A K-17 serotype specific Klebsiella phage JKP2 with biofilm reduction potential

Abstract

Klebsiella pneumoniae is an opportunistic pathogen responsible for nearly one-third of all Gram-negative infections. Increasing antibiotic resistance has pushed scientists to look for alternative therapeutics. Bacteriophages have emerged as one of the promising alternatives. In the current study, the Klebsiella phage JKP2 was isolated from a sewage sample and characterized against the K-17 serotype of K. pneumoniae. It produced bulls-eye-shaped clear plaques and has a latent period of 45 min with a burst size of 70 pfu/cell. It remained stable at tested pH (5 to 10) and temperatures (37 to 60 °C). Its optimum temperature for long-term storage is 4 °C and -80 °C. The JKP2 showed its infectivity against the K. pneumoniae K-17 serotype only. It controlled planktonic cells of K. pneumoniae 12 h post-incubation. At MOI-1, it efficiently eliminated 98% of 24 and 96% of 48-hour-old biofilm and 86% and 82% of mature biofilm of day 3 and 4, respectively. The JKP2 has an icosahedral capsid of 54 ± 0.5 nm with a short, non-contractile tail, measuring 12 ± 0.2 nm. It possesses a double-stranded DNA genome of 43.2 kbp with 54.1% GC content and encodes 54 proteins, including 29 with known functions and 25 with unknown functions. JKP2 was classified as Drulisvirus within the Autographiviridae family. It uses a T7-like direct terminal repeat strategy for genome packaging. JKP2 can be applied safely for therapeutic purposes as it does not encode an integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and mycotoxins.

Keywords: Antimicrobial resistance; Bacteriophage; Biofilm; Klebsiella phage, serotype; Klebsiella pneumoniae; Phage therapy; Whole genome.

Fig. 1

Plaque morphology of JKP2 after (A) 24 h, (B) 48 h and (C) 72 h of incubation at 37 °C on Kp-8890 lawn.

Fig. 2

Demonstrating growth reduction potential of JKP2 at MOI-1 and 0.1 compared to control. SEM can be observed by error bars.

Fig. 3

Time kill curve of JKP2 at MOI-1 and 0.1 compared to zero-time point and untreated growth control. SEM can be observed by error bars.

Fig. 4

Figure 4A and B are showing K. pneumoniae biofilm formation kinetics study by CV assay (A) and viable count assay (B). While figure C and D demonstrating the biofilm removal in log CFUs/mL after JKP2 treatment for 6, 12 and 24-hours for 1–4 days old biofilms at MOI-1 (C) and 0.1 (D) in comparison to zero-time point. The bars marked with * show a statistically significant reduction in biofilm at each treatment time point (6, 12, and 24-hour) compared to the zero-time point. The bars marked with ** indicate a statistically significant difference in biofilm reduction between the 6 and 12-hour treatment periods. While, the bars marked with *** demonstrate a statistically significant difference in biofilm reduction only after 12 and 24-hour treatment compared to the zero-time point. For determining statistical significance a threshold of a p-value <0.05 was employed.

Fig. 5

Biofilm removal in log CFUs/mL after JKP2 treatment for 6, 12 and 24-hours for 1–4 days old biofilms at MOI-1 and 0.1 in comparison to untreated biofilm growth control. Bar with * indicate statistically significant post treatment reduction in relation to untreated biofilm growth control.

Fig. 6

JKP2 stability after 1 and 2 h of heat treatment (A), long-term storage stability at various temperatures at different time intervals, and (B), the effect of various pH treatments on JKP2 stability (C). The mean titer after three independent experiments is shown as a bar graph. Error bars indicate SEM. Bar with * indicate significant reduction in titer relative to zero-time point (p value <0.05).

Fig. 7

One-step growth curve kinetics of Klebsiella phage JKP2. Abrupt increase in phage titer is evident after 45 min.

Fig. 8

Transmission electron micrographs of JKP2, showing icosahedral capsid with a short non-contractile tail.

Fig. 9

Linear genome map of JKP2 drawn with SnapGene 6.0. Putative ORFs and regulatory sequences are shown with different color arrows, representing functional modules of the genome. The direction of the arrows represents the transcription direction. Among 54 ORFs, 30 ORFs with known functions and 24 ORFs with hypothetical functions are shown here, 19 promotors and 14 rho independent terminators are also shown. Abbreviations: Prm; Promotor, Trm: Rho independent terminator

Fig. 10

Mauve multiple sequence alignment with JKP2 closet homologs. Block colors indicate areas of homology with no internal rearrangement. Block location above the central line indicates the forward orientation of all genes. White color gaps indicate novel regions.

Fig. 11

Phylogenetic tree of large terminase subunit (A) and whole-genome sequences (B) of JKP2 by MEGAX and VICTOR. UPGMA method with 1000 bootstrap value was employed for tree construction in MEGAX, while the default setting of VICTOR was used to construct a whole-genome tree.

Fig. 12

Comparative analysis of JKP2 with four closely related Klebsiella phages. Genome alignment was created by the VipTree tool. Highly conserved regions are shaded as pale pink. Arrows indicate the direction of transcription for the predicted ORFs. The termini of the JKP2 genome were entirely different relative to its closely related phages.

Fig. 13

Virfam generated proteomic tree of JKP2 based on the head-neck-tail module. JKP2 made a cluster with LKA1 and PhiKMV, T7-like Pseudomonas phages.

Fig. 14

Phylogenetic tree of a terminase large subunit of representative phages with known DNA packaging mechanism and JKP2. The tree was constructed by MEGAX, using the UPGMA method with a 100 bootstrap value. .