Fig. 3: Self-targeting HEPTs enhance killing of uropathogenic E. coli and K. pneumoniae isolates through release of colicin-like bacteriocins.
a Propagation of bacteria that survive WT phage treatment (e.g., phage-resistant subpopulations, pink) results in a failure to control bacterial growth. Self-targeting HEPTs can prevent or delay the growth of resistant subpopulations in situ by releasing complementary antimicrobial effectors, resulting in a two-pronged attack against a single bacterial target. b Genes encoding colicin E7 (green) or klebicin M (purple) were integrated within the structural gene cassette of phage scaffolds E2 or K1 to generate HEPTs targeting E. coli (E2::colE7) or K. pneumoniae (K1::kvarM). c, d Turbidity reduction assays combined with timepoint plating (red stars) demonstrated improved antimicrobial activity (i.e., regrowth was avoided or delayed) for E2::colE7 (c) and K1::kvarM (d) compared to WT phage treatment of uropathogenic E. coli and K. pneumoniae monocultures, respectively. e, f Combination treatment of an E. coli/K. pneumoniae co-culture with a self-HEPT (e) or cross-HEPT (f) cocktail was performed. Growing cultures of E. coli and K. pneumoniae were adjusted to OD600nm of 0.1, mixed at a ratio of 1:1, and infected with the indicated WT phages and/or HEPTs (5 × 107 PFU/mL). Optical density was monitored over 18 h of infection at 30 °C, followed by differential plating on chromogenic coliform agar (matching box and curve colors). hoc highly immunogenic outer capsid protein, gp gene product, OD600nm = optical density at 600 nm. Turbidity reduction data are technical triplicates shown as mean ± SD. Source data are provided as a Source data file.
