Designing an effective phage cocktail against Klebsiella pneumoniae covering metallo-β-lactamases producing multi-drug resistant clinical isolates

According to the ECDC in 2023, Greece presented a high rate of carbapenem-resistant Klebsiella pneumoniae (CRKP). Samples from the largest Athens' sewage treatment plant (n = 50), rectal swabs (n = 50), and stool (n = 10) from ICU and pediatric patients were screened for phages using the double-layer agar method. Plaque morphology, genomic features, biological properties (host range, phage adsorption time and rate, latent period length and burst size) and growth inhibition kinetics were determined for all isolated phages. Phage tissue distribution and efficacy against a dominant ST11 CRKP clinical isolate were assessed in murine thigh infection model. A cocktail from different phages was designed based on biological characteristics and host range spectrum. In total 7 distinct bacteriophages were isolated from different samples 5 Drulisvirus, 1 Webevirus and 1 Przondovirus with varied lytic activity against 40 (18-50) % of all isolates in host-range studies and 51 (41-85) % growth inhibition at the lowest MOI 0.00001 in growth curve experiments. The median (range) adsorption rate, time, latent phase and burst size in one-step growth experiments were 94 (88-97) %, 6 (2-10) min, 25 (15-40) min, and 63 (47-160) PFU/infected cell, respectively. Phages were rapidly distributed in different organs with high titers up to 8h and a 4log bacterial load reduction was found with the highest dose 10⁹ PFU/mouse. A cocktail of 5 phages was effective against 87.5 % of ESBL, NDM and VIM-producing isolates. Several phages with potent lytic activity and different host spectrum range against CRKP isolates were found. The 5-phage cocktail could be used for phage therapy of CRKP infections.