Complete genome sequence of Burkholderia cenocepacia bacteriophage Karil-mokiny-1

TL;DR

Scientists sequenced a new bacteriophage that can target a dangerous bacteria causing respiratory infections.

Contribution

The discovery and genome sequencing of a new bacteriophage with potential therapeutic use against Burkholderia cenocepacia.

Findings

The bacteriophage has a genome size of 70,144 bp and belongs to the taxonomic classification Irusalimvirus.

The genome lacks genes related to lysogeny, bacterial resistance, or virulence.

Abstract

Burkholderia cepacia complex causes life-threatening respiratory infections. Here, a bacteriophage with activity against B. cenocepacia was isolated from wastewater. It has a genome size of 70,144 bp and has the taxonomic classification Irusalimvirus. It has no genes associated with lysogeny, bacterial resistance, or virulence.

Tables

TABLE 1. Genomic characteristics of Karil-mokiny-1

Parameter	Value
Genome size (bp)	70,144
GC content (%)	51.5
Reads in assembly	1,868,306
Depth coverage (normalized reads)	3,476
CheckV quality and completeness score	Complete genome, 99.89%
Coding sequences	122
Hypothetical proteins	88
AMR, virulence, or lysogeny genes	None detected
Closest relative (accession no.)	Burkholderia phage BCSR52 (MW460246)
Average nucleotide identity (%)	70.7%

Full text

ANNOUNCEMENT

Burkholderia cepacia complex (BCC) is a grouping of 24 different species from the Burkholderia genus (1). BCC is an opportunistic human pathogen known for causing a wide array of infections in immunocompromised individuals (2), most notably respiratory infections in people with cystic fibrosis (CF) (3). This burden is exacerbated by BCC displaying intrinsic antimicrobial resistance (AMR), which limits antibiotic efficacy and complicates treatment (3). In the pursuit of alternative therapies to treat BCC infections, bacteriophages (phages) have seen renewed interest (3). Here, we report the genome sequence of Karil-mokiny-1, a phage isolated using a clinical isolate of Burkholderia cenocepacia. Karil-mokiny-kep-djiraly-karakaata-Wadjak (Karil-mokiny-1) was isolated from 5 mL of wastewater collected in Perth, Western Australia (“31.955839 S 115.793828 E”) following a 48-hour enrichment in ½ strength Luria-Bertani (LB) broth at 30°C, and was then triple-plaque purified using the double agar overlay method (4). Karil-mokiny-1 was propagated using a ½ strength Luria-Bertani (LB) overlay at 30°C according to a previously described “scrape” method and stored at 4°C in SM buffer (5). Phage DNA was extracted using the Qiagen (Hilden, Germany) DNeasy blood and tissue kit. High-titer phage lysate was treated with DNase I and RNase A to remove exogenous nucleic acids, and then treated with proteinase K for capsid destruction and release of phage DNA. Phage DNA was then purified in a DNeasy spin column and eluted into nuclease-free water (6). Samples were then sent to the Australian Genome Research Facility (Victoria, Australia), where DNA libraries were created using the NExtera XT kit (Illumina, San Diego, CA, USA), and whole-genome sequencing was performed using the Illumina NovaSeq 6000 (Illumina) platform. Sequencing generated 1,868,306 paired-end reads at 150 bps in length for assembly. Adapters and low-quality reads were removed using BBmap (v39.06) (HEADCROP:10) (7), after which reads were deduplicated, normalized (min = 5 target = 200), and merged. De novo assembly was completed using SPAdes (v 3.15.5) (-t 12 m 5 –only-assembler –careful -k 55,77,99,127) (8), and completeness was determined using CheckV (v1.0.3) (9). Read quality control and assembly as described above was completed using Phanta (https://github.com/JoshuaIszatt/phanta, v1.0.3). Annotations were completed using Pharokka (v1.14.6) with the Prokaryotic Virus Remote Homologous Groups (PHROGS) database (10). Antibiotic resistance and bacterial virulence genes were identified from the CARD (11) and VFDB databases, respectively (12), and t-RNAs were determined using ARAGON (11). Taxonomic assignment was completed using VIRIDIC (13), which determined the average nucleotide identity (ANI%) of Karil-mokiny-1 to its closest relatives (https://rhea.icbm.uni-oldenburg.de/viridic/). Default parameters were used for all software unless otherwise specified.

Karil-mokiny-1’s statistics are shown in Table 1. It possesses a coding-complete genome of 70,144 bp in length with a 51.55% GC content. Karil-mokiny-1 shares 70.7%% sequence homology with its closest relative, Burkholderia phage BCSR52, which was classified as a Bcepfunavirus (MW460246) at the time of analysis. Due to the low sequence homology between the two phages, we opted to perform ANI% calculations on BCSR52 and found that it shares only 47.9% of its genome with phage BcepF1, the exemplar Bcepfunavirus according to the ICTV (NC_009015). Despite being classified as a Bcepfunavirus, Karil-mokiny-1 and BCSR52 have significant genomic divergence from other reported phages, which suggests that the two phages likely represent a new viral genus within an unclassified family. Since the acceptance of this publication, BCSR52 has been classified as an Irusalimvirus, with which Karil-mokiny-1 now shares a genus.