Background In recent years, the inappropriate use of antibiotics has led to the widespread emergence of multidrug-resistant Klebsiella pneumoniae (MDR-K. pneumoniae), resulting in infections that are increasingly challenging to manage clinically. Bacteriophages (phages) are emerging as promising alternatives to antibiotics. This study aimed to isolate and characterize lytic phages targeting MDR-K. pneumoniae, providing biological resources and experimental data for phage-based control of MDR-K. pneumoniae infections. Results A lytic phage, designated vB_KpnA_Pkp-1 (Pkp-1), was successfully isolated. TEM revealed that Pkp-1 belongs to the Caudoviricetes class, featuring an icosahedral head (62 ± 2 nm) and a short tail (17 ± 1 nm), with plaques displaying clear centers and translucent halos. Pkp-1 exhibited strict specificity for porcine-derived ST967 K. pneumoniae isolates. Its optimal MOI was 0.00001, with a latent period of 25 min and a burst size of 108 PFU/cell. Pkp-1 demonstrated high stability at 40 °C–60 °C and pH 4.0–11.0, effectively inhibiting planktonic bacteria and suppressing/eradicating biofilms. Genomic analysis revealed a 38,455 bp dsDNA genome with 48 open reading frames (ORFs), functions of 30 proteins were predicted (e.g., DNA polymerase, tail fiber), while the remaining 18 proteins were annotated as hypothetical. Genomic analysis confirmed the absence of virulence, lysogeny-related, and antibiotic resistance genes. Pkp-1 shared high identity with phages phi1_146013 (98.71%) and P7124 (97.25%). Recombination analysis revealed 19 recombination events, with two specifically located within the tail protein gene. Notably, this tail protein gene exhibits significant divergence from those of other classified Kayfunavirus phages. Conclusion The lytic phage Pkp-1 represents a novel recombinant chimera within the Kayfunavirus genus, characterized by rapid replication, strict host specificity, environmental resilience, potent bactericidal activity, and biofilm clearance capability. The significant divergence of its tail protein from other Kayfunavirus phages suggests unique adaptive evolution. It represents a novel recombinant chimeric phage of the genus Kayfunavirus, with multiple recombination events in its genome. Its tail protein exhibits significant differences from those of other phages in the genus Kayfunavirus, indicating that it possesses adaptive evolutionary characteristics. These attributes position Pkp-1 as a potential biocontrol agent against MDR-K. pneumoniae infections, particularly in livestock and clinical settings. Further studies on in vivo efficacy and safety are warranted.

Multidimensional characterization of a novel porcine Klebsiella pneumoniae phage Pkp-1

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2026年1月10日

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Shenghui Wan,
Nana Li,
Yanfang Li,
Yan Liang,
Yonggang Qu

摘要

Background

In recent years, the inappropriate use of antibiotics has led to the widespread emergence of multidrug-resistant Klebsiella pneumoniae (MDR-K. pneumoniae), resulting in infections that are increasingly challenging to manage clinically. Bacteriophages (phages) are emerging as promising alternatives to antibiotics. This study aimed to isolate and characterize lytic phages targeting MDR-K. pneumoniae, providing biological resources and experimental data for phage-based control of MDR-K. pneumoniae infections.

Results

A lytic phage, designated vB_KpnA_Pkp-1 (Pkp-1), was successfully isolated. TEM revealed that Pkp-1 belongs to the Caudoviricetes class, featuring an icosahedral head (62 ± 2 nm) and a short tail (17 ± 1 nm), with plaques displaying clear centers and translucent halos. Pkp-1 exhibited strict specificity for porcine-derived ST967 K. pneumoniae isolates. Its optimal MOI was 0.00001, with a latent period of 25 min and a burst size of 108 PFU/cell. Pkp-1 demonstrated high stability at 40 °C–60 °C and pH 4.0–11.0, effectively inhibiting planktonic bacteria and suppressing/eradicating biofilms. Genomic analysis revealed a 38,455 bp dsDNA genome with 48 open reading frames (ORFs), functions of 30 proteins were predicted (e.g., DNA polymerase, tail fiber), while the remaining 18 proteins were annotated as hypothetical. Genomic analysis confirmed the absence of virulence, lysogeny-related, and antibiotic resistance genes. Pkp-1 shared high identity with phages phi1_146013 (98.71%) and P7124 (97.25%). Recombination analysis revealed 19 recombination events, with two specifically located within the tail protein gene. Notably, this tail protein gene exhibits significant divergence from those of other classified Kayfunavirus phages.

Conclusion

The lytic phage Pkp-1 represents a novel recombinant chimera within the Kayfunavirus genus, characterized by rapid replication, strict host specificity, environmental resilience, potent bactericidal activity, and biofilm clearance capability. The significant divergence of its tail protein from other Kayfunavirus phages suggests unique adaptive evolution. It represents a novel recombinant chimeric phage of the genus Kayfunavirus, with multiple recombination events in its genome. Its tail protein exhibits significant differences from those of other phages in the genus Kayfunavirus, indicating that it possesses adaptive evolutionary characteristics. These attributes position Pkp-1 as a potential biocontrol agent against MDR-K. pneumoniae infections, particularly in livestock and clinical settings. Further studies on in vivo efficacy and safety are warranted.