, with final assay concentration of 2.5%.