Isolation, characterization, and genome analysis of two novel virulent bacteriophages infecting extensively drug-resistant (XDR) clinical Klebsiella pneumoniae in Bangladesh

Abstract Background - Klebsiella pneumoniae , an opportunistic and difficult-to-treat pathogen, is a leading cause of morbidity and mortality associated with multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections. This study evaluates the potential of bacteriophages as an alternative to antibiotics against XDR isolates of K. pneumoniae. Methods - Thirty-one neonatal sepsis-causing Klebsiella spp. were collected, and K. pneumoniae were identified using both conventional and molecular techniques. Phenotypic and genotypic detection of ESBL (extended spectrum beta-lactamase) and carbapenem resistance was performed by disk diffusion and polymerase chain reaction (PCR). To isolate bacteriophages, sewage water samples were collected and enriched using reference host bacteria. Distinct morphological phage plaques were isolated by double layer agar assay (DLA). Characterization of bacteriophages was performed based on host range, temperature (4°C - 80°C upon 1 h incubation), pH (1-14 upon 1 h incubation), one-step growth curve, optimal multiplicity of infection (MOI), in vitro lytic ability and whole genome analysis. Results - Two novel lytic bacteriophages, Klebsiella phage Kpn BM7 and Klebsiella phage Kpn BU9, were isolated and characterized based on their competency to kill more than one clinical strain of K. pneumoniae . Both phages can steadily survive until a temperature of 40°C and from pH 5 to pH 11 without any significant reduction. The optimal MOI was 0.1 and 1, with short latent times of 10 min and 25 min and burst sizes of 85 PFU/cell and 12 PFU/cell. Whole-genome analysis reported that phages BM7 and BU9 belong to class Caudoviricetes. BM7 was 170558 bp long, a member of the Marfavirus genus and Marfavirus F48 species, and phage BU9 was 60450 bp long, reported as unclassified. Both phages did not harbor any lysogenic, toxin, or antimicrobial resistance genes. Conclusion - The isolated and established features of bacteriophages in this study can be a promising alternative solution to tackle XDR K. pneumoniae infection.