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.     

The Burkholderia cenocepacia peptidoglycan‐associated lipoprotein is involved in epithelial cell attachment and elicitation of inflammation

The Burkholderia cepacia complex (Bcc) is a group of Gram‐negative opportunistic pathogens causing infections in people with cystic fibrosis (CF). Bcc is highly antibiotic resistant, making conventional antibiotic treatment problematic. The identification of novel targets for anti‐virulence therapies should improve therapeutic options for infected CF patients. We previously identified that the peptidoglycan‐associated lipoprotein (Pal) was immunogenic in Bcc infected CF patients; however, its role in Bcc pathogenesis is unknown. The virulence of a pal deletion mutant (Δpal) in Galleria mellonella was 88‐fold reduced (p < .001) compared to wild type. The lipopolysaccharide profiles of wild type and Δpal were identical, indicating no involvement of Pal in O‐antigen transport. However, Δpal was more susceptible to polymyxin B. Structural elucidation by X‐ray crystallography and calorimetry demonstrated that Pal binds peptidoglycan fragments. Δpal showed a 1.5‐fold reduced stimulation of IL‐8 in CF epithelial cells relative to wild type (p < .001), demonstrating that Pal is a significant driver of inflammation. The Δpal mutant had reduced binding to CFBE41o^? cells, but adhesion of Pal‐expressing recombinant E. coli to CFBE41o^? cells was enhanced compared to wild‐type E. coli (p < .0001), confirming that Pal plays a direct role in host cell attachment. Overall, Bcc Pal mediates host cell attachment and stimulation of cytokine secretion, contributing to Bcc pathogenesis.     

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.     

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.     

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.     

Immunoproteomic Analysis of Proteins Expressed by Two Related Pathogens,Burkholderia multivorans and Burkholderia cenocepacia,during Human Infection

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.     

Proteomic analysis of uropathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most common of bacterial infections in humans. Although a number of Gram-negative bacteria can cause UTIs, most cases are due to infection by uropathogenic E. coli (UPEC). Genomic studies have shown that UPEC encode a number of specialized activities that allow the bacteria to initiate and maintain infections in the environment of the urinary tract. Proteomic analyses have complemented the genomic data and have documented differential patterns of protein synthesis for bacteria growing ex vivo in human urine or recovered directly from the urinary tracts of infected mice. These studies provide valuable insights into the molecular basis of UPEC pathogenesis and have aided the identification of putative vaccine targets. Despite the substantial progress that has been achieved, many future challenges remain in the application of proteomics to provide a comprehensive view of bacterial pathogenesis in both acute and chronic UTIs.     

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.     

The multifarious, multireplicon Burkholderia cepacia complex

The Burkholderia cepacia complex (Bcc) is a collection of genetically distinct but phenotypically similar bacteria that are divided into at least nine species. Bcc bacteria are found throughout the environment, where they can have both beneficial and detrimental effects on plants and some members can also degrade natural and man-made pollutants. Bcc bacteria are now recognized as important opportunistic pathogens that can cause variable lung infections in cystic fibrosis patients, which result in asymptomatic carriage, chronic infection or 'cepacia syndrome', which is characterized by a rapid decline in lung function that can include invasive disease. Here we highlight the unique characteristics of the Bcc, focusing on the factors that determine virulence.     

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.     

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 analysis of Burkholderia cepacia complex in the water bodies of West Lake, Hangzhou, China

A survey of Burkholderia cepacia complex (Bcc) species was conducted in water bodies of West Lake in China. A total of 670 bacterial isolates were recovered on selective media. Out of them, 39.6% (265 isolates) were assigned to the following species: Burkholderia multivorans, Burkholderia cenocepacia recA lineage IIIA, IIIB, Burkholderia stabilis, Burkholderia vietnamiensis, and Burkholderia seminalis while B. cenocepacia is documented as a dominant Bcc species in water of West Lake. In addition, all Bcc isolates tested were PCR negative for the cblA and esmR transmissibility marker genes except B. cenocepacia IIIB A8 which was positive for esmR genelater. The present study raises great concerns on the role of West Lake as a “reservoir” for potential Bcc pathogenic strains.     

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.     

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.     

Can flies help humans treat neurodegenerative diseases?

Neurodegenerative diseases are becoming increasingly common as life expectancy increases. Recent years have seen tremendous progress in the identification of genes that cause these diseases. While mutations have been found and cellular processes defined that are altered in the disease state, the identification of treatments and cures has proven more elusive. The process of finding drugs and therapies to treat human diseases can be slow, expensive and frustrating. Can model organisms such as Drosophila speed the process of finding cures and treatments for human neurodegenerative diseases? We pose three questions, (1) can one mimic the essential features of human diseases in an organism like Drosophila, (2) can one cure a model organisms of human disease and (3) will these efforts accelerate the identification of useful therapies for testing in mice and ultimately humans? Here we focus on the use of Drosophila to identify potential treatments for neurodegenerative diseases such as Huntington's and we discuss how well these therapies translate into mammalian systems. BioEssays 26:485–496, 2004. © 2004 Wiley Periodicals, Inc.     

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.     

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.     

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.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.