Burkholderia Phages and Control of Burkholderia-Associated Human, Animal, and Plant Diseases

Abstract

Gram-negative Burkholderia bacteria are known for causing diseases in humans, animals, and plants, and high intrinsic resistance to antibiotics. Phage therapy is a promising alternative to control multidrug-resistant bacterial pathogens. Here, we present an overview of Burkholderia phage characteristics, host specificity, genomic classification, and therapeutic potentials across medical, veterinary, and agricultural systems. We evaluate the efficacy and limitations of current phage candidates, the biological and environmental barriers of phage applications, and the phage cocktail strategy. We highlight the innovations on the development of targeted phage delivery systems and the transition from the exploration of clinical phage therapy to plant disease management, advocating integrated disease control strategies.

1. Introduction

Burkholderia sensu lato are Gram-negative bacteria within the family Burkholderiaceae, order Burkholderiales, and class Betaproteobacteria. Burkholderia sensu lato has been divided into Burkholderia sensu stricto and other six genera named Paraburkholderia, Caballeronia, Robbsia, Mycetohabitans, Trinickia, and Pararobbsia [1]. The genus Burkholderia sensu stricto currently comprises 36 validly published species (https://lpsn.dsmz.de/genus/burkholderia) (accessed on 10 May 2025). Burkholderia sensu stricto has wide metabolic versatility and adaptation to versatile lifestyles as free-living bacteria in soil or water and as commensals of plants, animals, or fungi [1]. The genus Burkholderia sensu stricto is phylogenetically divided into three major species complexes: Burkholderia cepacia complex (Bcc), Burkholderia pseudomallei complex (Bpc), and Burkholderia glumae complex (Bgc). Bcc includes B. cepacia, B. cenocepacia, B. ambifaria, B. contaminans, B. multivorans, B. stabilis, and B. vietnamiensis; Bpc includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, and B. singularis; and Bgc includes B. glumae, B. gladioli, and B. plantarii [2].

Although some members of the genus Burkholderia sensu stricto show biotechnological potentials of plant growth promotion, biocontrol, antibiotic production, biodegradation, and bioremediation, major members are pathogens to human, animals, and plants. Bcc members, such as B. cenocepacia and B. multivorans, are well-known pathogens that cause chronic pulmonary infections in cystic fibrosis (CF) [3,4]. Bpc members, such as B. pseudomallei, are etiological agents of melioidosis, a potentially fatal disease endemic to tropical regions [5]. Bgc members B. glumae and B. gladioli cause bacterial panicle blight (BPB) in rice, while B. plantarii causes rice seedling blight and grain rot [2]. Of particular concern to Bgc is their potential to be opportunistic human pathogens to various immunocompromised populations [6,7,8,9].

The genus Burkholderia sensu stricto is characterized by large and complex genomes, typically comprising multiple replicons (chromosomes and plasmids) that encode extensive repertoires of genes for environmental adaptation and metabolic plasticity [10]. The large and complex genomes facilitate environmental adaptation, allowing for the colonization of diverse niches, including soil ecosystems, aquatic environments, plant rhizospheres, and even intracellular compartments of eukaryotic hosts [11]. This genomic architecture also facilitates high levels of antibiotic resistance, posing serious challenges for both clinical treatment and agricultural disease control [11,12]. Of particular concern is the increasing prevalence of multidrug-resistant Burkholderia strains in clinical settings where therapeutic options are severely constrained [13]. In agriculture, overreliance on chemical pesticides has further driven resistance and raised environmental hazards. These challenges have spurred interest in alternative approaches, notably phage therapy [14].

Phages, also known as bacteriophages, are viruses that specifically infect and lyse bacteria, offering a targeted biocontrol strategy against Burkholderia infections. Phage action involves specific recognition of bacterial surface receptors, the injection of viral genetic material, and hijacking of the host’s cellular machinery for replication. This process culminates in cell lysis, releasing new phages to continue the cycle [15]. Due to the high specificity, phages offer an alternative to conventional antibiotics, particularly for managing multidrug-resistant strains. In clinical settings, phages have shown efficacy against multidrug-resistant strains where antibiotics fail [15,16,17,18,19]. For example, phage C34 targeting B. pseudomallei significantly reduced bacterial load and improved survival rates in infected mice [17]. In agriculture, phage NBP4-7 and jumbo phage S13 reduced BPB severity in rice by targeting key virulence factors like flagella [20,21].

Phage therapy presents a promising cross-disciplinary solution for managing Burkholderia-induced diseases in humans, animals, and plants. The advance of phage therapy in human and veterinary medicine provides an adaptation strategy for plant disease management. In clinical settings, phages are administered through intravenous, oral, and topical routes with formulations optimized for stability and therapeutic efficacy [22]. In agriculture, phages are typically applied via foliar sprays, seed treatments, or soil drenches depending on the crops and pathogens [23,24]. However, phage application in cropland is vulnerable to environmental inactivation and degradation by high temperature, UV radiation, drought, agrochemicals, and soil absorption. Recent development in delivery technologies, particularly phage encapsulation in nanocarriers, enhances phage viability and site-specific release [25]. These delivery technologies can be translated to protect phages targeting Bgc members in cropland [26].

Here, we review the advances in research on Burkholderia phages, particularly on Burkholderia phage therapeutic potential across host systems. We highlight the delivery innovations and cross-application strategies that may enhance the integration of phage therapy into sustainable disease management programs.

2. Pathogenic Burkholderia Species

2.1. Human and Animal Pathogens

The genus Burkholderia sensu stricto includes pathogenic species that pose significant threats to human and animal health (Table S1). Among these, members of Bcc such as B. cepacia, B. multivorans, and B. cenocepacia are well-known opportunistic pathogens. They are most frequently isolated from individuals with CF and chronic granulomatous disease, where they are associated with severe respiratory infections, including necrotizing pneumonia and septicemia [27,28]. B. dolosa and B. anthina have been linked to accelerated pulmonary decline and chronic obstructive pulmonary diseases, respectively [29,30]. Beyond respiratory infections, Bcc contributes to bloodstream infections, wound contaminations, and sepsis. B. stabilis and B. contaminans have been associated with nosocomial infections and bacteremia, posing challenges in hospital settings [31,32,33,34]. B. pseudomultivorans was first isolated from clinical CF sputum and rhizosphere soil [35]. Recently, B. pseudomultivorans was identified as the cause of sepsis in cats, suggesting zoonotic potential [36].

Within Bpc, B. pseudomallei causes melioidosis, a severe zoonotic disease endemic to Southeast Asia and northern Australia. It infects a broad-host range including humans, domestic animals, wildlife, and pets. Clinical presentations include pneumonia, sepsis, abscesses, and chronic infections [37,38]. B. mallei causes glanders, a zoonotic disease primarily affecting horses, donkeys, and mules. B. mallei was weaponized during World War I due to its high infectivity [39,40,41]. B. thailandensis is less virulent and typically causes opportunistic infections in immunocompromised individuals [42,43,44].

Members of the Bcc possess multiple virulence factors (Table 1) including biofilm formation, motility, quorum sensing, pili, LPS variation, secretion systems, and extracellular enzymes that enable colonization and immune evasion. They exhibit intrinsic resistance mechanisms such as efflux pumps, β-lactamases, low membrane permeability, modified LPS, and polymyxin resistance in some species [45,46,47,48,49,50,51]. Similarly, Bpc species like B. pseudomallei display virulence traits including biofilm formation, motility, intracellular survival, capsular polysaccharide, quorum sensing, diverse secretion systems (Type III, V, VI), and adhesins [14,52,53,54]. Both groups share resistance features, including multidrug efflux pumps and β-lactam resistance. Effective management typically requires carbapenems or β-lactam/β-lactamase inhibitor combinations. However, persistent infections and relapse are common due to biofilm formation and adaptive resistance [14].

2.2. Plant Pathogens

Several Bgc members (B. glumae, B. gladioli, and B. plantarii) and Bcc members (B. cepacia, B. orbicola, B. semiarida and B. sola) are recognized as plant pathogens, causing substantial agricultural losses (Table S1). B. glumae is a major pathogen responsible for BPB in rice, causing symptoms like aborted seeds, empty grains, and seedling rot, significantly reducing rice yield [55,56]. B. glumae also infects other crops such as pepper, eggplant, tomato, sesame, and perilla [50]. Notably, B. glumae has also been isolated from human clinical cases, indicating its potential for cross-kingdom pathogenicity [6,57]. Likewise, B. gladioli infects rice and a variety of other crops, causing grain rot and seedling blight. B. gladioli also acts as an opportunistic human pathogen, causing bacteremia, pneumonia, and lung infections in CF patients [58,59,60,61]. B. plantarii primarily infects rice, leading to seedling blight, grain rot, chlorosis, and stunting [62,63]. B. cepacia causes bulb rot in onions [64]. B. orbicola reduces bean seed germination and impairs insect survival [59]. B. semiarida and B. sola are associated with onion sour skin disease [65,66]. These pathogens exhibit biofilm formation, motility (flagella), quorum sensing systems, and secretion systems (including type III), and produce toxins like toxoflavin and extracellular enzymes that facilitate host tissue colonization and damage [67,68,69,70,71,72]. They possess resistance to conventional control measures, making them agriculturally significant threats.

Table 1.

Comparative summary of major pathogenic Burkholderia.

Species	Host Range	Key Virulence Factor	Resistance Trait	Zoonotic Risk	Reference
B. cepacia	Humans, occasionally animals, plants	Biofilm formation, motility, pili, lipopolysaccharide variation, quorum sensing (QS), extracellular enzymes	Efflux pumps, β-lactamases, low permeability, modified lipopolysaccharide	Opportunistic zoonotic risk	[45,46]
B. multivorans	Humans (CF)	Biofilm formation, motility, cable pili, QS-controlled virulence	Aminoglycoside, β-lactam resistance, efflux pumps, polymyxin resistance	No known zoonotic transmission	[14,46]
B. cenocepacia	Humans (CF, immunocompromised)	Biofilm formation, motility, QS-regulated proteases, cable pili, secretion systems, siderophore production	Efflux pumps, β-lactamases, polymyxin resistance	Potential zoonotic pathogen	[14,46,47,48]
B. dolosa	Humans (CF)	Biofilm and capsule formation, motility, adhesins and proteases, secretion systems	Extensive multidrug resistance, multiple efflux pumps, β-lactamases	No known zoonotic transmission	[49,50]
B. contaminans	Humans (nosocomial)	Biofilm formation, motility, hemolysins, antifungal activity, secretion systems	β-lactams, disinfectants, efflux pumps	Potential zoonotic pathogen	[33,46,51]
B. pseudomallei	Humans and animals	Biofilm formation, motility, intracellular survival, polysaccharides, QS, secretion systems, immune evasion	Aminoglycosides, macrolides, β-lactamases, efflux pumps, polymyxin resistance	Confirmed zoonotic agent	[14,52,53,54]
B. mallei	Equids, zoonotic to humans	Biofilm formation, motility, secretion systems, immune evasion, novel virulence proteins, modulation of ubiquitination, actin-cytoskeleton rearrangement	Aminoglycosides, β-lactams, efflux pumps	Confirmed zoonotic agent	[52,73,74,75]
B. thailandensis	Environment, immunocompromised hosts	Biofilm formation, motility, attenuated virulence, secretion systems, QS, siderophore (malleobactin) production	Limited resistance, efflux pumps, β-lactamases	Opportunistic zoonotic risk	[76,77,78]
B. glumae	Plants, rare human cases	Biofilm formation, motility, toxoflavin, lipase, QS, flagella, extracellular polysaccharides, lipase, secretion systems	Multidrug resistance, efflux pumps, β-lactamases	No known zoonotic transmission	[55,67,68,69]
B. gladioli	Plants, humans (CF, immunocompromised)	Biofilm formation, protein secretion systems (T2SS, T3SS), motility, proteases, toxoflavin, QS	β-lactams, aminoglycosides, multidrug efflux	Potential zoonotic pathogen	[70,71,72]

3. Characterization of Burkholderia Phages

3.1. Isolation

Burkholderia phages have been isolated from a wide range of environmental samples, including soil, water, plant tissues, compost, and clinical settings. Common isolation methods involve enrichment using selective media and plaque assays, where samples are mixed with host bacterial strains and plated onto agar to identify lytic and lysogenic phages through plaque formation [79,80]. For instance, Jungkhun et al. isolated 61 phages using direct plating and plaque assays, selecting NBP1-1, NBP4-7, and NBP4-8 as effective lytic agents against B. glumae [20]. Adachi et al. used filtration and ultracentrifugation to isolate phages BGPP-Ar, BGPP-Sa, and BGPP-Ya from water and puddles, demonstrating their potential for controlling bacterial seedling blight in rice [81]. Kanaizuka et al. obtained jumbo phages FLC8, FLC9, and FLC10 from fallen leaf compost, highlighting the natural abundance of Burkholderia phages in decaying plant material [82]. Jumbo phages Chiangavirus FLC6 and FLC8 infecting B. glumae were isolated from rice fields and compost samples [82,83]. Lessievirus BcepIL02 and Aptresvirus vB_BceM_AP3 infecting B. cenocepacia were obtained from soil sample planted with corns and irrigated fields [84,85]. These diverse isolations highlight the natural abundance and ecological adaptability of Burkholderia phages.

3.2. Morphology

Most Burkholderia phages possess icosahedral heads and exhibit either contractile or non-contractile (long or short) tails, morphologically classified into the families Myoviridae, Podoviridae, and Siphoviridae. For examples, jumbo Burkholderia phage FLC6 and non-jumbo Burkholderia phages NBP1-1, NBP4-7, and NBP4-8 infecting B. glumae possess icosahedral heads and contractile tails typical of the family Myoviridae [20,83]; Burkholderia phage Bp-AMP1 infecting B. pseudomallei has an icosahedral capsid and a short non-contractile tail typical of the family Podoviridae; Burkholderia phages phiE125 and phi1026b targeting B. mallei are characterized by icosahedral heads and long non-contractile tails typical of the family Siphoviridae [86,87,88]. Morphology-based phage classification depends on transmission electron microscopy to visualize phage particles and determine phage particle size, shape, and structural features [89].

3.3. Life Cycle

Burkholderia phages possess lytic or lysogenic life cycles (Table 2), impacting their use in phage therapy and biocontrol. Lytic Burkholderia phages hijack the host cellular machinery to replicate and lyse bacterial cells and are effective against pathogenic Burkholderia. For example, the jumbo phage Chiangavirus FLC6 shows strong lytic activity against B. glumae, B. plantarii, and even Ralstonia pseudosolanacearum, indicating broad-host range and cross-genus infectivity [83]. While promising, this broad-host range requires further validation through in vivo studies and testing against diverse environmental isolates, as current evidence are mainly derived from in vitro assays. Lysogenic Burkholderia phages integrate their genome into the host genome as prophages [90,91]. This lysogenic conversion drives horizontal gene transfer and phage–host co-evolution, where integrated phage genes may enhance bacterial virulence, stress tolerance, or antibiotic resistance. Most characterized Burkholderia phages within the family Peduoviridae, such as Kisquattuordecimvirus KS14, Kisquinquevirus KS5, and Tigrvirus phiE202, are lysogenic (Table 2). Interestingly, Ampunavirus phage Bp-AMP1 has a temperature-dependent life cycle, remaining lysogenic at 25 °C but switching to a lytic cycle at 37 °C [92,93]. This thermally controlled behavior suggests its potential in temperature-regulated phage therapies. Although temperate phages have limited direct therapeutic use, synthetic biology allows for conversion into obligate lytic forms by disrupting lysogeny-related genes (e.g., integrases, repressors), expanding their clinical and agricultural applications [94]. Overall, lytic Burkholderia phages with broad-host ranges are promising for phage therapy. Temperate Burkholderia phages may require genetic modification or lytic derivative selection to ensure therapeutic safety and efficacy.

3.4. Host Range and Specificity

Burkholderia phages exhibit diverse host specificities, ranging from narrow-host to broad-host. Narrow-host phages infect very limited strains within one species, such as Kayeltresvirus KL3 infecting only B. ambifaria LMG 17828, and temperate phages KS4 and KS9 infecting only two out of 24 tested Bcc strains. In contrast, broad-host phages can infect multiple bacterial species even genera, such as the jumbo phage FLC6, which can lyse multiple strains of B. glumae, B. plantarii, and Ralstonia pseudosolanacearum [83,90,95]. This specificity is primarily governed by tail fiber proteins (TFPs), which mediate phage–host interactions by recognizing bacterial surface receptors such as lipopolysaccharides (LPSs) and outer membrane proteins [96]. Variations in TFPs, including C-terminal extensions and single amino acid mutations, significantly impact host range [97]. Structural and genetic modifications in TFPs play a critical role in host adaptation. For example, Burkholderia phage AP3 possesses a unique 365-amino-acid C-terminal extension in its TFP that enhances its specificity for B. cenocepacia IIIA LPS variants, contributing to its narrow-host range [85]. Furthermore, engineered chimeric phages, such as Pseudomonas aeruginosa phage PaP1-rec1, acquire expanded host ranges through tail fiber gene swaps, demonstrating the potential of genetic modifications in customizing phage infectivity [98]. Recent advances, such as targeted point mutations (e.g., G→C in Acinetobacter phage Abp4-M) [99] and domain swapping (e.g., STyj5-1 with BD13 tail fibers) [100], have further expanded host ranges while maintaining adsorption efficiency. A rational therapeutic approach could involve phage cocktails, combining highly specific phages with engineered broad-range variants to balance efficacy and safety in treating multidrug-resistant infections.

3.5. Genomic Taxonomy

Bacteriophage taxonomy has evolved from a discipline based mainly on morphology to genome [101]. The morphology-based families Myoviridae, Podoviridae, and Siphoviridae were abolished and the order Caudovirales was replaced by the class Caudoviricetes to group all tailed bacterial and archaeal viruses with icosahedral capsids and a double-stranded DNA genome [102]. The advances of next-generation sequencing techniques promote the genome-based classification to generate a more accurate evolutionary framework and better reflection of the diversity and phylogeny of the abundant and diverse viruses, and establishment of new genome-based taxa recognized by the International Committee on Taxonomy of Viruses (ICTV) (Figure S1). Nowadays, ICTV uses a holistic approach to classify prokaryote viruses by considering morphotype, host, lifestyle, genome characteristics (such as size, mol% G + C), % protein homologs, overall DNA and protein similarity, and phylogeny based on core genes [101]. Prokaryote viruses belonging to the same taxonomy rank form a cohesive and monophyletic group. Two phages are assigned to the same species if their genomes are more than 95% identical at the nucleotide level over their full genome length, while 70% of nucleotide identity of the full genome length is the cut-off for genera [101]. Members of a viral family share a significant number of orthologous genes, forming a cohesive and monophyletic group based on common proteomes. The sequencing and analyzing of phage genomes revealed a much higher genomic diversity than had previously been considered, leaving a significant fraction of sequenced phages unclassified at the family level [101].

Almost all Burkholderia phages whose whole-genome sequences are deposited in the GenBank database of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) (accessed on 25 February 2025) belong to the class Caudoviricetes, except for Alphatectivirus BCE1, which belongs to the class Tectiliviricetes (Table 2). Another distinguished feature of Alphatectivirus BCE1 is the smallest genome size of 14,800 bp. Based on the genome size, Burkholderia phages belonging to the class Caudoviricetes are divided into two groups: jumbo Burkholderia phages and non-jumbo Burkholderia phages (Table 2, Figure 1). The genome size of the jumbo Burkholderia phages ranges from 225,545 bp to 321,833 bp. The genome size of the non-jumbo Burkholderia phages ranges from 32,090 bp to 72,415 bp.

Table 2.

Information of Burkholderia phages.

Phage Name	Morphotype	ICTV Taxonomy (Class > Order > Family > Genus)	Host	Lifestyle	GC Content (%)	Genome Length (bp)	Reference
BCE1	/	Tectiliviricetes > Kalamavirales > Tectiviridae > Alphatectivirus	B. cepacia	/	48.21	14,800	[103]
		Class Caudoviricetes
FLC6	Myovirus	Chimalliviridae > Chiangmaivirus	B. glumae; B. plantarii; Ralstonia pseudosolanacearum	Lytic	52.01	227,105	[83]
FLC8	Myovirus	Chimalliviridae > Chiangmaivirus	B. glumae; B. plantarii	Lytic	52.05	225,545	[82]
S13	Myovirus	Chimalliviridae > Chiangmaivirus	B. glumae; B. gladioli; B. multivorans; B. cenocepacia; B. dolosa;	Lytic	51.7	227,647	[21]
FLC9	Myovirus	Novel species 16 within a novel genus 8 *	B. glumae; B. plantarii	/	55.97	321,833	[82]
BcepSauron	Myovirus	Sarumanvirus	B. cenocepacia	Lytic	58.10	262,653	[104]
BcepSaruman	Myovirus	Sarumanvirus	B. cenocepacia	Unknown	58.14	263,735	/
BCSR5	Myovirus	Novel species 4 within a novel genus 2 *	B. cepacia	/	54.74	227,351	[105]
KL1	Siphovirus	Jondennisvirinae > Kilunavirus	B. cenocepacia	Lytic	54.61	42,832	[106]
BcepGomr	/	Novel species 7 within a novel genus 3 *	Burkholderia	Unknown	56.29	52,414	[106]
Bp-AMP2	Podovirus	Autographivirales > Autonotataviridae > Ampunavirus	B. pseudomallei	/	61.76	42,492	[92]
Bp-AMP1	Podovirus	Autographivirales > Autonotataviridae > Ampunavirus	B. pseudomallei; B. thaliandensis	Temperate	61.75	42,409	[92,93]
Bp AMP4	Podovirus	Autographivirales > Autonotataviridae > Ampunavirus	B. pseudomallei	/	61.79	42,112	[92]
Bp AMP3	Podovirus	Autographivirales > Autonotataviridae > Ampunavirus	B. pseudomallei	/	61.77	41,882	[92]
JG068	Podovirus	Autographivirales > Autonotataviridae > Mguuvirus	B. multivorans; B. cenocepacia; B. stabilis; B. dolosa	Lytic	60.69	41,604	[107]
Paku	/	Autographivirales > Autonotataviridae > Pakuvirus	B. cenocepacia	Temperate	61.86	42,727	[107]
Maja	Myovirus	Lindbergviridae > Gladiolivirus	B. gladioli	Temperate	54.50	68,393	[108]
BcepF1	Myovirus	Lindbergviridae > Bcepfunavirus	B. ambifaria	/	55.89	72,415	[106,109]
BCSR52	Myovirus	Lindbergviridae > Irusalimvirus	B. cepacia	/	51.45	70,038	/
WTB	Myovirus	Bglawtbvirus	B. gladiol i	Lytic	60.04	68,541	[110]
BCSR129	Myovirus	Novel species 10 within a novel genus 5 *	B. cepacia	Unknown	58.42	66,147	[105]
BcepB1A	Myovirus	Novel species 2 within a novel genus 1 *	B. cenocepacia	Lytic	54.45	47,399	[106]
BcepNazgul	Siphovirus	Casjensviridae > Nazgulvirus	B. cepacia	Lytic	60.64	57,455	[111]
AH2	Siphovirus	Casjensviridae > Ahduovirus	B. cenocepacia; B. gladioli	Lytic	61.31	58,065	[106,112]
PhiE255	Myovirus	Bcepmuvirus	B. thailandensis	Temperate	63.05	37,446	[91]
BcepMu	Myovirus	Bcepmuvirus	B. cenocepacia	Temperate	62.86	36,748	[18]
KS10	Myovirus	Novel species 25 within a novel genus 10 *	B. cenocepacia; B. stabilis; B. ambifaria	Temperate	62.87	37,635	[113]
phiX216	Myovirus	Peduoviridae > Tigrvirus	B. pseudomallei; B. mallei	Temperate	64.82	37,637	[114]
phi52237	Myovirus	Peduoviridae > Tigrvirus	B. pseudomallei	Temperate	64.82	37,639	[91]
BEK	Myovirus	Peduoviridae > Tigrvirus	B. pseudomallei	/	68.82	37,631	[85]
phiE202	Myovirus	Peduoviridae > Tigrvirus	B. mallei; B. pseudomallei	Temperate	65.43	35,741	[91]
phiE094	Myovirus	Peduoviridae > Tigrvirus	B. thailandensis; B. pseudomallei	Temperate	64.48	37,727	[115]
NBP1-1	Myovirus	Peduoviridae > Tigrvirus	B. glumae	Lytic	63.23	40,570	[20]
NBP4-7	Myovirus	Peduoviridae > Tigrvirus	B. glumae	Lytic	63.23	40,563	[20]
NBP4-8	Myovirus	Peduoviridae > Tigrvirus	B. glumae	Lytic	63.23	40,568	[20]
KL3	Myovirus	Peduoviridae > Kayeltresvirus	B. ambifaria	Temperate	63.23	40,555	[90]
PK23	Myovirus	Peduoviridae > Duodecimduovirus	B. pseudomallei	Temperate	65.12	35,343	[116]
phiE12_2	Myovirus	Peduoviridae > Duodecimduovirus	B. mallei	Temperate	64.62	36,690	[91]
FLC10	Myovirus	Peduoviridae > Kisquattuordecimvirus	B. glumae	Lytic	61.29	32,867	[82]
FLC5	Myovirus	Peduoviridae > Kisquattuordecimvirus	B. glumae; B. plantarii	Temperate	61.79	32,090	[117]
KS14	Myovirus	Peduoviridae > Kisquattuordecimvirus	B. multivorans; B. cenocepacia; B. dolosa; B. ambifaria	Temperate	62.28	32,317	[90]
vB BceM AP3	Myovirus	Peduoviridae > Aptresvirus	B. cenocepacia	Temperate	64.04	36,499	[85]
Mana	Myovirus	Peduoviridae > Aptresvirus	B. gladioli	/	64.31	38,038	[118]
KS5	Myovirus	Peduoviridae > Kisquinquevirus	B. multivorans; B. cenocepacia	Temperate	63.71	37,236	[90]
ST79	Myovirus	Peduoviridae > Nampongvirus	B. pseudomallei; B. mallei	Lytic	62.50	35,430	[119]
BcepMigl	Podovirus	Lessievirus	B. cenocepacia	/	65.51	62,952	/
Bcep22	Podovirus	Lessievirus	B. cenocepacia	Temperate	65.31	63,882	[84]
DC1	Podovirus	Lessievirus	B. cepacia; B. cenocepacia; B. stabilis	Temperate, unstably lysogenic	66.21	61,847	[120]
BcepIL02	Podovirus	Lessievirus	B. cenocepacia	Temperate	66.20	62,715	[84]
Mica	Myovirus	Micavirus	B. cenocepacia	Temperate	62.15	43,707	[121]
Bcep781	Myovirus	Naesvirus	B. cepacia	Lytic	63.33	48,247	[122]
Bcep43	Myovirus	Naesvirus	B. cepacia	Lytic	63.43	48,024	[122]
BcepNY3	/	Naesvirus	B. cenocepacia	/	63.64	47,382	/
Bcep1	Myovirus	Naesvirus	B. cenocepacia	Lytic	63.64	48,177	[122]
phiE058	Myovirus	Novel species 40 within a novel genus 16 *	B. mallei; B. pseudomallei; B. thailandensis	Temperate	64.12	44,121	[123]
PE067	Myovirus	Novel species 39 within a novel genus 16 *	B. pseudomallei; B. thailandensis	Temperate	64.48	43,649	[123]
BcepC6B	Podovirus	Ryyoungvirus	B. cepacia	Temperate	65.19	42,415	[122]
vB BmuP KL4	/	Kelquatrovirus	B. multivorans	/	63.18	42,250	/
Magia	Myovirus	Magiavirus	B. cenocepacia	Temperate	65.06	44,942	[124]
phiE125	Siphovirus	Stanholtvirus	B. mallei	Temperate	61.19	53,373	[86]
Phi644_2	Siphovirus	Stanholtvirus	B. mallei; B. pseudomallei	Temperate	60.45	48,674	[91]
PhiBP82.1	/	Stanholtvirus	B. pseudomallei	/	60.68	54,921	/
Phi1026b	Siphovirus	Stanholtvirus	B. mallei; B. pseudomallei	Temperate	60.68	54,865	[87]
phiBt	/	Stanholtvirus	B. pseudomallei	/	60.30	56,453	/
Bcep176	Siphovirus	Stanholtvirus	B. multivorans; B. cepacia	Temperate	61.54	44,856	[125]
KS9	Siphovirus	Stanholtvirus	B. pyrrocinia; B. cenocepacia	Temperate	60.68	39,896	[18,126]

* Genomic classification by VICTOR [127] in Figure 1; / data unavailable.

Sixty-one whole-genome sequences of Burkholderia phages within the class Caudoviricetes were used to generate a phylogenomic tree using the Genome BLAST Distance Phylogeny (GBDP) method implemented in VICTOR (https://ggdc.dsmz.de/victor.php) (accessed on 26 February 2025) [127], allowing genome-based classification. The 61 Burkholderia phages are classified into 54 species, 21 genera, and 3 families (Figure 1). Family 1 includes the jumbo Burkholderia phages Chiangmaivirus FLC6 and FLC8 within the ICTV family Chimalliviridae and an unclassified genus (phage FLC9), which infect B. glumae. Family 2 includes the jumbo Burkholderia phage Sarumanvirus infecting B. cenocepacia and an unclassified genus (phage BCSR5). Family 3 includes all non-jumbo Burkholderia phages belonging to 17 genomogenera, among which 8 genera were classified into 5 existing ICTV families (Jondennisvirinae, Autonotataviridae, Lindbergviridae, Casjensviridae, and Peduoviridae). In other words, one genome-based family contains all non-jumbo Burkholderia phages within the class Caudoviricetes, indicating a limited taxon range of the non-jumbo Burkholderia phages. Nonetheless, the G + C mol% of these non-jumbo Burkholderia phages ranging from 51.45% to 68.82% indicates considerable genomic diversities within the genome-based Family 3. Together, this phylogenomic overview highlights both evolutionary divergence and taxonomic coherence among Burkholderia phages.

The VICTOR phylogenomic overview (Figure 1) also shows the host ranges at three phage taxon levels. First, the host range of the Burkholderia phages within Family 3 covers the genus Burkholderia sensu stricto. Second, multiple Burkholderia phages within a virus species infects only one Burkholderia species. For example, four Burkholderia phages within Ampunavirus BpAMP1 infect B. pseudomallei; two Burkholderia phages within Stanholtvirus sv1026b infect B. pseudomallei; three Burkholderia phages within Tigrvirus phi52237 infect B. pseudomallei; and Naesvirus Bcep781 and Naesvirus Bcep43 composing a genomospecies infect B. cepacia. Third, multiple virus species within multiple genera can infect the same Burkholderia species. As just noted, Ampunavirus BpAMP1, Stanholtvirus sv1026b, and Tigrvirus phi52237 infect B. pseudomallei. Fourth, multiple virus genera can infect the same multiple species within a species complex. For example, Lessievirus and Naesvirus infect Bcc species B. cepacia and B. cenocepacia; Nazgulvirus BcepNazgul and Ahduovirus AH2 composing a genomogenus also infect B. cepacia and B. cenocepacia. Fifth, a virus genus can infect multiple Burkholderia species within multiple species complexes. For example, Bcepmuvirus infects B. thailandensis (Bpc) and B. cenocepacia (Bcc); Ampunavirus BpAMP1, Pakuvirus paku, and Mguuvirus JG068 composing a genomogenus infect B. pseudomallei (Bpc) and B. cenocepacia (Bcc); Gladiolivirus Maja, Bcepfunavirus BcepF1, Irusalimvirus BCSR52, and Bglawtbvirus WTB composing a genomogenus infect B. cepacia (Bcc), B. ambifaria (Bcc), and B. gladioli (Bgc). Tigrvirus, Kayeltresvirus, Duodecimduovirus, Kisquattuordecimvirus, Aptresvirus, Kisquinquevirus, and Nampongvirus composing a genomogenus infect B. pseudomallei (Bpc), B. thailandensis (Bpc), B. cenocepacia (Bcc), B. ambifaria (Bcc), B. glumae (Bgc), and B. gladioli (Bgc).

Together, the genetic variability of the Burkholderia phages holds significant promise for both medical and agricultural applications. The multiple virus species or genera targeting the same Burkholderia species or species complex supports the strategy of using phage cocktails to control the Burkholderia-associated human, animal, or plant diseases. The phage cocktails containing diverse Burkholderia phages may use multiple mechanisms to control Burkholderia pathogens and to avoid immune escape by the Burkholderia pathogens.

Figure 1.

Phylogenomic relationships among Burkholderia phages within the class Caudoviricetes. The balanced minimum-evolution tree inferred from intergenomic distances based on whole-genome sequence comparisons was generated using the Genome-BLAST Distance Phylogeny (GBDP) method implemented in VICTOR [127]. Branch support was inferred from 100 pseudo-bootstrap replicates via FASTME including SPR postprocessing [128]. Taxon boundaries at the species, genus, and family level were estimated with the OPTSIL program [129], the recommended clustering thresholds [127], and an F value (fraction of links required for cluster fusion) of 0.5 [130]. The branch lengths are scaled in terms of the GBDP distance formula d0. The tree was rooted at the jumbo phages and displayed using the online tool iTOL version 7 (https://itol.embl.de/) (accessed on 16 June 2025). Tree leaves were labeled with phage names (host) [nucleotide sequence accession numbers in GenBank] and genomic classification of phages into species, genus, and family. Phage host species within Burkholderia cepacia complex, Burkholderia pseudomallei complex, and Burkholderia glumae complex are highlighted in blue, green, and red, respectively.

4. Mechanism of Phage Action and Burkholderia Resistance

Phages infect Burkholderia host cells by recognizing and adsorbing to surface receptors, primarily LPS on the outer membrane. LPS consists of Lipid A (anchored in the membrane and responsible for endotoxicity), a core oligosaccharide with conserved inner and variable outer regions, and a highly variable O-antigen polysaccharide chain [21]. Additional receptors include capsular polysaccharides, flagella, and fimbriae [116]. After injection of genomes, phages hijack bacterial machinery to replicate and produce holins (membrane pore-forming proteins) and endolysins (peptidoglycan-degrading enzymes), which disrupt the cell envelope, leading to lysis and release of progeny phages [15,131].

Phage predation drives bacterial resistance primarily through modifications or loss of receptors. However, bacterial surface components are also critical to bacterial survival, motility, or virulence. As a result, receptor modifications frequently incur fitness costs including increased susceptibility to host immune factors and antibiotics [132]. For example, B. cenocepacia mutants with truncated LPS exhibit phage resistance but compromise serum resistance and increase sensitivity to colistin [133]. Similarly, infection by phage Bp-AMP1 can downregulate efflux pumps in B. thailandensis, increasing bacterial sensitivity to a broad range of antibiotics [134]. Beyond receptor modifications, Burkholderia has additional phage defense mechanisms, including excessive production of extracellular polysaccharides to physically shield receptors, activation of CRISPR-Cas systems to destroy invading phage genomes, and abortive infection systems that trigger programmed cell death to prevent phage propagation. However, these strategies also incur fitness trade-offs: overproduction of extracellular polysaccharide, reducing motility and nutrient uptake; CRISPR-Cas systems requiring metabolic resources and carrying a risk of autoimmunity; and abortive infection sacrificing the survival of individual cells [135,136]. These fitness trade-offs form the foundation of “phage steering” [133]. Strategically, phage–antibiotic synergy and phage therapy using phage cocktails can reduce bacterial resistance development and improve therapeutic outcomes. Combining phages with antibiotics such as meropenem can enhance bacterial clearance, reduce antibiotic doses, and delay resistance development [137]. Phage cocktails targeting diverse bacterial receptors and using evolving phages through directed adaptation improves treatment efficacy and mitigates Burkholderia resistance development (Figure 2).

Figure 2.

Mechanisms and application of Burkholderia phages in control of human, animal, and plant diseases. (A) Mechanism and evolutionary trade-offs: The lytic cycle—from adsorption to lysis—eliminates bacteria cells. In response, bacteria evolve resistance mechanisms such as receptor modification and CRISPR-Cas systems, which incur fitness costs and can increase sensitivity to antibiotics. (B) Human therapy: Phages are administered via oral, inhalable, intravenous, and topical routes. Phages can be used with antibiotics and medical device coatings. (C) Veterinary use: Phages are delivered via oral, inhalable, intravenous, intramuscular and topical routes. (D) Agricultural application: Phages are applied via foliar sprays, soil treatments, or seed coatings to control plant diseases.

5. Biotechnological Applications of Burkholderia Phages

The success of phage therapy against Burkholderia infections relies heavily on the selection of appropriate delivery strategies that can overcome biological and environmental barriers across different systems: humans, animals, and plants.

5.1. Medical and Veterinary Applications

In human medicine, phages targeting Burkholderia species are primarily administered via inhalation or intravenous injection, tailored to infection sites. Aerosolized delivery, especially through nose-only inhalation devices, has demonstrated efficacy in murine models by significantly reducing lung bacterial loads caused by B. cenocepacia [18]. This method provides direct access to the respiratory tract, a common infection site in CF patients, and ensures phage viability post-aerosolization despite mechanical and pH stress [133]. In clinical cases, intravenous phage therapy, such as the administration of phage BdPF16phi4281, has been used compassionately to treat B. dolosa infections, resulting in temporary bacterial load reductions [138]. However, systemic administration poses risks of immune clearance and antibiotic-related toxicity, underscoring the need for improved delivery formulations.

In veterinary medicine, although Burkholderia-specific phages are yet to be tested, analogs targeting other pathogens like Salmonella have shown promising outcomes via oral and topical delivery in broilers [139]. These methods provide scalable, practical models for future adaptation to treat Burkholderia infections in livestock, especially for gastrointestinal or dermal infections.

5.2. Agricultural Applications

Phage-based biocontrol presents a sustainable alternative to chemical pesticides for managing Burkholderia-associated plant diseases. Effective deployment, however, requires consideration of environmental factors such as UV exposure, high temperature, desiccation, and phage persistence in the phyllosphere and rhizosphere [23]. Several Burkholderia-specific phages have shown potential in agricultural disease management. Phages KS12 and AH2, targeting B. gladioli, significantly reduce tissue destruction in onion and mushroom using a quantitative ex planta maceration model [112]. Phage WTB (vB_BglM_WTB), a high-efficiency lytic phage, also targets B. gladioli, offering rapid suppression of infections and potential for field deployment [110]. For B. glumae, a key pathogen of rice, the jumbo phage S13 demonstrates a unique flagella-dependent infection mechanism. By selecting for non-flagellated, less virulent mutants, S13 reduces pathogenicity while directly lysing motile bacterial populations [21]. Similarly, compost-derived jumbo phages FLC8 and FLC9 display broad-host ranges and have achieved over 77% control of rice seedling rot in greenhouse assays, while FLC10 exhibits narrower efficacy [82]. Application methods for these phages vary based on the plant–pathogen context. Foliar sprays, commonly used against epiphytic pathogens, are suitable for applying phages like KS12 and AH2 to aerial plant parts. However, foliar applications of phages are vulnerable to rapid UV inactivation; phage viability may drop below 1% within hours under sunlight [140]. To address this problem, formulations with UV-protective agents and humectants are being developed to enhance phage persistence on leaves. Soil drenching offers an effective alternative for root-associated infections by delivering phages like FLC8, FLC9, and FLC10 directly to the rhizosphere. This approach exploits phage mobility in moist soil, improving contact with root pathogens [141]. Additionally, seed coating with phages, particularly using polymer-based carriers, provides early-stage protection during germination and colonization of the rhizosphere, enhancing defense against soil-borne Burkholderia [142].

5.3. Nanotechnology-Enhanced Delivery

Nanotechnology-based delivery systems are increasingly used in phage therapy to enhance survival, targeting, and controlled release of phages [139,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157]. Alginate and chitosan nanocarriers, leveraging their pH-responsive properties, effectively protect phages during gastrointestinal transit while promoting mucosal adhesion. This makes them ideal for oral delivery in humans and animals, as they shield phages from gastric acidity and enable targeted intestinal release [143]. However, most studies remain at the in vitro or proof-of-concept stage, and more in vivo validation is required.

Hydrogel matrices, such as alginate–CaCO₃ microcapsules, provide sustained phage release and have demonstrated efficacy in veterinary models by maintaining anti-Salmonella activity in poultry [139]. Their adaptability suggests potential application for Burkholderia-specific phages targeting both respiratory and gastrointestinal infections, though direct evidence for these specific phages is limited to date.

Other nanocarrier systems, such as liposomes, polymeric nanoparticles, nanofibers, and whey protein isolate-based films, expand phage therapy’s utility in clinical and agricultural settings [144,145,146,147]. These systems improve phage stability, controlled release, and adhesion to biological or environmental surfaces. Liposome encapsulation, for instance, protects phages in respiratory infections but requires optimization to address immune clearance and limited systemic circulation [148]. Notably, whey protein isolate-based films, especially when reinforced with chitosan nanofibers or nano-chitin, form biodegradable and biocompatible matrices ideal for encapsulating phages [149,150]. These composite systems support long-term storage, pH-responsive release, and enhanced adhesion, making them promising candidates for bioactive seed coatings and durable phage packaging in agricultural applications (Table 3).

Table 3.

Nanotechnology-enhanced phage delivery strategies.

Nanocarrier Type	Mechanism/Function	Human/Veterinary Use	Potentials in Agriculture	Translational Insight	References
Alginate/Chitosan	pH-responsive protection; mucosal adhesion	Oral delivery in gastrointestinal infections	Seed coating or root-targeted release	Protect phages during transit through acidic environments: adaptable to rhizosphere targeting	[143]
Hydrogels like Alginate–CaCO3	Sustained, slow release over time	Poultry models for Salmonella control	Soil drenching or foliar application	Long-lasting effect under variable field conditions; ideal for crop protection	[139]
Liposomes	Encapsulation for enhanced penetration	Oral delivery for gastrointestinal infections	Not yet applied	Protect phages from acid and enzymes while enabling slow release	[145]
Polymeric nanoparticles	Precision targeting; immune evasion	Under development	Experimental in agriculture	Enhance nanoparticle uptake by plant cells through foliar spray or irrigation water delivery to plant tissues	[146]
Nanofibers	High surface area; controlled release and adhesion	Wound dressing, tissue scaffolds for drug delivery	Leaf surface coating or seed coating	Provide gradual phage release, enhance adhesion to plant surfaces, and improve stability	[147]
Whey protein isolate-based films	Biopolymer matrix for encapsulation; moisture barrier, and controlled release	Not yet applied clinically; explored for probiotic and drug delivery	Edible coating, seed wraps, and phage packaging for crops	Enhance phage storage stability and enable slow release. Integration with nanofibers, chitosan, or nano-chitin expands potential for agricultural delivery systems	[150,151]
DL-lactic-co-glycolic acid microspheres (PLGA)	Encapsulate lyophilized (freeze-dried) phages for controlled release and protection	Biocompatible and approved for human use like inhalable phage delivery	Foliar or root delivery; greenhouse applications	Biodegradable, biocompatible, and tunable degradation rates (via lactide/glycolide ratios) for sustained phage release in crops	[152,153,154]
Lactose/lactoferrin 60:40 (w/w)	Carrier matrix for dry powder phage formulations; enhance stability and dispersibility	Used in inhalable dry powder formulations for pulmonary phage therapy	Spray-dried phage powders for crop protection	Protect phages during drying and storage; potential for integration into foliar sprays	[155]

6. Conclusions and Perspectives

The convergence of Burkholderia pathogens infecting plants, animals, and humans highlights their significance within the One Health framework. Some Burkholderia species exhibit cross-kingdom infectivity and share resistance mechanisms, such as efflux pumps, quorum sensing-regulated virulence, and biofilm formation. The zoonotic potential of Bpc species and the increasing clinical detection of Bcc strains from environmental and animal reservoirs emphasize the interconnectedness of ecosystems [5,45].

Lytic Burkholderia phages offer the foundation for phage therapy targeting Burkholderia-associated diseases in humans, animals, and plants. Genetically distinct Burkholderia phages belonging to different genera or even different families target the same Burkholderia species or multiple Burkholderia species causing the same disease, providing nature resources for phage cocktails with reduced risk of immune escape and resistance emergence. While temperate phages may be used after modification, either through the selection of lytic derivatives or synthetic design, advances in synthetic biology allow for the engineering of phages with defined host ranges, enabling a balance between therapeutic efficacy and biosafety against multidrug-resistant infections. Moreover, phage–antibiotic synergy can also stimulate increased phage activity and reduce the risk of resistance emergence, making it a valuable complement to phage cocktail strategy and synthetic biology in combating multidrug-resistant Burkholderia infections.

Effective phage therapy requires targeted delivery, environmental stability, and sustained activity. Appropriate delivery strategies can overcome biological and environmental barriers specific to each Burkholderia–host system. Lessons from clinical and veterinary applications, such as mucoadhesive polymers for gastrointestinal use and liposome encapsulation for respiratory infections, can be adapted for agricultural purposes. Encapsulation methods like alginate microbeads and alginate/chitosan composites protect phages from environmental stress and allow for controlled pH-responsive release. These formulations are compatible with diverse agricultural delivery modes, including foliar sprays, soil drenches, and seed coatings. Moreover, spray-dried phage powders and electrospun nanofiber matrices enable the production of stable, field-ready products. The natural mucoadhesive and biodegradable properties of alginate and chitosan enhance phage targeting and prolong antibacterial activity. Whey protein isolate-based films, especially those reinforced with chitosan nanofibers or nano-chitins, offer biodegradable and biocompatible matrices for long-term storage and sustained bioactivity.

Together, these nanotechnology-enabled delivery systems bridge the gap between laboratory research and real-world implementation of phage therapy. The development of controlled host range phage cocktails, refined application-specific delivery systems, field trials, and regulatory frameworks holds promises for establishing robust, sustainable, and scalable phage-based biocontrol strategies across various Burkholderia–host systems. This integrated approach aligns closely with the One Health perspective, offering an eco-friendly alternative to antibiotics and chemical pesticides for managing Burkholderia-associated diseases in clinical and agricultural contexts.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13081873/s1, Table S1. Pathogenic Burkholderia species. Figure S1. Comparison of morphology-based and genome-based phage classification. Conventional morphology-based phage taxonomy classifies phages by capsid shape and tail type using electron microscopy. Modern genome-based phage taxonomy classifies phages by genome sequencing, overall DNA and protein similarity, and phylogenetic analyses based on core genes and proteins. This transition improves classification in capturing phage diversity and evolutionary relationships. References [158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177] are cited in the Supplementary Materials.

Author Contributions

Conceptualization, B.W., D.D., Y.W., J.B., J.L., B.L. and Q.A.; methodology, B.W. and J.Z.; validation, B.W. and Q.A.; formal analysis, B.W. and J.Z.; resources, L.C., J.B. and J.L.; writing—original draft preparation, B.W., J.Z., L.C., M.I. and C.L.; writing—review and editing, B.L., J.L. and Q.A.; visualization, B.W., M.I. and Q.A.; supervision, B.L. and Q.A.; project administration, L.C., J.B., D.D., Y.W. and J.L.; funding acquisition, D.D., Y.W., J.B., B.L. and J.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the Ningbo Natural Science Foundation (2022J197), the Shanghai Agricultural Science and Technology Innovation Project (T2023101), and Industrial Technology Projects of Department of Agriculture and Rural Affairs of Zhejiang Province (2025-5) of China.