Interaction dynamics of phage and planktonic bacteria was tested in a 96-well microtiter plate (Tissue Culture Plate; Sarstedt) in triplicate, according to the protocol described by Vukotic et al. [22 (link)]. The K. pneumoniae Ni9 overnight culture was adjusted to 0.5 McFarland units and further diluted 100 times in 200 μL LB distributed in wells. Phages SJM3 and LASTA were applied separately, as well as in a cocktail suspension (mixture of 50:50), at final MOI 10 (growth control excluded from treatment with phage). At 3, 6, 9 and 24 h after the onset of the experiment, viable cell count was enumerated by spotting dilutions on LA plates [37 (link)]. A total of 80 colonies (ten from each time point for each phage) were collected, propagated and analyzed for sensitivity to both phages using spot testing [23 ]. Among colonies that appeared resistant, eleven were selected for further investigation of phage-resensitization phenomena and designated Ni92–Ni912. They were chosen from each time point and each infection setting.
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