Characterization of a Lytic Bacteriophage and Demonstration of Its Combined Lytic Effect with a K2 Depolymerase on the Hypervirulent Klebsiella pneumoniae Strain 52145

Abstract

Klebsiella pneumoniae is a nosocomial pathogen. Among its virulence factors is the capsule with a prominent role in defense and biofilm formation. Bacteriophages (phages) can evoke the lysis of bacterial cells. Due to the mode of action of their polysaccharide depolymerase enzymes, phages are typically specific for one bacterial strain and its capsule type. In this study, we characterized a bacteriophage against the capsule-defective mutant of the nosocomial K. pneumoniae 52145 strain, which lacks K2 capsule. The phage showed a relatively narrow host range but evoked lysis on a few strains with capsular serotypes K33, K21, and K24. Phylogenetic analysis showed that the newly isolated Klebsiella phage 731 belongs to the Webervirus genus in the Drexlerviridae family; it has a 31.084 MDa double-stranded, linear DNA with a length of 50,306 base pairs and a G + C content of 50.9%. Out of the 79 open reading frames (ORFs), we performed the identification of orf22, coding for a trimeric tail fiber protein with putative capsule depolymerase activity, along with the mapping of other putative depolymerases of phage 731 and homologous phages. Efficacy of a previously described recombinant K2 depolymerase (B1dep) was tested by co-spotting phage 731 on K. pneumoniae strains, and it was demonstrated that the B1dep-phage 731 combination allows the lysis of the wild type 52145 strain, originally resistant to the phage 731. With phage 731, we showed that B1dep is a promising candidate for use as a possible antimicrobial agent, as it renders the virulent strain defenseless against other phages. Phage 731 alone is also important due to its efficacy on K. pneumoniae strains possessing epidemiologically important serotypes.

Keywords: Klebsiella phage; Klebsiella pneumoniae; bacteriophage; capsule depoly-merase; capsule serotype; phage receptor, K2 serotype.

Figure 1

Characteristics of phage 731. (a,b) Individual plaques and clear spot (small pictures) on 52145-Δwca_K2 (K-) lawn (a) and on 53.8 (K33) lawn (b). Individual plaques were acquired by agar overlay method, clear spots were gained by dropping 10 µL of the concentrated phage suspension. Scale bars represent 10 mm. (c) One-step growth curves of phage 731 on the 52145-Δwca_K2 (K-) (gray) and on the 53.8 (K33) strain (black). The titer of the phage at different times are shown. Data are the mean of 3-3 independent experiments, error bars represent ± SD. (d) Electron micrograph of phage 731 stained with 1.5 w/v hosphor-tungstic acid. Scale bar represents 500 nm.

Figure 2

Synergistic effect of B1dep protein and phages 731 and 13 on 52145 lawn with the K2 capsule. Middle rows: 10 microliters of the purified recombinant B1dep protein was mixed and spotted along with 10-10 µL-s of different titers of phage 731 (left plate) or phage 13 (right plate) suspensions. Phage control dilutions (up) and B1dep control with dilution (bottom) were also spotted and are presented. Clear zones (dark here) indicate full lysis of the bacteria by the phages; semi-translucent (light dark here) halo zones are produced by B1dep. Cc = concentrated phage suspension (≈10⁹ PFU/mL, black text) or B1dep (≈800 ng, white text).

Figure 3

Whole genome-based phylogenetic relations of phage 731. Genome–BLAST distance phylogeny (GBDP) trees were inferred using the formula D4 and yielded average support of 17%. 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 yielded 26 species clusters, 2 clusters at the genus level, and 1 at the family level. Accession numbers are also indicated next to the phage names. Klebsiella phage 731 is marked with a grey arrow ().

Figure 4

Comparative genome analysis of the previously isolated Klebsiella phage B1 and phage 731. Annotations of the genomes were done by the RAST server and were visualized by Easyfig. Predicted gene function groups are indicated.

Figure 5

Molecular properties of the tail fiber protein coded by orf22 of phage 731. (a) Comparison of homologous proteins of related phages by NCBI COBALT, the order of the phage proteins was originally aligned by Jalview and is the following from the top: 731, GML-KpCol1, vB_KpnS-VAC4, vB_KpnS-VAC7, vB_Kpn-VAC111, vB_KpnS_15-38_KLPPOU149, pK8, MezzoGao, vB_KpnD_PeteCarol, PhiKpNIH-10, NJS2, NJS1, Sweeny, vB_KpnS_Domnhall, vB_KpnS_KingDDD, vB_KpnS_SegesCirculi, PWKp14, Sushi. Pages are detailed in Table 2. Conserved regions of orf22-coded tail fiber protein of phage 731 are shown below, generated by NCBI BLASTp. (b) 3D model of orf22-coded tail fiber protein by SWISS-MODEL: ribbon model of the trimerized protein viewed from the N-terminal (left), side (middle), C-terminal (right), and space-filling model (bottom).

Figure 6

Genome similarities and differences of phage 731 to the representatives of the K. pneumoniae phages of the Webervirus genus. (a) Comparison of phages Sushi, 731, vB_KpnS_SegesCirculi, vB_KpnS_Domnhall and vB_KpnS_KingDDD. (b) Comparison of phages NJS2, NJS1, 731, PhiKpNIH-10, MezzoGao and vB_KpnD_PeteCarol. (c) Comparison of phages GML-KpCol1, 731, Sweeny, vB_KpnS-VAC4, vB_KpnS-VAC7, vB_Kpn-VAC111, vB_KpnS_15-38_KLPPOU149, pK8 and PWKp14. Genomes of the phages are truncated to the tail regions, containing putative depolymerases. Phage names are indicated on the left. Capsule specificities, where known, are written on the right. Phage 731 is framed and orf22 is marked with an asterisk (*). The provided ORF numbering was used only in this study for practical reasons and is indicated on (a), predicted ORF functions are grouped, and their color codes are also indicated. Annotations of the genomes were performed by the RAST server and were visualized by Easyfig. Phages are listed in Table 2.

Figure 6

Genome similarities and differences of phage 731 to the representatives of the K. pneumoniae phages of the Webervirus genus. (a) Comparison of phages Sushi, 731, vB_KpnS_SegesCirculi, vB_KpnS_Domnhall and vB_KpnS_KingDDD. (b) Comparison of phages NJS2, NJS1, 731, PhiKpNIH-10, MezzoGao and vB_KpnD_PeteCarol. (c) Comparison of phages GML-KpCol1, 731, Sweeny, vB_KpnS-VAC4, vB_KpnS-VAC7, vB_Kpn-VAC111, vB_KpnS_15-38_KLPPOU149, pK8 and PWKp14. Genomes of the phages are truncated to the tail regions, containing putative depolymerases. Phage names are indicated on the left. Capsule specificities, where known, are written on the right. Phage 731 is framed and orf22 is marked with an asterisk (*). The provided ORF numbering was used only in this study for practical reasons and is indicated on (a), predicted ORF functions are grouped, and their color codes are also indicated. Annotations of the genomes were performed by the RAST server and were visualized by Easyfig. Phages are listed in Table 2.

Figure 6

Genome similarities and differences of phage 731 to the representatives of the K. pneumoniae phages of the Webervirus genus. (a) Comparison of phages Sushi, 731, vB_KpnS_SegesCirculi, vB_KpnS_Domnhall and vB_KpnS_KingDDD. (b) Comparison of phages NJS2, NJS1, 731, PhiKpNIH-10, MezzoGao and vB_KpnD_PeteCarol. (c) Comparison of phages GML-KpCol1, 731, Sweeny, vB_KpnS-VAC4, vB_KpnS-VAC7, vB_Kpn-VAC111, vB_KpnS_15-38_KLPPOU149, pK8 and PWKp14. Genomes of the phages are truncated to the tail regions, containing putative depolymerases. Phage names are indicated on the left. Capsule specificities, where known, are written on the right. Phage 731 is framed and orf22 is marked with an asterisk (*). The provided ORF numbering was used only in this study for practical reasons and is indicated on (a), predicted ORF functions are grouped, and their color codes are also indicated. Annotations of the genomes were performed by the RAST server and were visualized by Easyfig. Phages are listed in Table 2.

Figure 7

Hypothetical representation of the effect of phage B1 and 731 on K. pneumoniae 52145 wild-type and isogenic mutants. Upper left: wild type (52145); bottom left: LPS mutant (52145-ΔwaaL); upper right: CPS mutant (52145-Δwca_K2); bottom right: LPS-CPS double mutant (52145-Δwca_K2ΔwaaL). Phage efficiency is represented by the distance of the phage to the membrane. Outer membrane is represented as cross-segment, peaky spots are OMPs (irreversible receptors), LPS and long O antigen chains are represented by zig-zags, CPS is represented by the black dots.