Analysis of a novel phage as a promising biological agent targeting multidrug resistant Klebsiella pneumoniae

Abstract

The rise of deaths by resistant bacteria is a global threat to public health systems. Klebsiella pneumoniae is a virulent pathogen that causes serious nosocomial infections. The major obstacle to bacterial treatment is antibiotic resistance, which necessitates the introducing of alternative therapies. Phage therapy has been regarded as a promising avenue to fight multidrug-resistant (MDR) pathogens. In the current study, a novel phage vB_KpnP_KP17 was isolated from sewage, and its lytic potential was investigated against K. pneumoniae. The isolated phage vB_KpnP_kP17 was lytic to 17.5% of tested K. pneumoniae isolates. One step growth curve indicated a virulent phage with a short latent period (20 min) and large burst size (331 PFU/cell). Additionally, vB_KpnP_kP17 maintained its activity against planktonic cells over a wide range of pH, temperature and UV irradiation intervals. The potential of vB_KpnP_KP17 as antibiofilm agent was revealed by the biofilm inhibition assay. The isolated phage vB_KpnP_KP17 at multiplicity of infection (MOI) of 10 inhibited more than 50% of attached biofilms of tested K. pneumoniae isolates. The genome of vB_KpnP_kP17 was characterized and found to be a linear dsDNA of 39,936 bp in length and GC content of 52.85%. Additionally, the absence of toxicity, virulence and antibiotic resistance genes further confirms the safety of vB_KpnP_KP17 for clinical applications. These characteristics make vB_KpnP_KP17 of a potential therapeutic value to manage MDR K. pneumoniae infections. Additionally, the formulation of vB_KpnP_KP17 in a cocktail with other lytic phages or with antibiotics could be applied to further limit biofilm-producing K. pneumoniae infections.

Keywords: Klebsiella pneumoniae; Bacteriophage; Biofilm; Genomic characterization; Multidrug resistance; Phage therapy.

Fig. 1

Antibiotic sensitivity profile of K. pneumoniae clinical isolates. The susceptibility profile of K. pneumoniae isolates against antibiotics from different antibiotic classes is represented as a heatmap. Additionally, the MDR profile, modified MAR index, host range and susceptibility of K. pneumoniae isolates to vB_KpnP_KP17 are indicated. The efficiency of plating (EOP) was evaluated according to the degree of productivity as H (highly), L (low) and N (not) efficient

Fig. 2

Isolation and characterization of vB_KpnP_kP17. a Plaque overlay assay. Isolated phage vB_KpnP_KP17 produced clear plaques with translucent halos after overnight culture with its host KP17. b Transmission electron micrograph. The isolated phage vB_KpnP_KP17 was negatively stained with 2% (w/v) phosphotungstic acid, and the image was obtained under transmission electron microscopy at a scale bar of 100 nm

Fig. 3

Determination of environmental stability of VB_KpnP_KP17. a Temperature stability; b pH stability; and c UV stability

Fig. 4

Lytic activity of vB_KpnP_KP17. a One-step growth curve. The graph illustrates the initial number of invading virions and progenies released after lysis of host KP17. The phage latent period was 20 min and the phage burst size was 331 PFU/mL. b In vitro bacteriolytic activity. The isolated phage vB_KpnP_KP17 and indicator host KP17 were incubated at various MOIs (0.1, 1 and 10) for 12 h. The change in bacterial turbidity was recorded spectrophotometrically at 570 nm in a microtiter plate each 1 h for 12 h

Fig. 5

Antibiofilm activity of vB_KpnP_kP17 was detected by the crystal violet technique. a Percent of biofilm inhibition by vB_KpnP_KP17 at different MOIs (0.1, 1 and 10) on host bacteria KP17. b Percent of biofilm degradation by vB_KpnP_KP17 on tested K. pneumonia isolates KP12, KP17 and KP35

Fig. 6

Genomic circular map of vB_KpnP_kP17. The open reading frames (ORFs) are represented in different colors according to associated functions: hypothetical (grey); DNA packaging (green); DNA metabolism, replication and repair (blue); structure (red); lysis (light blue). The inner most circle represents the GC skew and the middle circle represents the GC content

Fig. 7

Comparative genomic analysis of vB_KpnP_KP17 and homologous phages. a Pairwise comparisons of coding sequence similarities were performed by the Easyfig program, and these gene similarities are displayed by percent identity. Red arrows indicate open reading frames and shaded blue bands represent homology between nucleotide sequences. b Proteomic comparison based on the bidirectional BLASTp hits is represented as a circular diagram

Fig. 7

Comparative genomic analysis of vB_KpnP_KP17 and homologous phages. a Pairwise comparisons of coding sequence similarities were performed by the Easyfig program, and these gene similarities are displayed by percent identity. Red arrows indicate open reading frames and shaded blue bands represent homology between nucleotide sequences. b Proteomic comparison based on the bidirectional BLASTp hits is represented as a circular diagram

Fig. 8

Phylogeny of the isolated phage vB_KpnP_KP17. a VIRDIC heat map was created to compare vB_KpnP_kP17 and closely related phages based on BLASTn top hits. b Genome-BLAST distance phylogenetic tree was generated by VICTOR using the complete genome sequence of vB_KpnP_kP17 and members of the Autographiviridae family

Fig. 8

Phylogeny of the isolated phage vB_KpnP_KP17. a VIRDIC heat map was created to compare vB_KpnP_kP17 and closely related phages based on BLASTn top hits. b Genome-BLAST distance phylogenetic tree was generated by VICTOR using the complete genome sequence of vB_KpnP_kP17 and members of the Autographiviridae family

Fig. 9

Viral proteomic tree based on genome-wide similarities. ViPTree of vB_KpnP_KP17 and other Klebsiella phages was represented as a circle. Taxonomic information of viral and host families is represented as colored inner and outer rings, respectively while the red star represents vB_KpnP_KP17

Fig. 10

Maximum likelihood trees based on amino acid sequences of vB_KpnP_KP17 and other Klebsiella phages. a Complete genome; b Terminase large subunit; c Major capsid protein; d RNA polymerase. The phylogeny trees were constructed by MEGA software using the neighbor-joining method

Fig. 10

Maximum likelihood trees based on amino acid sequences of vB_KpnP_KP17 and other Klebsiella phages. a Complete genome; b Terminase large subunit; c Major capsid protein; d RNA polymerase. The phylogeny trees were constructed by MEGA software using the neighbor-joining method

Fig. 10

Maximum likelihood trees based on amino acid sequences of vB_KpnP_KP17 and other Klebsiella phages. a Complete genome; b Terminase large subunit; c Major capsid protein; d RNA polymerase. The phylogeny trees were constructed by MEGA software using the neighbor-joining method