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. 2022 Nov 14;11(12):e00960-22. doi: 10.1128/mra.00960-22

Complete Genome Sequences of Four Pseudomonas aeruginosa Bacteriophages: Kara-mokiny 8, Kara-mokiny 13, Kara-mokiny 16, and Boorn-mokiny 1

Andrew Vaitekenas a,b, Anna S Tai c,d,e, Joshua P Ramsay f, Stephen M Stick b,g,h, Patricia Agudelo-Romero b,i,j,#, Anthony Kicic a,b,h,i,j,✉,#; WAERP‡; AREST CF‡
Editor: John J Dennehyk
PMCID: PMC9753604  PMID: 36374083

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen. Here, we report the isolation of four bacteriophages from wastewater. All four bacteriophages belong to the Myoviridae family. Kara-mokiny 8, 13, and 16 are of the Pbunavirus genus and have genomes between 65,527 and 66,420-bp. Boorn-mokiny 1 is of the Phikzvirus genus and has a 278,796-bp genome.

ANNOUNCEMENT

Pseudomonas aeruginosa is an opportunistic pathogen that frequently causes burn wound (1), nosocomial (2, 3), and airway infections (4–6). Treatment is complicated by its high intrinsic antibiotic resistance, biofilm formation, and ability to acquire resistance (7). Classified as a critical priority pathogen (8), P. aeruginosa is now a primary target for bacteriophage (phage) therapy. Here, we report complete genomic sequences of four lytic phages isolated from wastewater specific for P. aeruginosa.

Phages Kara-mokiny 8, Kara-mokiny 13, Kara-mokiny 16, and Boorn-mokiny 1 were isolated from a suburban wastewater treatment plant in Perth, Australia, using P. aeruginosa isolates M1C37, M1C74, M1C108 (derived from children with cystic fibrosis [CF]; AREST CF, Melbourne, Australia) and the PAO1 reference strain, respectively. Phages were purified by three rounds of single-plaque isolation and propagation on agar plates using their respective hosts (9). Phage genomes were extracted by DNase I and RNase A treatment before proteinase K capsid destruction and purification using the Qiagen (Hilden, Germany) DNeasy blood and tissue kit (10). DNA libraries were prepared using a Nextera XT kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina NovaSeq 6000 (Illumina) platform by the Australian Genomic Research Facility (Victoria, Australia), generating 150-bp paired-end reads. We used an ultradeep sequencing approach (coverage, >1,000×) which was complemented by a robust annotation and analysis pipeline (11). Software, versions and parameters used have been provided in a publicly available Docker image (https://github.com/JoshuaIszatt/phanatic; v1.0.3). Briefly, adapters and poor-quality sequences were removed by Trimmomatic (HEADCROP: 15) (12). Kara-mokiny 8, 13 and Boorn-mokiny 1 were then deduplicated (ac=f s = 10 e = 7 minidentity = 99) and normalized (min = 5 target = 500) and reads were merged (-ea mininsert = 125 minoverlap = 20) (13), followed by de novo assembly using SPAdes (-t 12 -m 5 –only-assembler –careful -k 21,33,55,77,99,127) (14). Kara-mokiny 16 reads surviving adapter trimming were assembled by Unicycler v0.4.8 in bold mode (15) and used as an untrusted contig for merged-read assembly using SPAdes v3.15.4 (14). Assembly quality and completeness were evaluated using Quast (16) and CheckV (17), respectively. Genomes were annotated using Prokka with the Prokaryotic Virus Remote Homologous Groups (PHROGS) database, and bacterial genes were searched for using Abricate (18). Nucleotide similarity searches using NCBI databases were performed with BLASTn (19). JSpecies (20) was also used to determine the average nucleotide identity (ANI) percentage of each phage to their closest relative. Default parameters were used in all software used unless specified.

The ANIs of Kara-mokiny 8, Kara-mokiny 13, and Kara-mokiny 16 indicate that they belong to the Pbunavirus genus, while Boorn-mokiny 1 belongs to the Phikzvirus genus (Table 1). Interestingly, Kara-mokiny 13 had an ANI of <95% (Table 1), indicating that it is a new phage species of Pbunavirus. The reported phages’ genomic and physical characteristics make them therapeutic candidates requiring further investigation.

TABLE 1.

Characteristics of isolated phages

Parameter Data for indicated phage
Kara-mokiny 8 Kara-mokiny 13 Kara-mokiny 16 Boorn-mokiny 1
Genome size (bp) 66,420 65,527 66,600 278,796
No. of reads used in assembly 187,247 183,933 250,250 794,559
Postnormalization genome coverage (×) 588 587 754 585
CheckV completeness (%) 100 99.06 100 100
CheckV quality High High High High
GC content (%) 55.68 55.20 54.76 36.91
No. of CDSa 91 92 93 355
No. of hypothetical proteins 55 56 55 268
No. of tRNAs 0 0 0 6
No. of antibiotic resistance genes 0 0 0 0
No. of bacterial virulence genes 0 0 0 0
Closest related phage (GenBank accession no.) Pseudomonas phage PASA16 (MT933737.1) Pseudomonas phage PAP8 (OL754588.1) Pseudomonas phage PA4 (MZ285878.1) Pseudomonas phage vB_PA32_GUMS (MN563784.1)
ANI (%) 97.26 94.29 97.18 98.82
GenBank accession no. OP310974 OP310979 OP310982 OP310983
Sequence read archive accession no. SRX17294654 SRX17295164 SRX17298365 SRX17298366
BioProject accession no. PRJNA862681 PRJNA862681 PRJNA862681 PRJNA862681
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a

CDS, coding DNA sequence.

Data availability.

Complete phage genomes were deposited in the GenBank and NCBI Sequence Read Archive databases under the accession numbers in Table 1.

ACKNOWLEDGMENTS

Member institutions of the Western Australian Epithelial Research Program (WAERP) include the Wal-Yan Respiratory Research Centre, Telethon Kids Institute, The University of Western Australia, Perth, Western Australia, Australia, and St John of God Hospital, Subiaco, Western Australia, Australia. Member institutions of the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) include the Wal-Yan Respiratory Research Centre, Telethon Kids Institute, The University of Western Australia, Perth, Western Australia, Australia; the Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, The University of Western Australia and Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia; Murdoch Children’s Research Institute, Melbourne, Victoria, Australia; and Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia.

We thank the participants, families, and other team members of AREST CF and WAERP.

This work was supported by a Perpetual Impact Grant and a WA Department of Health WACRF grant. A.V. is supported by an Australasian Cystic Fibrosis Postgraduate Studentship Grant, Cystic Fibrosis Western Australia Golf Classis Top-up Scholarship, Australian Government research training program stipend, and a Wesfarmers Centre of Vaccines and Infectious Diseases and Wal-Yan Respiratory Centre Top Up Scholarship. S.M.S. holds an NHMRC Investigator Grant, and A.K. is a Rothwell Family Fellow.

We also acknowledge that this project was conducted on the traditional homelands of the Noongar people, with phages isolated from waters across Noongar Wadjak. Phage WA thanks Sharon Gregory, who named the phages in this study in Wadjak Noongar language. Kara-mokiny kep-wari Wadjak (Kara-mokiny) translates as “good spider (from) still water pond Wadjak” and Koomba boorn-mokiny kep-wari Wadjak (Boorn-mokiny) translates as “big tree-like (from) still water pond Wadjak.” Wastewater samples were provided by the Water Corporation of Western Australia, from their Subiaco Wastewater Treatment Plant.

Contributor Information

Anthony Kicic, Email: Anthony.Kicic@telethonkids.org.au.

John J. Dennehy, Queens College CUNY

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Complete phage genomes were deposited in the GenBank and NCBI Sequence Read Archive databases under the accession numbers in Table 1.

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