Complete genome analysis of a Siphoviridae phage TSK1 showing biofilm removal potential against Klebsiella pneumoniae

Abstract

Multidrug-resistant Klebsiella pneumoniae is a nosocomial pathogen, produces septicemia, pneumonia and UTI. Excessive use of antibiotics contributes towards emergence of multidrug-resistance. Bacteriophage-therapy is a potential substitute of antibiotics with many advantages. In this investigation, microbiological and genome characterization of TSK1 bacteriophage and its biofilm elimination capability are presented. TSK1 showed narrow host range and highest stability at pH 7 and 37 °C. TSK1 reduced the growth of K. pneumoniae during the initial 14 hours of infection. Post-treatment with TSK1 against different age K. pneumoniae biofilms reduced 85-100% biomass. Pre-treatment of TSK1 bacteriophage against the biofilm of Klebsiella pneumoniae reduced > 99% biomass in initial 24 hr of incubation. The genome of TSK1 phage comprised 49,836 base pairs with GC composition of 50.44%. Total seventy-five open reading frames (ORFs) were predicted, 25 showed homology with known functional proteins, while 50 were called hypothetical, as no homologs with proved function exists in the genome databases. Blast and phylogenetic analysis put it in the Kp36 virus genus of family Siphoviridae. Proposed packaging strategy of TSK1 bacteriophage genome is headful packaging using the pac sites. The potential of TSK1 bacteriophage could be used to reduce the bacterial load and biofilm in clinical and non-clinical settings.

Figure 1

Plaque morphology. Plaques (clear surrounded by a large halo, 3 mm diameter) formed by TSK1 bacteriophage with k. pneumoniae lawn on double layer agar plate.

Figure 2

Linear genomic map of TSK1 bacteriophage obtained by Snapgene viewer. Arrow represent predicted ORFs, the direction of arrow represent direction of transcription. Different functional groups of TSK1 bacteriophage are denoted by different colors, according to their function.

Figure 3

The Phylogenetic tree of TSK1 bacteriophage terminase large subunit. The tree was constructed by using the UPGMA method with 2000 bootstrap replications. The terminase large subunit of Staphylococcus phage SA97 was used as an out-group. The GenBank accession numbers are also provided after phage names, in parentheses. This tree showed the relationship of TSK1 terminase large subunit with other closely and distant bacteriophages.

Figure 4

The Phylogenetic tree of TSK1 bacteriophage tail fiber protein. The tree was constructed by using the UPGMA method with 2000 bootstrap replications. The tail fiber protein of Lactococcus garvieae phage WP-2 was used as an out-group. The GenBank accession numbers are also provided after phage names, in parentheses. This tree showed the relationship of TSK1 tail fiber protein with other closely and distant bacteriophages.

Figure 5

Evaluation of biofilm establishment in polystyrene microtitre plate through CV assay and Viable cell count. Different days old biofilm analyzed through CV assay (A) and viable cell counting (B).

Figure 6

Comparison of percentage reduction in K. pneumoniae biofilms (1- to 3-day old) subjected to TSK1 challenge for 6 and 24 hours.

Figure 7

Study of Phage treatment on the pre-biofilm formation. (A) Bacteriophage TSK1 challenge was given at 0 times of biofilm formation and studied for 72 hr. (B) The percentage reduction of bacterial load compared with untreated control at different time intervals. Bacterial count was performed at 24, 48, and 72 hr post inoculation.