Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence

This is the final version. Available on open access from Nature Research via the DOI in this recordPhospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.Mahidol UniversityThailand Research Fun

Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se

Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction

This is the final version. Available on open access from Frontiers media via the DOI in this recordData Availability Statement: The datasets GENERATED for this study can be found in the SNBI SRA accession PRJNA599542.Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5′ end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.Mahidol University, ThailandThailand Research Fun

Induced Burkholderia prophages detected from the hemoculture: a biomarker for Burkholderia pseudomallei infection.

Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the environment but also in the human body. The use of phages for the diagnosis of melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is emerging as a promising novel approach, but our understanding of conditions under which Burkholderia prophages can be induced remains limited. Here, we first demonstrated the isolation of Burkholderia phages from the hemocultures of melioidosis patients. The B. pseudomallei-positive hemoculture bottles were filtered to remove bacteria, and then phages were isolated and purified by spot and double agar overlay plaque assays. Forty blood samples (hemoculture-confirmed melioidosis) were tested, and phages were found in 30% of the samples. Transmission electron microscopy and genome analysis of the isolated phages, vB_HM387 and vB_HM795, showed that both phages are Myoviruses. These two phages were stable at a pH of 5-7 and temperatures of 25-37°C, suggesting their ability to survive in human blood. The genome sizes of vB_HM387 and vB_HM795 are 36.3 and 44.0 kb, respectively. A phylogenetic analysis indicated that vB_HM387 has homologs, but vB_HM795 is a novel Myovirus, suggesting the heterogeneity of Burkholderia phages in melioidosis patients. The key finding that Burkholderia phages could be isolated from the blood of melioidosis patients highlights the potential application of phage-based assays by detecting phages in blood as a pathogen-derived biomarker of infection

The Role of the County Professional Council in Advanced Training and Professional Development of Class Teachers

Stručno usvršavanje je obvezan dio učiteljskog posla koji se provodi na četiri osnovne razine: individualnoj, školskoj, županijskoj i državnoj. Brojne su prednosti i nedostaci svakog oblika usavršavanja, a svi su oni na putu profesionalnog razvoja učitelja jednako važni i korisni. U radu je prikazan model stručnog usvršavanja učitelja na županijskoj razini (Županijsko stručno vijeće učitelja razredne nastave – Grad Sisak) pri čemu veliku ulogu imaju upravo županijski voditelji koji su poveznica između školske i državne razine stručnog usavršavanja učitelja.Advanced training is a mandatory part of teacher’s job and it is being carried out at four basic levels: individual, school, county and state level. There are numerous advantages and disadvantages of any form of training, and they are all equally important and useful in the course of professional development of teachers. This study presents the model of advanced training of class teachers at county level (County professional council of class teachers – the Town of Sisak), where the very county leaders, who are the link between the school and state level of advanced training of teachers, have a great role

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.ISSN:0099-2240ISSN:1098-533

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei.

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se