Burkholderia Species Research Articles

2167. Evaluation of Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy for Rapid and Reagent-Free Identification of Burkholderia spp.

Background Burkholderia cepacia complex including B. gladioli are opportunistic pathogenic bacteria affecting the immunocompromised population. For prognosis and appropriate treatment, rapid and accurate species identification is particularly important for those diagnosed with cystic fibrosis (CF). Conventional biochemical identification techniques are insensitive and problematic for identifying Burkholderia spp., leading to common misidentification or inconclusive results. Recent studies have successfully employed attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy for rapid, reagent-free and cost-effective microbial identification. In the present study, identification of Burkholderia spp. by this technique is investigated.MethodsA total of 59 isolates belonging to 7 species of Burkholderia were included in this study; all these isolates had been well-characterized by VITEK 2, 16S rRNA sequencing, random amplification of polymorphic DNA (recA typing) and/or matrix-assisted laser desorption/ionization time of flight mass spectrometry. ATR-FTIR spectra were acquired directly from colonies on 5% blood agar plates.ResultsA spectral database containing ATR-FTIR spectra of over 4300 bacterial isolates, encompassing over 70 genera and 190 species, was updated to include spectra of 39 isolates collected in this study and employed in the identification of the other isolates (n = 20). All isolates were correctly identified as Burkholderia by a multitier search approach. For Burkholderia species identification, spectra belonging to 39 isolates representative of all 7 species were used to construct a spectral database employed to identify the other 20 isolates [B. anthina (n = 2), B. gladioli (n = 8), B. multivorans (n = 7), and B. vietnamiensis (n = 3)]. Compared with VITEK 2 (30% correct species identification), ATR-FTIR spectroscopy correctly identified all but one isolate, resulting in overall correct species identification of 95%. Prospectively (10 months), 5 of 1100 isolates collected were identified as Burkholderia spp. by ATR-FTIR spectroscopy in concordance with VITEK 2.ConclusionATR-FTIR spectroscopy can provide the means of rapid Burkholderia spp. identification for appropriate treatment of those diagnosed with CF.Disclosures All authors: No reported disclosures.

Host-symbiont specificity determined by microbe-microbe competition in an insect gut.

Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.

Do Global Regulators Hold the Key to Production of Bacterial Secondary Metabolites?

The emergence of multiple antibiotic resistant bacteria has pushed the available pool of antibiotics to the brink. Bacterial secondary metabolites have long been a valuable resource in the development of antibiotics, and the genus Burkholderia has recently emerged as a source of novel compounds with antibacterial, antifungal, and anti-cancer activities. Genome mining has contributed to the identification of biosynthetic gene clusters, which encode enzymes that are responsible for synthesis of such secondary metabolites. Unfortunately, these large gene clusters generally remain silent or cryptic under normal laboratory settings, which creates a hurdle in identification and isolation of these compounds. Various strategies, such as changes in growth conditions and antibiotic stress, have been applied to elicit the expression of these cryptic gene clusters. Although a number of compounds have been isolated from different Burkholderia species, the mechanisms by which the corresponding gene clusters are regulated remain poorly understood. This review summarizes the activity of well characterized secondary metabolites from Burkholderia species and the role of local regulators in their synthesis, and it highlights recent evidence for the role of global regulators in controlling production of secondary metabolites. We suggest that targeting global regulators holds great promise for the awakening of cryptic gene clusters and for developing better strategies for discovery of novel antibiotics.

Transcriptome analysis of human monocytic cells infected with Burkholderia species and exploration of pentraxin-3 as part of the innate immune response against the organisms

BackgroundBurkholderia mallei (Bm) is a facultative intracellular bacterial pathogen causing highly-fatal glanders in solipeds and humans. The ability of Bm to thrive intracellularly is thought to be related to exploitation of host immune response-related genes and pathways. Relatively little is known of the molecular strategies employed by this pathogen to modulate these pathways and evade intracellular killing. This manuscript seeks to fill gaps in the understanding of the interface between Bm and innate immunity by examining gene expression changes during infection of host monocytes.MethodsThe transcriptome of Bm-infected human Mono Mac-6 (MM6) monocytes was profiled on Affymetrix Human Transcriptome GeneChips 2.0. Gene expression changes in Bm-infected monocytes were compared to those of Burkholderia thailandensis (Bt)-infected monocytes and to uninfected monocytes. The resulting dataset was normalized using Robust Multichip Average and subjected to statistical analyses employing a univariate F test with a random variance model. Differentially expressed genes significant at p < 0.001 were subjected to leave-one-out cross-validation studies and 1st and 3rd nearest neighbor prediction model. Significant probe sets were used to populate human pathways in Ingenuity Pathway Analysis, with statistical significance determined by Fisher’s exact test or z-score.ResultsThe Pattern Recognition Receptor (PRR) pathway was represented among significantly enriched immune response-related human canonical pathways, with evidence of upregulation across both infections. Among members of this pathway, pentraxin-3 was significantly upregulated by Bm- or Bt-infected monocytes. Pentraxin-3 (PTX3) was demonstrated to bind to both Bt and Burkholderia pseudomallei (Bp), but not Bm. Subsequent assays did not identify a role for PTX3 in potentiating complement-mediated lysis of Bt or in enhancing phagocytosis or replication of Bt in human monocytes.ConclusionWe report on the novel binding of PTX3 to Bt and Bp, with lack of interaction with Bm, suggesting that a possible evasive mechanism by Bm warrants further exploration. We determined that (1) PTX3 may not play a role in activating the lytic pathway of complement in different bacterial species and that (2) the opsonophagocytic properties of PTX3 should be investigated in different primary or immortalized cell lines representing host phagocytes, given lack of binding of PTX3 to MM6 monocytes.

Burkholderia pseudomallei, the causative agent of melioidosis, is rare but ecologically established and widely dispersed in the environment in Puerto Rico.

BackgroundBurkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated.Methodology/Principal findingsWe collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp.Conclusions/SignificanceB. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America.

A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

Bacterial rhizosphere community profile at different growth stages of Umorok (Capsicum chinense) and its response to the root exudates.

The role of microflora is an indispensable part of the living organisms. Plants actively recruit specific microbial community to establish favorable habitat with the distinct microbiome, essentially unique for each species, offering new opportunities for plant growth and productivity. Umorok, an indigenous chili variety of northeastern India, production is highly affected by various factors; therefore, rhizosphere bacteria and their relationship with the root exudates released were analyzed to demonstrate rhizosphere bacterial impact on plant growth and health. Culturable and metagenomic bacterial DNA was characterized and the chemical nature of the root exudate was analyzed using chemotaxis assay after its basic analysis in HPLC. Juvenile stage exhibited diverse bacterial species of gammaproteobacteria, alphaproteobacteria, and actinobacteria but lacked the betaproteobacteria while the microbial diversity was reduced in flowering and fruiting stages. However, every growth stage maintained a similar amount of bacterial population regardless of diversity. The population of Pseudomonas, Bacillus, and Burkholderia species was increased several folds in flowering and fruiting stage. Further, the chemotaxis assay unveiled the advantage of root exudate chemical composition for specific microbial recruitment. The chemical composition analysis of root exudates showed substantial variation in the concentration of organic acids, phenolics, and flavonoids that are favoring unique bacterial species. Thus, root exudates confer and limit the related microbial population besides typical plant-bacterial synergetic association. This study emphasized information about the type of microbial load present in each growth stage, which is essential to develop a microbial consortia package for Umorok overall crop improvement.

Cyclopropanol Warhead in Malleicyprol Confers Virulence of Human- and Animal-Pathogenic Burkholderia Species.

Burkholderia species such as B. mallei and B. pseudomallei are bacterial pathogens causing fatal infections in humans and animals (glanders and melioidosis), yet knowledge on their virulence factors is limited. While pathogenic effects have been linked to a highly conserved gene locus (bur/mal) in the B. mallei group, the metabolite associated to the encoded polyketide synthase, burkholderic acid (syn. malleilactone), could not explain the observed phenotypes. By metabolic profiling and molecular network analyses of the model organism B. thailandensis, the primary products of the cryptic pathway were identified as unusual cyclopropanol‐substituted polyketides. First, sulfomalleicyprols were identified as inactive precursors of burkholderic acid. Furthermore, a highly reactive upstream metabolite, malleicyprol, was discovered and obtained in two stabilized forms. Cell‐based assays and a nematode infection model showed that the rare natural product confers cytotoxicity and virulence.

Transcriptome analysis to understand the effects of the toxoflavin and tropolone produced by phytopathogenic Burkholderia on Escherichia coli.

The phytopathogenic Burkholderia species B. glumae and B. plantarii are the causal agents of bacterial wilt, grain rot, and seedling blight, which threaten the rice industry globally. Toxoflavin and tropolone are produced by these phytopathogens and are considered the most hostile biohazards with a broad spectrum of target organisms. However, despite their nonspecific toxicity, the effects of toxoflavin and tropolone on bacteria remain unknown. RNA-seq based transcriptome analysis was employed to determine the genome-wide expression patterns under phytotoxin treatment. Expression of 2327 and 830 genes was differentially changed by toxoflavin and tropolone, respectively. Enriched biological pathways reflected the down-regulation of oxidative phosphorylation and ribosome function, beginning with the inhibition of membrane biosynthesis and nitrogen metabolism under oxidative stress or iron starvation. Conversely, several systems such as bacterial chemotaxis, flagellar assembly, biofilm formation, and sulfur/taurine transporters were highly expressed as countermeasures against the phytotoxins. In addition, our findings revealed that three hub genes commonly induced by both phytotoxins function as the siderophore enterobactin, an iron-chelator. Our study provides new insights into the effects of phytotoxins on bacteria for better understanding of the interactions between phytopathogens and other microorganisms. These data will also be applied as a valuable source in subsequent applications against phytotoxins, the major virulence factor.

Semi-rational evolution of the 3-(3-hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA to improve rhamnolipid production in Pseudomonas aeruginosa and Burkholderia glumae.

The 3-(3-hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA is an essential enzyme involved in the biosynthesis of HAAs in Pseudomonas and Burkholderia species. RhlA modulates the aliphatic chain length in rhamnolipids, conferring distinct physicochemical properties to these biosurfactants exhibiting promising industrial and pharmaceutical value. A detailed molecular understanding of substrate specificity and catalytic performance in RhlA could offer protein engineering tools to develop designer variants involved in the synthesis of novel rhamnolipid mixtures for tailored eco-friendly products. However, current directed evolution progress remains limited due to the absence of high-throughput screening methodologies and lack of an experimentally resolved RhlA structure. In the present work, we used comparative modeling and chimeric-based approaches to perform a comprehensive semi-rational mutagenesis of RhlA from Pseudomonas aeruginosa. Our extensive RhlA mutational variants and chimeric hybrids between the Pseudomonas and Burkholderia homologs illustrate selective modulation of rhamnolipid alkyl chain length in both Pseudomonas aeruginosa and Burkholderia glumae. Our results also demonstrate the implication of a putative cap-domain motif that covers the catalytic site of the enzyme and provides substrate specificity to RhlA. This semi-rational mutant-based survey reveals promising 'hot-spots' for the modulation of RL congener patterns and potential control of enzyme activity, in addition to uncovering residue positions that modulate substrate selectivity between the Pseudomonas and Burkholderia functional homologs. DATABASE: Model data are available in the PMDB database under the accession number PM0081867.

Phenotypic and Genomic Analyses ofBurkholderia stabilisClinical Contamination, Switzerland

Abstract A recent hospital outbreak related to premoistened gloves used to wash patients exposed the difficulties of defining Burkholderia species in clinical settings. The outbreak strain displayed key B. stabilis phenotypes, including the inability to grow at 42°C; we used whole-genome sequencing to confirm the pathogen was B. stabilis. The outbreak strain genome comprises 3 chromosomes and a plasmid, sharing an average nucleotide identity of 98.4% with B. stabilis ATCC27515 BAA-67, but with 13% novel coding sequences. The genome lacks identifiable virulence factors and has no apparent increase in encoded antimicrobial drug resistance, few insertion sequences, and few pseudogenes, suggesting this outbreak was an opportunistic infection by an environmental strain not adapted to human pathogenicity. The diversity among outbreak isolates (22 from patients and 16 from washing gloves) is only 6 single-nucleotide polymorphisms, although the genome remains plastic, with large elements stochastically lost from outbreak isolates.

Phenotypic and Genomic Analyses of Burkholderia stabilis Clinical Contamination, Switzerland.

A recent hospital outbreak related to premoistened gloves used to wash patients exposed the difficulties of defining Burkholderia species in clinical settings. The outbreak strain displayed key B. stabilis phenotypes, including the inability to grow at 42°C; we used whole-genome sequencing to confirm the pathogen was B. stabilis. The outbreak strain genome comprises 3 chromosomes and a plasmid, sharing an average nucleotide identity of 98.4% with B. stabilis ATCC27515 BAA-67, but with 13% novel coding sequences. The genome lacks identifiable virulence factors and has no apparent increase in encoded antimicrobial drug resistance, few insertion sequences, and few pseudogenes, suggesting this outbreak was an opportunistic infection by an environmental strain not adapted to human pathogenicity. The diversity among outbreak isolates (22 from patients and 16 from washing gloves) is only 6 single-nucleotide polymorphisms, although the genome remains plastic, with large elements stochastically lost from outbreak isolates.

Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition.

Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens.IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

Characterization of Microbiota in Endometrial Fluid and Vaginal Secretions in Infertile Women with Repeated Implantation Failure.

Studies suggest that persisting intrauterine bacterial infectious conditions such as chronic endometritis potentially impair the embryo implantation process. The microbial environment in the female reproductive tract, however, remains largely undetermined in infertile patients with a history of repeated implantation failure (RIF). Using next-generation sequencing, we aimed to characterize the microbiota in the endometrial fluid (EF) and vaginal secretions (VS) in women with RIF. Twenty-eight infertile women with a history of RIF and eighteen infertile women undergoing the first in vitro fertilization-embryo transfer attempt (the control group) were enrolled in the study. On days 6-8 in the luteal phase of the natural, oocyte-pickup, or hormone replacement cycle, the paired EF and VS samples were obtained separately. Extracted genomic DNA was pyrosequenced for the V4 region of 16S ribosomal RNA using a next-generation sequencer. The EF microbiota had higher α-diversity and broader bacterial species than the VS microbiota both in the RIF and control groups. The analysis of the UniFrac distance matrices between EF and VS also revealed significantly different clustering. Additionally, the EF microbiota, but not the VS microbiota, showed significant variation in community composition between the RIF group and the control group. Burkholderia species were not detected in the EF microbiota of any samples in the control group but were detectable in a quarter of the RIF group. To our best knowledge, this is the first study investigating the microbiota in the paired EF and VS samples in infertile women with RIF.

The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods

Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 from B. pseudomallei. For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia.

A novel selective medium for the isolation of Burkholderia mallei from equine specimens

BackgroundBurkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations (e.g. horses, donkeys, and mules). B. mallei usually grows slowly on most culture media, and this property makes it difficult to isolate from clinical specimens. One of the problems is that B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites. The aim of this study was to develop a new selective agar for the laboratory diagnosis of glanders. We formulated a new agar, named BM agar, to enrich B. mallei growth, but inhibit the growth of other bacteria and fungi based on their antimicrobial profiles. We compared the growth of B. mallei on BM with Xie’s and PC agars, the two previously described selective agars for B. mallei.ResultsBM agar could sufficiently grow almost all of the tested B. mallei strains within 72 h: only one out of the 38 strains grew scantly after 72 h of incubation. BM agar was further tested with other Burkholderia species and various bacterial species commonly found in the nasal cavities and on the skin of horses. We have found that other Burkholderia species including B. pseudomallei and B. thailandensis can grow on BM agar, but non-Burkholderia species cannot. Furthermore, the specificities of the three selective agars were tested with or without spiking B. mallei culture into clinical specimens of non-sterile sites collected from healthy horses. The results showed that BM agar inhibited growths of fungi and other bacterial species better than PC and Xie’s agars. We have also found that growth of B. mallei on BM agar was equivalent to that on 5% horse blood agar and was significantly greater than those on the other two agars (P < 0.05).ConclusionsWe believe that BM agar can be used to efficiently isolate B. mallei from mixed samples such as those typically collected from horses and other contaminated environments.

Metagenomics-Guided Survey, Isolation, and Characterization of Uranium Resistant Microbiota from the Savannah River Site, USA.

Despite the recent advancements in culturomics, isolation of the majority of environmental microbiota performing critical ecosystem services, such as bioremediation of contaminants, remains elusive. Towards this end, we conducted a metagenomics-guided comparative assessment of soil microbial diversity and functions present in uraniferous soils relative to those that grew in diffusion chambers (DC) or microbial traps (MT), followed by isolation of uranium (U) resistant microbiota. Shotgun metagenomic analysis performed on the soils used to establish the DC/MT chambers revealed Proteobacterial phyla and Burkholderia genus to be the most abundant among bacteria. The chamber-associated growth conditions further increased their abundances relative to the soils. Ascomycota was the most abundant fungal phylum in the chambers relative to the soils, with Penicillium as the most dominant genus. Metagenomics-based taxonomic findings completely mirrored the taxonomic composition of the retrieved isolates such that the U-resistant bacteria and fungi mainly belonged to Burkholderia and Penicillium species, thus confirming that the chambers facilitated proliferation and subsequent isolation of specific microbiota with environmentally relevant functions. Furthermore, shotgun metagenomic analysis also revealed that the gene classes for carbohydrate metabolism, virulence, and respiration predominated with functions related to stress response, membrane transport, and metabolism of aromatic compounds were also identified, albeit at lower levels. Of major note was the successful isolation of a potentially novel Penicillium species using the MT approach, as evidenced by whole genome sequence analysis and comparative genomic analysis, thus enhancing our overall understanding on the uranium cycling microbiota within the tested uraniferous soils.

Intracellular Burkholderia Symbionts induce extracellular secondary infections; driving diverse host outcomes that vary by genotype and environment

Symbiotic associations impact and are impacted by their surrounding ecosystem. The association between Burkholderia bacteria and the soil amoeba Dictyostelium discoideum is a tractable model to unravel the biology underlying symbiont-endowed phenotypes and their impacts. Several Burkholderia species stably associate with D. discoideum and typically reduce host fitness in food-rich environments while increasing fitness in food-scarce environments. Burkholderia symbionts are themselves inedible to their hosts but induce co-infections with secondary bacteria that can serve as a food source. Thus, Burkholderia hosts are “farmers” that carry food bacteria to new environments, providing a benefit when food is scarce. We examined the ability of specific Burkholderia genotypes to induce secondary co-infections and assessed host fitness under a range of co-infection conditions and environmental contexts. Although all Burkholderia symbionts intracellularly infected Dictyostelium, we found that co-infections are predominantly extracellular, suggesting that farming benefits are derived from extracellular infection of host structures. Furthermore, levels of secondary infection are linked to conditional host fitness; B. agricolaris infected hosts have the highest level of co-infection and have the highest fitness in food-scarce environments. This study illuminates the phenomenon of co-infection induction across Dictyostelium associated Burkholderia species and exemplifies the contextual complexity of these associations.

Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens

A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5 cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.

Development of biofilm-penetrant antimicrobial delivery system to counter Burkholderia infections

Melioidosis is caused by the Gram-negative, intracellular bacterium Burkholderia pseudomallei. The epidemiology of the disease is unclear- it is limited to tropical regions, where monitoring can be sparse and further complicated by the multifaceted nature of the condition. The most common indication of acute melioidosis is pneumonia (51 %) that can progress to acute fulminant sepsis with multifocal infiltrates with high rates of mortality. Meliodosis has been estimated to cause 89 000 deaths annually with an estimated mortality rate of approximately 50 %. Due to ease of the dissemination of the pathogen, its ability to form biofilms, the high degree of antimicrobial resistance and paucity of effective treatments, controlling the infection is a major challenge. Our approach is to develop nanoparticles capable of effectively delivering antimicrobials to biofilms formed by Burkholderia species. Formulations of a proprietary lipidic delivery agent, CM2, have been tested against two species: B. cepacia UCB717 that has no capsule and B. thailandensis (that is used as a surrogate for B. pseudomallei), including strains with and without capsules both of which readily form biofilms. Confocal laser scanning microscopy was used to monitor the uptake of the nanoparticles and the MIC and MBEC of CM2 alone (and analogues), in combination with a panel of antimicrobials and a novel oligonucleotide antimicrobial, termed a Transcription Factor Decoy (TFD), were measured. The most efficacious combinations will be formulated for delivery by inhalation prior to testing in animal models.