Genomic Analysis of Prophages from Klebsiella pneumoniae Clinical Isolates

Abstract

Klebsiella pneumoniae is an increasing threat to public health and represents one of the most concerning pathogens involved in life-threatening infections. The resistant and virulence determinants are coded by mobile genetic elements which can easily spread between bacteria populations and co-evolve with its genomic host. In this study, we present the full genomic sequences, insertion sites and phylogenetic analysis of 150 prophages found in 40 K. pneumoniae clinical isolates obtained from an outbreak in a Portuguese hospital. All strains harbored at least one prophage and we identified 104 intact prophages (69.3%). The prophage size ranges from 29.7 to 50.6 kbp, coding between 32 and 78 putative genes. The prophage GC content is 51.2%, lower than the average GC content of 57.1% in K. pneumoniae. Complete prophages were classified into three families in the order Caudolovirales: Myoviridae (59.6%), Siphoviridae (38.5%) and Podoviridae (1.9%). In addition, an alignment and phylogenetic analysis revealed nine distinct clusters. Evidence of recombination was detected within the genome of some prophages but, in most cases, proteins involved in viral structure, transcription, replication and regulation (lysogenic/lysis) were maintained. These results support the knowledge that prophages are diverse and widely disseminated in K. pneumoniae genomes, contributing to the evolution of this species and conferring additional phenotypes. Moreover, we identified K. pneumoniae prophages in a set of endolysin genes, which were found to code for proteins with lysozyme activity, cleaving the β-1,4 linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan network and thus representing genes with the potential for lysin phage therapy.

Keywords: K. pneumoniae genomes; bacteriophage; bioinformatics; comparative genomics; genomic analysis; phage endolysins; phylogeny; prophages; sequence annotation and comparison.

Figure 1

(A) Box plot showing the total, intact, incomplete, questionable, and defective (incomplete + questionable) prophages of Klebsiella pneumoniae. (B) Bars graph showing the distribution of total prophages (intact, incomplete, and questionable) in K. pneumoniae strains.

Figure 2

Distribution of 104 intact prophages integrating K. pneumoniae genomes by in silico determined family. (A) Prevalence of prophages in K. pneumoniae genomes by family. (B) Box plot of average genome size of prophages according to family.

Figure 3

Pan-genome of K. pneumoniae prophages. The size of the core genome (continuous line) and pan-genome (dashed line) as more genomes are added.

Figure 4

Phylogenetic tree of prophage genomic sequences. Tree was constructed using the Jukes–Cantor substitution model in PHYML 3.3.20180621 (Geneious Prime version 2021.1.1). Tree was analysed and annotated using Interactive Tree Of Life (iTOL) v6 [51]. Tree branches represent Myoviridae (green); Siphoviridae (blue); and Podoviridae (red). Shaded circles represent clusters with identities higher than 50%. Myoviridae (green); Siphoviridae (blue); and mixed cluster (yellow).

Figure 5

Phylogenetic trees of prophage endolysins based on (A) nucleotides and (B) amino acids sequences. Genomic tree was constructed using the Jukes–Cantor substitution model and the proteomic tree was constructed using the Le Gascuel substitution model in PHYML 3.3.20180621 (Geneious Prime version 2021.1.1). Trees were analysed and annotated using Interactive Tree Of Life (iTOL) v6 [51]. Tree branches represent Myoviridae (green); Siphoviridae (blue); Podoviridae (red); and Ackermannviridae (black) families (in silico determined). Grey-shaded circles represent clusters with identities higher than 50%. Small, shaded circles represent endolysins groups. Lysozymes/muramidases (orange); Chitinases (yellow); and Endopeptidases (light blue).