Isolation, characterization, and genomic analysis of a novel bacteriophage vB_Kp_XP4 targeting hypervirulent and multidrug-resistant Klebsiella pneumoniae

Xiaocui Peng ^1,2 Hongxia Zhang ² Xiaoyu Li ^1,2 Jianliang Chang ^1,2 Chengxiu Lv ³ Changhong Zhang ² Bu Whang ² Jun Zhao ^1,2 Zhang Wei ^4* Zhihua Zhang ^2*

• ¹ Department of postgraduate of Hebei North University, Zhangjiakou, China
• ² Respiratory and Critical Care Medicine Department, The First Affiliated Hospital of Hebei North University, Zhangjiakou, China
• ³ Department of Clinical Laboratory, Zibo First Hospital, Zibo, China
• ⁴ Central Laboratory, The First Affiliated Hospital of Hebei North University, Zhangjiakou, China

This study aimed to isolate a novel bacteriophage with specific lytic activity against hypervirulent and multidrug-resistant Klebsiella pneumoniae. The bacteriophage, named Klebsiella phage vB_Kp_XP4, was isolated from river water using the double-layer agar plate method with the clinically isolated strain P4 as the host. Morphological analysis by transmission electron microscopy revealed an icosahedral head and a long, flexible tail, categorizing the phage as a tailed phage. Klebsiella phage vB_Kp_XP4 demonstrated specificity by lysing one hypervirulent strain and one multidrug-resistant strain of K. pneumoniae among 21 clinical isolates. Stability was maintained across a wide pH range (4-11) and temperatures up to 70°C.The optimal multiplicity of infection (MOI) was determined to be 0.1, with a latent period of less than 10 minutes. Whole-genome sequencing revealed a linear double-stranded DNA genome of 44,344 bp with a G+C content of 53.80%, containing 54 coding sequences without any lysogenic, virulence, or antibiotic resistance genes. Phylogenetic analysis indicated that Klebsiella phage vB_Kp_XP4 represents a new species within the genus Drulisvirus, family Autographiviridae. The high stability, specificity, potent lytic activity of phage vB_Kp_XP4, the absence of undesirable genes, along with the ability to prolong the survival time of Galleria mellonella larvae infected with the host bacteria (P < 0.001), suggest its potential for clinical applications against K. pneumoniae infections. The presence of multiple halos during plaque formation further enhances its research value. The complete genome sequence has been submitted to GenBank under accession number PP663283.