Harnessing Lytic Phages for Biofilm Control in Carbapenem-Resistant Klebsiella pneumoniae Causing Urinary Tract Infection

Mandal, D. K.; Upadhaya, E.; Dahal, P.; Adhikari, G.; Pandey, R.; Rehaman, A.; Timilsina, S.; Sapkota, P.; Amagain, S.; Pun, D.; Khadka, K.; Gorathoki, K.; Neupane, B.; Chaudhary, S.; Thapa, S.; Khadka, B.; Dhungana, G.; Giri, G. R.; Nepal, R.; Napit, R.; Pradhan, P.; Manandhar, K. D.; Malla, R.

doi:10.1101/2025.10.01.679830

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bioRxiv · 10.1101/2025.10.01.679830

Harnessing Lytic Phages for Biofilm Control in Carbapenem-Resistant Klebsiella pneumoniae Causing Urinary Tract Infection

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Abstract

BackgroundKlebsiella pneumoniae is a major opportunistic pathogen with rising multidrug resistance and biofilm-related infections. Molecular and phage characterization is crucial to understand resistance mechanisms and explore alternative therapies such as phage therapy. MethodsWe performed whole-genome sequencing and antibiotic susceptibility testing of hospital-isolated Klebsiella pneumoniae (KP6697). MLST, plasmid replicon analysis, and resistance gene identification were conducted using bioinformatics. Phage isolation, electron microscopy-based morphological and biofilm analysis, and evaluation of lytic activity, stability, and host range were performed. Phage genome sequencing and annotation identified functional genes. ResultsThe host strain Klebsiella pneumoniae (KP6697) was multidrug-resistant, exhibiting resistance to 18 of 22 tested antibiotics, and genome analysis identified ST16 with eight plasmid replicons and 23 resistance genes, including blaCTX-M-15, blaNDM-5, and blaOXA-181. Functional annotations revealed extensive metabolic versatility and a rich repertoire of genes for biofilm formation, quorum sensing, secretion systems, and stress response. A lytic phage, Phage_KP6697_Omshanti, was isolated and classified as a Caudoviricetes member with a 45.3kb genome encoding lysis, replication, and structural genes. It demonstrated short latency, high burst size, thermal and pH stability, and broad host range against CRKP and other MDR strains. Importantly, microscopy confirmed its ability to inhibit and degrade biofilms at multiple stages, highlighting strong therapeutic potential. ConclusionComprehensive analysis of carbapenem-resistant K. pneumoniae (KP6697) revealed multidrug resistance and strong biofilm formation. The lytic phage Phage_KP6697_Omshanti, with depolymerase and endolysin activity, disrupted biofilms, and its stability, high burst size, and genomic traits suggest potential as an anti-CRKP agent, especially with antibiotics IMPORTANCEKlebsiella pneumoniae is increasing multidrug resistance and robust biofilm formation pose severe clinical challenges, limiting treatment options. Understanding the molecular basis of its resistance and exploiting bacteriophages with strong biofilm-disrupting properties provide promising alternative therapeutic strategies. This study highlights the isolation and genomic characterization of a lytic phage with potent anti-biofilm activity against carbapenem-resistant K. pneumoniae, underscoring its potential in combating resistant infections.

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Mandal, D. K., Upadhaya, E., Dahal, P., Adhikari, G., Pandey, R., Rehaman, A., Timilsina, S., Sapkota, P., Amagain, S., Pun, D., Khadka, K., Gorathoki, K., Neupane, B., Chaudhary, S., Thapa, S., Khadka, B., Dhungana, G., Giri, G. R., Nepal, R., Napit, R., Pradhan, P., Manandhar, K. D., Malla, R.. 2025-10-01. Harnessing Lytic Phages for Biofilm Control in Carbapenem-Resistant Klebsiella pneumoniae Causing Urinary Tract Infection. https://doi.org/10.1101/2025.10.01.679830

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