# Isolation, characterization, and genomic analysis of novel bacteriophage AEV23 against multidrug-resistant Klebsiella pneumoniae isolated from Cystic Fibrosis patients

## TL;DR

This study isolates and characterizes a new bacteriophage effective against drug-resistant Klebsiella pneumoniae, a pathogen affecting Cystic Fibrosis patients.

## Contribution

The paper introduces a novel lytic bacteriophage with high therapeutic potential against MDR K. pneumoniae.

## Key findings

- The phage showed high stability under various stress conditions and effectively removed biofilms.
- Genomic analysis revealed a 55,637-bp linear DNA genome with no virulence or lysogenic genes.
- The phage had a latent period of 30 minutes and a burst size of 98 PFU/cell.

## Abstract

Klebsiella pneumoniae is a well-known opportunistic pathogen in humans and can cause chronic obstructive pulmonary disease (COPD) in Cystic Fibrosis (CF) patients. This pathogen has developed resistance to β-lactam antibiotics due to the expression of extended-spectrum beta-lactamase (ESBL) genes, leading to current treatment challenges in these patients. Bacteriophages are alternative and effective treatment options against multidrug-resistant (MDR) pathogens. In this study, a lytic bacteriophage was isolated from untreated sewage, tested against multidrug-resistant K. pneumoniae strains, and evaluated for its therapeutic potential in vitro.

This study presents the microbiological, physicochemical, and genomic characterization of a virulent bacteriophage. The phage was studied by electron microscopy, host range analysis, multiplicity of infection (MOI) determination, adsorption rate measurement, burst size calculation, latent period assessment, stability testing to temperature, chloroform, pH and salt stress, and biofilm removal ability evaluation demonstrated by SEM; the bacteriophage genome was studied by complete genome sequencing.

The phage exhibited a broad and highly specific host range for K. pneumoniae strains. Its stability under stress conditions, including changes in temperature, pH, salt concentration, and exposure to chloroform, was 64.63%, 52.79%, 68.36%, and 98.92%, respectively. The one-step growth curve results demonstrated that the bacteriophage had a latent period of 30 min and a burst size of approximately 98 plaque-forming units per infected cell (PFU/cell). Adsorption assays revealed that 92% of isolated phages adsorbed to bacterial cells within 5 min. Additionally, the bacteriophage showed inhibitory activity against bacterial growth at an MOI 1. The biofilm removal assay demonstrated that the phage eliminated over 93% of the cellular biomass, as confirmed by scanning electron microscopy (SEM). Whole-genome analysis showed that it belongs to the Loughboroughvirus genus. The phage has a linear, double-stranded DNA genome with a length of 55,637 bp and a GC content of 45.9%. The genome encodes 76 open reading frames (ORFs), including structural proteins, DNA metabolism enzymes, lysis modules, and packaging proteins. No tRNAs, lysogenic genes, or virulence factors were detected in the genome.

Characterization of phage VB_KpM-AEV23 demonstrated its high host specificity against K. pneumoniae, making it a suitable candidate for phage therapy applications.