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Isolation of a PRD1-like Phage Uncovers the Carriage of Three Putative Conjugative Plasmids in Clinical Burkholderia Contaminans

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10 April 2023

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Abstract
The Burkholderia cepacia complex (Bcc) are a group of increasingly multi-drug resistant opportunistic bacteria that can cause severe pulmonary infections. This resistance is driven through a combination of intrinsic factors and the carriage of a broad range of conjugative plasmids harbouring virulence determinants. Therefore, novel treatments are required to not only treat Bcc infection but also to prevent further spread of these virulence determinants. In the search for phages infective for two clinical Bcc isolates, CSP1 phage, a PRD1-like phage was isolated. CSP1 phage was found to require pilus machinery commonly encoded on conjugative plasmids to facilitate infection of multiple Gram-negative bacteria genera including Escherichia and Pseudomonas. Whole genome sequencing and characterisation of one of the clinical Burkholderia isolates revealed it to be Burkholderia contaminans. B. contaminans 5080 was found to contain a genome of over 8 Mbp encoding multiple intrinsic resistance factors, such as efflux pump systems, but more interestingly, carried three novel plasmids encoding multiple putative virulence factors for increased host fitness, including antimicrobial resistance. Even though PRD1-like phages are broad host range, their use in novel antimicrobial treatments shouldn’t be dismissed, as the dissemination potential of conjugative plasmids is extensive. Continued survey of clinical bacterial strains is
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Subject: Biology and Life Sciences  -   Immunology and Microbiology

1. Introduction

The rising occurrence and spread of antibiotic resistance are of great concern to public health. It is predicted that by 2050, antibiotic resistance will cause 10 million global premature fatalities and cost approximately 100 trillion USD in damages (1). Gram-negative bacteria are of particular concern due to their innate cell wall structure, incidence of horizontal gene transfer and acquisition of resistance determinants (2). One such group of organisms are the Burkholderia cepacia complex (Bcc), containing over 24 species that cause severe and chronic pulmonary infections in immunocompromised individuals, such as those with cystic fibrosis (3). The Bcc are renowned for their innate resistance to a wide range of antibiotics, including aminoglycosides, β-lactams, chloramphenicol, quinolones, tetracyclines and trimethoprim. They can also persist within nutrient limited environments and are often found to contaminate sterile products such as intravenous drugs, saline and other medical solutions (4). The Bcc ability to persist in unfavourable conditions and possess resistance to antimicrobials is mainly attributed to their innate cellular resistance mechanisms but also can be acquired through the uptake of mobile genetic elements (MGEs).
In addition to the intrinsically encoded antibiotic resistance determinants, including at least 19 known antibiotic extrusions efflux pumps providing resistance to chloramphenicol, tetracyclines, trimethoprim and some quinolone antibiotics in addition to resistance conferred by enzymatic modification of such as inducible β- lactamase enzymes including PenA1 that possess a broad hydrolytic spectrum, degrading ampicillin, aztreonam and ceftazidime (5), the Bcc also gain various antimicrobial resistance and metabolism factors from the horizontal gene transfer of MGEs. Plasmids, transposons, integrons and genomic islands have been found within Bcc strains that encode not only for antimicrobial resistance but also addition carbon and lipid metabolism, arsenic and toluene resistance and other virulence factors (6, 7). Dissemination of these resistance determinants from environmental into clinical settings is of concern, as environmental reservoirs are hotspots for antibiotic resistance genes and opportunistic bacteria such as Burkholderia sp. and Pseudomonas sp. (8). The spread of these determinants through MGEs such as plasmids is becoming more apparent, and the discovery of novel antimicrobials is now lagging the prevalence of antibiotic resistant bacteria. Last resort treatments are quickly becoming futile. However, one promising pipeline for novel antimicrobials are bacteriophages (also known as phages), bacterial specific viruses that rapidly infect and lyse their host.
Phages are inherently bactericidal in nature, and it is thought that they can be harnessed to treat multi-drug resistant bacterial infections. Since phages are also very selective in their host, they are harmless to commensal microbiome species or eukaryotic cells (4). Phage therapy research has regained momentum in the recent decades as a result of the increase in antibiotic resistant bacteria, with a focus on isolating and characterising phages that can be of suitable use for alternative therapies. However, a drawback of using phages is that they can be lysogenic, integrating into the host genome and in some cases can encode additional host advantages (9). Therefore, suitable phages need to be purely lytic to avoid any chance of in efficient bacterial lysis. Despite effort, the pool of phages infective against Bcc organisms is not well developed compared to other pathogenic bacteria. Therefore, this study aimed to isolate and characterise phages infective for two clinical Bcc isolates.
Here we report the isolation of a PRD1-like phage, CSP1 phage, which is a part of a genus of phages that utilise pilus machinery encoded by conjugative plasmids to infect multiple genera of Gram-negative bacteria. Whole genome sequencing of one clinical Burkholderia isolate identified it as Burkholderia contaminans and was found to carry three conjugative plasmids. One of the plasmids was related to previously isolated Incompatibility (Inc) group P found within Gram-negative bacteria including Burkholderia, Pseudomonas and Acidovorax. These plasmids contained putative antimicrobial resistance genes amongst other fitness determinants. While CSP1 phage (and other PRD1-like phages) are broad host range and often overlooked compared to strain specific phages, they could provide additional utility to phage therapies by specifically targeting pathogenic bacteria carrying and spreading antimicrobial resistance via conjugative plasmids.

2. Results and Discussion

2.1. Isolation of PRD1-like phage that infects clinical Burkholderia cepacia complex strains

Screening of pond water samples from Victoria, Australia resulted in the visualisation of plaques on two clinical isolates of Burkholderia cepacia complex (as determined by MALDI-TOF) isolated from a patient with Bcc bacteraemia in a hospital in metropolitan Melbourne, Victoria, Australia. Whole genome sequencing and assembly of the phage (CSP1 phage) using Illumina technology revealed a 14,942 bp linear dsDNA genome with a G+C content of 48.4% (Figure 1A). Whole genome BLASTn against the GenBank database indicated CSP1 phage displayed ~97.85% identity (and 100% coverage) to PRD1 phage, and similarly high identity to group of previously characterised phages known as PRD1-like phages (10). Intergenomic similarities and a phylogenetic tree were generated to compare the homology between CSP1, PRD1 and the other PRD1-like phages indicating CSP1 shared between between 93.35% and 97.87% intergenomic identity with PRD1 and other PRD1-like phages including PR5, PR772, PR3, L17 and PR4 (Figure 1B). Generation of a DNA polymerase phylogenetic tree also showed agreement with the intergenomic similarity analysis (Figure 1C). CSP1 phage forms a distinct clade with L17, PR3, PR772, PR5, PR4 and PRD1 phages. CSP1 phage DNA polymerase also shares homology to a group of Vibrio phages. PRD1 and PR4 phages represent two individual species currently classified by the International Committee on Taxonomy of Viruses (ICTV) in the Alphatectivirus genus. The other phages (L17, PR3, PR5, PR772 and LNA9) are greater than 95% similar to PRD1 phage and are known as PRD1-like phages. Since CSP1 is also >95% identical to PRD1 phage, CSP1 phage is also classified as a PRD1-like phage.
Annotation of the CSP1 phage genome revealed it is flanked by 110 bp inverted terminal repeats and encodes 30 putative open reading frames (ORFs) organised in characteristic modules (Figure 1A; Table 1). The gene products were assigned putative functions based on similarity to known domains based on the conserved domain database (CDD) and the Pfam database (11). Flanking the genome next to the inverted repeats are orf1, orf2, and orf29 and orf30. These genes encode products essential for phage replication including a terminal protein, DNA polymerase, and two single-stranded DNA binding proteins, respectively. PRD1 phage utilises a unique protein-primed DNA replication mechanism where the terminal protein is covalently bound to the 5’ end of the genome and initiates replication of the rest of the genome, with the inverted repeats serving as replication origins (12).
The virion structure of PRD1 phage has been extensively characterised (13-17). The capsid consists of 240 copies of the major capsid protein (orf14) arranged in a pseudo T=25 triangulation (15) with the minor capsid protein (orf21) stretching the capsid and cementing the edges of the icosahedron (13). On each capsid vertex but the twelfth are receptor binding proteins (orf4) and spike proteins (orf6) (13). The receptor binding and spike proteins attach to the penton base protein (orf5), protruding from capsid vertices and are responsible for host receptor recognition (14). On the twelfth vertex is the packaging complex that consists of the DNA packaging ATPase (orf11) and DNA packaging proteins (orf9, orf13 and orf15). This complex spans from the inner membrane to the outer capsid and is involved in both packaging of newly synthesised virus particles and injection of the viral genome into the host cell by formation of a tubular structure (15, 16). The tubular structure is derived from the inner membrane and consists of numerous proteins encoded by orf7, orf11, orf14 and orf18 (17).
Finally, CSP1 phage contains a predicted four gene lysis cluster comprising of endolysin (orf3), holin (orf24) and Rz/Rz1 (orf25/orf26) transglycosylases (18). The endolysin (orf3) has 1,4-β- N-acetylmuramidase activity while the Rz/Rz1 transglycosylases form a complex that spans the cytoplasmic membrane to outer membrane and aids in holin function. Holin proteins accumulate and form lesions in the cytoplasmic membrane that allow for the endolysin to reach the periplasm and degrade peptidoglycan, ultimately causing host cell lysis (18).

2.2. CSP1 phage relies on the sex pilus for successful infection

PRD1 phage has been shown to infect Gram-negative bacteria (i.e. Pseudomonas, Escherichia, Klebsiella) that harbour incompatibility (Inc) group P, W, or N plasmids (10). Given the genomic similarity between CSP1 and PRD1 phages, we reasoned CSP1 phage would share this property. To test this, we examined the ability of CSP1 phage to lyse strains of E. coli DH5α and P. aeruginosa PAO1 carrying derivatives of the conjugative IncP plasmid RP1 (ie., pUB307 and pUB1601). CSP1 phage could lyse E. coli DH5α and P. aeruginosa PAO1 only if they carried a conjugative IncP plasmid (Table 2). We further explored the ability of CSP1 phage to lyse the strains when they carried conjugation defective IncP plasmids due to transposon deletions. When the strains with the conjugation defective plasmids were challenged by the addition of CSP1 phage no lysis was observed suggesting that CSP1 phage requires an intact and fully functional mating complex to infect the Gram-negative bacteria.

2.3. Whole genome sequence confirms Bcc isolate as Burkholderia contaminans

Lysis of Burkholderia by a PRD1-like phage has not been described previously. As CSP1 phage appeared to rely on the conjugation machinery encoded by a conjugative plasmid for infection, this suggested our clinical Burkholderia sp. isolates were carrying a conjugative plasmid(s). To investigate we performed whole genome sequencing on Burkholderia sp. 5080 utilising both Illumina short-read and Oxford Nanopore long-read sequencing. The complete genome assembly was represented by six circularised contigs; three chromosomes and three plasmids (Table 3). Bcc typically contain multi-chromosome (also known as replicons) genomes as seen with this isolate, and are hypothesised to be essential for their fitness and persistence in a wide range of environments (19).
Preliminary attempts as species-level classification using 16S rRNA sequence identity, like previous MALDI-TOF identification, could not distinguish Burkholderia sp. 5080 from other members of the Bcc with matches of >99.8% identity to B. cepacia, B. cenocepacia and B. contaminans isolates, among others (Figure 2A). The Bcc are genetically very similar (and almost biochemically identical) and therefore 16S rRNA comparison has limitations in resolving species classification (19). To determine the species more definitively, we utilised whole-genome similarity analyses including average nucleotide identity (ANI) and genome DNA-DNA hybridisation (GDDH). ANI analysis determined that our isolate, Burkholderia sp. 5080, was most closely related to B. contaminans SK875 and B. contaminans ZCC, sharing 99.98% and 99.96% average nucleotide identity, respectively. The ANI results also better defined the groupings, with clearer clustering of each species group and fewer outliers (Figure 2B). Further comparison of the B. contaminans cluster showed that Burkholderia sp. 5080 shared 97.7% and 96.2% GDDH with B. contaminans SK875 and B. contaminans ZCC, respectively. Whole genome phylogenetic analysis ultimately agreed with the ANI and GDDH analyses, resolving each clade or species cluster and showed little substitutions between the genomes (Supplementary Figure 1). Furthermore, analysis with the GTDB-Tk pipeline correlated the identification as B. contaminans. Combined, these results indicate that the Burkholderia sp. 5080 isolate can be classified as Burkholderia contaminans and will be referred to as such moving forward.
B. contaminans 5080 encodes 7,655 predicted coding-sequences (CDS) across the three replicons. Functional categorisation of these using cluster of orthologous groups (COGs) resulted in ~88% CDS’s with at least one COG prediction, with some proteins containing multiple COG functions (Supplementary Figure 2). Given the role of efflux pumps in antimicrobial resistance within the Bcc (23) we inspected the genome and identified 117 CDS with predicted efflux pump-related function (Supplementary Table 1). Overall, the three B. contaminans 5080 replicons were found to encode at least 35 putative efflux pump systems: 17 in replicon 1, 10 in replicon 2 and 8 in replicon 3. Most of these efflux systems fell into the RND family efflux pump family, however there were also MATE, ABC and MFS family efflux pumps present (Figure 8). Some examples of these pumps included the OqxAB and CeoAB multidrug efflux pumps able to provide resistance to fluroquinolones and trimethoprim (20-22), the AcrAB-TolC efflux pump that gives rise to β-lactams (23), MexAB-OprM efflux pump that confers resistance to meropenem and ciprofloxacin (24) and ArpABC efflux pump involved in tetracycline, carbenicillin, chloramphenicol and streptomycin resistance (25). Other genes associated with antimicrobial resistance were also present including tetAR encoding for tetracycline resistance, penA and ampC that are β-lactamases that hydrolyse carbapenem antibiotics (26) and omp38 that encodes a porin protein able to lower cell membrane permeability to gain resistance to carbapenems and cephalosporins (27). Since Bcc organisms can occupy a large range of ecological niches, it is no surprise a large component of the genome has beneficial functions for the bacteria to adapt and persist in these environments and tolerate various antimicrobial compounds (28).

2.4. B. contaminans contains three conjugative plasmids

B. contaminans 5080 contained three unique DNA plasmids of 95.8 kb, 70.78 kb and 49.76 kb in size here named pBCO-1, pBCO-2 and pBCO-3, respectively (Table 4). Preliminary analysis revealed plasmid pBCO-3 belongs to the IncPβ family whereas pBCO-1 and pBCO-2 are uncharacterised. IncP plasmids are broad host range, conjugative plasmids that have been encountered in a wide range of Gram-negative bacteria (29). pBCO-1 and pBCO-2 are both also suspected to be conjugative as they encode the virB operons (30).
The first plasmid, pBCO-1, was found to share high identity (~99%) across a 71-kb region, the plasmid backbone, with three previously uncharacterised plasmid sequences from strains B. contaminans ZCC, B. contaminans XL73 and Burkholderia sp. FXe9 (Table 4, Figure 3). These strains were isolated from environmental locations (soil or plant-associated rhizosphere) in China between 2017-2021 (Table 4). pBCO-1 was found to contain an additional 25,471 bp sequence containing a nested transposon, here named Tn6611. Tn6611 is flanked by 38 bp inverted repeat sequences which are further flanked by 5 bp target site duplication sequences (TGGAA), a characteristic feature of a transposition event. This transposon contains a transposition module related to the Tn3-family and an accessory module of unknown function. Within the accessory module is an additional insertion of IS256 and appears to flank a remnant of another transposase, suggesting multiple insertions that have generated the structure seen in Tn6611.
Adjacent to the transposition module is an insertion of another intact transposon here named Tn6610 (Figure 3). Tn6610 belongs to the Tn5053 res-site hunter transposon family and is flanked a 25 bp inverted repeats. This transposon has a disrupted transposition module as an insertion of IS21 occurs in tniQ, that is essential for transposition (31, 32). Nonetheless Tn6610 appears to have inserted in a res site of the carrier transposon (Tn6611) and is flanked by 5 bp target site duplications (GTCAG) suggesting its placement within Tn6611 occurred prior to the acquisition of IS21 or transposed via tniQ being supplied in trans. The accessory module of Tn6610 is unique as res-site hunter family transposons traditionally contain either an integron or mercury(II) resistance module (31). In this case, Tn6610 instead contained a cluster of genes encoding an ABC type phosphonate transporter system (phnCDE), for phosphonate uptake and transport. Downstream of phnCDE is ptxD, a phosphonate dehydrogenase that degrades phosphonate to phosphate (33). Phosphonates are used in antibiotics such as fosfomycin (34) and fungicides and herbicides containing glyphosate (35). While phnCDE are typically of chromosomal origin (36), it is seen that phnCDE and ptxD were transferred through a transposon and inserted within pBCO-1.
The second plasmid, pBCO-2, shared >99% sequence similarity with five uncharacterised plasmids in GenBank (Table 4, Figure 3). Annotation of the pBCO-2 plasmid genes revealed that majority encode proteins of unknown function. Genes encoding proteins of known function appeared to be involved in plasmid maintenance and stability as well as type II and type IV secretion systems responsible for conjugative pilus and secretion machinery. Comparison of pBCO-2 with its close relatives suggests that the plasmids are conserved with no insertions of transposable elements (Figure 3).
The third plasmid, pBCO-3, belongs to the well characterised IncPβ group, sharing >99% homology with other IncPβ plasmids (Table 4, Figure 3). IncP plasmids are broad host range and have been isolated from a range of Gram-negative bacteria. The plasmids differ based on insertions of MGEs integrated between the plasmid modules (37). A truncated IS91 is present that is missing a portion of the 5’ end of the element, probably due to an intermolecular deletion. Adjacent to the IS91 are three genes, one that encodes a hydroxyacylglutathione hydrolase family protein, GloB, one that encodes a sterol desaturase family protein (SDFP) and one gene with an unknown function. There are also an additional two genes, one that encodes a vicinal oxygen chelate (VOC) family protein that is a putative GloA protein and an uroporphyrin-III methyltransferase that is involved in the cobalamin and siroheme biosynthetic pathway (38). GloA and GloB are known to detoxify the glycolysis by-product methylglyoxal and are associated with antibiotic resistance, biofilm formation and cell growth (39, 40). Their placement within this region is unknown but could have been part of a larger transposon with IS91. Analysis of the regions flanking the Tn6612 revealed that to the left was the IncP backbone whereas the right had a remnant tniQ gene and a complete tniR gene from a res-site hunter transposon. Immediately adjacent to tniR was a deleted form of parA which is commonly encountered in IncP plasmids and is known to be required for transposition of res-site hunter transposons (32). The remainder of the res hunter transposon has been deleted.
Based on the finding of the three plasmids, and the data available in Genbank, it can be presumed that pBCO-1 and pBCO-2 are narrow host range and replicate in Burkholderia species as they have only been detected in Burkholderia sp. (Table 4). The plasmids in GenBank homologous to pBCO-1 were isolated from environmental sources whereas those related to pBCO-2 were from clinical environments (Table 4). The third plasmid pBCO-3 belongs to the IncPβ group which are well established broad host range plasmids that can replicate in a diverse range of Gram-negative bacteria. All three plasmids identified in B. contaminans are presumed to be conjugative and have acquired genes from environmental and medical settings via MGEs such as transposons. These plasmids can further disseminate these genes in other Burkholderia strains and even more broadly to other Gram-negative bacteria.

3. Conclusions

This study describes the isolation of a PRD1-like phage, CSP1, the first time this phage has been shown to infect two members of the Burkholderia cepacia complex. CSP1, like PRD1, was shown to require the pilus machinery of a conjugative IncP plasmid for infection. Further investigation of one the clinical Burkholderia isolates revealed it B. contaminans harbouring three conjugative plasmids, one of which was identified as an IncP plasmid. We believe it is this conjugative plasmid, pBCO-3, that is providing the CSP1 receptor for infection. Both the chromosomes and plasmids of Burkholderia contaminans contained over 35 efflux pump systems and genes encoding for multidrug resistance, metal ion transport and host virulence, providing fitness advantages within both environmental and clinical settings. The core regions of the plasmids were highly conserved within the Burkholderia genus and other Gram-negative bacteria such as Pseudomonas and Acidovorax, highlighting the broad spread of these plasmids. The dissemination of these plasmids and their unique insertion regions containing virulence genes are of great concern with the rise of multi-drug resistant bacteria. While phages such as PRD1 and CSP1 can be very broad host range, they provide a novel treatment alternative, particularly in use with other strain specific phages to target virulent clinical pathogens.

4. Materials and methods

4.1. Bacterial strains and media

Bacterial cultures were grown in LB media (1% tryptone, 0.5% yeast extract and 1% sodium chloride ± 1.2% agar) from Oxoid (ThermoFisher Scientific, Australia). All cultures were grown aerobically at 37°C, and liquid cultures were incubated with shaking. The transposon mutagenesis strains used in this study were previously characterised in Petrovski and Stanisich (31).

4.2. Isolation and purification of phages

Pond water samples (1 ml) from Victoria, Australia were filtered using a 0.22-μm pore size cellulose acetate membrane filter. 1 ml of these samples were then incubated with the three Burkholderia strains (Table 1) in 10 ml cultures overnight. Following incubations, the cultures were pelleted by centrifugation at 6,000 x g for 10 min and filtration through a 0.22-μm cellulose acetate membrane filter. Fresh lawn plates of the strains were prepared and 100 μl aliquots of the filtrates were applied onto the lawn plates and allowed to dry. Plates were incubated and visually inspected for the presence of the plaques the following day. Single plaques were purified through three rounds of dilution and re-isolation to ensure purity.

4.3. Phage DNA isolation, genome sequencing and analysis

Purified phage particles were polyethylene glycol (PEG) precipitated and followed by a proteinase K treatment to extract DNA as described previously (41). Isolated phage DNA (100 ng) was prepared using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB) followed by sequencing on an Illumina MiSeq v3 600-cycle kit with 300 bp paired-end reads. Raw data were filtered with fastp v0.23.2 (42), with default settings,. The phage genome was assembled with SPAdes v3.15.4 (43). Raw sequence reads were manually inspected in CLC Genomics WorkBench v9.5.5 (Qiagen) using the duplicated sequence’s function to detect regions of high starting position coverage to determine the genome termini (44). The phage genomes were manually screened for putative open reading frames (ORFs) with a minimum size of 100 bp using Geneious Prime 2022.2.1 and Glimmer (45). Sequence similarity searches were conducted using the predicted amino acid sequences against the GenBank database and the BLASTp algorithm was used (46, 47). Conserved domains and motifs were identified using the conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and the Pfam database (http://pfam.sanger.ac.uk) (11). For phage comparisons, VIRIDIC (48) was used to predict intergenomic similarities with the heatmap generated by GraphPad Prism 9.

4.4. Bacterial and plasmid DNA isolation and genome sequencing

DNA was extracted from 5 ml of log-phage bacterial culture using the Monarch® HMW DNA Extraction Kit (New England Biolabs) according to manufacturer’s instructions. For short-read Illumina sequencing, DNA (100 ng) was prepared using the NEBNext® UltraTM II DNA Library Prep Kit (New England Biolabs) and sequenced on an Illumina MiSeq V3 600-cycle kits (Illumina) to generate 300 bp paired-end reads. For long-read Oxford Nanopore sequencing, DNA (1 μg) was prepared using the Ligation Sequencing Kit (LSK-109) with Native Barcoding Expansion (NBD104), loaded onto a MinION/GridION SpotON flow cell (R9.4.1) and sequenced on a GridION (Oxford Nanopore). Basecalling was performed on-device in super accurate mode. Long-read data was assembled using Flye v2.9 (49) and then polished with short-read data using Polypolish v0.5.0 (50).

4.5. Genome annotation

For whole genome characterisation, gene annotation was completed by Prokka 1.14.5 and the genome was visualised using ClicO FS (51). For comparison to other species, the Burkholderia Genome DB (https://www.burkholderia.com/) was used to collate other Bcc genomes and 16s rRNA genes. For 16s rRNA phylogenetic analysis, A BLASTn search was conducted using Burkholderia sp. 5080 first 16s rRNA gene within chromosome 1. The top BLASTn hits were collated and the first 16s rRNA gene from the chromosome 1 was extracted for analysis. The collated 16s rRNA genes were then aligned with Geneious Prime (45) plug-in MUSCLE v3.8.425 (52) and a maximum-likelihood tree using the alignment was generated in MEGA11 (53). Average nucleotide identities (ANI) and a Neighbour-joining whole genome phylogenetic tree were generated using Kostas Lab tools (54), the heatmap of the ANI matrix was generated in GraphPad Prism 9. DNA:DNA hybridization (DDH) was calculated using the Genome-to-Genome Distance Calculator (55). Classification was also performed using the Type Strain Genome Server (TYGS) (55) and the classify-wf workflow with GTDB-Tk v.2.2.6 using the TDB-Tk reference data version r207 (56). Core gene analysis of B. contaminans 5080 was completed using Roary Galaxy v3.11.2 (57) with default parameters and EggNOG-mapper Galaxy v2.1.9 (58) using eggnog v5.0 database was used to assign COGs functions. ARBicate Galaxy v1.0.1 (https://github.com/tseemann/ABRicate) and the CARD database (https://card.mcmaster.ca/) were used to screen for virulence factors. The plasmids were annotated using both Prokka and BLASTp for determination of CDS function. Plasmid map comparisons were generated using Clinker v0.0.25 (59). EggNOG-mapper Galaxy v2.1.9 (58) using eggnog v5.0 database was used to assign COGs functions. ARBicate Galaxy v1.0.1 (https://github.com/tseemann/ABRicate) and the CARD database (https://card.mcmaster.ca/) were used to screen for virulence factors. The plasmids were annotated using both Prokka and BLASTp for determination of CDS function. Plasmid map comparisons were generated using Clinker v0.0.25 (59).

Author Contributions

Conceptualization, C.R.S., S.P. and S.B.; formal analysis, C.R.S., S.P. and S.B.; investigation, C.R.S.; data curation, C.R.S., S.P. and S.B.; writing—original draft preparation, C.R.S., S.P. and S.B.; writing—review and editing, C.R.S., S.P. and S.B.; supervision, S.P. and S.B.; resources, S.P. All authors have read and agreed to the published version of the manuscript.

Funding

C.R.S. was supported by a La Trobe University postgraduate award and a Defence Science Institute (DSI) RHD student grant.

Data Availability Statement

The nucleotide sequences for the bacteria and phage sequenced and assembled in this study are available on NCBI Genbank under the following accession numbers: Burkholderia phage vB-Bco-CSP1, OQ674210; Burkholderia contaminans 5080, CP120947- CP120952.

Acknowledgements

We thank Dr Mark Chan for providing the Burkholderia cepacia complex strains. We thank the La Trobe University Genomics Platform for their 2200 TapeStation service.

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Figure 1. Characterisation of Burkholderia phage CSP1. A) Genome map of CSP1 phage. Features are colour coded based on predicted function. B) Whole genome comparison of CSP1 phage with other Alphatectivirus phages. C) Maximum likelihood phylogenetic tree tree of CSP1 phage with top GenBank matches using the DNA polymerase gene. The percentage of trees in which the associated taxa clustered together is shown next to the branches (of 100 bootstraps).
Figure 1. Characterisation of Burkholderia phage CSP1. A) Genome map of CSP1 phage. Features are colour coded based on predicted function. B) Whole genome comparison of CSP1 phage with other Alphatectivirus phages. C) Maximum likelihood phylogenetic tree tree of CSP1 phage with top GenBank matches using the DNA polymerase gene. The percentage of trees in which the associated taxa clustered together is shown next to the branches (of 100 bootstraps).
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Figure 2. Figure 2. Whole genome and phylogenetic comparisons of Bcc strains. A) 16s rRNA maximum-likelihood phylogenetic tree of the top GenBank hits against B. contaminans 5080. Bootstrap values are shown next to the branches. B) Average nucleotide identity heatmap of whole Bcc genomes. In both A and B, B. contaminans 5080 isolated from this study is highlighted by blue shading.
Figure 2. Figure 2. Whole genome and phylogenetic comparisons of Bcc strains. A) 16s rRNA maximum-likelihood phylogenetic tree of the top GenBank hits against B. contaminans 5080. Bootstrap values are shown next to the branches. B) Average nucleotide identity heatmap of whole Bcc genomes. In both A and B, B. contaminans 5080 isolated from this study is highlighted by blue shading.
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Figure 3. Plasmid map comparisons of B. contaminans 5080 plasmids A) pBCO-1 , B) pBCO-2 and 3) pBCO-3 with closest GenBank matches. Unique transposon insertions in pBCO-1 and pBCO-3 are shown in expanded form. CDS in wholeplasmid comparisons are displayed by various colours depending on their homology and nucleotide identities are showed by a grey-scale key and CDS in transposon insertion sequences and represented named arrows, remnant genes are denoted by prime notations (e.g., tniQ’). Inverted repeats are shown by vertical lines with small arrows indicating the orientation of the repeat and 5 bp target duplication sequences are shown adjacent to the inverted repeat. All putative mobile elements are indicated with a bracket with the name of the element. The plasmid from B. contaminans FXe9 (unnamed2) was omitted due to the presence of a large duplication in the plasmid sequence.
Figure 3. Plasmid map comparisons of B. contaminans 5080 plasmids A) pBCO-1 , B) pBCO-2 and 3) pBCO-3 with closest GenBank matches. Unique transposon insertions in pBCO-1 and pBCO-3 are shown in expanded form. CDS in wholeplasmid comparisons are displayed by various colours depending on their homology and nucleotide identities are showed by a grey-scale key and CDS in transposon insertion sequences and represented named arrows, remnant genes are denoted by prime notations (e.g., tniQ’). Inverted repeats are shown by vertical lines with small arrows indicating the orientation of the repeat and 5 bp target duplication sequences are shown adjacent to the inverted repeat. All putative mobile elements are indicated with a bracket with the name of the element. The plasmid from B. contaminans FXe9 (unnamed2) was omitted due to the presence of a large duplication in the plasmid sequence.
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Table 1. ORF annotations of CSP1 genome with predicted functions.
Table 1. ORF annotations of CSP1 genome with predicted functions.
ORF Predicted function Size
(bp)		 Coordinates Conserved protein
domain		 E-value
1 Terminal protein 780 233 – 1,012 - 0.0
2 DNA polymerase 1662 1,016 – 2,677 pfam03175 0.0
3 N-acetylmuramidase endolysin 450 2,680 – 3,129 pfam11860 2e-118
4 Receptor binding protein 1776 3,128 – 4,903 - 0.0
5 Penton protein 381 4,907 – 5,287 pfam08948 6e-89
6 Spike protein 1026 5,287 – 6,312 pfam08949; pfam08948 0.0
7 Assembly protein 261 6,331 – 6,591 - 2e-56
8 Assembly protein 207 6,581 – 6,787 - 2e-38
9 DNA packaging
efficiency factor		 501 6,787 – 7,287 - 4-104
10 Assembly protein 324 7,321 – 7,644 - 1e-70
11 DNA packaging ATPase 645 7,680 – 8,323 cd01127 6e-168
12 Hypothetical protein 129 8,335 – 8,463 - 9e-20
13 DNA packaging protein 129 8,463 – 8,591 - 4e-21
14 Major capsid protein 1188 8,598 – 9,785 pfam09018 0.0
15 DNA packaging protein 144 9,804 – 9,947 - 1e-24
16 Hypothetical protein 84 9,959 – 10,042
17 Hypothetical protein 123 10,047 – 10,169 - 3e-19
18 DNA delivery protein 273 10,171 – 10,443 - 2e-55
19 DNA delivery protein 165 10,443 – 10,607 pfam11087 6e-27
20 DNA delivery protein 206 10,620 – 10,825 pfam11087 1e-04
21 Minor capsid protein 255 10,835 – 11,089 pfam09300 1e-52
22 DNA delivery protein 624 11,204 – 11,827 - 4e-145
23 Hypothetical protein 354 11,838 – 12,191 pfam09301 5e-79
24 Transglycosylase (Rz) 810 12,192 – 13,001 pfam01464; cd00254 0.0
25 Putative Rz1 465 12,539 – 13,003
26 Holin 354 12,998 – 13,351 pfam16083 4e-80
27 Hypothetical protein 363 13,345 – 13,707 - 5e-86
28 Hypothetical protein 234 13,670 – 13,903 - 1e-58
29 ssDNA-binding protein 285 13,862 – 14,146 - 1e-62
30 ssDNA-binding protein 483 14,219 – 14,701 - 4e-108
Table 2. List of bacterial strains tested for CSP1 phage infection.
Table 2. List of bacterial strains tested for CSP1 phage infection.
Strain Description CSP1 lysis a Conjugation b
Burkholderia sp. 5080 Wild-type strain + NA
Burkholderia sp. 3720 Wild-type strain + NA
E. coli Lab strain - -
E. coli (pUB307) Lab strain with an IncP plasmid pUB307 + +
E. coli (pUB1601::Tn502) pUB1601 Tn502 deletant mutant - -
E. coli (pUB307::Tn502) pUB307 Tn502 deletant mutant - -
P. aeruginosa Lab strain - -
P. aeruginosa (pUB307) Lab strain with an IncP plasmid + +
a + indicates lysis; - indicates no lysis. b + indicates ability to conjugate; - indicates inability to conjugate. E. coli strains were DH5α; P. aeruginosa were PAO1
Table 3. Properties of Burkholderia sp. 5080 genome.
Table 3. Properties of Burkholderia sp. 5080 genome.
Replicon 1 Replicon 2 Replicon 3 Plasmid 1 Plasmid 2 Plasmid 3
Size (bp) 3,799,460 3,244,835 1,529,354 95,791 70,779 49,756
G+C content (%) 66.2 66.7 65.8 58.9 62.2 65.6
CDS 3, 429 2, 853 1, 304 124 85 67
rRNA 12 3 3 - - -
tRNA 64 11 6 - 1 -
Table 4. Plasmids pBCO-1 and pBCO-2 and their close relatives.
Table 4. Plasmids pBCO-1 and pBCO-2 and their close relatives.
Plasmid Strain Size (bp) Country Source Year Accession
pBCO-1 Burkholderia contaminans 5080 95,791 Australia Clinical 2018 CP120950
unnamed2 Burkholderia contaminans ZCC 71,607 China Soil 2017 CP042168
unnamed2 Burkholderia contaminans XL73 71,703 China Cucumber rhizosphere 2018 CP046611
unnamed2 Burkholderia sp FXe9 10,5779 China Soil 2021 CP101282
pBCO-2 Burkholderia contaminans 5080 70,779 Australia Clinical 2018 CP120951
plasmid IV Burkholderia stabilis E 70,766 Switzerland* Contaminated wipes 2017 LR025745
unnamed2 Burkholderia contaminans NML151067 70,795 Canada Sputum 2015 CP102471
unnamed2 Burkholderia contaminans NML151013 70,795 Canada Sputum 2015 CP102466
unnamed2 Burkholderia contaminans toggle1 77,157 Taiwan Blood -human 2018 CP073666
unnamed Burkholderia aenigmatica CMCC(B)23010 448,767 China Unknown 2019 CP091649
* Genbank entry does not indicate the country of isolation, country of the research group is displayed.
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