Complete genome sequences of three Pseudomonas aeruginosa jumbo bacteriophages discovered in Kenya

ABSTRACT

The genomes of three Pseudomonas aeruginosa Phikzvirus bacteriophages isolated in Kenya are described. The genomes of phages vB_PaePAO1-KEN19, vB_Pae3705-KEN49, and vB_Pae10145-KEN51, respectively, had lengths of 278,921, 280,231, and 280,173 bp, with 36.93%, 36.84%, and 36.86% GC content, containing 419, 417, and 417 coding sequences (including seven tRNAs in each genome).

ANNOUNCEMENT

Rising multidrug-resistant Pseudomonas aeruginosa infections compels the use of phages to augment flagging antibiotics, and several clinical trials have shown efficacy (1–3). We collected virulent P. aeruginosa phages to build durable therapeutic cocktails. The genomes of three unique giant (jumbo) phages isolated in Kenya are described: vB_PaePAO1-KEN19 (KEN19), vB_Pae3705-KEN49 (KEN49), and vB_Pae10145-KEN51 (KEN51).

The phages were isolated in 2021 from sewage samples and runoff in densely populated areas of Nairobi at the geographic coordinates of −1.367662, 36.725424 for KEN19 and 1.258099, 36.863883 for KEN49 and KEN51. Phages were enriched by incubating with P. aeruginosa strains PAO1 (KEN19), MRSN 3705 (KEN49), and ATCC 10145 (KEN51) in Trypticase Soy Broth by shaking at 220 rpm overnight at 37°C. Three rounds of single-plaque isolation ensured purity. Broth-propagated phages at 10⁸ pfu/mL were extracted using QIAamp DNA Mini Kits (Qiagen, Germantown, MD, USA) following DNase + RNase and Proteinase K treatments to yield at least 20 µg/mL. KAPA HyperPlus Kits (Roche Diagnostics, Indianapolis, IN, USA) were used for library preparation. Sequencing was performed on a MiSeq (Illumina, Inc., San Diego, CA, USA) using Reagent Kit v3 (600 cycles, 300-bp reads). The quality of paired-end reads (157,956, 285,978, and 392,269 for KEN19, KEN49, and KEN51, respectively) was evaluated with FastQC (4) version 0.11.9, reads were trimmed by Trimmomatic (5) version 0.39, and genomes were assembled using Unicycler (6) version 0.5.0. Since undetermined by PhageTerm (7) version 1.0.12, termini were manually identified 5,491-bp upstream of the terminase large subunit coding sequence (CDS) start. Phage lifestyle was predicted using BACPHLIP (8) version 0.9.3, and protein CDSs were annotated using the Pharokka pipeline (9–19) version 1.4.0. Amino acid sequence similarity searches used DIAMOND (20, 21) version 2.0.4. Unless otherwise noted, default parameters were used for all analyses.

Genome lengths, GC content, and the number of predicted CDSs for KEN19, KEN49, and KEN51 are provided in Table 1. Linear comparison showed high similarity of gene organization (Fig. 1). Each phage encoded seven predicted tRNA genes, postulated to compensate for phage-host codon usage disparity caused by GC content differences (P. aeruginosa host GC%: 65%–67%) (22). They also encoded virion-associated and non-virion RNA polymerases found in other phiKZ-like viruses (23). Using Mash (19) version 2.3 alignment against INPHARED (18) version 1.7, the phages were classified in a group of jumbo phages with myoviral morphology in the genus Phikzvirus that produces a nucleus-like compartment to protect replication by excluding bacterial defense factors (24–26). Phages of this group exhibit lytic activity against up to 50% of a P. aeruginosa diversity panel and are included in commercial therapeutic cocktails in Russia and Georgia (27). There are 20 Phikzvirus phage genomes in the NCBI Nucleotide Database with 98%–99% identity to KEN19, KEN49, and KEN51, indicating these phages belong to the same viral species (28).

TABLE 1.

Genome characteristics of three P. aeruginosa jumbo phages isolated in Kenya

Phage name	Enrichment strain	Genome length	GC%	No. of CDS	Genus	GenBank accession no.	No. of raw reads	Coverage	SRA accession number
KEN19	P. aeruginosa PAO1	278,921	36.93	419	Phikzvirus	PP456878	157,956	171×	SRX23539368
KEN49	P. aeruginosa MRSN 3705	280,231	36.84	417	Phikzvirus	PP456877	285,978	206×	SRX23539367
KEN51	P. aeruginosa ATCC 10145	280,173	36.86	417	Phikzvirus	PP456879	392,269	206×	SRX23539369

Fig 1.

Genome organization of the three Kenyan Phikzvirus phages. (A) KEN19; (B) KEN49; (C) KEN51. This genome comparison was visualized using gggenomes version 1.0.0 (https://thackl.github.io/gggenomes/).

BACPHLIP scored the KEN19, KEN49, and KEN51 genomes at 93%, 85%, and 83%, respectively, compared with a lytic lifestyle threshold of 95% (8). Putative proteins had no homology to those involved in lysogeny or gene transfer, nor to bacterial proteins in antibiotic resistance (14) or virulence (15). KEN19, KEN49, and KEN51 appear to be exclusively lytic and are candidates for therapeutic application.

DATA AVAILABILITY

The KEN19, KEN49, and KEN51 genome BioProject accession number is PRJNA1069762, BioSample accession numbers are SAMN39622049, SAMN39622093, and SAMN39622050, GenBank accession numbers are PP456878, PP456877, and PP456879, and the NCBI Sequence Read Archive accession numbers are SRX23539368, SRX23539367, and SRX23539369, respectively.