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.

Identification of a newly isolated lytic bacteriophage against K24 capsular type, carbapenem resistant Klebsiella pneumoniae isolates

“…The burst size of vB_KpnS_Kp13 (~220 phage particles/infected cell) was not extremely high compared with two phages in the Siphoviridae 36 and Podoviridae 35 families, but they assured an effective bactericidal potential as vB_KpnS_Kp13 could prevent proliferation of its target bacterium from an MOI of 0.01. Compared to phage TSK1 21 , this value was lower by 2 orders of magnitude and therefore was more effective. There is no relevant efficacy studies published in vivo or in vitro using K. pneumoniae phages such as JY917 (accession MG894052.1), MezzoGao (accession MF612072.1), Sushi (accession KT001920.1), NJS1 (accession MH445453.1), 1513 (accession KP658157.1), KP36 (accession JF501022.1), and NJR15 (accession MH633487.1).…”

“…A characteristic biofilm degradation potential was also visualized by the classical assay and Confocal Laser Scanning Microscopy (CLSM). Few works detailing biofilm degradation are available, but similarly to phage Z 36 and TSK1 21 , the phage vB_KpnS_Kp13 showed drastically degraded biofilm, reducing the biomass by ~73% 48 h post-treatment. Analysis of biofilm formation is essential as it is an important survival strategy of bacteria on biotic and abiotic surfaces, potentially inhibiting antibiotic efficacy and protecting bacteria from host responses 21 .…”

“…This feature makes them suitable therapeutic agents without influencing the normal microbiota of the patient 20 . Certain bacteriophages also have the capacity to target and destroy defined capsular types and weaken biofilm typically produced by Klebsiella pneumoniae 21 .…”

“…The biomass of the control K. pneumoniae 533 biofilm was 13.12 μm 3 /μm 2 , in contrast to the vB_KpnS_Kp13 treated biofilm where this value was 4.76 μm 3 /μm 2 Table 2). The ORF analysis (GeneMark) of the complete vB_KpnS_Kp13 genome revealed 5 major functional clusters 21 , hypothetically responsible for coding determinants of (i) DNA replication/modification/transcriptional regulations: DNA N-6-adenine methyltransferase (ORF72), ssDNA-binding protein (ORF1), DNA terminase (large subunit = ORF24 and small subunit = ORF 25), (ii) structure and packaging: portal protein (ORF23), major capsid protein (ORF21), membrane protein (ORF32 and ORF73) and head morphogenesis protein (ORF22), (iii) host lysis: holin (ORF65), endolysin (ORF64), (iv) tail structure: tail fiber proteins (ORF4, ORF5 and ORF6), tail assembly (ORF10) and tail length tap measure proteins (ORF9) and (v) hypothetical or unknown functions.…”

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“…A number of in vitro experiments have identified the possibility of bacterial resistance arising as a result of phage therapy (Kumari, Harjai & Chhibber, 2010a;Gu et al, 2012;Cao et al, 2015;Chadha, Katare & Chhibber, 2016;Tabassum et al, 2018). To reduce the emergence of phageresistant strains of K. pneumoniae during treatment, research has begun to explore combination therapy either using phage cocktails, or by combining phage treatment with antibacterial drugs.…”