Transport of Nanoparticles and Tobramycin-loaded Liposomes in Burkholderia cepacia Complex Biofilms

Due to the intrinsic resistance of Burkholderia cepacia complex (Bcc) to many antibiotics and the production of a broad range of virulence factors, lung infections by these bacteria, primarily occurring in cystic fibrosis (CF) patients, are very difficult to treat. In addition, the ability of Bcc organisms to form biofilms contributes to their persistence in the CF lung. As Bcc infections are associated with poor clinical outcome, there is an urgent need for new effective therapies to treat these infections. In the present study, we investigated whether liposomal tobramycin displayed an increased anti-biofilm effect against Bcc bacteria compared to free tobramycin. Single particle tracking (SPT) was used to study the transport of positively and negatively charged nanospheres in Bcc biofilms as a model for the transport of liposomes. Negatively charged nanospheres became immobilized in close proximity of biofilm cell clusters, while positively charged nanospheres interacted with fiber-like structures, probably eDNA. Based on these data, encapsulation of tobramycin in negatively charged liposomes appeared promising for targeted drug delivery. However, the anti-biofilm effect of tobramycin encapsulated into neutral or anionic liposomes did not increase compared to that of free tobramycin. Probably, the fusion of the anionic liposomes with the negatively charged bacterial surface of Bcc bacteria was limited by electrostatic repulsive forces. The lack of a substantial anti-biofilm effect of tobramycin encapsulated in neutral liposomes could be further investigated by increasing the liposomal tobramycin concentration. However, this was hampered by the low encapsulation efficiency of tobramycin in these liposomes.     

Virulence of Burkholderia cepacia complex strains in gp91phox-/- mice

In cystic fibrosis (CF), infection with Burkholderia cepacia complex (Bcc) strains may cause long-term asymptomatic airway colonization, or severe lung infection leading to rapid pulmonary decline. To assess the virulence of Bcc strains, we established a lung infection model in mice with a null allele of the gene involved in X-linked chronic granulomatous disease (CGD). CGD mice, challenged intratracheally with 10(3) cells of the epidemic Burkholderia cenocepacia strain J2315, died within 3 days from sepsis after bacteria had multiplied to 3.3 x 10(8) cells. Infected mice developed neutrophil-dominated lung abscesses. Other B. cenocepacia strains and a B. cepacia strain were less virulent and one B. multivorans and one B. vietnamensis CF isolate were both avirulent. Bcc mutants, defective in exopolysaccharide synthesis or quorum sensing revealed diminished or no abscess formation and mortality. Immunofluorescence staining of Bcc-infected murine and CF lung tissues revealed colocalization of Bcc and neutrophils, suggesting Bcc persistence within neutrophils in CGD and CF. In vitro, Bcc cells were rapidly killed during aerobic neutrophil phagocytosis; however, the pathogens survived in neutrophils with blocked nicotinamide adenine dinucleotide phosphate oxidase activity and under anaerobic conditions. We conclude that the Bcc infection model in CGD mice is well suited for the assessment of Bcc virulence.     

Matrix-assisted laser desorption ionisation-time-of of-flight mass spectrometry of intact cells allows rapid identification of Burkholderia cepacia complex

The present study examined the potential of intact-cell matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid identification of Burkholderia cepacia complex (Bcc) bacteria using an Applied Biosystems 4700 Proteomics Analyser. Two software packages were used to analyse mass profiles based on densitometric curves and peak positions. The 75 strains examined, represented the nine established Bcc species and some commonly misidentified species, closely related or biochemically similar to Bcc and relevant in the context of cystic fibrosis microbiology. All Bcc strains clustered together, separated from non-Bcc strains. Within Bcc, most Bcc strains grouped in species specific clusters, except for Burkholderia anthina and Burkholderia pyrrocinia strains which constituted a single cluster. The present study demonstrates that MALDI-TOF MS is a powerful approach for the rapid identification of Bcc bacteria.     

The various lifestyles of the Burkholderia cepacia complex species: a tribute to adaptation

The Burkholderia cepacia complex (Bcc) is composed of 17 closely related species. These bacteria are widely but heterogeneously distributed in the natural environment, such as soil, water and rhizosphere. Bcc strains are able to colonize various ecological niches by adopting versatile lifestyles, including saprophytism and (positive or deleterious) association with eukaryotic cells. Bcc strains have proven to be very efficient in biocontrol, plant growth promotion and bioremediation. However, they also are important opportunistic pathogens that can cause severe respiratory infections among individuals suffering from cystic fibrosis or chronic granulomatous disease. Therefore, considering that the distinction between plant beneficial and clinical strains is not obvious, biotechnological applications of Bcc strains are currently not allowed. This minireview provides an overview of the wide range of lifestyles that Bcc bacteria can adopt, leading to glimpses into their tremendous adaptation potential and highlighting remaining questions concerning potential implicated mechanisms.     

Bacteria of the <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> complex are cyanogenic under biofilm and colonial growth conditions

Background  

The Burkholderia cepacia complex (Bcc) is a collection of nine genotypically distinct but phenotypically similar species. They show wide ecological diversity and include species that are used for promoting plant growth and bio-control as well species that are opportunistic pathogens of vulnerable patients. Over recent years the Bcc have emerged as problematic pathogens of the CF lung. Pseudomonas aeruginosa is another important CF pathogen. It is able to synthesise hydrogen cyanide (HCN), a potent inhibitor of cellular respiration. We have recently shown that HCN production by P. aeruginosa may have a role in CF pathogenesis. This paper describes an investigation of the ability of bacteria of the Bcc to make HCN.     

The antibacterial properties of docosahexaenoic omega-3 fatty acid against the cystic fibrosis multiresistant pathogen Burkholderia cenocepacia

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens that cause multiresistant pulmonary infections in patients with cystic fibrosis (CF). In this study, we evaluated the in vitro antimicrobial efficacy of eight unsaturated fatty acids against Burkholderia cenocepacia K56-2, a CF epidemic strain. Docosahexaenoic acid (DHA) was the most active compound. Its action can be either bacteriostatic or bactericidal, depending upon the concentration used. The effect of DHA was also evaluated on two others B.?cenocepacia clinical isolates and compared with one representative member of all the 17 Bcc species. To test whether DHA could have a therapeutic potential, we assessed its efficacy using a Galleria mellonella caterpillar model of B.?cenocepacia infection. We observed that the treatment of infected larvae with a single dose of DHA (50 mM) caused an increase in the survival rate as well as a reduced bacterial load. Moreover, DHA administration markedly increases the expression profile of the gene encoding the antimicrobial peptide gallerimycin. Our results demonstrate that DHA has in vitro and in vivo antibacterial activity against Bcc microorganisms. These findings provide evidence that DHA may be a useful nutraceutical for the treatment of CF patients with lung infections caused by antibiotic multiresistant Bcc microorganisms.     

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     

Use of the gyrB gene to discriminate among species of the Burkholderia cepacia complex

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can cause serious infections in lungs of cystic fibrosis patients. The Bcc comprises at least nine species that have been discriminated by a polyphasic taxonomic approach. In this study, we focused on the gyrB gene, universally distributed among bacteria, as a new target gene to discriminate among the Bcc species. New PCR primers were designed to amplify a gyrB DNA fragment of about 1900 bp from 76 strains representative of all Bcc species. Nucleotide sequences of PCR products were determined and showed more than 400 polymorphic sites with high sequence similarity values from most isolates of the same species. Phylogenetic tree analysis revealed that most of the 76 gyrB sequences grouped, forming clusters, each corresponding to a given Bcc species.     

Sponge-associated microbial Antarctic communities exhibiting antimicrobial activity against Burkholderia cepacia complex bacteria

The aerobic heterotrophic bacterial communities isolated from three different Antarctic sponge species were analyzed for their ability to produce antimicrobial compounds active toward Cystic Fibrosis opportunistic pathogens belonging to the Burkholderia cepacia complex (Bcc). The phylogenetic analysis performed on the 16S rRNA genes affiliated the 140 bacterial strains analyzed to 15 genera. Just three of them (Psychrobacter, Pseudoalteromonas and Arthrobacter) were shared by the three sponges. The further Random Amplified Polymorphic DNA analysis allowed to demonstrate that microbial communities are highly sponge-specific and a very low degree of genus/species/strain sharing was detected. Data obtained revealed that most of these sponge-associated Antarctic bacteria and belonging to different genera were able to completely inhibit the growth of bacteria belonging to the Bcc. On the other hand, the same Antarctic strains did not have any effect on the growth of other pathogenic bacteria, strongly suggesting that the inhibition is specific for Bcc bacteria. Moreover, the antimicrobial compounds synthesized by the most active Antarctic bacteria are very likely Volatile Organic Compounds (VOCs), a finding that was confirmed by the SPME-GC-MS technique, which revealed the production of a large set of VOCs by a representative set of Antarctic bacteria. The synthesis of these VOCs appeared to be related neither to the presence of pks genes nor the presence of plasmid molecules. The whole body of data obtained in this work indicates that sponge-associated bacteria represent an untapped source for the identification of new antimicrobial compounds and are paving the way for the discovery of new drugs that can be efficiently and successfully used for the treatment of CF infections.     

Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology

Interaction with plants around their roots and foliage forms the natural habitat for a wide range of gram-negative bacteria such as Burkholderia, Pseudomonas and Ralstonia. During these interactions many of these bacteria facilitate highly beneficial processes such as the breakdown of pollutants or enhancement of crop growth. All these bacterial species are also capable of causing opportunistic infections in vulnerable individuals, especially people with cystic fibrosis (CF). Here we will review the current understanding of the Burkholderia cepacia complex (Bcc) as a group of model opportunistic pathogens, contrasting their clinical epidemiology with their ecological importance. Currently, the B. cepacia complex is composed of nine formally named species groups which are all difficult to identify using phenotypic methods. Genetic methods such as 16S rRNA and recA gene sequence analysis have proven useful for Bcc species identification. Multilocus sequence typing (MLST) is also emerging as a very useful tool for both Bcc strain and species identification. Historically, Burkholderia cenocepacia was the most dominant Bcc pathogen in CF, however, probably as a result of strict infection control practices introduced to control the spread of this species, its prevalence has been reduced. Burkholderia multivorans is the now the most dominant Bcc infection encountered in the UK CF population, a changing epidemiology that also appears to be occurring in the US CF population. The distribution of Bcc species residing in the natural environment may vary considerably with the type of environment examined. Clonally identical Bcc strains have been found to occur in the natural environment and cause infection. The contamination of medical devices, disinfectants and pharmaceutical formulations has also been directly linked to several outbreaks of infection. In the last 10 years considerable progress has been made in understanding the natural biology and clinical infections caused by this fascinating group of bacteria.     

Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates

AIMS: Burkholderia cepacia complex (Bcc) isolates causing pulmonary infection in cystic fibrosis (CF) patients grow within an acidic environment in the lung. As exposure to acid pH has been shown to increase intracellular inorganic polyphosphate (polyP) formation in some bacteria, we investigated the inter-relationship between acidic pH and polyP accumulation in Bcc isolates. METHODS AND RESULTS: The formation of polyP by one Burkholderia cenocepacia clinical isolate was initially examined at a range of pH values by measuring total intracellular polyP accumulation and phosphate uptake. The pattern of polyP accumulation corresponded with the pattern of phosphate uptake with the maximum for both occurring at pH 5.5. Phosphate uptake and formation of polyP by this isolate was further determined over 48 h at pH 5.5, 6.5 and 7.5; formation of polyP was maximal at pH 5.5 at all time points studied. Sixteen of 17 additional clinical and environmental Bcc isolates examined also exhibited maximum phosphate uptake at pH 5.5. CONCLUSIONS: Both clinical and environmental Bcc isolates, of five genomovars, show enhanced formation of polyP in an acidic environment. Given both the speculated role of polyP in pathogenesis, cell signalling and biofilm formation and the acidic nature of the CF lung, this may be of considerable clinical importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth of Bcc in an acidic environment, such as that found in the lungs of CF patients may be influenced in part by polyP accumulation.     

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 degrees 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.     

Characterization of Burkholderia cepacia complex from cystic fibrosis patients in China and their chitosan susceptibility

A survey of Burkholderia cepacia complex (Bcc) species was conducted in sputum from cystic fibrosis (CF) patients in China. One hundred and four bacterial isolates were recovered on B. cepacia selective agar and 42 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates from CF sputum was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the 42 Bcc isolates belong to B. cepacia, B. cenocepacia and B. contaminans while predominant Bcc species was B. cenocepacia. This is the first report of B. contaminans from CF sputum in China. In addition, results from this study showed that chitosan solution at 10, 25, 50 and 100 μg/ml markedly inhibited the growth of the 16 representative isolates from the three different Bcc species, which indicated that chitosan was a potential bactericide against Bcc bacteria.     

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.     

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.     

Isolation of Burkholderia cepacia complex genomovars from waters

The aim of this study was to develop a selective enrichment broth as an aid for the isolation of Burkholderia cepacia complex (Bcc) bacteria from water. To allow growth of all nine genomovars, mixtures of two carbon sources had to be used, i.e. L-arabinose/D-cellobiose or L-arabinose/L-threonine. Selectivity was provided by polymyxin B and 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390). Following enrichment, Bcc bacteria were isolated on a diagnostic O/F agar supplemented with gentamicin. A preliminary bio-diversity study on 28 surface waters yielded five different genomovars, i.e. B. cepacia (genomovar I), B. multivorans, B. cenocepacia, B. vietnamiensis and B. anthina. Drinking waters did not contain Bcc bacteria. However, the genomovar pattern from a given sample varied with the enrichment broth used.     

Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid

The Burkholderia cepacia complex (Bcc) consists of 17 closely related species of opportunistic bacterial pathogens, which are particularly problematic for cystic fibrosis patients and immunocompromised individuals. Bcc genomes consist of multiple replicons, and each strain sequenced to date has three chromosomes. In addition to genes thought to be essential for survival, each chromosome carries at least one rRNA operon. We isolated three mutants during a transposon mutagenesis screen that were non-pathogenic in a Caenorhabditis elegans infection model. It was demonstrated that these mutants had lost chromosome 3 (c3), and that the observed attenuation of virulence was a consequence of this. We constructed a c3 mini-replicon and used it to cure c3 from strains of several Bcc species by plasmid incompatibility, resulting in nine c3-null strains covering seven Bcc species. Phenotypic characterization of c3-null mutants revealed that they were attenuated in virulence in multiple infection hosts (rat, zebrafish, C. elegans, Galleria mellonella and Drosophila melanogaster), that they exhibited greatly diminished antifungal activity, and that c3 was required for d-xylose, fatty acid and pyrimidine utilization, as well as for exopolysaccharide production and proteolytic activity in some strains. In conclusion, we show that c3 is not an essential chromosomal element, rather a large plasmid that encodes virulence, secondary metabolism and other accessory functions in Bcc bacteria.     

Cangene gold medal award lecture - Genomic analysis and modification of Burkholderia cepacia complex bacteriophages

The Burkholderia cepacia complex (Bcc) is a group of 17 Gram-negative predominantly environmental bacterial species that cause potentially fatal opportunistic infections in cystic fibrosis (CF) patients. Although its prevalence in these individuals is lower than that of Staphylococcus aureus and Pseudomonas aeruginosa , the Bcc remains a serious problem in the CF community because of the pathogenicity, transmissibility, and inherent antibiotic resistance of these organisms. An alternative treatment for Bcc infections that is currently being developed is phage therapy, the clinical use of viruses that infect bacteria. To assess the suitability of individual phage isolates for therapeutic use, the complete genome sequences of a panel of Bcc-specific phages were determined and analyzed. These sequences encode a broad range of proteins with a gradient of relatedness to phage and bacterial gene products from Burkholderia and other genera. The majority of these phages were found not to encode virulence factors, and despite their predominantly temperate nature, a proof-of-principle experiment has shown that they may be modified to a lytic form. Both the genomic characterization and subsequent engineering of Bcc-specific phages are fundamental to the development of an effective phage therapy strategy for these bacteria.     

Pathogenicity, virulence factors, and strategies to fight against Burkholderia cepacia complex pathogens and related species

The Burkholderia cepacia complex (Bcc) is a group of 17 closely related species of the β-proteobacteria subdivision that emerged in the 1980s as important human pathogens, especially to patients suffering from cystic fibrosis. Since then, a remarkable progress has been achieved on the taxonomy and molecular identification of these bacteria. Although some progress have been achieved on the knowledge of the pathogenesis traits and virulence factors used by these bacteria, further work envisaging the identification of potential targets for the scientifically based design of new therapeutic strategies is urgently needed, due to the very difficult eradication of these bacteria with available therapies. An overview of these aspects of Bcc pathogenesis and opportunities for the design of future therapies is presented and discussed in this work.     

Highly purified lipopolysaccharides from Burkholderia cepacia complex clinical isolates induce inflammatory cytokine responses via TLR4-mediated MAPK signalling pathways and activation of NFkappaB

In cystic fibrosis (CF), bacteria of the Burkholderia cepacia complex (Bcc) can induce a fulminant inflammation with pneumonitis and sepsis. Lipopolysaccharide (LPS) may be an important virulence factor associated with this decline but little is known about the molecular pathogenesis of Bcc LPS. In this study we have investigated the inflammatory response to highly purified LPS from different Bcc clinical isolates and the cellular signalling pathways employed. The inflammatory response (TNFalpha, IL-6) was measured in human MonoMac 6 monocytes and inhibition experiments were used to investigate the Toll-like receptors and associated adaptor molecules and pathways utilized. LPS from all clinical Bcc isolates induced significant pro-inflammatory cytokines and utilized TLR4 and CD14 to mediate activation of mitogen-activated protein kinase pathways, IkappaB-alpha degradation and NFkappaB activation. However, LPS from different clinical isolates of the same clonal strain of Burkholderia cenocepacia were found to induce a varied inflammatory response. LPS from clinical isolates of Burkholderia multivorans was found to activate the inflammatory response via MyD88-independent pathways. This study suggests that LPS alone from clinical isolates of Bcc is an important virulence factor in CF and utilizes TLR4-mediated signalling pathways to induce a significant inflammatory response.