Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection

Abstract

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

Keywords: Klebsiella pneumoniae; K5; capsule; depolymerase; hypervirulent; phage.

Figure 1

Morphology of strains. A Colony morphology of strain B16. B Colony morphology of strain KP181. C The results of the modified Hodge test for B16. KPC-2 was acquired from B16 through plasmid conjugation. As shown by the red circle, B16 hydrolysed meropenem, resulting in the growth of the strain ATCC25922 as an apple-like indentation in the inhibition zone.

Figure 2

Morphology of the phage. A Plaque of P1011 using strain B16 as a host. P1011 plaques were observed at 12 h, 36 h and 60 h. As time progressed, the halo gradually became larger. B No plaques were generated by the double plate method using KP181 as the host. C The halo of P1011 formed on the lawn of KP181 according to the spotting test. D Transmission electron micrograph of phage P1011. The scale bar is 100 nm.

Figure 3

Biological characteristics of phages. A One-step growth curve of P1011. The sample was collected at certain intervals (every 2 min from 0 to 10 min and every 10 min from 10 to 100 min). B The optimal multiplicity of infection (MOI) of P1011 was evaluated. C pH stability of P1011. D Thermal stability of P1011.

Figure 4

Phage genome comparison. A Whole-genome comparison of ten phages highly homologous to P1011. The comparison was performed through the Genome-BLAST Distance Phylogeny (GBDP) method by VICTOR using Formula D0. Phage P1011 is marked with a red circle. B Viral proteomic trees of phages. A viral proteomic tree of P1011 was constructed with 34 phages (those with greater than 50% genomic similarity). P1011 is marked with a red pentacle.

Figure 5

Protein phylogenetic analysis and comparison. A Phylogenetic analysis of the large terminase subunit. The phylogenetic analysis was conducted using the neighbour-joining method with 1000 bootstrap replicates in MEGA X. B Protein alignment of K5 depolymerase. Amino acid sequence alignment of the putative depolymerase P1011 with a previously reported K5 depolymerase.

Figure 6

Protein expression and activity. A SDS‒PAGE of recombinant depolymerase. Lane 1, protein marker; Lane 2, purified depolymerase dep1011. B Depolymerase activity against KP181. First, 50 ng of dep1011 or PBS was spotted on the lawn of KP181. C Bacterial sediment of wild-type strains and strains treated with depolymerase. 1, Bacterial sediment of B16; 2, bacterial sediment of B16 treated with depolymerase; 3, bacterial sediment of KP181; 4, bacterial sediment of KP181 treated with depolymerase.

Figure 7

Therapeutic efficacy of phage and depolymerase. A Survival of mice infected with B16 and treated with P1011 and dep1011. B Survival of mice infected with KP181 and treated with P1011 and dep1011. C Viable bacteria in the blood of mice infected with B16 and in the treatment group. D Viable bacteria in the liver of mice infected with B16 and in the treatment group. E Viable bacteria in the lungs of mice infected with B16 and in the treatment group. F Viable bacteria in the blood of mice infected with KP181 and in the treatment group. G Viable bacteria in the liver of mice infected with KP181 and in the treatment group. H Viable bacteria in the lungs of mice infected with KP181 and in the treatment group.

Figure 8

Pathological analysis of mice infected with B16 and the treatment group. H&E-stained liver and lung tissues are shown. The scale bar is 50 μm.

Figure 9

Pathological analysis of KP181-infected and treated mice. H&E-stained liver and lung tissues are shown. The scale bar is 50 μm.