Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis. A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source. The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy. On both media an exopolysaccharide having the following repeating unit was produced: -->5)-beta-Kdop-(2-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1--> This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B. pseudomallei, the microbial agent causing melioidosis. In addition to this, when grown on the mannitol-rich medium, B. cepacia strain BTS13 produced another polysaccharide that was established to be levan: -->6)-beta-D-Fruf-(2-->. The content of levan was about 20% (w/w) of the total amount of polymers. The ability of B. cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.     

Species Abundance and Diversity of Burkholderia cepacia Complex in the Environment

Despite considerable interest in studying Burkholderia cepacia complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of B. cepacia isolates. To better describe the ecology and diversity of B. cepacia complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a probe consisting of three repeats of a B. cepacia complex-specific probe, each separated by a phosphoramidite spacer. In addition, a duplex PCR targeting B. cepacia complex-specific recA and 16S rRNA sequences was developed to enable a fast and reliable diagnostic assay for members of the complex. When applied to maize rhizosphere samples, colony hybridization results were in good agreement with those of most-probable-number duplex PCR, both indicating a >100-fold fluctuation of abundance between individual plants. Using restriction analysis of recA for a total of 285 confirmed isolates of the B. cepacia complex, up to seven B. cepacia complex species were identified; however, their diversity and abundance were not evenly distributed among individual plants, and several allelic variants were commonly found from the same rhizosphere sample. These results indicate that not only complex communities of B. cepacia complex species and closely related strains of the same species may coexist at high population levels but also species composition and abundance may dramatically vary between individual plants.     

A Quorum-Quenching Approach To Investigate the Conservation of Quorum-Sensing-Regulated Functions within the Burkholderia cepacia Complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.     

Culture-Based and Non-Growth-Dependent Detection of the Burkholderia cepacia Complex in Soil Environments

Burkholderia cepacia complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear. We sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards, and gardens. A total of 91 sites was sampled in three large U.S. cities. In the first phase of the study, putative Bcc isolates were recovered on Burkholderia cepacia selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22°C. Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and recA gene sequences. Among the 1,013 bacterial isolates examined, 68 were identified as Bcc; 14 (15%) of 91 sampled sites yielded Bcc isolates. In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences. Either 82 or 93% of the soil samples were positive for at least one Bcc genomovar, depending on the PCR assay system used. Cloning and sequencing were performed to check the specificity of the PCR assays. Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bcc. The four soil samples from which these clones were generated did not yield isolates identified as Bcc. Based on PCR detection, Bcc appears to be prevalent in soil from urban and suburban environments. Culture-based recovery of Bcc may underestimate environmental populations.     

Garlic Revisited: Antimicrobial Activity of Allicin-Containing Garlic Extracts against Burkholderia cepacia Complex

The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics.     

Drosophila melanogaster as a Model Host for the Burkholderia cepacia Complex

Background

Colonization with bacterial species from the Burkholderia cepacia complex (Bcc) is associated with fast health decline among individuals with cystic fibrosis. In order to investigate the virulence of the Bcc, several alternative infection models have been developed. To this end, the fruit fly is increasingly used as surrogate host, and its validity to enhance our understanding of host-pathogen relationships has been demonstrated with a variety of microorganisms. Moreover, its relevance as a suitable alternative to mammalian hosts has been confirmed with vertebrate organisms.

Methodology/Principal Findings

The aim of this study was to establish Drosophila melanogaster as a surrogate host for species from the Bcc. While the feeding method proved unsuccessful at killing the flies, the pricking technique did generate mortality within the populations. Results obtained with the fruit fly model are comparable with results obtained using mammalian infection models. Furthermore, validity of the Drosophila infection model was confirmed with B. cenocepacia K56-2 mutants known to be less virulent in murine hosts or in other alternative models. Competitive index (CI) analyses were also performed using the fruit fly as host. Results of CI experiments agree with those obtained with mammalian models.

Conclusions/Significance

We conclude that Drosophila is a useful alternative infection model for Bcc and that fly pricking assays and competition indices are two complementary methods for virulence testing. Moreover, CI results indicate that this method is more sensitive than mortality tests.     

Structural and Functional Characterization of Diffusible Signal Factor Family Quorum-Sensing Signals Produced by Members of the Burkholderia cepacia Complex

Previous work has shown that Burkholderia cenocepacia produces the diffusible signal factor (DSF) family signal cis-2-dodecenoic acid (C₁₂:Δ², also known as BDSF), which is involved in the regulation of virulence. In this study, we determined whether C₁₂:Δ² production is conserved in other members of the Burkholderia cepacia complex (Bcc) by using a combination of high-performance liquid chromatography, mass spectrometry, and bioassays. Our results show that five Bcc species are capable of producing C₁₂:Δ² as a sole DSF family signal, while four species produce not only C₁₂:Δ² but also a new DSF family signal, which was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5). In addition, we demonstrate that the quorum-sensing signal cis-11-methyl-2-dodecenoic acid (11-Me-C₁₂:Δ²), which was originally identified in Xanthomonas campestris supernatants, is produced by Burkholderia multivorans. It is shown that, similar to 11-Me-C₁₂:Δ² and C₁₂:Δ², the newly identified molecule 11-Me-C₁₂:Δ^2,5 is a potent signal in the regulation of biofilm formation, the production of virulence factors, and the morphological transition of Candida albicans. These data provide evidence that DSF family molecules are highly conserved bacterial cell-cell communication signals that play key roles in the ecology of the organisms that produce them.The Burkholderia cepacia complex (Bcc) comprises a group of currently 17 formally named bacterial species that, although closely related, are phenotypically diverse (17, 22, 23). Strains of the Bcc are ubiquitously distributed in nature and have been isolated from soil, water, the rhizosphere of plants, industrial settings, hospital environments, and infected humans. Some Bcc strains have emerged as problematic opportunistic pathogens in patients with cystic fibrosis or chronic granulomatous disease, as well as in immunocompromised individuals (17). The clinical outcome of Bcc infections ranges from asymptomatic carriage to a fulminant and fatal pneumonia, the so-called “cepacia syndrome” (12, 17). Although all Bcc species have been isolated from both environmental and clinical sources, B. cenocepacia and B. multivorans are most commonly found in clinical samples (16).Many bacterial pathogens have evolved a cell-cell communication mechanism known as quorum sensing (QS) to coordinate the expression of virulence genes. In spite of their genetic differences, most Bcc species produce N-acylhomoserine lactone (AHL) QS signals (25). More recently, another QS signal molecule, cis-2-dodecenoic acid (BDSF), has been identified in B. cenocepacia (3). Subsequent studies showed that BDSF plays a role in the regulation of bacterial virulence (6, 20). Interestingly, the two QS systems appear to act in conjunction in the regulation of B. cenocepacia virulence, as a set of the AHL-controlled virulence genes are also positively regulated by BDSF (6). Furthermore, mutation of Bcam0581, which is required for BDSF biosynthesis, results in substantially retarded energy production and impaired growth in minimal medium (6), highlighting the dual roles of the QS system in the physiology of and infection by B. cenocepacia.BDSF is a structural analogue of cis-11-methyl-2-dodecenoic acid, which is a QS signal known as diffusible signal factor (DSF) originally identified from the plant bacterial pathogen Xanthomonas campestris pv. campestris (2, 24). Evidence is accumulating that DSF-type fatty acid signals represent a new family of QS signals, which are widespread among Gram-negative bacteria (10, 24). For example, DSF and seven structural derivatives were identified in supernatants of Stenotrophomonas maltophilia (8, 11), 12-methyl-tetradecanoic acid was shown to be produced by Xylella fastidiosa (18), and cis-2-decenocic acid was found to be synthesized by Pseudomonas aeruginosa (5). In addition, DSF-like activity has also been reported in a range of Xanthomonas species, including X. oryzae pv. oryzae and X. axonopodis pv. citri (1, 2, 4, 24), but the chemical structures of these DSF analogues remain to be determined. Unlike other known QS signals, such as AHL and AI-2 family signals, DSF and its analogues, including BDSF, are fatty acids and these fatty acid signals were collectively designated DSF family signals for the convenience of discussion (10). Considering the fact that the list of DSF family signal is expanding, we propose to designate cis-11-methyl-2-dodecenoic acid (DSF) 11-Me-C₁₂:Δ² and cis-2-dodecenoic acid (BDSF) C₁₂:Δ². This nomenclature is based on one of the fatty acid nomenclatures (13, 19) where the methyl (Me) substitution and its position are indicated first (for example, 11-Me indicates a methyl group on C-11 of the fatty acid carbon chain), followed by the length of the fatty acid carbon chain (C₁₂ represents a 12-carbon fatty acid chain), and then the position of the double bond in the fatty acid chain (Δ² indicates a double bond in the cis configuration at site 2, i.e., between C-2 and C-3 of the fatty acid carbon chain). In this way, it is convenient to say that 11-Me-C₁₂:Δ² and C₁₂:Δ² have identical 12-carbon fatty acid chains with a cis bond at the same site but differ in a methyl substitution on C-11. Following this nomenclature system, 12-methyl-tetradecanoic acid and cis-2-decenocic acid can be referred to as 12-Me-C₁₄ and C₁₀:Δ², respectively.DSF family signals have emerged as important factors in the regulation of virulence and biofilm formation in a wide range of bacterial pathogens (10). In this study, we have investigated the production of the DSF family signals in nine Bcc species. It is demonstrated that C₁₂:Δ² is conserved in members of the Bcc and that 11-Me-C₁₂:Δ² and a novel DSF family signal were also produced by some, but not all, of the Bcc strains investigated. This new signal was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5) by nuclear magnetic resonance (NMR) analysis and mass spectrometry. We have also investigated the biological significance of 11-Me-C₁₂:Δ^2,5 in intraspecies and interspecies communication.     

Structural study of the exopolysaccharide produced by a clinical isolate of Burkholderia cepacia

The primary structure of the exopolysaccharide produced by a clinical isolate of the bacterium Burkholderia cepacia was studied by means of methylation analysis, selective degradation, NMR spectroscopy, and electrospray mass spectrometry. The resulting data showed that the parent repeating unit of the exopolysaccharide is a highly branched heptasaccharide with the following structure: Two acetyl groups are present per repeating unit, as noncarbohydrate substituents.     

Burkholderia cepacia Complex Phage-Antibiotic Synergy (PAS): Antibiotics Stimulate Lytic Phage Activity

The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.     

A physical genome map of the Burkholderia cepacia type strain

Burkholderia cepacia (basonym Pseudomonas cepaci a), the type speciesof the new genus Burkholderia , is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 Spel , 11 Swal , 11 Pacl , 11 Pmel and six l-Ceul sites, resulting in an average resolution of 1O5 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia . The phylogenetically related species Burkholderia glumae , Burkholderia pickettii , Burkholderia solanacearum , Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.     

A novel aminopeptidase from Burkholderia cepacia specific for acidic amino acids

An aminopeptidase specific for the N-terminal acidic residue (BcepAP) was purified from the cell extract of Burkholderia cepacia svr as a homotrimeric (subunit mass 66 kDa) molecule. It was identified as an unassigned peptidase of family M61. The only other member characterized so far from this family is a broad-specificity aminopeptidase of Sphingomonas capsulata (ScapAP) with preference for Gly or Ala residues. However, BcepAP exhibited narrow specificity and the preferred substrate was a peptide with an N-terminal Asp or Glu residue, which is quite unusual. The proteins assigned to this family were grouped separately on the basis of their homology to either BcepAP or ScapAP. It led the conclusion that BcepAP is a prototype of a new PepM61 subfamily, with a representative in other Proteobacteria , and to the prediction that members of the family share the ability to cleave N-terminal acidic residues of peptide substrates.     

Iron acquisition mechanisms of the Burkholderia cepacia complex

The Burkholderia cepacia complex (Bcc) is comprised of at least 10 closely related species of Gram-negative proteobacteria that are associated with infections in certain groups of immunocompromised individuals, particularly those with cystic fibrosis. Infections in humans tend to occur in the lungs, which present an iron-restricted environment to a prospective pathogen, and accordingly members of the Bcc appear to possess efficient mechanisms for iron capture. These bacteria specify up to four different types of siderophore (ornibactin, pyochelin, cepabactin and cepaciachelin) that employ the full repertoire of iron-binding groups present in most naturally occurring siderophores. Members of the Bcc are also capable of utilising some exogenous siderophores that they are not able to synthesise. In addition to siderophore-mediated mechanisms of iron uptake, the Bcc possess mechanisms for acquiring iron from haem and from ferritin. The Bcc therefore appear to be well-equipped for life in an iron-poor environment. An erratum to this article can be found at     

Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates

Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37°C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.     

洋葱伯克霍尔德菌脂肪酶：一种应用前景广阔的生物催化剂

洋葱伯克霍尔德菌脂肪酶对有机溶剂（醇）、热、氧化剂、表面活性剂、去污剂、蛋白酶等具有良好的抗性,在有机合成、对映体拆分、非水相催化等领域应用十分广泛。综述了洋葱伯克霍尔德菌脂肪酶的发酵生产、分离纯化、基因克隆与表达、固定化与生物印迹、蛋白质结构解析及应用研究等,并展望了其未来发展方向,以期为该工业酶的研发与广泛应用提供参考。     

Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.     

Biochemical and Functional Studies on the Burkholderia cepacia Complex bceN Gene,Encoding a GDP-D-Mannose 4,6-Dehydratase

This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. V_max and K_m values of 1.5±0.2 µmol.min⁻¹.mg⁻¹ and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl₂ per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated K_i of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.     

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Burkholderia cepacia D1.

Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced by Burkholderia cepacia D1 at 30°C in nitrogen-free culture solutions containing n-butyric acid and/or n-valeric acid. When n-valeric acid was used as the sole carbon source, the 3HV fraction in copolyester increased from 36 to 90 mol% as the concentration of n-valeric acid in the culture solution increased from 1 to 20 g/l. The addition of n-butyric acid to the culture solution resulted in a decrease in the 3HV fraction in copolyester. The copolymers biosynthesized by this method were mixtures of random copolymers having a wide variety of composition of the 3HV component. The melting points of the fractionated copolymers show a concave curve with the minimum at the 3HV content of ≈40 mol%. The a-parameter of lattice indices of the P(3HB) crystal for the fractionated copolymers largely increased as the 3HV composition increased. Biodegradability of the copolymer increased with the lower content of 3HV composition and/or the lower crystallinity.