Phage Host Specificity Evaluation

In order to discern the host specificity of phage, spot test and liquid assay were performed. The phage was tested against different clinical isolates of K. pneumoniae (BC936, E474, U2016, BC1415, BC1994) and E. coli (U3790, U3176, U1007, U1024, U2354) obtained from Sundaram Medical Foundation, Chennai, India. In addition, the phage was also tested against reference strains of E. coli (MG1655), Acenitobacter baumannii, Enterobacter cloacae, Pseudomonas aeruginosa and Enterococcus faecalis. Spot assay was done as reported earlier (Zhao et al. 2019 (link)). 300 µl of fresh bacterial culture was added to soft agar, overlaid on nutrient agar plate and allowed to solidify. 5 µl of the purified phage lysate (10¹² PFU/ml) were spotted on to the plate and incubated at 37 °C overnight. For liquid culture based evaluation, 180 µl of culture (0.05 OD) was mixed with phage (10⁸ PFU/ml) and incubated at 37 °C (Xie et al. 2018 (link)). The bacterial growth was monitored by measuring absorbance at 595 nm at 0, 30, 60, 90, 120 and 240 min using Tecan Infinite® F50 Microplate reader (TECAN, Männedorf, Switzerland) and the growth curve was plotted. The experiment was carried out in triplicates.