When clearing zones were observed, the antibacterial activity of the LOXO-101 phages against each bacterial host was assessed based on the minimum phage concentration required to form a completely transparent zone. Investigation of ZZ1 antimicrobial activity against AB09V at different temperatures The antibacterial activity of ZZ1 against A. baumannii AB09V was evaluated by serial dilution spot testing at different temperatures. Phage stock (5 μl) from a dilution series was spotted onto a lawn of AB09V in top agar. The plates were examined for cell lysis after overnight incubations at 25°C, 30°C, 35°C, 37°C,

39°C, 40°C, and 42°C. The optimal antibacterial temperature was determined by comparing the minimum phage concentration required to form a completely transparent zone. Phage adsorption and growth curve An overnight culture of strain AB09V (1 ml) was inoculated into fresh medium (100 ml) and incubated with shaking at 37°C for MLN2238 ic50 approximately 1 h to yield a cell density of approximately 7.0 × 107 CFU/ml (at an OD600 of 0.15). A 1 ml

sample of a nutrient broth suspension of the phage ZZ1 at an approximate MOI of 10 was added to this culture. Samples were periodically withdrawn and immediately chilled while being further diluted to measure total phage activity (including infected bacterial cells and free phages) by the double-layered-agar plate technique. Bacterial viable counts were determined before the bacteria were mixed with the phage and were assessed periodically. Burst size was estimated from triplicate experiments find more using the equation described by Jiang et al. [27]. Each experiment was performed three times, and the results are reported as the mean of three observations ± standard deviation (SD). Stability Resistance to different pH values at 37°C was determined according to the methods described by Verma et al. [28]. The pH of

the nutrient broth Lepirudin was adjusted with either 1 M HCl or 1 M NaOH to obtain a pH within the range of 2–11. A total of 100 μl of bacteriophage suspension (4.7 × 1011 PFU/ml) was inoculated into 10 ml of pH-adjusted medium. After incubation for 1 h at 37°C, the surviving phages were diluted and counted immediately using the soft agar overlay method at 37°C. Moreover, according to the methods described by Capra et al. [29], the stability of ZZ1 at various temperatures (50°C, 60°C, 70°C, and 80°C) was checked by incubating the phage (3.2 × 1010 PFU/ml) at the indicated temperature for 1 h at pH 7.0 in nutrient broth; the surviving phages were then counted using the soft agar overlay method at 37°C. Morphology of phage and its host strain AB09V cells were infected with ZZ1 during the exponential growth phase (OD600 = 0.35) at an MOI of approximately 100 and incubated at 37°C for 5 min in nutrient broth medium. The mixture was fixed with 1% glutaraldehyde at 0°C for 60 min and then centrifuged (4500 × g, 3 min).