Magnetically modified bacteriophage-triggered ATP release activated EXPAR-CRISPR/Cas14a system for visual detection of Burkholderia pseudomallei.

Burkholderia pseudomallei, widely distributed in tropical and subtropical ecosystems, is capable of causing the fatal zoonotic disease melioidosis and exhibiting a global trend of dissemination. Rapid and sensitive detection of B. pseudomallei is essential for environmental monitoring as well as infection control. Here, we developed an innovative biosensor for quantitatively detecting B. pseudomallei relies on ATP released triggered by bacteriophage-induced bacteria lysis. The lytic bacteriophage vB_BpP_HN01, with high specificity, is employed alongside magnetic nanoparticles assembly to create a biological receptor, facilitating the capture and enrichment of viable target bacteria. Following a brief extraction and incubation process, the captured target undergoes rapid lysis to release contents including ATP. The EXPAR-CRISPR cascade reaction provides an efficient signal transduction and dual amplification module that allowing the generated ATP to guide the signal output as an activator, ultimately converting the target bacterial amount into a detectable fluorescence signal. The proposed bacteriophage affinity strategy exhibited superior performance for B. pseudomallei detection with a dynamic range from 10^2 to 10^7 CFU mL-1, and a LOD of 45 CFU mL-1 within 80 min. Moreover, with the output signal compatible across various monitoring methods, this work offers a robust assurance for rapid diagnosis and on-site environmental monitoring of B. pseudomallei.

Rapid Clinical Screening of Burkholderia pseudomallei Colonies by a Bacteriophage Tail Fiber-Based Latex Agglutination Assay.

Melioidosis is a life-threatening disease in humans caused by the Gram-negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 h, a rapid diagnosis of melioidosis is critical for ensuring that an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and the whole genome was sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an area of melioidosis endemicity over 11 months. The 94TF-LAA took less than 5 min to produce positive agglutination, demonstrating 98% (95% confidence interval [CI] of 94.2% to 99.59%) sensitivity and 83% (95% CI of 75.64% to 88.35%) specificity compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen. IMPORTANCE Rapid diagnosis of melioidosis is essential for ensuring that optimal antibiotic courses are prescribed to patients and thus warrants the development of cost-effective and easy-to-use tests for implementation in underresourced areas such as northeastern Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust and easy to manufacture, with many tail fibers (such as 94TF investigated here) capable of targeting a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many areas where melioidosis is endemic.

Modelling the spatiotemporal complexity of interactions between pathogenic bacteria and a phage with a temperature-dependent life cycle switch.

We apply mathematical modelling to explore bacteria-phage interaction mediated by condition-dependent lysogeny, where the type of the phage infection cycle (lytic or lysogenic) is determined by the ambient temperature. In a natural environment, daily and seasonal variations of the temperature cause a frequent switch between the two infection scenarios, making the bacteria-phage interaction with condition-dependent lysogeny highly complex. As a case study, we explore the natural control of the pathogenic bacteria Burkholderia pseudomallei by its dominant phage. B. pseudomallei is the causative agent of melioidosis, which is among the most fatal diseases in Southeast Asia and across the world. We assess the spatial aspect of B. pseudomallei-phage interactions in soil, which has been so far overlooked in the literature, using the reaction-diffusion PDE-based framework with external forcing through daily and seasonal parameter variation. Through extensive computer simulations for realistic biological parameters, we obtain results suggesting that phages may regulate B. pseudomallei numbers across seasons in endemic areas, and that the abundance of highly pathogenic phage-free bacteria shows a clear annual cycle. The model predicts particularly dangerous soil layers characterised by high pathogen densities. Our findings can potentially help refine melioidosis prevention and monitoring practices.

Bacteriophage-associated genes responsible for the widely divergent phenotypes of variants of Burkholderia pseudomallei strain MSHR5848.

PURPOSE: Burkholderia pseudomallei, the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation. METHODOLOGY: Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics.Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R. CONCLUSION: Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei.

Temperature-dependent virus lifecycle choices may reveal and predict facets of the biology of opportunistic pathogenic bacteria.

Melioidosis, a serious illness caused by Burkholderia pseudomallei, results in up to 40% fatality in infected patients. The pathogen is found in tropical water and soil. Recent findings demonstrated that bacterial numbers can be regulated by a novel clade of phages that are abundant in soil and water. These phages differentially infect their bacterial hosts causing lysis at high temperatures and lysogeny at lower temperatures. Thus seasonal and daily temperature variations would cause switches in phage-bacteria interactions. We developed mathematical models using realistic parameters to explore the impact of phages on B. pseudomallei populations in the surface water of rice fields over time and under seasonally changing environmental conditions. Historical records were used to provide UV radiation levels and temperature for two Thailand provinces. The models predict seasonal variation of phage-free bacterial numbers correlates with the higher risk of melioidosis acquisition during the "warm and wet" season. We find that enrichment of the environment may lead to irregular large amplitude pulses of bacterial numbers that could significantly increase the probability of disease acquisition. Our results suggest that the phages may regulate B. pseudomallei populations throughout the seasons, and these data can potentially help improve the melioidosis prevention efforts in Southeast Asia.

Complete genome sequence of a novel lytic bacteriophage isolated from Ralstonia solanacearum.

A lytic podophage RSPI1 was isolated from tobacco field soil collected in Fujian Province, South China using host bacterium Ralstonia solanacearum Tb15-14. Whole genome sequencing of this phage was performed using the high-throughput Ion Torrent PGM Sequencer. The complete genome of RSPI1 was 43,211 bp in length with a mean DNA G + C content of 61.5%. A total of 48 open reading frames were identified with lengths ranging from 132 bp to 5,061 bp, of which, 11, 12 and 25 were identified as functional, structural and unknown genes, respectively. A BLAST analysis revealed that this phage genome had a query cover of 78-79% and a highest identity of 84% with four podophages that infect Burkholderia pseudomallei. Two neighbor-joining phylogenetic trees were constructed using phage DNA polymerase I and tail fiber protein sequences and showed that this phage is closely related to Burkholderia phage Bp-AMP1, and also related to several phages that infect Ralstonia solanacearum. These findings indicate that RSPI1 is a novel phage that infects the notorious plant pathogen Ralstonia solanacearum.

Identification of the Burkholderia pseudomallei bacteriophage ST79 lysis gene cassette.

AIMS: To identify and characterize the lysis gene cassette from the bacteriophage ST79 that lyses Burkholderia pseudomallei. METHODS AND RESULTS: Approximately 1·5 kb of ST79 lysis genes were identified from the phage genome data. It was composed of holin, peptidase M15A or endolysin, lysB and lysC. Each gene and its combinations were cloned into Escherichia coli and the lytic effects were measured. Co-expression of holin and peptidase M15A showed the highest lysis activity. Expression of holin, lysB/C or holin-peptidase M15A-lysB/lysC lysed the E. coli membrane, whereas peptidase M15A alone did not. The predicted transmembrane structures of holin and lysB/C indicated that they could be inserted into the bacterial membrane to form pores, affecting cell permeability and causing lysis. CONCLUSION: This is the first report of an investigation into the lysis genes of B. pseudomallei's lytic phage using E. coli as a model. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia pseudomallei, a Gram-negative bacterium causing an infectious disease, is intrinsically resistant to several antibiotics, and a vaccine is not available. The lysis genes of ST79, the first reported lytic bacteriophage of B. pseudomallei, were characterized. The development of ST79 as an alternative treatment for skin ulceration, for example, or to be used as a gene cloning tool for B. pseudomallei may be possible with this knowledge.

Melioidosis parotitis in children

A recent paper published in JVATiTD reporting a child in Hainan with parotitis caused by Burkholderia pseudomallei misleadingly described parotitis as a rare manifestation of melioidosis. In fact, it is one of the commonest forms of paediatric melioidosis seen in other parts of Southeast Asia, although interestingly not in Australia.

Pediatric suppurative parotitis caused by Burkholderia pseudomallei

Suppurative parotitis caused by Burkholderia pseudomallei has been rarely found outside endemic areas. Case presentation: Herein, we report the recovery of Burkholderia pseudomallei from the pus of a suppurative parotitis observed in a 12-year-old boy who lived in Hainan province, China. Specimens of necrotic tissue were collected and sections were stained with hematoxylin and eosin. Pus sample was also collected for bacteriological examination. The suppurative inflammation was observed in the necrotic tissue section and Burkholderia pseudomallei were detected in the sample. Conclusion: In this adolescent, Burkholderia pseudomallei infection was present in the parotid, which consists of the first report of this bacterium in a parotitis case acquired in China.

LYTIC CAPABILITY OF BACTERIOPHAGES (FAMILY MYOVIRIDAE) ON BURKHOLDERIA PSEUDOMALLEI.

Burkholderia pseudomallei, a gram-negative bacillus found in soil and water, is the causative agent of melioidosis. It can produce a biofilm, which increases resistance to antibacterial agents. Bacteriophages (phages) have been suggested as alternative antibacterial agents. In this study, the ability of six phages (family Myoviridae) to lyse B. pseudomallei isolates was examined using a microplate phage virulence assay. The six phages were more efficient in lysing soil than clinical B. pseudomallei isolates. Phage ST79 had the highest lytic capability, independent of inoculating phage quantity with a 4-log reduction of bacterial numbers after a 4 hour treatment. Three modified derivatives of ST79 were developed by multiple passages on phage-resistant B. pseudomallei isolates, leading to an increase in lytic capability from 62% to 80%. Phage ST79 at a multiplicity of infection (MOI) of 10 significantly reduces biofilm formation determined by a colorimetric method. The recovery of B. pseudomallei growth following phage treatment needs to be overcome if these lytic phages are to be used as biocontrol agents of B. pseudomallei in the environment.

Identification of a predicted trimeric autotransporter adhesin required for biofilm formation of Burkholderia pseudomallei.

The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity.

BACKGROUND: Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237. RESULTS: We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains. CONCLUSIONS: The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.

Isolation and characterization of a novel podovirus which infects burkholderia pseudomallei.

Burkholderia pseudomallei is a saprophytic soil bacterium and the etiological agent that causes melioidosis. It is naturally resistant to many antibiotics and therefore is difficult to treat. Bacteriophages may provide an alternative source of treatment. We have isolated and characterised the bacteriophage ΦBp-AMP1. The phage is a member of the Podoviridae family and has a genome size of ~ 45 Kb. Molecular data based on the gene which encodes for the phage tail tubular protein suggests that the phage is distinct from known phages but related to phages which infect B. thailandensis and Ralstonia spp. The phage ΦBp-AMP1 is the first B. pseudomallei podovirus to be isolated from the environment rather than being induced from a bacterial culture. It has a broad host range within B. pseudomallei and can infect all 11 strains that we tested it on but not related Burkholderia species. It is heat stable for 8 h at 50°C but not stable at 60°C. It may potentially be a useful tool to treat or diagnose B. pseudomallei infections as it can lyse several strains of clinical relevance.

Novel lytic bacteriophages from soil that lyse Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative saprophytic bacterium that causes severe sepsis with a high mortality rate in humans and a vaccine is not available. Bacteriophages are viruses of bacteria that are ubiquitous in nature. Several lysogenic phages of Burkholderia spp. have been found but information is scarce for lytic phages. Six phages, ST2, ST7, ST70, ST79, ST88 and ST96, which lyse B. pseudomallei, were isolated from soil in an endemic area. The phages belong to the Myoviridae family. The range of estimated genome sizes is 24.0-54.6 kb. Phages ST79 and ST96 lysed 71% and 67% of tested B. pseudomallei isolates and formed plaques on Burkholderia mallei but not other tested bacteria, with the exception of closely related Burkholderia thailandensis which was lysed by ST2 and ST96 only. ST79 and ST96 were observed to clear a mid-log culture by lysis within 6 h when infected at a multiplicity of infection of 0.1. As ST79 and ST96 phages effectively lysed B. pseudomallei, their potential use as a biocontrol of B. pseudomallei in the environment or alternative treatment in infected hosts could lead to benefits from phages that are available in nature.

Mimotope identification from monoclonal antibodies of Burkholderia pseudomallei using random peptide phage libraries.

This study used random peptide libraries, displayed by bacteriophage T7 and M13, to identify mimotopes from four monoclonal antibodies (mAbs) specific to Burkholderia pseudomallei. Bound phages, selected from fourth-round panning with each mAb, were tested for binding specificity with each mAb using ELISA, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Overall, 75 of 90 phages (83.3%) were ELISA-positive with each mAb. Mimotopes from all 75 phages (100%) were found to match protein sequences of Burkholderia spp. from GenBank. The predominant mimotopes were TP-GRTRVT found in 13.3%, LTPCGRTxD (8%), AREVTLL (6.7%), NxVxKVVSR (5.3%), PCAPRSS (4%), LGRVLAN (4%), RNPKKA (2.7%) and CPYPR (2.7%). The following GenBank-matched proteins (i.e. the hypothetical proteins) were located at the outer membrane of Burkholderia spp.: BPSL2046 of B. pseudomallei K96243 (matched with mimotope CGRTxD), BpseP_02000035 (matched with LGRVLAN), BPSS0784 of B. pseudomallei K96243 (matched with CPYPR), BURPS1710b_1104 of B. pseudomallei 1710b (matched with CARQY) and TonB-dependent siderophore receptor of B. cenocepacia H12424 (matched with CVRxxLTPC and TPCRxRT). These phage mimotopes and matched proteins may have the potential for further use as diagnostic reagent and immunogen against melioidosis. These results demonstrate that phage-display technique has the potential for rapidly identifying phage mimotopes that interact with B. pseudomallei mAbs.

Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B. pseudomallei 1026b.

Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated phi1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage phi1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the phi1026b genome was revealed by comparison with bacteriophage phiE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The phi1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in phiE125. On the other hand, phi1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against phiE125 reacted with the tail of phi1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.

[Effect of cultivation temperature on the extrachromosomal hereditary factors of the causative agent of melioidosis]. / Vliianie temperatury kul'tivirovaniia na vnekhromosomnye faktory nasledstvennosti vozbuditelia melioidoza.

The cultivation temperature of Burkholderia pseudomallei has been shown to determine both the direction of morphological dissociation and the prophage induction rate. Inheriting plasmid replicons was found to depend on the temperature conditions during the growth of these bacteria. No influence of B. pseudomallei plasmids pPM1 and pCM2 on the lysogenic state of the bacterial cell and the formation of different B. pseudomallei variants was noted.

[Identification of the bacterium Pseudomonas mallei using Pseudomonas pseudomallei bacteriophages]. / Identifikatsiia bakterii Pseudomonas mallei s pomoshch'iu bakteriofagov Pseudomonas pseudomallei.

Phage production by Pseudomonas pseudomallei and Pseudomonas mallei strains has been studied. 32 P. pseudomallei bacteriophages have been isolated. Their spectrum of lytic action against P. pseudomallei, P. mallei and other Pseudomonas sp. has been defined. It has been shown that P. pseudomallei bacteriophages PP19, PP23, PP33 may be used for identification P. mallei among related Pseudomonas.