Complete Genome Sequences of Bacteriophages Kaya, Guyu, Kopi, and TehO, Which Target Clinical Strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important nosocomial opportunistic pathogen that is able to live in a wide range of environments (1). The motile rod-shaped bacterium can cause lethal infections, such as sepsis in immunocompromised hosts and hospitalized patients (e.g., burn wounds), and infects a wide range of organs, including the lungs, urinary tract, and kidneys. Some strains of P. aeruginosa exhibit extensive drug resistance to available antibiotics, and the species has hence been listed as a priority 1 pathogen by the WHO (2, 3). Therefore, novel antibiotics or clinical therapeutic options are needed. One strategy is the use of therapeutic bacteriophages (4, 5). Here, we report the complete genome sequences of four lytic bacteriophages (Kaya, Guyu, Kopi, and TehO) that have been isolated using clinical multidrug-resistant strains of P. aeruginosa.

Water was collected in January 2020 from a river in Haining, China (120.605111°E, 30.481146°N). The water was filtered (pore size, 0.45 μm) before phage enrichment using cultures of P. aeruginosa. P. aeruginosa host strains were grown in lysogeny broth (LB) at 37°C overnight with agitation; the strains used to isolate each phage are provided in Table 1. Phages were obtained from clear single plaques and grown in the presence of the bacterial host in LB overnight. Bacterial cells were removed by centrifugation, and the supernatant was filtered through a 0.22-μm membrane (6). Nucleic acids were extracted using the Biomed virus rapid DNA/RNA kit (Beijing, China) according to the manufacturer’s instructions. Sequencing libraries were prepared using the NEBNext Ultra II DNA library prep kit for Illumina, and the genomes were sequenced using the Illumina HiSeq platform. The average read length obtained was 150 bp. The assembly pipeline Unicycler v0.4.8 (7) was used to conduct quality control of raw reads, assemble the genomes, and determine the completion of the assembled genomes. Genome annotation was completed using the CPT Galaxy and Web Apollo interfaces (8). tRNAs were predicted using ARAGORN v2.36 (9) and tRNA-scan-SE v2.0 (10). Open reading frames (ORFs) were predicted using GeneMarkS v4.28 (11), Glimmer v3.0 (12), and MetaGeneAnnotator v1.0 (13) and were then manually validated using BLAST v2.9.0 searches (14) against the NCBI nonredundant and Swiss-Prot databases (15). Pairwise nucleotide alignments between the phages were evaluated using NCBI blastn. Default parameters were used unless stated otherwise.

The characteristics of all four phage genomes are listed in Table 1. The phages are novel but are close relatives of each other, with their genes showing the mosaicism typical of bacterial viruses (Fig. 1). No genes were found to encode toxins or antibiotic resistance factors according to blastn searches against the Bacterial Virulence Factor Database (VFDB) (16). The phages were categorized as lytic using PhageAI (17). The most closely related phages of Kaya and Guyu are Xanthomonas phage Samson (GenBank accession number MN062187) and Pseudomonas phage PaMx42 (JQ067092), with genome coverage between 90% and 92% at sequence identities between 85% and 97%. Kopi and TehO are most closely related to Stenotrophomonas phage vB_SmaS-DLP2 (KR537871) and Pseudomonas phage vB_Pae-Kakheti25 (JQ307387), with sequence coverage between 91% and 95% at 95.48% to 97.89% sequence identity. With these sequence similarities, Kaya, Guyu, Kopi, and TehO are predicted to be Siphoviridae of the order Caudovirales.

This work was supported by the National Natural Science Foundation of China (32011530116).