Isolation and Characterization of vB_kpnM_17-11, a Novel Phage Efficient Against Carbapenem-Resistant Klebsiella pneumoniae

Abstract

Phages and phage-encoded proteins exhibit promising prospects in the treatment of Carbapenem-Resistant Klebsiella pneumoniae (CRKP) infections. In this study, a novel Klebsiella pneumoniae phage vB_kpnM_17-11 was isolated and identified by using a CRKP host. vB_kpnM_17-11 has an icosahedral head and a retractable tail. The latent and exponential phases were 30 and 60 minutes, respectively; the burst size was 31.7 PFU/cell and the optimal MOI was 0.001. vB_kpnM_17-11 remained stable in a wide range of pH (4-8) and temperature (4-40°C). The genome of vB_kpnM_17-11 is 165,894 bp, double-stranded DNA (dsDNA), containing 275 Open Reading Frames (ORFs). It belongs to the family of Myoviridae, order Caudovirales, and has a close evolutionary relationship with Klebsiella phage PKO111. Sequence analysis showed that the 4530 bp orf022 of vB_kpnM_17-11 encodes a putative depolymerase. In vitro testing demonstrated that vB_kpnM_17-11 can decrease the number of K. pneumoniae by 10⁵-fold. In a mouse model of infection, phage administration improved survival and reduced the number of K. pneumoniae in the abdominal cavity by 10⁴-fold. In conclusion, vB_kpnM_17-11 showed excellent in vitro and in vivo performance against K. pneumoniae infection and constitutes a promising candidate for the development of phage therapy against CRKP.

Keywords: Klebsiella pneumonia; animal model; depolymerase; phage; phage therapy.

Figure 1

Plaques and transmission electron microscopy of phage vB_kpnM_17-11. (A) Plaque morphology of phage vB_kpnM_17-11 on a bacterial lawn of K. pneumoniae 17-11 in nutrient broth with 0.5% agar (scale bar = 1 cm). (B) Transmission electron microscopy of phage vB_kpnM_17-11. (C) Contractive and non-contractive tails. Dashed arrow for non-contractile tail; Solid arrow for contractile tail.

Figure 2

One-step growth curve of vB_kpnM_17-11. The PFU of each infected cell is shown at different time points. Each data point represents the mean of three independent experiments, and the error bar represents the standard deviation.

Figure 3

Serotype identification of k. pneumoniae. The CD1-VR2-CD2 region of the wzc gene was amplified by PCR, sequenced and compared to determine the serotype of k. pneumoniae lysed by vB_kpnM_17-11. K19 represents the inherent sequence of CD1-VR2-CD2 region of wzc gene of k. pneumoniae serotype K19, and 17-11, B8, H27, and S43 are four strains that can be lysed by vB_kpnM_17-11 respectively.

Figure 4

Thermal and pH stability of vB_kpnM_17-11. (A) Temperature stability of phages vB_kpnM_17-11. When incubated at different temperatures for 60 min, the phages could survive at 4°C, 25°C, 37°C and 40°C. At 50°C, the phage concentration decreased by 80%. Phages could not survive at 60°C and 70°C. Data were obtained from three independent experiments and are shown as mean ± standard deviation. (B) PH stability of phages vB_kpnM_17-11. vB_kpnM_17-11 was incubated at the indicated pH conditions for 60 min. vB_kpnM_17-11 is relatively stable under neutral condition. The phage titer decreased significantly under acidic or alkaline conditions. vB_kpnM_17-11 titer extreme acid-base conditions cannot be detected (ND, Not Detected). Data were obtained from three independent experiments and are shown as mean ± standard deviation.

Figure 5

Genome map of phage vB_kpnM_17-11. The black circle in the middle represents the GC content; the violet circle represents GC skew. Different colors represent genes with different functions: peach for host lysis module, green for morphology module, orange for nucleotide metabolism and replication, blue for packaging module, red for specific protein, gray for hypothetical protein.

Figure 6

Multiple genome alignments of phage vB_kpnM_17-11, PKO111 and KP1. Red and blue represent the forward and reverse matches, respectively. The darker the color, the higher similarity.

Figure 7

Phylogenetic tree of vB_kpnM_17-11 based on MCP. Phylogenetic tree of phages established using MEGA-X software by the neighbor-joining method with 1000 bootstrap replicates. The red dot highlights the vB_kpnM_17-11.

Figure 8

The prediction domines and phylogenetic tree of depolymerase Dep022. (A) Domains of depolymerase Dep022 predicted using HHpred software. HHpred prediction results show that DEP022 has three different domains. (B) Phylogenetic tree of depolymerase. Phylogenic tree of depolymerases established using MEGA-X software by the neighbor-joining method with 1000 bootstrap replicates. The red dot highlights the Dep022 depolymerase of vB_kpnM_17-11.

Figure 9

Sterilization experiment in vitro and in vivo. (A) Bacteriostatic curve of phage in vitro. PBS, vB_kpnM_17-11 or PB were added to 50 mL exponential phase K. pneumoniae 17-11. The value of OD₅₉₅ was measured every 30 min from 0 min to 180 min. (B) Bactericidal efficiency of phage in vitro. ****P < 0.0001, two-sided Mann–Whitney test. (C) Survival of mice after infection by K. pneumoniae 17-11. **P < 0.01 (Gehan-Breslow-Wilcoxon test). (D) Abdominal lavage fluid bacterial load. Mice were infected with K. pneumoniae 17-11 intraperitoneally and treated with phage. Peritoneal lavage was harvested at 24 h post infection and CFU were quantified. ****P < 0.0001, two-sided Mann–Whitney test.