Characterization of novel Klebsiella pneumoniae phages isolated from hospital wastewater in Mumbai, India

Abstract

The escalating antimicrobial resistance in Klebsiella pneumoniae underscores the urgent need for alternative therapies. Phage therapy represents a promising approach owing to its specificity, phage self-replication, and potential synergy with antibiotics. Yet, the narrow host range of phages and rapid emergence of resistance necessitate the discovery of novel phages with diverse specificities, which can be facilitated by screening geographically distant environmental reservoirs. Here, we describe four novel Klebsiella bacteriophages belonging to the subfamily Studiervirinae , isolated from hospital wastewater in Mumbai, India. We characterized their host range, infection kinetics, physicochemical stability, complete genome sequences, phylogenetic placement, and encoded depolymerases to evaluate their potential as candidates for precision phage therapy against K. pneumoniae . Phages were isolated via enrichment on clinical K. pneumoniae and sequenced using a hybrid approach. Following genomic annotation and in silico depolymerase prediction, phages were characterized by transmission electron microscopy, one-step growth curves, and thermal/pH tolerance assays. The capsular specificity of native phages and recombinant depolymerases was evaluated via spot assays against 198 clinical K. pneumoniae isolates. The four phages were classified as novel species within the subfamily Studiervirinae (genera Przondovirus and Teetrevirus ), exhibiting strictly lytic lifestyles and favorable safety profiles devoid of antimicrobial resistance genes. They demonstrated strict specificity for clinically prominent capsular types (KL2, KL22, KL25, KL35, and KL47), with phage 1627 uniquely displaying dual specificity for both KL2 and KL22. All phages showed robust stability across broad pH (4–11) and temperature (4–50 °C) ranges. Expression of the recombinant depolymerase yielded preliminary evidence of targeted capsular degradation via spot assays, supporting in silico structural predictions of potential in vitro lytic activity. These findings suggest that cross-regional environmental screening is a promising strategy for identifying phages effective against local multidrug-resistant clinical pathogens. Moreover, these newly described phage species and their putative depolymerases represent a promising addition to the expanding therapeutic arsenal against K. pneumoniae infections.