Characterization and comprehensive genome analysis of novel bacteriophage, vB_Kpn_ZCKp20p, with lytic and anti-biofilm potential against clinical multidrug-resistant Klebsiella pneumoniae

Abstract

IntroductionThe rise of infections by antibiotic-resistant bacterial pathogens is alarming. Among these, Klebsiella pneumoniae is a leading cause of death by hospital-acquired infections, and its multidrug-resistant strains are flagged as a global threat to human health, which necessitates finding novel antibiotics or alternative therapies. One promising therapeutic alternative is the use of virulent bacteriophages, which specifically target bacteria and coevolve with them to overcome potential resistance. Here, we aimed to discover specific bacteriophages with therapeutic potential against multiresistant K. pneumoniae clinical isolates.Methods and ResultsOut of six bacteriophages that we isolated from urban and medical sewage, phage vB_Kpn_ZCKp20p had the broadest host range and was thus characterized in detail. Transmission electron microscopy suggests vB_Kpn_ZCKp20p to be a tailed phage of the siphoviral morphotype. In vitro evaluation indicated a high lytic efficiency (30 min latent period and burst size of ∼100 PFU/cell), and extended stability at temperatures up to 70°C and a wide range of (2-12) pH. Additionally, phage vB_Kpn_ZCKp20p possesses antibiofilm activity that was evaluated by the crystal violet assay and was not cytotoxic to human skin fibroblasts. The whole genome was sequenced and annotated, uncovering one tRNA gene and 33 genes encoding proteins with assigned functions out of 85 predicted genes. Furthermore, comparative genomics and phylogenetic analysis suggest that vB_Kpn_ZCKp20p most likely represents a new species, but belongs to the same genus as Klebsiella phages ZCKP8 and 6691. Comprehensive genomic and bioinformatics analyses substantiate the safety of the phage and its strictly lytic lifestyle.ConclusionPhage vB_Kpn_ZCKp20p is a novel phage with potential to be used against biofilm-forming K. pneumoniae and could be a promising source for antibacterial and antibiofilm products, which will be individually studied experimentally in future studies.

Comparative genomics and proteomics analysis of phages infecting multi-drug resistant Escherichia coli O177 isolated from cattle faeces

“…Given that the genomes of E. coli O177 phages were smaller than 200 kbp, they were classified as small phages. These findings were consistent with previous studies, which have reported small phages infecting other pathogenic bacteria such as E. coli, K. pneumoniae, P. aeruginosa, and Salmonella species [10][11][12][13][14]25 . Although TEM analysis revealed that E. coli O177 phages had similar morphotype 20 , BLASTn and PhageAI analysis classified these phages under the subfamilies Stephanstirmvirinae, Tevenvirinae, and Vequintavirinae within the Caudoviricetes class.…”

Relationship between biofilm formation and antibiotic resistance of Klebsiella pneumoniae and updates on antibiofilm therapeutic strategies

“…Compared to the control group, treatment with phage led to a distorted biofilm morphology and bacterium death. Phage vB_Kpn_ZCKp20p isolated from urban and medical sewage showed the ability to lyse biofilm-producing MDR K. pneumoniae isolates without cytotoxicity to human skin fibroblasts, and could inhibit biofilm formation and disrupt mature biofilms (Zaki et al, 2023). Polyvalent phage KL-2146 can infect NDM producing Klebsiella and antibiotic-sensitive K. pneumoniae 13, 883, which was demonstrated to effectively disrupt biofilms possessing multiple Klebsiella strains (Gilcrease et al, 2023).…”

The Lytic Activity of Bacteriophage ZCSE9 against Salmonella enterica and Its Synergistic Effects with Kanamycin

“…ARAGORN v1.2.41 was used to identify tRNA genes [45]. The presence of temperate genetic markers, bacterial virulence, and AMR genes was determined using PHageBACterioPHage LIfstyle Predictor (BACPHLIP v0.9.6) and PhageLeads to evaluate the phage's potential for therapeutic applications [46][47][48]. The circular genomic map was drawn using CGView on the PROKSEE server [49].…”

“…More phages filtered from the NCBI virus database (filtration criteria was Jerseyvirus and complete RefSeq genome) were analyzed using CoreGenes 5.0 to identify terminase genes conserved through the genus and family levels [55]. Terminase, a large subunit (TerL, signature gene), was used to construct a protein-based phylogenetic tree using the phylogeny.fr web-based tool [48,56]. The TerL tree was constructed to verify the assignment of phage ZCSE9 to the genus of the closely related matches according to intergenomic similarity analysis.…”