Isolation and Characterization of a Novel Lytic Bacteriophage against the K2 Capsule-Expressing Hypervirulent Klebsiella pneumoniae Strain 52145, and Identification of Its Functional Depolymerase

Abstract

Klebsiella pneumoniae is among the leading bacteria that cause nosocomial infections. The capsule of this Gram-negative bacterium is a dominant virulence factor, with a prominent role in defense and biofilm formation. Bacteriophages, which are specific for one bacterial strain and its capsule type, can evoke the lysis of bacterial cells, aided by polysaccharide depolymerase enzymes. In this study, we isolated and characterized a bacteriophage against the nosocomial K. pneumoniae 52145 strain with K2 capsular serotype. The phage showed a narrow host range and stable lytic activity, even when exposed to different temperatures or detergents. Preventive effect of the phage in a nasal colonization model was investigated in vivo. Phlyogenetic analysis showed that the newly isolated Klebsiella phage B1 belongs to the Webervirus genus in Drexlerviridae family. We identified the location of the capsule depolymerase gene of the new phage, which was amplified, cloned, expressed, and purified. The efficacy of the recombinant B1dep depolymerase was tested by spotting on K. pneumoniae strains and it was confirmed that the extract lowers the thickness of the bacterium lawn as it degrades the protective capsule on bacterial cells. As K. pneumoniae strains possessing the K2 serotype have epidemiological importance, the B1 phage and its depolymerase are promising candidates for use as possible antimicrobial agents.

Keywords: K2 serotype; Klebsiella pneumoniae; bacteriophage; capsule depolymerase; tail fibers.

Figure 1

Characteristics of the B1 phage. (a) Electron micrograph of B1 phage stained with 1.5 w/v phospho-tungstic acid (top) and plaques on 52145 WT lawn (bottom). Scale bars represent 200 nm and 10 mm, respectively. Individual plaques were acquired by agar overlay method. (b) One-step growth curve of phage B1. The plaque-forming unit (PFU) per infected cell at different times are shown. Data are the mean of three independent experiments.

Figure 2

Time-kill assay of phage B1 against Klebsiella pneumoniae 52145. Overnight bacterial culture was diluted (10³ CFU/mL) and infected with phage B1 at different multiplications of infection (MOIs). Non-phage-treated bacterial cultures acted as a positive control (■), lysogeny broth (LB) without bacteria acted as a negative control (▲). Each data point is the mean from three experiments. Standard deviations (±SD) are shown as vertical lines.

Figure 3

Adaptation limits of phage B1. (a,b) Heat tolerance. (c) Detergent tolerance. Data are presented in PFU by time. Each data point is the mean from three experiments. Standard deviations (±SD) are shown as vertical lines.

Figure 4

Whole genome-based phylogenetic relations of phage B1. Genome–BLAST distance phylogeny (GBDP) trees were inferred using the formula D4 and yielded average support of 9%. The numbers above branches are GBDP pseudo-bootstrap support values from 100 replications. The branch lengths of the resulting VICTOR trees are scaled in terms of the respective distance formula used. The OPTSIL clustering (results indicated after the @ mark) yielded 31 species clusters (S), 3 clusters at the genus level (G), and 2 at the family level (F). Accession numbers are also indicated next to the phage names. Klebsiella phage B1 is marked with a black arrow ().

Figure 5

Representation of the homology similarities and differences of K. pneumoniae K2 serotype-specific phages and one reference phage of the Webervirus genus. Phage B1 (K2 specific, Siphoviridae), GH-K3 (K2 specific, Siphoviridae), KP36 (K63 specific, Siphoviridae, reference phage of the Webervirus genus), KpV74 (K2 specific, Podoviridae), and KpV763 (K2 specific, Podoviridae). Orf61 of phage B1 is marked with an asterisk (*). Homology was only restricted to the N-terminal between the depolymerase genes of B1 and KP36, as well as between GH-K3 and KP36, while no homology could be revealed to the two K2 specific podophage sequences. Annotations of the genomes were performed by the RAST server and were visualized by Easyfig.

Figure 6

Capsule-degrading activity of B1dep. Spot tests of the purified recombinant B1dep protein on 52145 WT lawn. (A) Turbid spot of concentrated crude KRX lysate, resembling the plaque halo. (B) Spots of dilution series of purified B1dep. A total of 10 µL was spotted from every dilution. Scale bar represents 10 mm.

Figure 7

Comparison the therapeutic and preventive effect of B1 phage in frame of survival and bodyweight in a murine nasal infection model. The graph shows body mass of mice from the start of nasal cavity infection over 16 days. Body mass in groups of therapeutic phage administrations (B1 phage 1 h, 3 h, 6 h, 12 h, 24 h) and in the K. pneumoniae 52145 infection positive control (K. pn. 52145) are shown as the mean of four mice from the same group. Body mass graphs of mice in the preventive phage administered group (−1 h) are indicated individually (B1phage −1 m1, m2, m3, m4).