Method of Generating Phage Clones with Expanded Activity

A method of developing phages having antibacterial activity in expanded range of multidrug resistant (MDR) strains of a bacterial species, wherein the bacterial species is Pseudomonas aeruginosa or Klebsiella pneumoniae, the method comprising steps of;

• i. selecting at least two genetically similar phages, which have complementary lytic activity against a bacterial species, and determining the titer of each phage;
• ii. mixing the phages at about the same ratio of each phage titer to make a phage cocktail to yield an input cocktail of 1×105-1×1020 pfu/mL, and optionally about 1×1010 pfu/mL;
• iii. adding a volume of 10-20 MDR strains and a phage-susceptible strain of a bacterial species, which are individually cultured overnight in wells of a multi-well plate filled with a certain volume of bacterial culture media, in one strain per one column (or row) manner;
• iv. performing a serial dilution of the phage cocktail of step ii (100 to 10−7 or to 10−11) in bacterial culture media;
• v. adding each of the serial dilutions of step iv into a row (or a column) of the wells containing the bacterial culture of step iii;
• vi. incubating the plate(s) with shaking, optionally at 37° C. overnight;
• vii. pooling the lysate from the bacterial culture wells showing reduced bacterial growth or bacterial lysis;
• viii. clearing the pooled lysates, optionally by vortex mixing with CHCl3 and centrifugation;
• ix. repeating steps iii to viii with the cleared lysate at least 5, 7, 10 or 15 rounds;
• x. while performing step ix, periodically, optionally every 5 rounds, testing plaque forming activity with the cleared lysate, using a double-layered agar plate with a pan-phage-resistant strain;
• xi. collecting individual phage plaques from the double-layered agar plate;
• xii. performing steps iii to vii with each collected individual phages;
• and
• xiii. selecting phages showing expanded host range when compared with parent phages;
• wherein the method optionally further comprises
• xiv. testing the selected phages against genetically diverse strain global diversity set;
• xv. selecting phages from the phages of step xiv, which show expanded host range when compared with parent phages in step xiv;
• xvi. isolating and purifying the phage particles, and sequencing their DNAs;
• and
• xvii. testing phage stability with at least three clones of the selected phages;
• by re-testing them against the strain global diversity set to assess host range.