Comprehensive Genomic Analysis of Klebsiella pneumoniae and Its Temperate N-15-like Phage: From Isolation to Functional Annotation

Abstract

Antibiotic resistance to Klebsiella pneumoniae poses a major public health threat, particularly in intensive care unit (ICU) settings. The emergence of extensively drug-resistant (XDR) strains complicates treatment options, requiring a deeper understanding of their genetic makeup and potential therapeutic targets. This research delineated an extensively drug-resistant (XDR) Klebsiella pneumoniae strain obtained from an ICU patient and telomeric temperate phage derived from hospital effluent. The bacteria showed strong resistance to multiple antibiotics, including penicillin (≥16 μg/mL), ceftriaxone (≥32 μg/mL), and meropenem (≥8 μg/mL), which was caused by SHV-11 beta-lactamase, NDM-1 carbapenemase, and porin mutations (OmpK37, MdtQ). The strain was categorized as K46 and O2a types and carried virulence genes involved in iron acquisition, adhesion, and immune evasion, as well as plasmids (IncHI1B_1_pNDM-MAR, IncFIB) and eleven prophage regions, reflecting its genetic adaptability and resistance dissemination. The 172,025 bp linear genome and 46.3% GC content of the N-15-like phage showed strong genomic similarities to phages of the Sugarlandvirus genus, especially those that infect K. pneumoniae. There were structural proteins (11.8%), DNA replication and repair enzymes (9.3%), and a toxin-antitoxin system (0.4%) encoded by the phage genome. A protelomerase and ParA/B partitioning proteins indicate that the phage is replicating and maintaining itself in a manner similar to the N15 phage, which is renowned for maintaining a linear plasmid prophage throughout lysogeny. Understanding the dynamics of antibiotic resistance and pathogen development requires knowledge of phages like this one, which are known for their temperate nature and their function in altering bacterial virulence and resistance profiles. The regulatory and structural proteins of the phage also provide a model for research into the biology of temperate phages and their effects on microbial communities. The importance of temperate phages in bacterial genomes and their function in the larger framework of microbial ecology and evolution is emphasized in this research.

Keywords: Klebsiella pneumoniae; functional annotation; phage; temperate.

Figure 1

A circular phylogenetic tree illustrating the evolutionary relationships of K. pneumoniae Kpn_R01 and its 34 closest strains. The tree was inferred from 16S rRNA gene sequences using the maximum likelihood (ML) tree highlighting the phylogenetic placement of Kpn_R01 (marked with a red dot) among closely related strains. Clustering patterns indicate potential global dissemination, with strains originating from diverse geographic locations. The close association between clinical and environmental isolates underscores the role of environmental reservoirs in bacterial transmission. The tree was inferred using the ML method with the Tamura–Nei model [52], and branch support was assessed with 500 bootstrap replicates [53]. The initial heuristic search compared a neighbor-joining (NJ) tree [54] and a maximum parsimony (MP) tree, selecting the one with the best log-likelihood score. Evolutionary analyses were conducted in MEGA12 [55].

Figure 2

Isolation and characterization of Klebsiella phage Kpn_R1. (A) Plaque morphology of Klebsiella phage Kpn_R1 on a bacterial lawn, showing distinct, clear plaques. (B) Transmission electron microscopy (TEM) image of Klebsiella phage Kpn_R1, revealing a polyhedral head (53.7 nm in diameter) and a wavy tail (178.3 nm in length). Based on morphology, the phage is classified within the Demerecviridae family according to the International Committee on Taxonomy of Viruses (ICTV). Scale bar: 200 nm.

Figure 3

A circular genome map of Klebsiella phage Kpn_R1 generated using PhageScope. The outermost rings represent annotated coding sequences (CDSs) classified by function, while the inner rings display GC content and GC skew. Red markers indicate putative virulence-associated or antibiotic resistance-related genes. The color gradient represents the variation in GC content across the genome.

Figure 4

The phylogenetic analysis of Klebsiella phage Kpn_R1 was conducted using ViPTree, a web server that generates viral proteomic trees based on genome-wide sequence similarities computed by tBLASTx. The circular proteomic tree illustrates the evolutionary relationships of Kpn_R1, highlighting its closest related phages forming distinct clusters (gray).

Figure 5

Proteomic similarity map of Klebsiella phage Kpn_R1 and related phages. The alignment shows protein sequence similarities between Klebsiella phage Kpn_R1 (Seq1) and its closest relatives: Klebsiella phage vB_Kpn_IME260, Klebsiella phage Sugarland, and Escherichia phage N15. Conserved regions are depicted by connected syntenic blocks, with the strongest similarities shown in pink. The distinct structural organization of Escherichia phage N15 highlights its evolutionary divergence.