Bacteriophage.
Phage treatment in liquid cultures. A 96-well plate was filled with 200 μl of TSB in each well, additionally containing 106 CFU/ml PAO1-GFP or 108 CFU/ml PA14-WT alone, or together with or without 106 PFU/ml of phages (MOIPAO1 = 1). These initial population sizes were chosen since they allowed the two strains to co-exist and grow over 48 hours. The plate was then put in a Tecan i-control infinite M200 PRO plate reader at 37°C under agitation for 48 hours. After 6, 24 and 48 hours, the untreated samples were transferred into Eppendorf tubes and centrifuged at 4°C, 8000 rpm for 15 minutes, resuspended in 200 μl of PBS, serially diluted and plated on either TSA or LB Agar gentamicin 10 μg/ml plates. For the treated samples, the same protocol was followed except that after centrifugation, the supernatants containing phages were kept in the fridge at 4°C. They were further diluted in NaCl 0.9% to count the PFU/ml. The pellets containing the bacteria were then washed 3 times with 1 ml of fresh PBS at 8000 rpm, 4°C for 5 minutes to remove all the potential phages remaining in the pellet. Finally, the phage resistance rate of PAO1-GFP was calculated after 24 and 48 hours by plating them on TSA plates containing approximately 1010 PFU (plaque forming units) of pre-absorbed phages. If PAO1-GFP were growing in co-culture with PA14-WT, plates additionally containing 10 μg/ml gentamicin were used. To evaluate resistance rates, the CFU/ml of PAO1-GFP growing on plates saturated with phages was then compared to the CFU/ml growing on plates with no phage.