Genomic diversity and clade clustering of Burkholderia pseudomallei and B. thailandensis prophages with soil-derived phages

Patoo Withatanung; Veerachat Muangsombut; Sujintana Janesomboon; Vanaporn Wuthiekanun; Premjit Amornchai; Sorujsiri Chareonsudjai; Dave J Baker; Martha RJ Clokie; Edouard E Galyov; Ozan Gundogdu; Sunee Korbsrisate

doi:10.1016/j.isci.2026.114658

. 2026 Jan 9;29(2):114658. doi: 10.1016/j.isci.2026.114658

Genomic diversity and clade clustering of Burkholderia pseudomallei and B. thailandensis prophages with soil-derived phages

Patoo Withatanung ¹, Veerachat Muangsombut ¹, Sujintana Janesomboon ¹, Vanaporn Wuthiekanun ², Premjit Amornchai ², Sorujsiri Chareonsudjai ³, Dave J Baker ⁴, Martha RJ Clokie ⁵, Edouard E Galyov ⁵, Ozan Gundogdu ⁶, Sunee Korbsrisate ^1,^7,^∗

PMCID: PMC12860723 PMID: 41630905

Summary

Most studies on bacteriophages (phages) of the Gram-negative bacterium Burkholderia pseudomallei rely on in silico predictions and thus underestimate the true diversity of phages. Analysis of the whole genome sequences of culturable prophages induced from B. pseudomallei and B. thailandensis, along with their free Burkholderia phages isolated from soils in Thailand, identified six novel groups of Burkholderia phages, surpassing in silico expectations. The analysis also indicated that soil-dwelling phages may have originated from lysogenic B. pseudomallei strains. Free phages isolated from soil showed high nucleotide similarity to prophage sequences in B. pseudomallei, including phages previously cultured from melioidosis patients’ hemocultures, indicating that similar phage types occur in both environmental and clinical sources. Phylogenomic analysis also revealed close genomic relatedness between prophages from B. thailandensis and B. pseudomallei, although the biological significance remains unknown. Together, these findings refine our understanding of the genomic diversity and ecological patterns of Burkholderia phages.

Subject areas: Agricultural soil science, Microbiology, Natural sciences, Soil ecotoxicology

Graphical abstract

Highlights

•
Genome analysis of 82 culturable Burkholderia phages revealed novel phage clusters
•
Soil-dwelling phages may have originated from lysogenic B. pseudomallei strains
•
Blood-isolated phages linked to prophage sequences in the B. pseudomallei genome
•
Close genetic similarity among B. pseudomallei, B. thailandensis, and BTCV prophages

Agricultural soil science; Microbiology; Natural sciences; Soil ecotoxicology

Introduction

The genus Burkholderia comprises bacteria with diverse ecological and clinical roles, ranging from environmental symbionts to highly virulent pathogens. Burkholderia pseudomallei, a Gram-negative bacterium, is classified as a virulent pathogen that causes a life-threatening tropical infectious disease called melioidosis. The disease is endemic in Northern Australia and Southeast Asia, with an estimated 165,000 infections annually,¹ and mortality rates reaching 40%.² B. pseudomallei is a soil saprophyte commonly found in soil and water in endemic areas.³ However, the environmental conditions that govern its persistence in soil and water remain unclear.

Humans and animals become infected through contact with contaminated soil or water (especially through skin abrasions/wounds) or via air.⁴ Unlike B. pseudomallei, Burkholderia thailandensis is less pathogenic for humans, and most B. thailandensis isolates do not produce a capsule, a recognized virulence determinant of B. pseudomallei.⁵ Interestingly, a capsulated B. thailandensis strain that produces B. pseudomallei-like capsules (BTCV) was recently discovered, highlighting their close genetic similarity.⁶ In Thailand, where melioidosis is endemic, B. pseudomallei and B. thailandensis are uncommon in the same fields. B. pseudomallei is predominantly isolated in the Northeast, whereas B. thailandensis is more commonly found in the East and Central regions, although it is also present in the Northeast too. BTCV is uncommon, but is closely associated with B. thailandensis.⁶

Lysogeny is a state in which a bacteriophage (phage) persists within a bacterial host, either through integration into the host chromosome or by maintaining itself as an extrachromosomal element.⁷ Such integrated phages are known as prophages or temperate phages, and they often encode genes that provide advantages to their host, such as improved fitness, enhanced virulence, and adaptability to challenging environments.⁸^,⁹ Lysogens (bacteria that carry prophage) can remain in the lysogenic cycle for many generations but can switch to the lytic cycle at any time, leading to bacterial lysis and the release of hundreds of progenies. The decision between lytic and lysogenic cycles is influenced by multiple factors, including host cell stress, metabolic state, and phage regulatory networks.¹⁰ Most B. pseudomallei and B. thailandensis strains are lysogenic, and mulitple prophages exist in their genomes.¹¹ Interestingly, temperate phages from B. pseudomallei can also infect and integrate into B. thailandensis where they induce phenotypic changes in infected strains, such as increased serum resistance.¹² The significance of prophage abundance, diversity, and their role in Burkholderia spp. pathogenesis and survival in the environment warrants further investigation.

Indeed, our previous work showed that free phages could be isolated from the environment. The observed Burkholderia podovirus AMP1 was isolated from soil in a melioidosis-endemic region in Thailand.¹³ Subsequent analyses suggested that its temperature-dependent characteristics may influence both B. pseudomallei virulence and detectability in environmental and clinical samples.¹⁴ Moreover, Yordpratum et al.¹⁵ identified six Burkholderia myoviruses from soil samples, indicating the presence of Burkholderia phage population in soil environments. Furthermore, our past research revealed that B. pseudomallei and its free phages could be co-isolated from soil samples.¹⁶

Not only from environmental sources, phages can be identified in clinical samples. Recent research study demonstrated that B. pseudomallei phages could be cultured from hemoculture-confirmed melioidosis patients. Data suggest that these phages are induced from bacteria within circulating blood.¹⁷ These blood-isolated phages carry putative bacterial virulence genes such as virulence-associated protein E (vapE), suggesting they may play a role in modulating B. pseudomallei virulence.¹⁷ The specific factors that trigger prophage induction from the B. pseudomallei genome remain unclear. Additionally, the role of prophages in Burkholderia adaptation to environmental conditions and human pathogenesis is yet to be fully understood. At present, most studies on Burkholderia prophages rely on in silico analysis. Since not all Burkholderia isolates undergo whole-genome sequencing, many natural strains may harbor uncharacterized prophages. Environmental prophage diversity likely exceeds current in silico predictions, as shown by spontaneous prophage induction studies in environmental bacteria.¹⁸ Isolation and whole-genome sequencing of prophages induced from Burkholderia and their free phages collected from soil in the endemic area of melioidosis are essential for elucidating their diversity, origin, and the relationships between phages in environmental and clinical settings, as well as identifying genetic determinants that may contribute to bacterial adaptation, environmental persistence, and pathogenic potential.

To address this, soil samples were collected from the northeast of Thailand where melioidosis is endemic for the isolation of both B. pseudomallei and their phages. Additionally, B. thailandensis, and BTCV were collected from soils in the central and northeast of Thailand. A total of 100 Burkholderia bacteria were treated with mitomycin C (MMC) to induce prophages; this yielded 66 culturable prophages. These prophages together with 16 free culturable Burkholderia phages isolated from soil were analyzed by whole-genome sequencing. Comparative genomic analyses mapped the prophages and free phages into various clades, revealing substantial Burkholderia phage diversity and distinct evolutionary pathways. Remarkably, a novel B. pseudomallei phage group was identified, indicating that environmental prophage diversity exceeds current in silico predictions. This study expands current understanding of Burkholderia phage diversity and highlights genomic relationships between phages from environmental sources and those previously detected in clinical isolates.

Results

B. pseudomallei and their phages are prevalent in soil of endemic area

A total of 100 soil samples were collected from two provinces (Ubon Ratchathani and Khon Kaen provinces), which are ∼287 km apart, in the northeast of Thailand where melioidosis is endemic (Figure 1A). Forty of fifty soil samples collected from Ubon Ratchathani were culture-positive for B. pseudomallei (80% isolation rate), whereas none of the 50 soil samples from Khon Kaen province tested positive for B. pseudomallei by culture.

Genome-wide comparative analysis and clade classification of 82 *Burkholderia* phages

(A) Map of Thailand showing the geographic distribution of *Burkholderia* phage clades across different provinces. *Burkholderia* phages are classified into clades BP1-BP2 and BT1-BT7, each represented by distinct colored circles. Cyan blue shading denotes provinces in the Northeastern region, and yellow shading denotes provinces in the Central region. Provinces are numbered (1–14) and listed below the map. *B. thailandensis*-associated phages show greater phage diversity than *B. pseudomallei*-associated phages. Red asterisks indicate that all phages in the clade are novel, while blue asterisks indicate that some phages in the clade are novel.

(B) Whole-genome comparative analysis of 82 *Burkholderia* phages, comprising 16 free phages isolated from soil and 66 MMC-induced phages, was used to construct a phylogenetic tree (tree scale = 1 nucleotide substitution per site). Colored dots adjacent to each phage name indicate the isolation source: soil-isolated (black), *B. pseudomallei*-induced (red), *B. thailandensis*-induced (blue), and BTCV-induced (green). The analysis resolved the phages into eight major clusters (numbered 1–8). Clusters 2–7 were composed predominantly of *B. thailandensis*-associated phages (clades BT2-BT7), whereas cluster 1 contained only *B. pseudomallei*-associated phages (clade BP1). Cluster 8 was the largest and most diverse, encompassing both *B. pseudomallei*-associated (clade BP2) and *B. thailandensis*-associated phages (clade BT1).

In addition, the same 100 soil samples were tested for the presence of Burkholderia phages that could form plaques on B. pseudomallei isolate UB4. Of the fifty soil samples collected from Ubon Ratchathani province, 10 samples yielded 13 Burkholderia phages (20% isolation rate), with three samples yielding two phages each. Among these, 8 soil samples harbored both B. pseudomallei and free phages, whereas 2 soil samples contained only free phages. Whereas, 3/50 soil samples from Khon Kaen tested positive for Burkholderia phages (6% isolation rate), yielding a total of 16 soil-isolated free phages for further characterization.

Prophages could be induced from soil-isolated B. pseudomallei

The 40 B. pseudomallei collected from Ubon Ratchathani were induced using MMC to examine them for phage content. We found that phages could be induced from 21 out of 40 (52.5%) isolates. Among these 21 phage-positive samples, two isolates produced two distinct plaque morphologies, suggesting that these samples contained two different culturable phages. Therefore, a total of 23 MMC-induced B. pseudomallei phages were included for further study.

BTCV showed a higher proportion of prophage induction than B. pseudomallei and B. thailandensis under our experimental conditions

To compare the MMC-induced prophages from B. pseudomallei and related species, 40 B. thailandensis and 20 BTCV isolates were also induced using MMC. Prophages could be induced from 21 out of 40 (52.5%) B. thailandensis isolates, with one isolate producing two distinct plaque morphologies. Interestingly, 17 out of 20 (85.0%) BTCV isolates were prophage-inducible, with one isolate producing three distinct plaque morphologies and two isolates producing two distinct plaque morphologies. As a result, a total of 43 prophages were obtained, composed of 22 from B. thailandensis and 21 from BTCV.

Induced Burkholderia prophages and soil-isolated free phages could be classified into eight distinct groups

To comprehensively assess the diversity and genetic relationships of MMC-induced prophages from B. pseudomallei (23 phages), B. thailandensis (22 phages), BTCV (21 phages) and 16 soil-isolated culturable phages, whole-genome sequencing and analysis were undertaken. As shown in Tables 1 and S1, the genomes of all 82 Burkholderia phages ranged from 33.9 to 58.2 kb and encoded 42 to 86 ORFs. PhageTerm analysis did not detect fixed genome termini in any of the 82 isolates and consistently indicated circularly permuted genomes with terminal redundancy (Table S2). Because these patterns prevent the identification of a unique physical start position, the genomes were subsequently arranged in a standardized orientation based on conserved gene module organization, allowing the consistent visualization and comparison across clades.

Table 1.

Summary of 82 Burkholderia phages analyzed in this study

Clades	Phages^a	Numbers of phages	Types of phages	Genome sizes	ORFs	% GC	Nucleotide blast against phages	% CV	% ID
BP1	vB_SBP9^S1, vB_SBP39^S1, vB_SBP33^S1	3	MMC-Bps	46654–48001	69–71	57.9–58.1	phiE125	3	85
BP1	vB_FUBS1^S1, vB_FKKS2^S2, vB_FUB47a^S1	3	Free phage	46654–48001	69–71	57.9–58.1	phiE125	3	85
BP2	vB_SBP20^S1, vB_SBP7^S1, vB_SBP10a^S1	3	MMC-Bps	33895–37648	45–49	64.6–65.3	phiX216	65–94	97.26–99.35
	vB_FUB26^S1, vB_FUB21a^S1, vB_FUB5a^S1, vB_FUB41^S1	4	Free phage	33895–37648	45–49	64.6–65.3	phiX216	65–94	97.26–99.35
	vB_SBP30^S1, vB_SBP26^S1, vB_SBP38^S1, vB_SBP40a^S1, vB_SBP18^S1, vB_SBP15^S1, vB_SBP21^S1, vB_SBP36^S1, vB_SBP40b^S1, vB_SBP34^S1, vB_SBP27^S1	11	MMC-Bps	35144–37474	42–50	64.6–65.4	phiE12-2	61–100	95.49–100
	vB_FUB61^S1, vB_FKKS3^S2	2	Free phage	35144–37474	42–50	64.6–65.4	phiE12-2	61–100	95.49–100
	vB_SBP10b^S1, vB_SBP23^S1, vB_SBP24^S1, vB_SBP8^S1, vB_SBP29^S1, vB_SBP16^S1	6	MMC-Bps	34409–36696	47–49	64.5–64.6	phiE12-2	99–100	99.9–99.96
	vB_FUB21b^S1, vB_FUB47b^S1, vB_FUB29^S1, vB_FUBS2^S1, vB_FUB5b^S1, vB_FUB33^S1, vB_FKKS1^S1	7	Free phage	34409–36696	47–49	64.5–64.6	phiE12-2	99–100	99.9–99.96
BT1	vB_E188^S3	1	MMC-Bth	36779–39199	49–50	64.6–65.1	phiX216	79–91	93.49–98.3
BT1	vB_SR4^S4, vB_E555^S5, vB_PL2b^S6, vB_SR7^S4, vB_SR3^S4, vB_PL15^S6, vB_CC4^S7, vB_CB1^S8, vB_BA^S1	9	MMC-BTCV	36779–39199	49–50	64.6–65.1	phiX216	79–91	93.49–98.3
BT2	vB_E159^S1, vB_E436^S9, vB_E177^S3, vB_E169^S1	4	MMC-Bth	44120–45068	67–68	63.7–64.1	phiE131	100	100
BT2	vB_E159^S1, vB_E436^S9, vB_E177^S3, vB_E169^S1	4	MMC-Bth	44120–45068	67–68	63.7–64.1	phiPE067	73–75	96.54–98.63
BT3	vB_E332^S10	1	MMC-Bth	42358	52	65.3	phiBcepC6B	75	86.22
BT4	vB_PL1^S6	1	MMC-Bth	56536–57352	81	63.9–64.1	phiMagia	40–42	90.1–90.18
BT4	vB_E192a^S11	1	MMC-BTCV	56536–57352	81	63.9–64.1	phiMagia	40–42	90.1–90.18
BT5	vB_CB2b^S8	1	MMC-Bth	36108–45829	49–59	62.7–64.1	phiE255	80–91	95.54–100
BT5	vB_E427^S12, vB_E430^S12, vB_E433^S12, vB_E274^S1	4	MMC-BTCV	36108–45829	49–59	62.7–64.1	phiE255	80–91	95.54–100
BT6	vB_D1^S13	1	MMC-Bth	36279–38191	49–50	62.5	phiKS10	70–74	83.26–83.3
BT6	vB_PR2c^S9	1	MMC-BTCV	36279–38191	49–50	62.5	phiKS10	70–74	83.26–83.3
BT7A	vB_E192b^S11, vB_E264^S14, vB_E175^S1, vB_E184^S3, vB_E228^S1, vB_E354^S10	6	MMC-Bth	43711–44671	60–63	61–61.5	phiE264.1	86–100	95.7–100
BT7A	vB_PR5^S9, vB_PR2a^S9, vB_CB2a^S8, vB_PL2a^S6	4	MMC-BTCV	43711–44671	60–63	61–61.5	phiE264.1	86–100	95.7–100
BT7B	vB_E253^S14, vB_E438^S15, vB_E174^S1, vB_E173^S1	4	MMC-Bth	52117–58216	70–86	60.8–61.3	phiE125	64–97	95.1–99.97
BT7B	vB_SR1^S4, vB_RY5^S16, vB_RY3^S16, vB_PR1^S9, vB_PR2b^S9	5	MMC-BTCV	52117–58216	70–86	60.8–61.3	phiE125	64–97	95.1–99.97

Open in a new tab

Sources of bacteria or free-phage (provinces): ^S1Ubon Ratchathani; ^S2Khon Kaen; ^S3Sisaket; ^S4Saraburi; ^S5Cambodia; ^S6Phitsanulok; ^S7Chachoengsao; ^S8Chonburi; ^S9Prachinburi; ^S10Nakhon Phanom; ^S11Yasothon; ^S12Nakhon Nayok; ^S13Southern; ^S14Pathum Thani; ^S15Nakhon Ratchasima; ^S16Rayong.

Whole-genome nucleotide alignment using MAFFT identified eight distinct clusters, with cluster 8 being the largest, comprising 43 phages (52.44%) (Figure 1B). Consistent clustering into the same eight groups was independently confirmed using VICTOR, VIRIDIC, and VirClust (Figures S1–S3; Tables S3, S4, and S5), supporting the robustness of the clade assignments.

Within cluster 8 are prophages induced from B. pseudomallei, B. thailandensis, BTCV, and soil-isolated free phages. This highlights the fluidity of the host for these phages and shows interplay between lysogenic and environmental phages when shaping microbial communities. In addition, MMC-induced B. pseudomallei prophages were common and accounted for 46.5% of the cluster, followed by soil-isolated free phages (30.2%). All soil-isolated free phages showed more than 95% average nucleotide identity (ANI) to prophage sequences integrated in B. pseudomallei genomes, suggesting that these soil-isolated free phages could have been activated from prophages sequences in the genomes of B. pseudomallei.

As depicted in Figure 1B, MMC-induced B. thailandensis prophages accounted for only 2.3% of cluster 8 and were found across six of the eight clusters, indicating distinct genomic characteristics and broader diversification compared with B. pseudomallei prophages. In contrast, BTCV-induced prophages comprised 20.9% of cluster 8, showing closer genetic relationships with MMC-induced B. pseudomallei prophages. The remaining BTCV-induced prophages were distributed across four of the eight clusters.

Induced B. pseudomallei prophages and soil-isolated free phages map to two clades

To better understand the evolution of 23 MMC-induced B. pseudomallei prophages, a phylogenetic tree was constructed. Since all 16 soil-isolated free phages showed more than 95% similarity to prophage sequences on B. pseudomallei genomes, these free phages were combined in the analysis. According to the whole-genome alignments and gene synteny analysis, the B. pseudomallei-associated phages were classified into two main clades: BP1 and BP2 (Table 1). Clade BP2 was the largest, comprising 84.6% (33/39) of the phages, while clade BP1 accounted for 15.4% (6/39). Soil-isolated free phages were distributed across all clades, indicating significant genetic overlap between soil environmental phages and prophages present on the B. pseudomallei genomes. Representative plaque morphology of soil-isolated free phages (vB_FUB21a, vB_FUB61, and vB_FUBS2) showed uniformly sized, clear plaques measuring approximately 1.5–2.0 mm in diameter on B. pseudomallei UB4 (Figure 2). In contrast, MMC-induced prophage (vB_SBP39) formed larger (∼3.0 mm) and noticeably more turbid plaques under the same assay conditions. No semi-transparent halos surrounding plaques were observed for either free phages or MMC-induced prophage under the assay conditions tested. Thus, plaque appearance differed between free phages and inducible prophages, with free-phage plaques being smaller and clearer on the UB4 host.

Genomic and morphological characterization of *B. pseudomallei*-associated phages

(A) Synteny analysis of representative phages from clades BP1 and BP2. Four conserved genome modules were compared: head, tail, lysis, and accessory modules. Arrows represent ORFs, with colors denoting homologous gene clusters. Gray shading between genomes indicates amino acid identity levels (0%–100%). Clade BP2 differs in genes located downstream of the integrase.

(B) Transmission electron micrographs and plaque morphologies of representative phages from each clade. Scale bars, 100 nm and 5–10 mm for TEM and plaque diameters, respectively. Plaque morphologies are shown after multiple rounds of single-plaque purification.

(C) Whole-proteome-based phylogenetic tree of 86 *Burkholderia* phages, comprising 39 *B. pseudomallei* phages from this study (red dots, B. pseudomallei-induced; black dots, soil-isolated) and 47 *Burkholderia* phages from public databases. Branch colors represent phage source: red, *Burkholderia* phages from this study; black, *Burkholderia* phages from public databases. Colored bars on the left denote predicted viral families (orange, *Autographiviridae*; gray, other families), while colored bars on the right indicate host lineages (green, *Pseudomonadota*). Background shading highlights clades containing both phages from this study and related phages from public databases: blue for clade BP1 and yellow for clade BP2. Unshaded clades consist exclusively of phages from external clades. Percentages indicate the proportion of phages from this study in each clade relative to the total number of *B. pseudomallei*-associated phages from this study.

Whole-genome alignments using Clinker (Figure 2A) revealed conserved structural regions across clade BP2, with notable genomic variations near the integrase gene (Int), indicating potential hotspots for genetic recombination. Clade BP1 displayed a unique gene composition, lacking shared proteins with clade BP2, suggesting a distinct lineage adapted to specialized niches or alternative hosts.

According to TEM analysis (Figure 2B), all phages exhibited isometric heads but differed in tail morphology: clade BP1 representative had long, non-contractile tails, while clade BP2 representative featured long, contractile tails. Genome-based classification using ICTV-aligned frameworks confirmed that clades BP1 and BP2 belong to the class Caudoviricetes. Their placement was supported by three independent analyses, Virus Classification and Tree Building Online Resource (VICTOR) (Table S3), VIRIDIC (Table S4), and VirClust (Table S5), which consistently grouped BP2 with reference members of Peduoviridae; Tigrvirus, exhibiting >60% intergenomic similarity and forming a stable genus-level cluster. In contrast, BP1 displayed >70% internal similarity but lacked a closely related reference genome in GenBank, indicating an unclassified lineage within Caudoviricetes.

Comprehensive phylogenetic analysis of B. pseudomallei prophages and soil-isolated free phages with public database sequences

To contextualize the genomic diversity of B. pseudomallei phages, a phylogenetic analysis of 86 sequences comprised of 23 B. pseudomallei induced prophages and 16 free phages from this study, along with 47 Burkholderia phage sequences from public databases (Table S6) was undertaken. The resulting tree (Figure 2C) showed that previously reported B. pseudomallei prophages deposited in public databases clustered within clade BP2, however, clade BP1 was identified as a novel, distinct clade. Phages outside clades BP1-2 were primarily from other Burkholderia species, not B. pseudomallei. These results suggest that clade BP2 is the predominant cluster for B. pseudomallei prophages. Notably, the previously reported B. pseudomallei phages, such as AMP1¹³ and ST79,¹⁵ do not cluster within either clade BP1 or BP2 suggesting that these phages may have distinct evolutionary lineages. To further explore this, genomic organization and synteny analysis were conducted as follows:

Clade BP1: a novel group of B. pseudomallei phages. Clade BP1 comprises six phages, representing 15.4% (6/39) of the isolated B. pseudomallei phages. Phages in clade BP1 showed minimal similarity to any previously reported phages. The closest match was Burkholderia phage phiE125, with only 3.0% coverage (Table 1), indicating a novel group of Burkholderia phages. Figure 3A highlights structural genes of clade BP1.

Genomic and synteny analysis of *B. pseudomallei*-associated phage clades

(A–D) Representative phages from each clade are shown in (A) BP1 (vB_SBP39), (B) BP2 (vB_FUB5a), (C) BP2 (vB_SBP40a), and (D) BP2 (vB_FKKS1). Upper panels: Genome visualization using DNAplotter shows the phage genome as a dark gray circle organized into eight functional modules (colored bars). The outer scale denotes genome position in bases, with 0 as the origin of replication. The inner circle displays GC skew (yellow: positive; purple: negative), and the outermost circle shows GC content (yellow: above average; purple: below average). Lower panels: Synteny analysis compares four genome modules: head, tail, lysis, and accessory, between phages in each clade and their corresponding reference phages from the GenBank database (clade BP2: phages E094 and X216; clade BP2: phage 52237; clade BP2: phage E12-2). ORFs are shown as arrows, colored by homologous gene clusters, with gray shading indicating amino acid identity (0%–100%). Genes with notable variations among phages within each clade are annotated (highlighted in yellow). Phages in clade BP2 differs in the set of genes located downstream of the integrase.

Clade BP2: a common group of B. pseudomallei phages. Clade BP2 consists of 33 phages, representing 84.6% (33/39) of all B. pseudomallei phages in this study. Publicly available Burkholderia phages, such as phiX216 (JX681814) and phiE202 (NC_009234), also cluster within BP2, indicating conserved genomic features (Table 1), suggesting close genetic relationships.

Structural and replication genes were identified and illustrated in the circular genome maps (Figures 3B–3D). A key genomic difference from clade BP1 is the presence of putative toxin-antitoxin system (TAS)-like genes in clade BP2. These genes were identified based on their 100% amino acid sequence identity of encoded proteins to previously reported TAS components, including a RelE/ParE family toxin (e.g., MBK3339294.1, B. pseudomallei; AJX76198.1, Burkholderia phage phiE094; QWY84944.1, Burkholderia phage PK23) and a probable antitoxin homologous to addiction module antidote proteins (e.g., ACQ98486.1, B. pseudomallei MSHR346; QWY84945.1, Burkholderia phage PK23). Functional domain prediction using InterPro classified these ORFs within the IPR014056 (RelE/ParE-like toxins) and IPR014057 (antitoxin-like proteins) families. Structural homology analysis via HHpred further supported these annotations, revealing high-confidence matches to the HigB toxin (PDB: 6AF4_C, probability = 99.4%) and the putative antitoxin HigA3 (PDB: 6LTZ_A, probability = 95.3%) (Table S7). Furthermore, these phages harbor four lysis-associated genes, compared to one in clade BP1, suggesting enhanced lysis mechanisms for environmental adaptability. Synteny analysis (Figures 3B–3D) highlights conserved structural and replication genes across clade BP2, indicating shared origins.

B. thailandensis and BTCV prophages were grouped into seven clades

Total 43 prophage genome sequences induced from B. thailandensis and BTCV were also investigated. Phage classification into seven distinct clades (BT1-BT7) was primarily based on genome-wide alignment and synteny patterns (Table 1). Clade BT1 (10 phages) and clade BT7 (19 phages) were the largest, comprising 23.3% and 44.2%, respectively. BTCV-induced prophages were predominantly concentrated in clades BT1 and BT7, whereas B. thailandensis-induced phages were distributed across all seven clades, indicating greater genomic diversity. Whole-genome alignments (Figure 4A) revealed significant differences among the seven phage clades, suggesting distinct evolutionary pathways and functional specializations that reflect the adaptability and diverse ecological roles of B. thailandensis and BTCV phages.

Genomic and morphological characterization of MMC-induced *B. thailandensis*- and BTCV phages

(A) Synteny analysis of representative phages from clades BT1-BT7. Four conserved genome modules are compared: head, tail, lysis, and accessory modules. Arrows represent ORFs, with colors denoting homologous gene clusters. Gray shading between genomes indicates amino acid identity levels (0%–100%).

(B) Transmission electron micrographs and plaque morphologies of representative phages from each clade (BT1-BT7). Scale bars, 50–100 nm and 5–10 mm for TEM and plaque diameters, respectively. Plaque morphologies are shown after multiple rounds of single-plaque purification.

(C) Whole-proteome-based phylogenetic tree of 91 *Burkholderia* phages, comprising 43 *B. thailandensis*/BTCV-associated phages from this study (blue dots, *B. thailandensis*-induced; green dots, BTCV-induced) and 48 *Burkholderia* phages from public databases. Branch colors represent phage source: red, *Burkholderia* phages from this study; black, *Burkholderia* phages from public databases. Colored bars on the left denote predicted viral families (orange, *Autographiviridae*; gray, other families), while colored bars on the right indicate host lineages (green, *Pseudomonadota*). Background shading highlights clades containing both phages from this study and related phages from public databases: BT1 (blue), BT2 (orange), BT3 (green), BT4 (purple), BT5 (pink), BT6 (dark orange), and BT7 (yellow). Unshaded clades consist exclusively of phages from external clades. Percentages indicate the proportion of phages from this study in each clade relative to the total number of *B. thailandensis*-associated phages from this study.

Following plaque assays using the corresponding Burkholderia host strains listed in Tables 2 and S1, MMC-induced prophages from B. thailandensis and BTCV produced plaques with distinct but partially overlapping morphologies (Figure 4B). Plaques formed by the BTCV phage vB_CC4 (clade BT1) were turbid, with diameters of approximately 1.5–2.0 mm. In contrast, plaques formed by B. thailandensis phages vB_E159 (clade BT2) and vB_E274 (clade BT5) were small, clear pinpoint plaques, generally less than 1.0 mm in diameter. Notably, B. thailandensis phage vB_E332 (clade BT3) formed turbid plaques of approximately 2.0 mm in diameter with diffuse margins, giving a halo-like appearance. Meanwhile, B. thailandensis phage vB_E192a (clade BT4) generated markedly turbid plaques with poorly defined edges, typically ranging from 1.0 to 2.0 mm in diameter. In comparison, B. thailandensis phage vB_D1 (clade BT6) and BTCV phage vB_PR2b (clade BT7) consistently formed clear plaques of approximately 1.0 mm in diameter. Overall, none of the MMC-induced B. thailandensis and BTCV phages produced true halo plaques under the assay conditions, with the exception of phage vB_E332 (clade BT3), which exhibited a halo-like appearance.

Table 2.

Bacterial strains used in this study

Bacteria	Numbers of isolates	Isolates	Sources	Reference
B. pseudomallei	41	K96243	Clinical	Wuthiekanun et al.¹⁹
B. pseudomallei	41	UB1, UB2, UB3, UB4, UB5, UB6, UB7, UB8, UB9, UB10, UB11, UB12, UB13, UB14, UB15, UB16, UB17, UB18, UB19, UB20, UB21, UB22, UB23, UB24, UB25, UB26, UB27, UB28, UB29, UB30, UB31, UB32, UB33, UB34, UB35, UB36, UB37, UB38, UB39, UB40	Soil	This study
Typical B. thailandensis	40	DW503	Soil	Burtnick et al.²⁰
		D1	Soil	Jitprasutwit et al.²¹
		E152, E153, E154, E158, E159, E169, E173, E174, E175, E177, E184, E188, E192, E201, E202, E205, E207, E228, E234, E246, E253, E264, E27, E274, E332, E352, E354, E360, E421, E426, E427, E430, E433, E435, E436, E438, E440, E441	Soil	Hantrakun et al.⁶
B. thailandensis Capsule Variant (BTCV)	20	E555	Soil	Sim et al.⁵
		SBXCB001, SBXCB002, SBXCC001, SBXCC003, SBXCC004, SBXPL001, SBXPL002, SBXPL003, SBXPL015, SBXPR001, SBXPR002, SBXPR005, SBXRY003, SBXRY005, SBXSR001, SBXSR003, SBXSR004, SBXSR007	Soil	Hantrakun et al.⁶
		WBXUBA33005104	Water	Hantrakun et al.⁶

Open in a new tab

TEM analysis (Figure 4B) showed that although all MMC-induced B. thailandensis and BTCV prophages exhibited isometric heads, they displayed diverse tail morphologies. Clades BT1, BT2, and BT5 had long, contractile tails; BT4 and BT6 also appeared to have long tails, although the contractile sheath was not clearly visible in the micrographs; BT3 had short tails; and BT7 had long, non-contractile tails. Genome-based classification using ICTV-aligned analyses (VIRIDIC, VICTOR, and VirClust) further resolved these clades within the class Caudoviricetes. BT5 showed >90% intergenomic similarity to reference Peduoviridae; Bcepmuvirus genomes (e.g., phiE255), consistent with placement in this genus. BT3 clustered with members of Ryyoungvirus, whereas BT4 shared ∼60% similarity with Magiavirus. BT7 (including phiE125 and phiE264-1) formed a coherent group affiliated with Stanholtvirus. In contrast, BT1, BT2, and BT6 exhibited internal similarity >70% but lacked close matches to established genera, indicating unclassified lineages within Caudoviricetes.

Comprehensive phylogenetic analysis of B. thailandensis and BTCV prophages across clades and public databases

The phylogenetic analysis of 91 sequences, including 43 B. thailandensis and BTCV prophages identified in this study, along with 48 public Burkholderia phages sequences (Table S6), revealed diversity extending beyond the seven major clades (BT1-BT7). As illustrated in Figure 4C, most B. thailandensis and BTCV phages clustered within BT1-BT7, while phages from other Burkholderia species were distributed outside these clades. Clade BT1 aligns with B. pseudomallei phage phiX216, while BT2 shows no close similarity to known Burkholderia phages. Clades BT3 and BT6 align with B. cepacia complex (BCC) phages BcepC6B and KS10, respectively, while clade BT4 resembles B. cenocepacia phage Magia. Clades BT5 and BT7 include phages like B. thailandensis phiE255 and phiE125, respectively. These results suggest shared origins and conserved elements between B. thailandensis, B. pseudomallei, and BCC phages. The genomic organization and synteny of B. thailandensis and BTCV phages were comparatively analyzed within clades as follows:

Clade BT1: the only BT clade showing similarity to B. pseudomallei phages. Clade BT1 comprises 10 phages (Table 1), representing 23.3% (10/43) of B. thailandensis and BTCV phages. Genome mapping and synteny (Figure S4) resemble B. pseudomallei clade BP2 (Figures 3B–3D), and BLAST analysis shows 79%–91% coverage and 93.5%–98.3% identity with phage phiX216 (Table 1), suggesting a shared ancestor. Putative toxin-antitoxin systems (TAS) were identified in clade BT1 phages, showing 100% coverage and 100% nucleotide identity to TAS sequences found in B. pseudomallei clade BP2, suggesting they may encode homologous functional modules. Of the 10 phages in clade BT1, nine are from BTCV and only one from B. thailandensis, suggesting that BTCV may act as a reservoir for B. pseudomallei-like phages that can be transferred to other Burkholderia species, such as soil-dwelling B. thailandensis, which often coexists with BTCV and facilitates genetic exchange in the same environments.

Clade BT2 comprised four B. thailandensis-derived phages (vB_E159, vB_E169, vB_E177, and vB_E436), representing 9.3% of the 43 B. thailandensis and BTCV prophages (Table 1). Phages in this clade shared 73%–75% coverage and 96.5%–98.6% nucleotide identity with B. thailandensis phage PE067, while one phage (vB_E159) showed complete coverage and identity to phage E131 (Table 1). Synteny analysis (Figure 5A) revealed conserved structural gene organization within the clade. Across BT2 phages, conserved accessory genes with annotations included a CI-type repressor and a site-specific integrase, both features commonly associated with lysogeny regulation.

Genomic and synteny analysis of phages in *B. thailandensis* clades BT2, BT3, and BT4

(A–C) Representative phages from each clade are shown in (A) BT2 (vB_E159), (B) BT3 (vB_E332), and (C) BT4 (vB_E192a). Upper panels: Genome visualization using DNAplotter shows the phage genome as a dark gray circle organized into eight functional modules (colored bars). The outer scale denotes genome position in bases, with 0 as the origin of replication. The inner circle displays GC skew (yellow: positive; purple: negative), and the outermost circle shows GC content (yellow: above average; purple: below average). Lower panels: Synteny analysis compares four genome modules: head, tail, lysis, and accessory, between phages in each clade and their corresponding reference phages from the GenBank database (Clade BT2: phages PE067 and E131; Clade BT3: phage BcepC6B; Clade BT4: phage Magia). ORFs are shown as arrows, colored by homologous gene clusters, with gray shading indicating amino acid identity (0%–100%). Genes with notable variations among phages within each clade are annotated (highlighted in yellow).

Clade BT3 consisted of a single phage (vB_E332) derived from MMC-induced B. thailandensis E332, representing 2.3% (1/43) of all B. thailandensis and BTCV phages (Table 1). It shared 75% genome coverage and 86.2% nucleotide identity with B. cepacia complex phage BcepC6B (Table 1). Phage vB_E332 (Figure 5B) was found to possess a gene encoding a putative lytic transglycosylase with 91.9%–95.5% amino acid sequence identity to annotated proteins from Burkholderia (e.g., WP_119337678.1) and phage homologs (e.g., YP_009800740.1). Domain analysis identified a conserved soluble lytic transglycosylase (SLT) domain (Pfam: PF01464). Structural prediction by HHpred supported this classification, showing 99.6% similarity to the lytic murein transglycosylase from Pseudomonas aeruginosa (PDB: 5OHU_A) (Table S7). Synteny analysis (Figure 5B) comparing vB_E332 with BcepC6B revealed conserved gene organization, with regional variations that likely reflect evolutionary divergence between the two phages.

Clade BT4 consisted of two phages, vB_E192a derived from B. thailandensis and vB_PL1 derived from BTCV, representing 4.7% (2/43) of all B. thailandensis and BTCV phages (Table 1). They shared 40%–42% genome coverage and 90.1%–90.2% nucleotide identity with B. cenocepacia phage Magia (Table 1). Phage vB_E192a (Figure 5C) contained several annotated accessory genes showing high amino acid sequence identity (98.1%–100%) to previously reported Burkholderia proteins. These included a putative phosphoadenosine phosphosulfate (PAPS) reductase (98.1% identity to WP_180986649.1) containing the conserved PF01507 domain; a putative metallopeptidase (99.5% identity to WP_043296378.1) with a PF18894 domain; and a putative acyltransferase (100% identity to WP_158338970.1) classified within the acyltransferase 3 family (IPR050879). HHpred predictions further supported similarities to known protein structures (Table S7). Synteny analysis (Figure 5C) revealed partial conservation of structural genes with phage Magia, while variation in accessory and replication-associated genes reflect evolutionary divergence within the clade.

Across clades BT2-BT4, a relatively low proportion of genes encoded known structural proteins, whereas a high proportion corresponded to hypothetical proteins: 67.2% (45/67 ORFs) in BT2, 61.5% (32/52 ORFs) in BT3, and 64.2% (52/81 ORFs) in BT4 (Figures 5A–5C). These observations highlighted the presence of numerous uncharacterized ORFs, although their functional roles remained undetermined and will require future experimental investigation.

Clade BT5 consisted of five phages (Table 1), one derived from MMC-induced BTCV and the others from B. thailandensis, representing 11.6% (5/43) of all B. thailandensis and BTCV phages. Phages in this clade shared 80%–91% genome coverage and 95.5%–100% nucleotide identity with B. thailandensis phage phiE255 (Table 1). Phage vB_E274 (Figure 6A) contained annotated regulatory genes showing 100% amino acid identity to previously reported proteins in Burkholderia phages and members of the pseudomallei group. These included a putative GemA-like protein (WP_015985046.1) belonging to the GemA family (IPR009363; Pfam: PF06252), and a middle operon regulator (MOR; WP_015985055.1) possessing the PF08765 domain. HHpred analyses further supported structural similarity to known proteins (Table S7). Synteny analysis (Figure 6A) showed high conservation with B. thailandensis phage phiE255, reflecting shared origins. Additional genes detected in clade BT5 phages, including an acetyltransferase and an efflux RND transporter periplasmic adaptor, reflected genetic variation within the clade, although their functional relevance remains undetermined.

Genomic and synteny analysis of phages in *B. thailandensis* clades BT5 and BT6

(A and B) Representative phages from each clade are shown in (A) BT5 (vB_E274) and (B) BT6 (vB_D1). Upper panels: Genome visualization shows the phage genome as a dark gray circle organized into eight functional modules (colored bars). The outer scale denotes genome position in bases, with 0 as the origin of replication. The inner circle displays GC skew (yellow: positive; purple: negative), and the outermost circle shows GC content (yellow: above average; purple: below average). Lower panels: Synteny analysis compares four genome modules: head, tail, lysis, and accessory, between phages in each clade and their corresponding reference phages from the GenBank database (clade BT5: phage E255; clade BT6: phage KS10). ORFs are shown as arrows, colored by homologous gene clusters, with gray shading indicating amino acid identity (0%–100%). Genes with notable variations among phages within each clade are annotated (highlighted in yellow).

Clade BT6 consisted of two phages, vB_D1 derived from B. thailandensis and vB_PR2c derived from BTCV, representing 4.6% (2/43) of all B. thailandensis and BTCV phages (Table 1). These phages shared 70%–74% genome coverage and 83.3% nucleotide identity with B. cepacia complex phage KS10 (Table 1). Interestingly, no annotated accessory or lysogeny-associated genes were identified in phage vB_D1. Synteny analysis (Figure 6B) revealed partial gene conservation between B. cepacia complex phage KS10 and B. thailandensis phage vB_D1, indicating shared evolutionary origins.

Clade BT7: the most common B. thailandensis phage group. Clade BT7 comprised 19 phages (Table 1), representing 44.2% (19/43) of all B. thailandensis and BTCV phages, making it the largest group. It included 9 phages from BTCV and the rest from B. thailandensis. Phages in clade BT7A shared 86%–100% coverage and 95.7%–100% nucleotide identity with B. thailandensis phage phiE264.1,¹¹ while clade BT7B shared 64%–97% coverage and 95.1%–99.9% identity with phage phiE125 (Table 1). Synteny analysis (Figure S5) revealed strong conservation of structural gene regions across clades BT7A and BT7B, indicating shared origins, while showing variability in accessory genes. Regulatory genes, including CII and CII repressors, were identified in vB_PR2a, a BTCV prophage in clade BT7A (Figure S5). Phage vB_RY3 (Figure S5) was found to encode two putative accessory genes with proteins showing 100% amino acid identity to known Burkholderia proteins, including a putative RelE-like toxin (WP_004549735.1), classified under the RelE/ParE toxin family (IPR052747; Pfam: PF05016) and supported by HHpred structural similarity to the RelE toxin from Escherichia coli (PDB: 2KHE_A, 99.5% probability). A putative phosphoadenosine phosphosulfate (PAPS) reductase (WP_009896489.1), containing conserved PF01507 and IPR050128 domains, was also identified, with HHpred predicting structural similarity to the PAPS reductase from Vibrio vulnificus (PDB: 6VPU_C, 99.9% probability) (Table S7). The specific biological roles of these genes within the prophage context remain to be experimentally verified.

Discussion

In this study, we analyzed the diversity of prophages induced from selected strains of B. pseudomallei, B. thailandensis, and BTCV, together with Burkholderia phages isolated from soils in the northeast of Thailand. Soil sampling was conducted at two different sites, one from the province of Khon Kaen and one from Ubon Ratchathani. Although B. pseudomallei and culturable free phages were recovered from soil collected in Ubon Ratchathani province, B. pseudomallei was not cultured from any of the 50 soil samples collected in Khon Kaen province. However, culturable Burkholderia phages were detected in samples from both provinces. This discrepancy may be attributed to several factors, such as differences in environmental conditions and nutrient-depleting agricultural practices that may impact the viability and distribution of both bacteria and phages in the soil.²²^,²³ In addition, the location of soil sampling in Ubon Ratchathani province had been identified (personal communication) previously as a hot spot of B. pseudomallei whereas the sampling site at Khon Kaen province was randomly selected and not known as an abundant source of such bacteria. Notably, we cannot rule out the possibility that the positive phage/negative B. pseudomallei isolation may result from several reasons, such as (1) prophages being activated from B. pseudomallei genome, leading to bacterial lysis and thus the bacteria could not be cultured, or (2) the culturable free phages may have B. thailandensis or other, yet unknown, bacterial species as their bacterial host.

The geographical distribution analysis of phages isolated from soil showed that phages identified in both provinces belong to the clades BP1 (a novel group that did not show homology to any other phages deposited into the GenBank database) and BP2, which represent phages commonly found across multiple B. pseudomallei isolates. This finding indicates that no significant differences were found in terms of phage clade distribution between the two endemic regions of B. pseudomallei. The consistency of phage clade presence across these areas suggests a stable and persistent phage population, supporting their potential use as indicators for monitoring B. pseudomallei spread in the future. This suggestion is supported by previous reports that environmental phage surveillance is useful for tracking Salmonella typhi in endemic settings.²⁴ However, further investigation with additional sampling sites should be undertaken to more extensively investigate this in conjunction with the bacterial detection data and environmental context.

Pertinent to the 82 MMC-induced Burkholderia phages and soil-isolated free phages, intergenomic clustering identified eight distinct clusters, with cluster 8 being the largest (52.44%). Cluster 8 was composed of prophages induced from B. pseudomallei, B. thailandensis, and BTCV, as well as soil-isolated free phages, indicating the interplay between lysogenic and environmental phages in shaping microbial communities. Of note, this study identified for the first time that all soil-isolated free phages showed more than 95% average nucleotide identity to prophage sequences in B. pseudomallei genomes, indicating that these soil-dwelling phages may have originated from B. pseudomallei genomes. However, the induction factors in the soil environment that contribute to prophage induction require further investigation, particularly as environmental cues have been shown to trigger prophage induction in soil bacteria.²⁵

The phylogenetic tree constructed from 39 B. pseudomallei phages, including MMC-induced prophages (23 phages) and free phages directly isolated from soil (16 phages) revealed that these phages could be classified into two clades: BP1 and BP2. Nucleotide BLAST analysis revealed that phages in clade BP1 are a novel group of Burkholderia phages that have not been reported before, supporting the notion that Burkholderia prophages diversity exceeds current in silico predictions.

The identification of putative toxin-antitoxin system (TAS)-like gene pairs in phage clade BP2 raises interesting questions regarding their potential biological roles. Although predicted products of these genes share 100% amino acid sequence identity with previously reported TAS components from Burkholderia genomes and phages, and exhibit conserved domain signatures, their actual functions within the phage context remain unresolved. TAS are genetic modules typically composed of a stable toxin and a labile antitoxin. They were originally described in the context of plasmid maintenance via post-segregational killing (or “plasmid addiction”).²⁶ However, TAS are now recognized to play broader roles beyond plasmid stability, including contributing to phage stability,²⁷ regulating lysogeny,²⁸ and modulating bacterial stress responses,²⁹ as has been observed in other phage-host systems. However, without experimental validation, their roles in enhancing host adaptability remain speculative. Further functional studies, such as gene expression profiling or mutational analysis, are needed to determine whether these modules operate as bona fide TAS or serve distinct phage-specific functions.

Interestingly, we found from this study that MMC-induced B. pseudomallei prophages in clade BP2 share similarities (>97% ANI) with phage vB_HM387 which was isolated from the hemoculture of melioidosis patients¹⁷ indicating that prophages were not only induced and detected in vitro but also in a clinical sample from a melioidosis patient. The factor(s) in the blood of melioidosis patients that trigger release of prophage particles from bacteria and mediate their appearance in the blood are largely unknown. Consequently, the impact of phage presence in vivo on the pathogenesis or severity of melioidosis requires further investigation.

Strikingly, none of the culturable free phages and induced prophages of B. pseudomallei collected from soil in this study carried a gene encoding the virulence-associated protein E (vapE) that was previously reported in the genome of B. pseudomallei phage vB_HM795 collected from the hemoculture of melioidosis patient.¹⁷ Another key observation in this study is that the previously reported Burkholderia phages isolated from northeast of Thailand including ST79,¹⁵ and AMP1-like phages¹³ were not found in this study. The possible explanation may be due to several reasons such as differences of B. pseudomallei host strains and the locations of soil sampling. Anyhow, this indicates that diversity of B. pseudomallei is present in the environment.

In comparison with B. pseudomallei phages, 43 MMC-induced B. thailandensis and BTCV prophages were analyzed and could be classified into seven clades (BT1-BT7). This is the first study to report that, under our experimental conditions, BTCV displayed a higher proportion of MMC-inducible prophages than B. pseudomallei or B. thailandensis. BTCV-induced prophages were concentrated in clades BT1 and BT7, whereas B. thailandensis-induced phages were distributed across all seven clades. Clade BT7 is the most common phage group in B. thailandensis. Only clade BT1 showed high sequence similarity to B. pseudomallei clade BP2.

Plaque morphology analysis showed that most B. pseudomallei phages in this study produced clear or turbid plaques without detectable halo formation under the conditions tested, consistent with previously described B. pseudomallei phages such as AMP1,¹³ ST79,¹⁵ and C34,³⁰ which also form clear plaques without halos. In contrast, the B. pseudomallei phage vB_BpP_HN01³¹ has previously been reported to develop a pronounced halo after extended incubation, a feature associated with capsular polysaccharide-degrading activity.³² The lack of halo formation in our B. pseudomallei phages may reflect the absence or limited activity of capsule-degrading enzymes, as no clear homologs of known depolymerases or tail spike-associated hydrolases were identified in genomes of our isolated Burkholderia phages. However, enzyme activity may be condition-dependent and influenced by the host strain used for plaque assays, and therefore cannot be entirely excluded.

For B. thailandensis and BTCV, plaque morphologies were more variable, ranging from clear pinpoint plaques to larger turbid plaques. Notably, the Burkholderia phage vB_E332 in clade BT3 exhibited a halo-like appearance. This distinctive phenotype may be associated with the presence of a predicted glycoside hydrolase family protein encoded in its genome (Figure 5B). Enzymes of the glycoside hydrolase family have been reported to cleave glycosidic bonds in bacterial capsular components, a function that has been associated with halo formation.³² However, functional validation is required to confirm the role of glycoside hydrolase activity in generating the halo-like appearance observed for phage vB_E332.

In B. thailandensis phages, several annotated accessory genes were identified across multiple clades. In clade BT4, a gene encoding a putative lytic transglycosylase was detected, showing similarity to enzymes involved in peptidoglycan-associated processes. Additional annotated genes in this clade included those encoding phosphoadenosine phosphosulfate (PAPS) reductase, metallopeptidase, and acyltransferase, each containing conserved domains characteristic of their respective protein families. Regulatory genes such as gemA and mor were also identified. Although these genes exhibit conserved domain signatures and high sequence similarity to known proteins, their specific roles in the B. thailandensis phage context remain unknown and will require further experimental investigation.

This study showed that both B. pseudomallei phage clades and four out of seven B. thailandensis phage clades (BT1, BT2, BT5, and BT7) were identified in Ubon Ratchathani province, an area of high endemicity for B. pseudomallei. The high phage diversity observed in Ubon Ratchathani, particularly the presence of multiple B. thailandensis and B. pseudomallei phage clades, indicates a complex and dynamic phage-bacteria interaction in this area. Potential ecological interactions between prophages and bacteria may facilitate gene flow between these closely related Burkholderia species, although the underlying mechanisms and frequency of such events remain unclear. Observations in other bacterial systems show that mobile genetic elements can mediate cross-species gene transfer, but whether similar processes occur in Burkholderia requires further investigation. For examples, B. thailandensis phages clades BT3 BT4 and BT6 share genomic links with B. cepacia complex (BCC) and B. cenocepacia phages, highlighting their connections and potential for cross-species genetic exchange. A similar phenomenon has been seen in Vibrio cholerae, where V. cholerae El Tor acquired the classical CTX prophage from V. cholerae O141 in chitin-rich aquatic environments,³³ highlighting how prophages can mediate gene transfer across strains in nature. Likewise, in Streptococcus pyogenes, prophages carrying toxin genes can integrate into diverse strains, contributing to the spread of virulence factors, antibiotic resistance, and shaping bacterial population dynamics.³⁴

In conclusion, B. pseudomallei, B. thailandensis, and BTCV carried prophages on their genomes that could be induced with MMC to generate different culturable phage particles for characterization. Whole-genome sequencing and genome analysis indicated that the induced Burkholderia prophages are diverse with some phages shared among B. pseudomallei, B. thailandensis, and BTCV. B. pseudomallei prophage clade BP1 is a novel group which that has not been reported before, whereas clade BP2 is similar to previously identified phages. Phylogenomic analysis revealed strong genetic similarity between B. thailandensis and B. pseudomallei prophages, reflecting their close evolutionary relatedness. The wide distribution of B. thailandensis phages suggests substantial ecological diversity. BTCV phages exhibited genomic similarity to prophages from both species, consistent with their intermediate phylogenomic position. While these patterns may indicate broader genomic connectivity among Burkholderia lineages, the underlying mechanisms and biological relevance remain unclear and will require future functional investigation.

Limitations of the study

The number of phages recovered in this study was influenced by both environmental and methodological factors. Soil samples were collected from only two melioidosis-endemic sites, where isolation outcomes may vary with seasonal conditions, soil composition, and agricultural practices. Importantly, this work focused specifically on culturable, MMC-inducible prophages and therefore did not capture non-inducible or defective elements. Because whole-genome sequences of most host strains, including all BTCV isolates, were unavailable, in silico prophage prediction could not be carried out. As a result, the isolation frequencies presented here reflect only prophages detectable under the MMC-induction conditions used.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Dr. Sunee Korbsrisate (sunee.kor@mahidol.ac.th).

Materials availability

This study did not generate new unique reagents.

Data and code availability

Data: Phage genome sequences have been deposited in the NCBI database, with accession numbers; vB_SBP9: PQ789808; vB_SBP39: PV072743; vB_FUBS1: PV072773; vB_FKKS2: PV072744; vB_SBP33: PV072774; vB_FUB47a: PV072775; vB_SBP20: PQ789809; vB_FUB26: PV987627; vB_FUB21a: PV072745; vB_SBP7: PV111787; vB_SBP10a: PV976088; vB_FUB5a: PQ789810; vB_FUB41: PV987629; vB_SBP30: PQ789829; vB_SBP26: PV976089; vB_FUB61: PV072746; vB_SBP38: PV987637; vB_SBP40a: PQ789811; vB_SBP18: PV987632; vB_SBP15: PV111788; vB_SBP21: PV111789; vB_FKKS3: PV976091; vB_SBP36: PV111790; vB_SBP40b: PV987638; vB_SBP34: PQ789812; vB_SBP27: PV987635; vB_FUB21b: PV976092; vB_FUB47b: PV987630; vB_FKKS1: PQ789813; vB_SBP10b: PV987631; vB_SBP23: PV987633; vB_FUB29: PV976093; vB_FUBS2: PV987626; vB_SBP24: PV987634; vB_SBP8: PV976090; vB_SBP29: PV987636; vB_FUB5b: PV976094; vB_FUB33: PV987628; vB_SBP16: PV072747; vB_E188: PQ789830; vB_SR4: PV072748; vB_E555: PQ789814; vB_PL2b: PV072749; vB_SR7: PV072750; vB_SR3: PV072751; vB_PL15: PV072752; vB_CC4: PV072753; vB_CB1: PQ789815; vB_BA: PV072754; vB_E159: PQ789816; vB_E436: PQ789831; vB_E177: PQ789817; vB_E169: PV072755; vB_E332: PQ789818; vB_PL1: PV072756; vB_E192a: PQ789819; vB_E427: PQ789820; vB_CB2b: PV072757; vB_E430: PV072758; vB_E433: PV072759; vB_E274: PQ789821; vB_D1: PQ789822; vB_PR2c: PV072760; vB_PR5: PV072761; vB_E192b: PV072762; vB_PR2a: PQ789823; vB_CB2a: PV072763; vB_E264: PV072772; vB_E175: PV072769; vB_E184: PV072770; vB_E228: PV072771; vB_PL2a: PV072768; vB_E354: PQ789824; vB_SR1: PQ789825; vB_RY5: PV072764; vB_RY3: PQ789826; vB_PR1: PV072765; vB_PR2b: PQ789827; vB_E253: PV072766; vB_E438: PQ789832; vB_E174: PQ789828; vB_E173: PV072767. All data are publicly available as of the date of publication.

Code: This paper does not report original code.

Additional information: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This project is funded by the National Research Council of Thailand (NRCT) and Mahidol University (N42A660430) and the Royal Society NMG\R1\191196 - Newton Mobility Grants 2019. V.W. is supported by The Wellcome Trust of Great Britain (grant no. 089275/Z/09/Z). P.W., V.M., S.J., and S.K. are supported by the Faculty of Medicine Siriraj Hospital, Mahidol University. Finally, we acknowledge the constructive suggestions from Dr. Prasert Auewarakul, Faculty of Medicine Siriraj Hospital, Mahidol University.

Author contributions

Conceptualization, P.W. and S.K.; methodology, P.W. and S.K.; investigation, P.W., V.M., S.J., P.A., D.J.B., and O.G.; writing-original draft, P.W. and S.K.; writing-review & editing, P.W., V.W., M.R.J.C., E.E.G., O.G., and S.K.; funding acquisition, P.W., O.G., and S.K.; resources, V.W. and S.C.; supervision, O.G, and S.K.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains

All bacterial strain	See Table 2	N/A
Phages	This study	See Tables 1 and S1

Chemicals, peptides, and recombinant proteins

Luria-Bertani broth	Criterion™, Hardy Diagnostics, USA	Cat# C9271
Agar powder	TM MEDIA®	REF#1201
Sodium chloride (NaCl)	VWR Chemicals BDH®	CAS# 7647-14-5
Calcium Chloride Dihydrate	Vivantis Technologies	CAS# 10035-04-8
Magnesium Sulfate Heptahydrate (MgSO4 · 7H2O)	VWR Chemicals BDH®	CAS# 10034-99-8
Mitomycin C	Sigma Aldrich, USA	Cat# 50-07-7
Formvar/carbon-coated copper grids (200 mesh)	Sigma Aldrich, USA	Cat# TEM-FCF200CU50
DNase I	Thermo Fisher Scientific	Cat# EN0521
RNase A	Thermo Fisher Scientific	Cat# EN0531
Proteinase K	Thermo Fisher Scientific	Cat# EN0491
Tris	Vivantis Technologies	Cat# PR0612
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich, USA	Cat# E5134
Sodium dodecyl sulfate (SDS)	Sigma Aldrich, USA	Cat# L3771
Phenol	Thermo Fisher Scientific	CAS# 108-95-2
Chloroform	Thermo Fisher Scientific	CAS# 67-66-3
Isoamyl alcohol	Fisher Chemical™	Cat# BPA3934
Ethanol	VWR Chemicals BDH®	Cat# VWR20821
Sodium acetate	Sigma Aldrich, USA	Cat# S8750

Critical commercial assays

Illumina DNA Library Prep kit	Illumina, Inc., USA	https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/illumina-dna-prep.html
Latex agglutination test	Duval et al.³⁵	https://www.tm.mahidol.ac.th/micro-immuno/portfolio-item/latex/

Deposited data

Whole genome sequences of phages	This paper	GenBank accession numbers see Table S1

Software and algorithms

Sickle v1.33	Joshi et al.³⁶	https://github.com/najoshi/sickle; RRID: SCR_006800
Trimmomatic v0.39	Bolger et al.³⁷	usadellab.org; RRID: SCR_011848
SPAdes v3.6.0	Bankevich et al.³⁸	https://github.com/ablab/spades; RRID: SCR_000131
Unicycler v0.4.8	Wick et al.³⁹	https://github.com/rrwick/Unicycler; RRID: SCR_024380
Prokka v1.12	Seemann⁴⁰	https://github.com/tseemann/prokka; RRID: SCR_014732
BLASTP	NCBI	https://blast.ncbi.nlm.nih.gov/Blast.cgi
PhageTerm v1.0.12	Garneau et al.⁴¹	https://sourceforge.net/projects/phageterm/
HHPred v2.08	Zimmermann et al.⁴²	https://toolkit.tuebingen.mpg.de/tools/hhpred; RRID: SCR_010276
InterPro v106.0	Blum et al.⁴³	https://www.ebi.ac.uk/interpro/; RRID: SCR_006695
Artemis v17.0.1	Rutherford et al.⁴⁴	https://github.com/sanger-pathogens/Artemis; RRID: SCR_004267
MAFFT v7.505	Katoh et al.⁴⁵	https://mafft.cbrc.jp/alignment/server/index.html; RRID: SCR_011811
Tree of Life (iTOL) v6.5.8	Letunic et al.⁴⁶	https://itol.embl.de/
Clinker v0.0.25	Gilchrist et al.⁴⁷	https://github.com/gamcil/clinker; RRID: SCR_016140
VipTree v1.1.3	Nishimura et al.⁴⁸	https://github.com/yosuken/ViPTreeGen
VICTOR v1	Meier-Kolthoff et al.⁴⁹	https://ggdc.dsmz.de/victor.php
VIRIDIC v1.1	Moraru et al.⁵⁰	https://rhea.icbm.uni-oldenburg.de/viridic/
VirClust v2	Moraru C.⁵¹	https://rhea.icbm.uni-oldenburg.de/virclust/

Open in a new tab

Experimental model and study participant details

This research did not employ experimental models, and it did not involve animals, human subjects, plants, cell lines, or primary cell cultures.

Method details

Biosafety approval

B. pseudomallei isolates were grown and tested under Biosafety Level 2 Enhanced (BSL-2+) containment procedures at the Department of Microbiology, Faculty of Medicine, Khon Kaen University. This research project was approved by the Institutional Biosafety Committees of the Faculty of Medicine Siriraj Hospital, Mahidol University and the Faculty of Medicine, Khon Kaen University. The approval certificate numbers are SI 2020-003 and IBCKKU no. 2/2563, respectively.

Burkholderia spp. culture condition

B. pseudomallei, B. thailandensis and BTCV were cultured in Luria-Bertani (LB) broth (Criterion™, Hardy Diagnostics, USA) at 37°C. Mid-log phase cultures were obtained by inoculating 1% of an overnight culture into fresh LB broth and incubating at 37°C for 4 h until reaching an OD600–0.18 (∼10⁸ CFU/mL).

Soil sampling and B. pseudomallei isolation

Soil samples were collected from rice paddy fields in Ubon Ratchathani (50 samples) and Khon Kaen (50 samples) provinces, located in the northeast of Thailand, for B. pseudomallei isolation according to a previously described method.¹⁶ Briefly, 5 g of soil was mixed with 30 mL of sterile distilled water and incubated at 40°C for 48 h. After sedimentation, the supernatant was transferred to Ashdown’s selective enrichment broth⁵² and incubated for another 48 h. Enriched samples were plated onto Ashdown’s agar. Suspected B. pseudomallei colonies were identified using biochemical assays and confirmed using a latex agglutination test.³⁵

B. thailandensis and BTCV isolation

B. thailandensis (40 isolates) and BTCV (20 isolates) were isolated from soils collected from both northeastern and central Thailand (Table S1) as reported in the previous study by Hantrakul et al.⁶ These bacteria were kindly provided by Prof. Direk Limmathurotsakul, MORU, Thailand.

Burkholderia phages isolation from soil

To isolate culturable phages, 2 g of soil was suspended in 2x LB broth supplemented with CaCl₂ and inoculated with B. pseudomallei UB4 as a bacterial host for enrichment. The UB4 isolate was selected as the enrichment bacterial host because it does not contain any detectable inducible prophages under mitomycin C (MMC; Sigma Aldrich, USA) treatment, reducing the likelihood of interference from host-derived phages during isolation. Cultures were incubated at 37°C for 24 h before centrifugation and were filtered through a 0.45 μm membrane to remove bacterial cells. Filtrates were screened for phage presence using a spot assay.¹⁷ Clear zones on the cultured medium indicated phage activity. Phages were then purified using the double agar overlay plaque assay.¹⁷

Prophage induction from Burkholderia spp.

B. pseudomallei, B. thailandensis, and BTCV were cultured to the exponential phase in LB broth, followed by the addition of 1 μg/mL mitomycin C (MMC; Sigma Aldrich, USA) and incubation at 37°C for 6 h. After centrifugation, the supernatant was filtered and screened for induced prophages by a spot assay against a panel of B. thailandensis isolates including E159, E174, E264, E348, D1, and DW503 (Table S1). This assay was performed to identify susceptible host strains for each phage. Positive MMC-induced prophages identified by the spot assay were subsequently confirmed by the double agar overlay plaque assay.¹⁷

Phage nomenclature

Phage names in this study were assigned following the convention recommended by Adriaenssens and Brister (2017),⁵³ in which phages are designated with the prefix vB_ (“virus of bacteria”), followed by an abbreviation of the bacterial host and an isolate-specific identifier. MMC-induced prophages derived from B. thailandensis and BTCV were named using the format vB_<bacterial isolate>, such as vB_E188 and vB_E555, indicating phages induced from B. thailandensis isolate E188 and BTCV isolate E555, respectively. Prophages induced from soil-isolated B. pseudomallei strains were designated vB_SBP<isolateID>, for example vB_SBP39, referring to a prophage induced from B. pseudomallei isolate 39. Free phages recovered from soil using B. pseudomallei as the enrichment host were named using the prefix vB_F<phage isolate>, such as vB_FUB33 and vB_FKKS1, representing free phages isolated from soil samples collected in Ubon Ratchathani (UB33) and Khon Kaen (KKS1), respectively.

Transmission electron microscopy (TEM)

Phage morphology was examined by TEM after negative staining.⁵⁴ Purified phage suspensions (10⁸ particles/mL) were fixed with 2.5% glutaraldehyde and applied to Formvar/carbon-coated copper grids (200 mesh) (Sigma Aldrich, USA). After adsorption for 5 min, the grids were stained with 1% uranyl acetate. Images were captured using a JEM-1400 transmission electron microscope (JEOL, Japan) operating at 80 kV.

Phage DNA extraction, sequencing and genome comparative analysis

Phage genomic DNA was prepared following the same workflow described in our previous study,¹⁷ utilizing the Illumina DNA Library Prep kit (Illumina, Inc., USA) for library construction and the Illumina MiSeq for sequencing. Raw sequencing reads were processed by trimming low-quality bases with Sickle v1.33³⁶ and Trimmomatic v0.39.³⁷ Quality-filtered reads were then assembled de novo using both SPAdes v3.6.0³⁸ and Unicycler v0.4.8.³⁹ Open reading frames (ORFs) were predicted and annotated using Prokka v1.12⁴⁰ against a custom database tailored to Caudoviricetes. To assess genome termini and packaging mechanisms, all assemblies were analyzed with PhageTerm v1.0.12.⁴¹ As no fixed termini were detected, genomes were oriented following established phage genomics conventions by placing the terminase (Ter) gene at the start position when present. For clades whose closest reference genomes initiated elsewhere, orientation was refined using conserved synteny with related phages in public databases. This standardized orientation was applied prior to all comparative and collinearity analyses.

Functional annotations of predicted proteins were assigned through homology searches against BLASTP (http://www.ncbi.nlm.nih.gov/BLAST), HHPred v2.08⁴² and InterPro v106.0.⁴³ Circular phage genome maps were visualized using Artemis v17.0.1.⁴⁴ To infer evolutionary relationships, whole-genome sequences were aligned using MAFFT v7.505⁴⁵ under the auto-alignment setting, producing nucleotide alignments based on translated amino acid sequences. Phylogenetic trees were constructed using the neighbor-joining method⁵⁵ and visualized with Interactive Tree of Life (iTOL) v6.5.8.⁴⁶ For comparative genome analysis, Clinker v0.0.25⁴⁷ was employed to examine gene synteny and modular conservation. To classify phages at the family level and explore proteomic diversity, hierarchical clustering was conducted using the Viral Proteomic Tree method (VipTree) v1.1.3.⁴⁸

To validate taxonomic assignments and genomic relationships, three additional ICTV-aligned genome-based tools were applied: VICTOR v1,⁴⁹ VIRIDIC v1.1,⁵⁰ and VirClust v2.⁵¹ Intergenomic similarities and clustering patterns were evaluated against reference Burkholderia phages in GenBank. Taxonomic boundaries followed ICTV genomic thresholds: species ≥95% intergenomic similarity (≈Genome-BLAST Distance Phylogeny method (GBDP) ≤ 0.05), genus/subclade 70–95% (≈ GBDP ≤0.25), and family/clade 40–70% (≈ GBDP ≤0.45), in accordance with the updated ICTV classification framework.⁵⁶

Quantification and statistical analysis

No inferential statistical analyses were performed in this study. Sample sizes (n) represent the number of independent soil samples, bacterial isolates (Table 2), or phage genomes analyzed (Tables 1 and S1). Reported proportions and percentages were calculated directly from observed counts within each dataset (e.g., number of phages per clade divided by the total number analyzed) without replication-based statistical testing. Plaque morphology characteristics, including plaque size, clarity, and the presence or absence of halo-like features, were evaluated qualitatively based on repeated plaque assays. Phylogenomic clustering were assessed using whole-genome sequence comparisons, as described in the method details. All bioinformatic analyses were performed using publicly available software, with software versions described in the method details and key resources table.

Published: January 9, 2026

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2026.114658.

Supplemental information

Document S1. Figures S1–S5

mmc1.pdf^{(2.9MB, pdf)}

Table S1. Genomic and host information of 82 Burkholderia phages analyzed in this study, including accession numbers, sources, propagation hosts, genomic features, and nucleotide BLAST results

mmc2.xlsx^{(17.5KB, xlsx)}

Table S2. Classification of genomic termini and DNA packaging mechanisms determined using PhageTerm for 82 tailed phages

mmc3.xlsx^{(16.3KB, xlsx)}

Table S3. Taxonomic clustering of the analyzed phages was determined using the Genome BLAST Distance Phylogeny (GBDP) approach as implemented in VICTOR, applying formulas D0, D4, and D6 for intergenomic distance calculation, related to Figure S1

mmc4.xlsx^{(19KB, xlsx)}

Table S4. Pairwise intergenomic similarity matrix of Burkholderia phages calculated with VIRIDIC, related to Figure S2

mmc5.xlsx^{(71.5KB, xlsx)}

Table S5. Protein-based clustering of Burkholderia phages generated with VirClust, related to Figure S3

mmc6.xlsx^{(49.1KB, xlsx)}

Table S6. Accession information of Burkholderia phage sequences retrieved from public NCBI databases

mmc7.xlsx^{(11.4KB, xlsx)}

Table S7. Predicted functions of phage accessory genes based on BLASTP, InterPro, and HHpred analyses

mmc8.xlsx^{(14.3KB, xlsx)}

References

1.Limmathurotsakul D., Golding N., Dance D.A.B., Messina J.P., Pigott D.M., Moyes C.L., Rolim D.B., Bertherat E., Day N.P.J., Peacock S.J., Hay S.I. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat. Microbiol. 2016;1 doi: 10.1038/nmicrobiol.2015.8. [DOI] [PubMed] [Google Scholar]
2.Chantratita N., Phunpang R., Yarasai A., Dulsuk A., Yimthin T., Onofrey L.A., Coston T.D., Thiansukhon E., Chaisuksant S., Tanwisaid K., et al. Characteristics and one year outcomes of melioidosis patients in Northeastern Thailand: A prospective, multicenter cohort study. Lancet Reg. Health. Southeast Asia. 2023;9 doi: 10.1016/j.lansea.2022.100118. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Dance D.A. Melioidosis: the tip of the iceberg? Clin. Microbiol. Rev. 1991;4:52–60. doi: 10.1128/CMR.4.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Wiersinga W.J., Virk H.S., Torres A.G., Currie B.J., Peacock S.J., Dance D.A.B., Limmathurotsakul D. Melioidosis. Nat. Rev. Dis. Primers. 2018;4 doi: 10.1038/nrdp.2017.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Sim B.M.Q., Chantratita N., Ooi W.F., Nandi T., Tewhey R., Wuthiekanun V., Thaipadungpanit J., Tumapa S., Ariyaratne P., Sung W.K., et al. Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates. Genome Biol. 2010;11:R89. doi: 10.1186/gb-2010-11-8-r89. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Hantrakun V., Thaipadungpanit J., Rongkard P., Srilohasin P., Amornchai P., Langla S., Mukaka M., Chantratita N., Wuthiekanun V., Dance D.A.B., et al. Presence of B. thailandensis and B. thailandensis expressing B. pseudomallei-like capsular polysaccharide in Thailand, and their associations with serological response to B. pseudomallei. PLoS Negl. Trop. Dis. 2018;12 doi: 10.1371/journal.pntd.0006193. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Pfeifer E., Bonnin R.A., Rocha E.P.C. Phage-plasmids spread antibiotic resistance genes through infection and lysogenic conversion. mBio. 2022;13 doi: 10.1128/mbio.01851-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Naureen Z., Dautaj A., Anpilogov K., Camilleri G., Dhuli K., Tanzi B., Maltese P.E., Cristofoli F., De Antoni L., Beccari T., et al. Bacteriophages presence in nature and their role in the natural selection of bacterial populations. Acta Biomed. 2020;91 doi: 10.23750/abm.v91i13-S.10819. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Nordstrom H.R., Griffith M.P., Rangachar Srinivasa V., Wallace N.R., Li A., Cooper V.S., Shields R.K., Van Tyne D. Harnessing the diversity of Burkholderia spp. prophages for therapeutic potential. Cells. 2024;13:428. doi: 10.3390/cells13050428. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Oppenheim A.B., Kobiler O., Stavans J., Court D.L., Adhya S. Switches in bacteriophage lambda development. Annu. Rev. Genet. 2005;39:409–429. doi: 10.1146/annurev.genet.39.073003.113656. [DOI] [PubMed] [Google Scholar]
11.Ronning C.M., Losada L., Brinkac L., Inman J., Ulrich R.L., Schell M., Nierman W.C., Deshazer D. Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements. BMC Microbiol. 2010;10:202. doi: 10.1186/1471-2180-10-202. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Sariya L., Prempracha N., Keelapan P., Chittasophon N. Bacteriophage isolated from Burkholderia pseudomallei causes phenotypic changes in Burkholderia thailandensis. Sci. Asia. 2006;32:83–91. doi: 10.2306/scienceasia1513-1874.2006.32.083. [DOI] [Google Scholar]
13.Gatedee J., Kritsiriwuthinan K., Galyov E.E., Shan J., Dubinina E., Intarak N., Clokie M.R.J., Korbsrisate S. Isolation and characterization of a novel podovirus which infects Burkholderia pseudomallei. Virol. J. 2011;8:366. doi: 10.1186/1743-422X-8-366. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Shan J., Korbsrisate S., Withatanung P., Adler N.L., Clokie M.R.J., Galyov E.E. Temperature dependent bacteriophages of a tropical bacterial pathogen. Front. Microbiol. 2014;5:599. doi: 10.3389/fmicb.2014.00599. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Yordpratum U., Tattawasart U., Wongratanacheewin S., Sermswan R.W. Novel lytic bacteriophages from soil that lyse Burkholderia pseudomallei. FEMS Microbiol. Lett. 2011;314:81–88. doi: 10.1111/j.1574-6968.2010.02150.x. [DOI] [PubMed] [Google Scholar]
16.Withatanung P., Chantratita N., Muangsombut V., Saiprom N., Lertmemongkolchai G., Klumpp J., Clokie M.R.J., Galyov E.E., Korbsrisate S. Analyses of the distribution patterns of Burkholderia pseudomallei and associated phages in soil samples in Thailand suggest that phage presence reduces the frequency of bacterial isolation. PLoS Negl. Trop. Dis. 2016;10 doi: 10.1371/journal.pntd.0005005. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Withatanung P., Janesomboon S., Vanaporn M., Muangsombut V., Charoensudjai S., Baker D.J., Wuthiekanun V., Galyov E.E., Clokie M.R.J., Gundogdu O., Korbsrisate S. Induced Burkholderia prophages detected from the hemoculture: a biomarker for Burkholderia pseudomallei infection. Front. Microbiol. 2024;15 doi: 10.3389/fmicb.2024.1361121. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Dougherty P.E., Nielsen T.K., Riber L., Lading H.H., Forero-Junco L.M., Kot W., Raaijmakers J.M., Hansen L.H. Widespread and largely unknown prophage activity, diversity, and function in two genera of wheat phyllosphere bacteria. ISME J. 2023;17:2415–2425. doi: 10.1038/s41396-023-01547-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Wuthiekanun V., Amornchai P., Saiprom N., Chantratita N., Chierakul W., Koh G.C.K.W., Chaowagul W., Day N.P.J., Limmathurotsakul D., Peacock S.J. Survey of antimicrobial resistance in clinical Burkholderia pseudomallei isolates over two decades in Northeast Thailand. Antimicrob. Agents Chemother. 2011;55:5388–5391. doi: 10.1128/AAC.05517-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Burtnick M., Bolton A., Brett P., Watanabe D., Woods D. Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. Microbiology (N. Y). 2001;147:111–120. doi: 10.1099/00221287-147-1-111. [DOI] [PubMed] [Google Scholar]
21.Jitprasutwit S., Thaewpia W., Muangsombut V., Lulitanond A., Leelayuwat C., Lertmemongkolchai G., Korbsrisate S. Effect of acidic pH on the invasion efficiency and the type III secretion system of Burkholderia thailandensis. J. Microbiol. 2010;48:526–532. doi: 10.1007/s12275-010-0078-x. [DOI] [PubMed] [Google Scholar]
22.Hantrakun V., Rongkard P., Oyuchua M., Amornchai P., Lim C., Wuthiekanun V., Day N.P.J., Peacock S.J., Limmathurotsakul D. Soil nutrient depletion is associated with the presence of Burkholderia pseudomallei. Appl. Environ. Microbiol. 2016;82:7086–7092. doi: 10.1128/AEM.02538-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Palasatien S., Lertsirivorakul R., Royros P., Wongratanacheewin S., Sermswan R.W. Soil physicochemical properties related to the presence of Burkholderia pseudomallei. Trans. R. Soc. Trop. Med. Hyg. 2008;102:S5–S9. doi: 10.1016/S0035-9203(08)70003-8. [DOI] [PubMed] [Google Scholar]
24.Hooda Y., Islam S., Kabiraj R., Rahman H., Sarkar H., da Silva K.E., Raju R.S., Luby S.P., Andrews J.R., Saha S.K., Saha S. Old tools, new applications: Use of environmental bacteriophages for typhoid surveillance and evaluating vaccine impact. PLoS Negl. Trop. Dis. 2024;18 doi: 10.1371/journal.pntd.0011822. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Jacoby M.L., Hogg G.D., Assaad M.R., Williamson K.E. Seasonal trends in lysogeny in an Appalachian oak-hickory forest soil. Appl. Environ. Microbiol. 2024;90 doi: 10.1128/aem.01408-23. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Jurėnas D., Fraikin N., Goormaghtigh F., Van Melderen L. Biology and evolution of bacterial toxin-antitoxin systems. Nat. Rev. Microbiol. 2022;20:335–350. doi: 10.1038/s41579-021-00661-1. [DOI] [PubMed] [Google Scholar]
27.Peltier J., Hamiot A., Garneau J.R., Boudry P., Maikova A., Hajnsdorf E., Fortier L.C., Dupuy B., Soutourina O. Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile. Commun. Biol. 2020;3:718. doi: 10.1038/s42003-020-01448-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Kelly A., Arrowsmith T.J., Went S.C., Blower T.R. Toxin-antitoxin systems as mediators of phage defence and the implications for abortive infection. Curr. Opin. Microbiol. 2023;73 doi: 10.1016/j.mib.2023.102293. [DOI] [PubMed] [Google Scholar]
29.Singh G., Yadav M., Ghosh C., Rathore J.S. Bacterial toxin-antitoxin modules: classification, functions, and association with persistence. Curr. Res. Microb. Sci. 2021;2 doi: 10.1016/j.crmicr.2021.100047. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Guang-Han O., Leang-Chung C., Vellasamy K.M., Mariappan V., Li-Yen C., Vadivelu J. Experimental Phage therapy for Burkholderia pseudomallei infection. PLoS One. 2016;11 doi: 10.1371/journal.pone.0158213. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Wang Y., Li X., Dance D.A.B., Xia H., Chen C., Luo N., Li A., Li Y., Zhu Q., Sun Q., et al. A novel lytic phage potentially effective for phage therapy against Burkholderia pseudomallei in the tropics. Infect. Dis. Poverty. 2022;11:87. doi: 10.1186/s40249-022-01012-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Latka A., Maciejewska B., Majkowska-Skrobek G., Briers Y., Drulis-Kawa Z. Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process. Appl. Microbiol. Biotechnol. 2017;101:3103–3119. doi: 10.1007/s00253-017-8224-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Udden S.M.N., Zahid M.S.H., Biswas K., Ahmad Q.S., Cravioto A., Nair G.B., Mekalanos J.J., Faruque S.M. Acquisition of classical CTX prophage from Vibrio cholerae O141 by El Tor strains aided by lytic phages and chitin-induced competence. Proc. Natl. Acad. Sci. USA. 2008;105:11951–11956. doi: 10.1073/pnas.0805560105. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.McShan W.M., McCullor K.A., Nguyen S.V. The bacteriophages of Streptococcus pyogenes. Microbiol. Spectr. 2019;7 doi: 10.1128/microbiolspec.gpp3-0059-2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Duval B.D., Elrod M.G., Gee J.E., Chantratita N., Tandhavanant S., Limmathurotsakul D., Hoffmaster A.R. Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei. Am. J. Trop. Med. Hyg. 2014;90:1043–1046. doi: 10.4269/ajtmh.14-0025. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Joshi N., Fass J.N. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (Version 1.33). [Software] 2011. https://github.com/najoshi/sickle
37.Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prjibelski A.D., et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012;19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Wick R.R., Judd L.M., Gorrie C.L., Holt K.E. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 2017;13 doi: 10.1371/journal.pcbi.1005595. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics (Oxford, England) 2014;30:2068–2069. doi: 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]
41.Garneau J.R., Depardieu F., Fortier L.C., Bikard D., Monot M. PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci. Rep. 2017;7:8292. doi: 10.1038/s41598-017-07910-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Zimmermann L., Stephens A., Nam S.Z., Rau D., Kübler J., Lozajic M., Gabler F., Söding J., Lupas A.N., Alva V. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J. Mol. Biol. 2018;430:2237–2243. doi: 10.1016/j.jmb.2017.12.007. [DOI] [PubMed] [Google Scholar]
43.Blum M., Andreeva A., Florentino L.C., Chuguransky S.R., Grego T., Hobbs E., Pinto B.L., Orr A., Paysan-Lafosse T., Ponamareva I., et al. InterPro: the protein sequence classification resource in 2025. Nucleic Acids Res. 2025;53:D444–D456. doi: 10.1093/nar/gkae1082. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Rutherford K., Parkhill J., Crook J., Horsnell T., Rice P., Rajandream M.A., Barrell B. Artemis: sequence visualization and annotation. Bioinformatics. 2000;16:944–945. doi: 10.1093/bioinformatics/16.10.944. [DOI] [PubMed] [Google Scholar]
45.Katoh K., Rozewicki J., Yamada K.D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2019;20:1160–1166. doi: 10.1093/bib/bbx108. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Letunic I., Bork P. Interactive Tree of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019;47:W256–W259. doi: 10.1093/nar/gkz239. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Gilchrist C.L.M., Chooi Y.H. Clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics (Oxford, England) 2021;37:2473–2475. doi: 10.1093/bioinformatics/btab007. [DOI] [PubMed] [Google Scholar]
48.Nishimura Y., Yoshida T., Kuronishi M., Uehara H., Ogata H., Goto S. VViPTree: the viral proteomic tree server. Bioinformatics (Oxford, England) 2017;33:2379–2380. doi: 10.1093/bioinformatics/btx157. [DOI] [PubMed] [Google Scholar]
49.Meier-Kolthoff J.P., Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics (Oxford, England) 2017;33:3396–3404. doi: 10.1093/bioinformatics/btx440. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Moraru C., Varsani A., Kropinski A.M. VIRIDIC-A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses. 2020;12:1268. doi: 10.3390/v12111268. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Moraru C. VirClust-A tool for hierarchical clustering, core protein detection and annotation of (prokaryotic) viruses. Viruses. 2023;15:1007. doi: 10.3390/v15041007. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Ashdown L.R. An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens. Pathology. 1979;11:293–297. doi: 10.3109/00313027909061954. [DOI] [PubMed] [Google Scholar]
53.Adriaenssens E., Brister J.R. How to name and classify your phage: An informal guide. Viruses. 2017;9:70. doi: 10.3390/v9040070. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Nale J.Y., Shan J., Hickenbotham P.T., Fawley W.N., Wilcox M.H., Clokie M.R.J. Diverse temperate bacteriophage carriage in Clostridium difficile 027 strains. PLoS One. 2012;7 doi: 10.1371/journal.pone.0037263. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Saitou N., Nei M. TThe neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987;4:406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
56.Turner D., Shkoporov A.N., Lood C., Millard A.D., Dutilh B.E., Alfenas-Zerbini P., van Zyl L.J., Aziz R.K., Oksanen H.M., Poranen M.M., et al. Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee. Arch. Virol. 2023;168:74. doi: 10.1007/s00705-022-05694-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

Document S1. Figures S1–S5

mmc1.pdf^{(2.9MB, pdf)}

Table S1. Genomic and host information of 82 Burkholderia phages analyzed in this study, including accession numbers, sources, propagation hosts, genomic features, and nucleotide BLAST results

mmc2.xlsx^{(17.5KB, xlsx)}

Table S2. Classification of genomic termini and DNA packaging mechanisms determined using PhageTerm for 82 tailed phages

mmc3.xlsx^{(16.3KB, xlsx)}

mmc4.xlsx^{(19KB, xlsx)}

Table S4. Pairwise intergenomic similarity matrix of Burkholderia phages calculated with VIRIDIC, related to Figure S2

mmc5.xlsx^{(71.5KB, xlsx)}

Table S5. Protein-based clustering of Burkholderia phages generated with VirClust, related to Figure S3

mmc6.xlsx^{(49.1KB, xlsx)}

Table S6. Accession information of Burkholderia phage sequences retrieved from public NCBI databases

mmc7.xlsx^{(11.4KB, xlsx)}

Table S7. Predicted functions of phage accessory genes based on BLASTP, InterPro, and HHpred analyses

mmc8.xlsx^{(14.3KB, xlsx)}

Data Availability Statement

Code: This paper does not report original code.

Additional information: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

[bib1] 1.Limmathurotsakul D., Golding N., Dance D.A.B., Messina J.P., Pigott D.M., Moyes C.L., Rolim D.B., Bertherat E., Day N.P.J., Peacock S.J., Hay S.I. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat. Microbiol. 2016;1 doi: 10.1038/nmicrobiol.2015.8. [DOI] [PubMed] [Google Scholar]

[bib2] 2.Chantratita N., Phunpang R., Yarasai A., Dulsuk A., Yimthin T., Onofrey L.A., Coston T.D., Thiansukhon E., Chaisuksant S., Tanwisaid K., et al. Characteristics and one year outcomes of melioidosis patients in Northeastern Thailand: A prospective, multicenter cohort study. Lancet Reg. Health. Southeast Asia. 2023;9 doi: 10.1016/j.lansea.2022.100118. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Dance D.A. Melioidosis: the tip of the iceberg? Clin. Microbiol. Rev. 1991;4:52–60. doi: 10.1128/CMR.4.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Wiersinga W.J., Virk H.S., Torres A.G., Currie B.J., Peacock S.J., Dance D.A.B., Limmathurotsakul D. Melioidosis. Nat. Rev. Dis. Primers. 2018;4 doi: 10.1038/nrdp.2017.107. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Sim B.M.Q., Chantratita N., Ooi W.F., Nandi T., Tewhey R., Wuthiekanun V., Thaipadungpanit J., Tumapa S., Ariyaratne P., Sung W.K., et al. Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates. Genome Biol. 2010;11:R89. doi: 10.1186/gb-2010-11-8-r89. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6.Hantrakun V., Thaipadungpanit J., Rongkard P., Srilohasin P., Amornchai P., Langla S., Mukaka M., Chantratita N., Wuthiekanun V., Dance D.A.B., et al. Presence of B. thailandensis and B. thailandensis expressing B. pseudomallei-like capsular polysaccharide in Thailand, and their associations with serological response to B. pseudomallei. PLoS Negl. Trop. Dis. 2018;12 doi: 10.1371/journal.pntd.0006193. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7.Pfeifer E., Bonnin R.A., Rocha E.P.C. Phage-plasmids spread antibiotic resistance genes through infection and lysogenic conversion. mBio. 2022;13 doi: 10.1128/mbio.01851-22. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Naureen Z., Dautaj A., Anpilogov K., Camilleri G., Dhuli K., Tanzi B., Maltese P.E., Cristofoli F., De Antoni L., Beccari T., et al. Bacteriophages presence in nature and their role in the natural selection of bacterial populations. Acta Biomed. 2020;91 doi: 10.23750/abm.v91i13-S.10819. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9.Nordstrom H.R., Griffith M.P., Rangachar Srinivasa V., Wallace N.R., Li A., Cooper V.S., Shields R.K., Van Tyne D. Harnessing the diversity of Burkholderia spp. prophages for therapeutic potential. Cells. 2024;13:428. doi: 10.3390/cells13050428. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Oppenheim A.B., Kobiler O., Stavans J., Court D.L., Adhya S. Switches in bacteriophage lambda development. Annu. Rev. Genet. 2005;39:409–429. doi: 10.1146/annurev.genet.39.073003.113656. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Ronning C.M., Losada L., Brinkac L., Inman J., Ulrich R.L., Schell M., Nierman W.C., Deshazer D. Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements. BMC Microbiol. 2010;10:202. doi: 10.1186/1471-2180-10-202. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Sariya L., Prempracha N., Keelapan P., Chittasophon N. Bacteriophage isolated from Burkholderia pseudomallei causes phenotypic changes in Burkholderia thailandensis. Sci. Asia. 2006;32:83–91. doi: 10.2306/scienceasia1513-1874.2006.32.083. [DOI] [Google Scholar]

[bib13] 13.Gatedee J., Kritsiriwuthinan K., Galyov E.E., Shan J., Dubinina E., Intarak N., Clokie M.R.J., Korbsrisate S. Isolation and characterization of a novel podovirus which infects Burkholderia pseudomallei. Virol. J. 2011;8:366. doi: 10.1186/1743-422X-8-366. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14.Shan J., Korbsrisate S., Withatanung P., Adler N.L., Clokie M.R.J., Galyov E.E. Temperature dependent bacteriophages of a tropical bacterial pathogen. Front. Microbiol. 2014;5:599. doi: 10.3389/fmicb.2014.00599. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15.Yordpratum U., Tattawasart U., Wongratanacheewin S., Sermswan R.W. Novel lytic bacteriophages from soil that lyse Burkholderia pseudomallei. FEMS Microbiol. Lett. 2011;314:81–88. doi: 10.1111/j.1574-6968.2010.02150.x. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Withatanung P., Chantratita N., Muangsombut V., Saiprom N., Lertmemongkolchai G., Klumpp J., Clokie M.R.J., Galyov E.E., Korbsrisate S. Analyses of the distribution patterns of Burkholderia pseudomallei and associated phages in soil samples in Thailand suggest that phage presence reduces the frequency of bacterial isolation. PLoS Negl. Trop. Dis. 2016;10 doi: 10.1371/journal.pntd.0005005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] 17.Withatanung P., Janesomboon S., Vanaporn M., Muangsombut V., Charoensudjai S., Baker D.J., Wuthiekanun V., Galyov E.E., Clokie M.R.J., Gundogdu O., Korbsrisate S. Induced Burkholderia prophages detected from the hemoculture: a biomarker for Burkholderia pseudomallei infection. Front. Microbiol. 2024;15 doi: 10.3389/fmicb.2024.1361121. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.Dougherty P.E., Nielsen T.K., Riber L., Lading H.H., Forero-Junco L.M., Kot W., Raaijmakers J.M., Hansen L.H. Widespread and largely unknown prophage activity, diversity, and function in two genera of wheat phyllosphere bacteria. ISME J. 2023;17:2415–2425. doi: 10.1038/s41396-023-01547-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 19.Wuthiekanun V., Amornchai P., Saiprom N., Chantratita N., Chierakul W., Koh G.C.K.W., Chaowagul W., Day N.P.J., Limmathurotsakul D., Peacock S.J. Survey of antimicrobial resistance in clinical Burkholderia pseudomallei isolates over two decades in Northeast Thailand. Antimicrob. Agents Chemother. 2011;55:5388–5391. doi: 10.1128/AAC.05517-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib33] 20.Burtnick M., Bolton A., Brett P., Watanabe D., Woods D. Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. Microbiology (N. Y). 2001;147:111–120. doi: 10.1099/00221287-147-1-111. [DOI] [PubMed] [Google Scholar]

[bib34] 21.Jitprasutwit S., Thaewpia W., Muangsombut V., Lulitanond A., Leelayuwat C., Lertmemongkolchai G., Korbsrisate S. Effect of acidic pH on the invasion efficiency and the type III secretion system of Burkholderia thailandensis. J. Microbiol. 2010;48:526–532. doi: 10.1007/s12275-010-0078-x. [DOI] [PubMed] [Google Scholar]

[bib19] 22.Hantrakun V., Rongkard P., Oyuchua M., Amornchai P., Lim C., Wuthiekanun V., Day N.P.J., Peacock S.J., Limmathurotsakul D. Soil nutrient depletion is associated with the presence of Burkholderia pseudomallei. Appl. Environ. Microbiol. 2016;82:7086–7092. doi: 10.1128/AEM.02538-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 23.Palasatien S., Lertsirivorakul R., Royros P., Wongratanacheewin S., Sermswan R.W. Soil physicochemical properties related to the presence of Burkholderia pseudomallei. Trans. R. Soc. Trop. Med. Hyg. 2008;102:S5–S9. doi: 10.1016/S0035-9203(08)70003-8. [DOI] [PubMed] [Google Scholar]

[bib21] 24.Hooda Y., Islam S., Kabiraj R., Rahman H., Sarkar H., da Silva K.E., Raju R.S., Luby S.P., Andrews J.R., Saha S.K., Saha S. Old tools, new applications: Use of environmental bacteriophages for typhoid surveillance and evaluating vaccine impact. PLoS Negl. Trop. Dis. 2024;18 doi: 10.1371/journal.pntd.0011822. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 25.Jacoby M.L., Hogg G.D., Assaad M.R., Williamson K.E. Seasonal trends in lysogeny in an Appalachian oak-hickory forest soil. Appl. Environ. Microbiol. 2024;90 doi: 10.1128/aem.01408-23. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 26.Jurėnas D., Fraikin N., Goormaghtigh F., Van Melderen L. Biology and evolution of bacterial toxin-antitoxin systems. Nat. Rev. Microbiol. 2022;20:335–350. doi: 10.1038/s41579-021-00661-1. [DOI] [PubMed] [Google Scholar]

[bib24] 27.Peltier J., Hamiot A., Garneau J.R., Boudry P., Maikova A., Hajnsdorf E., Fortier L.C., Dupuy B., Soutourina O. Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile. Commun. Biol. 2020;3:718. doi: 10.1038/s42003-020-01448-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 28.Kelly A., Arrowsmith T.J., Went S.C., Blower T.R. Toxin-antitoxin systems as mediators of phage defence and the implications for abortive infection. Curr. Opin. Microbiol. 2023;73 doi: 10.1016/j.mib.2023.102293. [DOI] [PubMed] [Google Scholar]

[bib26] 29.Singh G., Yadav M., Ghosh C., Rathore J.S. Bacterial toxin-antitoxin modules: classification, functions, and association with persistence. Curr. Res. Microb. Sci. 2021;2 doi: 10.1016/j.crmicr.2021.100047. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib27] 30.Guang-Han O., Leang-Chung C., Vellasamy K.M., Mariappan V., Li-Yen C., Vadivelu J. Experimental Phage therapy for Burkholderia pseudomallei infection. PLoS One. 2016;11 doi: 10.1371/journal.pone.0158213. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib28] 31.Wang Y., Li X., Dance D.A.B., Xia H., Chen C., Luo N., Li A., Li Y., Zhu Q., Sun Q., et al. A novel lytic phage potentially effective for phage therapy against Burkholderia pseudomallei in the tropics. Infect. Dis. Poverty. 2022;11:87. doi: 10.1186/s40249-022-01012-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib29] 32.Latka A., Maciejewska B., Majkowska-Skrobek G., Briers Y., Drulis-Kawa Z. Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process. Appl. Microbiol. Biotechnol. 2017;101:3103–3119. doi: 10.1007/s00253-017-8224-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib30] 33.Udden S.M.N., Zahid M.S.H., Biswas K., Ahmad Q.S., Cravioto A., Nair G.B., Mekalanos J.J., Faruque S.M. Acquisition of classical CTX prophage from Vibrio cholerae O141 by El Tor strains aided by lytic phages and chitin-induced competence. Proc. Natl. Acad. Sci. USA. 2008;105:11951–11956. doi: 10.1073/pnas.0805560105. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 34.McShan W.M., McCullor K.A., Nguyen S.V. The bacteriophages of Streptococcus pyogenes. Microbiol. Spectr. 2019;7 doi: 10.1128/microbiolspec.gpp3-0059-2018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] 35.Duval B.D., Elrod M.G., Gee J.E., Chantratita N., Tandhavanant S., Limmathurotsakul D., Hoffmaster A.R. Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei. Am. J. Trop. Med. Hyg. 2014;90:1043–1046. doi: 10.4269/ajtmh.14-0025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] 36.Joshi N., Fass J.N. Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (Version 1.33). [Software] 2011. https://github.com/najoshi/sickle

[bib37] 37.Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib38] 38.Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prjibelski A.D., et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012;19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib39] 39.Wick R.R., Judd L.M., Gorrie C.L., Holt K.E. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 2017;13 doi: 10.1371/journal.pcbi.1005595. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib40] 40.Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics (Oxford, England) 2014;30:2068–2069. doi: 10.1093/bioinformatics/btu153. [DOI] [PubMed] [Google Scholar]

[bib41] 41.Garneau J.R., Depardieu F., Fortier L.C., Bikard D., Monot M. PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci. Rep. 2017;7:8292. doi: 10.1038/s41598-017-07910-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib42] 42.Zimmermann L., Stephens A., Nam S.Z., Rau D., Kübler J., Lozajic M., Gabler F., Söding J., Lupas A.N., Alva V. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J. Mol. Biol. 2018;430:2237–2243. doi: 10.1016/j.jmb.2017.12.007. [DOI] [PubMed] [Google Scholar]

[bib43] 43.Blum M., Andreeva A., Florentino L.C., Chuguransky S.R., Grego T., Hobbs E., Pinto B.L., Orr A., Paysan-Lafosse T., Ponamareva I., et al. InterPro: the protein sequence classification resource in 2025. Nucleic Acids Res. 2025;53:D444–D456. doi: 10.1093/nar/gkae1082. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib44] 44.Rutherford K., Parkhill J., Crook J., Horsnell T., Rice P., Rajandream M.A., Barrell B. Artemis: sequence visualization and annotation. Bioinformatics. 2000;16:944–945. doi: 10.1093/bioinformatics/16.10.944. [DOI] [PubMed] [Google Scholar]

[bib45] 45.Katoh K., Rozewicki J., Yamada K.D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2019;20:1160–1166. doi: 10.1093/bib/bbx108. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib46] 46.Letunic I., Bork P. Interactive Tree of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019;47:W256–W259. doi: 10.1093/nar/gkz239. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib47] 47.Gilchrist C.L.M., Chooi Y.H. Clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics (Oxford, England) 2021;37:2473–2475. doi: 10.1093/bioinformatics/btab007. [DOI] [PubMed] [Google Scholar]

[bib48] 48.Nishimura Y., Yoshida T., Kuronishi M., Uehara H., Ogata H., Goto S. VViPTree: the viral proteomic tree server. Bioinformatics (Oxford, England) 2017;33:2379–2380. doi: 10.1093/bioinformatics/btx157. [DOI] [PubMed] [Google Scholar]

[bib49] 49.Meier-Kolthoff J.P., Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics (Oxford, England) 2017;33:3396–3404. doi: 10.1093/bioinformatics/btx440. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib50] 50.Moraru C., Varsani A., Kropinski A.M. VIRIDIC-A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses. 2020;12:1268. doi: 10.3390/v12111268. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib51] 51.Moraru C. VirClust-A tool for hierarchical clustering, core protein detection and annotation of (prokaryotic) viruses. Viruses. 2023;15:1007. doi: 10.3390/v15041007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib52] 52.Ashdown L.R. An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens. Pathology. 1979;11:293–297. doi: 10.3109/00313027909061954. [DOI] [PubMed] [Google Scholar]

[bib53] 53.Adriaenssens E., Brister J.R. How to name and classify your phage: An informal guide. Viruses. 2017;9:70. doi: 10.3390/v9040070. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib54] 54.Nale J.Y., Shan J., Hickenbotham P.T., Fawley W.N., Wilcox M.H., Clokie M.R.J. Diverse temperate bacteriophage carriage in Clostridium difficile 027 strains. PLoS One. 2012;7 doi: 10.1371/journal.pone.0037263. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib55] 55.Saitou N., Nei M. TThe neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987;4:406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]

[bib56] 56.Turner D., Shkoporov A.N., Lood C., Millard A.D., Dutilh B.E., Alfenas-Zerbini P., van Zyl L.J., Aziz R.K., Oksanen H.M., Poranen M.M., et al. Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee. Arch. Virol. 2023;168:74. doi: 10.1007/s00705-022-05694-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Genomic diversity and clade clustering of Burkholderia pseudomallei and B. thailandensis prophages with soil-derived phages

Patoo Withatanung

Veerachat Muangsombut

Sujintana Janesomboon

Vanaporn Wuthiekanun

Premjit Amornchai

Sorujsiri Chareonsudjai

Dave J Baker

Martha RJ Clokie

Edouard E Galyov

Ozan Gundogdu

Sunee Korbsrisate

Summary

Graphical abstract

Highlights

Introduction

Results

B. pseudomallei and their phages are prevalent in soil of endemic area

Figure 1.

Prophages could be induced from soil-isolated B. pseudomallei

BTCV showed a higher proportion of prophage induction than B. pseudomallei and B. thailandensis under our experimental conditions

Induced Burkholderia prophages and soil-isolated free phages could be classified into eight distinct groups

Table 1.

Induced B. pseudomallei prophages and soil-isolated free phages map to two clades

Figure 2.

Comprehensive phylogenetic analysis of B. pseudomallei prophages and soil-isolated free phages with public database sequences

Figure 3.

B. thailandensis and BTCV prophages were grouped into seven clades

Figure 4.

Table 2.

Comprehensive phylogenetic analysis of B. thailandensis and BTCV prophages across clades and public databases

Figure 5.

Figure 6.

Discussion

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model and study participant details

Method details

Biosafety approval

Burkholderia spp. culture condition

Soil sampling and B. pseudomallei isolation

B. thailandensis and BTCV isolation

Burkholderia phages isolation from soil

Prophage induction from Burkholderia spp.

Phage nomenclature

Transmission electron microscopy (TEM)

Phage DNA extraction, sequencing and genome comparative analysis

Quantification and statistical analysis

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases