Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex

The Burkholderia cepacia complex (BCC) comprises a group of bacteria associated with opportunistic infections, especially in cystic fibrosis patients. B. cenocepacia J2315, of the transmissible ET12 lineage, contains a type III secretion (TTS) gene cluster implicated in pathogenicity. PCR and hybridisation assays indicate that the TTS gene cluster is present in all members of the BCC except B. cepacia (formerly genomovar I). The TTS gene clusters of B. cenocepacia J2315 and B. multivorans are similar in organisation but have variable levels of gene identity. Nucleotide sequence data obtained for the equivalent region of the B. cepacia genome indicate the absence of TTS structural genes due to a rearrangement likely to involve more than one step.     

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.     

A BCAM0223 Mutant of Burkholderia cenocepacia Is Deficient in Hemagglutination, Serum Resistance, Adhesion to Epithelial Cells and Virulence

Burkholderia cepacia complex (Bcc) bacteria are a problematic group of microorganisms causing severe infections in patients with Cystic Fibrosis. In early stages of infection, Bcc bacteria must be able to adhere to and colonize the respiratory epithelium. Although this is not fully understood, this primary stage of infection is believed to be in part mediated by a specific type of adhesins, named trimeric autotransporter adhesins (TAAs). These homotrimeric proteins exist on the surface of many Gram negative pathogens and often mediate a number of critical functions, including biofilm formation, serum resistance and adherence to an invasion of host cells. We have previously identified in the genome of the epidemic clinical isolate B. cenocepacia J2315, a novel cluster of genes putatively encoding three TAAs (BCAM0219, BCAM0223 and BCAM0224). In this study, the genomic organization of the TAA cluster has been determined. To further address the direct role of the putative TAA BCAM0223 in B. cenocepacia pathogenicity, an isogenic mutant was constructed via insertional inactivation. The BCAM0223::Tp mutant is deficient in hemagglutination, affected in adherence to vitronectin and in biofilm formation and showed attenuated virulence in the Galleria mellonella model of infection. Moreover, the BCAM0223::Tp mutant also showed a significant reduction in its resistance to human serum as well as in adherence, but not in invasion of, cultured human bronchial epithelial cells. Altogether these results demonstrate that the BCAM0223 protein is a multifunctional virulence factor that may contribute to the pathogenicity of B. cenocepacia.     

Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia

Chronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Bcc (Burkholderia cepacia complex) is the major cause of morbidity and mortality among CF (cystic fibrosis) patients. PA-IIL (lecB gene), a soluble lectin from Ps. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologues have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centres, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA (B. cenocepacia lectin A), was obtained. The presence of native BclA in B. cenocepacia extracts was checked using a proteomic approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with methyl alpha-D-mannoside demonstrates a dissociation constant (K(d)) of 2.75 x 10(-6) M. The X-ray crystal structure of the complex with methyl alpha-D-mannoside was determined at 1.7 A (1 A=0.1 nm) resolution. The lectin forms homodimers with one binding site per monomer, acting co-operatively with the second dimer site. Each monomer contains two Ca2+ ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site and oligomerization mode indicate that the proteins should have different roles in the bacteria.     

Diversity and distribution of Burkholderia cepacia complex in the rhizosphere of rice and maize

A survey of Burkholderia cepacia complex (Bcc) species was conducted in agricultural fields within Hangzhou, China. Out of the 251 bacterial isolates recovered on the selective media from the rhizosphere of rice and maize, 112 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates 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 majority belong to B. cepacia, Burkholderia cenocepacia recA lineage IIIB, Burkholderia vietnamiensis and Burkholderia pyrrocinia. Burkholderia cenocepacia and B. vietnamiensis dominated the rhizosphere of maize and rice, respectively, indicating that species composition and abundance of Bcc may vary dramatically in different crop rhizospheres. In addition, one isolate (R456) formed a single discrete cluster within the phylogenetic analysis of the Bcc recA gene, and it may belong to a new genomovar.     

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.     

A putative type III secretion gene cluster is widely distributed in the Burkholderia cepacia complex but absent from genomovar I

Using probes constructed from Ralstonia solanacearum and Burkholderia pseudomallei, putative type III secretion (TTS) genes were identified in Burkholderia cepacia J2315 (genomovar III). A cosmid clone containing DNA with homology to five TTS genes was sub-cloned and regions were sequenced in order to design oligonucleotides for polymerase chain reaction assays. These indicated that two putative TTS genes (bcscQ and bcscV) were present in all members of the B. cepacia complex with the exception of strains from genomovar I. Southern blot assays confirmed this observation, suggesting that the lack of a TTS gene cluster may define a major difference between B. cepacia genomovar I and other members of the B. cepacia complex, including genomovar III. In contrast to TTS gene clusters in other bacteria, a putative gene homologous to the virB1 gene of Brucella suis was located directly downstream of bcscQR.     

Differential modulation of innate immune cell functions by the Burkholderia cepacia complex: Burkholderia cenocepacia but not Burkholderia multivorans disrupts maturation and induces necrosis in human dendritic cells

Burkholderia cepacia complex (BCC) bacteria cause pulmonary infections that can evolve into fatal overwhelming septicemia in chronic granulomatous disease or cystic fibrosis patients. Burkholderia cenocepacia and Burkholderia multivorans are responsible for the majority of BCC infections in cystic fibrosis patients, but B. cenocepacia is generally associated with a poorer prognosis than B. multivorans. The present study investigated whether these pathogens could modulate the normal functions of primary human monocyte-derived dendritic cells (DCs), important phagocytic cells that act as critical orchestrators of the immune response. Effects of the bacteria on maturation of DCs were determined using flow cytometry. DCs co-incubated for 24 h with B. cenocepacia, but not B. multivorans, had reduced expression of costimulatory molecules when compared with standard BCC lipopolysaccharide-matured DCs. B. cenocepacia, but not B. multivorans, also induced necrosis in DCs after 24 h, as determined by annexin V and propidium iodide staining. DC necrosis only occurred after phagocytosis of live B. cenocepacia; DCs exposed to heat-killed bacteria, bacterial supernatant or those pre-treated with cytochalasin D then exposed to live bacteria remained viable. The ability of B. cenocepacia to interfere with normal DC maturation and induce necrosis may contribute to its pathogenicity in susceptible hosts.     

Intracellular trafficking and replication of Burkholderia cenocepacia in human cystic fibrosis airway epithelial cells

We investigated the trafficking of Burkholderia cenocepacia, an opportunistic respiratory pathogen of persons with cystic fibrosis (CF), in immortalized CF airway epithelial cells in vitro. Our results indicate that bacteria enter cells in a process involving actin rearrangement. Whereas both live and heat-killed bacteria reside transiently in early endosomes, only live bacteria escape from late endosomes to colocalize in vesicles positive for lysosomal membrane marker LAMP1, endoplasmic reticulum (ER) membrane marker calnexin, and autophagosome marker monodansylcadavarine (MDC). Twenty-four hours after infection, microcolonies of live bacteria were observed in the perinuclear area colocalizing with calnexin. In contrast, after ingestion, dead bacteria colocalized with late endosome marker Rab7, and lysosome markers LAMP1 and cathepsin D, but not with calnexin or MDC. Six to eight hours after ingestion of dead bacteria, degraded bacterial particles were observed in the cytoplasm and in vesicles positive for cathepsin D. These results indicate that live B. cenocepacia gain entry into human CF airway cells by endocytosis, escape from late endosomes to enter autophagosomes that fail to fuse with mature lysosomes, and undergo replication in the ER. This survival and replication strategy may contribute to the capacity of B. cenocepacia to persist in the lungs of infected CF patients.     

PCR-based identification and characterization of Burkholderia cepacia complex bacteria from clinical and environmental sources

AIMS: To study the genotypic identification and characterization of the 119 Burkholderia cepacia complex (Bcc) strains recovered from clinical and environmental sources in Japan and Thailand. METHODS AND RESULTS: Based on the results of analysis by 16S rDNA RFLP generated after digestion with DdeI, the Bcc strains were differentiated into two patterns: pattern 1 (including Burkholderia vietnamiensis) and pattern 2 (including B. cepacia genomovar I, Burkholderia cenocepacia and Burkholderia stabilis). All strains belonged to pattern 2 except for one strain. In the RFLP analysis of the recA gene using HaeIII, strains were separated into eight patterns designated as A, D, E, G, H, I, J and K, of which pattern K was new. Burkholderia cepacia epidemic strain marker (BCESM) encoded by esmR [corrected] and the pyrrolnitrin biosynthetic locus encoded by prnC were present in 22 strains (18%) and 88 strains (74%) from all sources, respectively. All esmR-positive [corrected] strains belonged to B. cenocepacia, whereas most prnC-positive strains belonged to B. cepacia genomovar I. CONCLUSIONS: Strains derived from clinical sources were assigned to B. cepacia genomovar I, B. cenocepacia, B. stabilis and B. vietnamiensis. The majority of Bcc strains from environmental sources (77 of a total 95 strains) belonged to B. cepacia genomovar I, whereas the rest belonged to B. cenocepacia. On the basis of genomovar-specific PCR and prnC RFLP analysis, strains belonging to recA pattern K were identified as B. cepacia genomovar I. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the genotypic identification of a collection of the Bcc strains from Japan and Thailand. RFLP analysis of the prnC gene promises to be a useful method for differentiating Burkholderia pyrrocinia from B. cepacia genomovar I strains.     

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.     

An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals that is endemic in subtropical areas. B. pseudomallei is a facultative intracellular pathogen that may invade and survive within eukaryotic cells for prolonged periods. After internalization, the bacteria escape from endocytic vacuoles into the cytoplasm of infected cells and form membrane protrusions by inducing actin polymerization at one pole. It is believed that survival within phagocytic cells and cell-to-cell spread via actin protrusions is required for full virulence. We have studied the role of a putative type III protein secretion apparatus (Bsa) in the interaction between B. pseudomallei and host cells. The Bsa system is very similar to the Inv/Mxi-Spa type III secretion systems of Salmonella and Shigella. Moreover, B. pseudomallei encodes proteins that are very similar to Salmonella and Shigella Inv/Mxi-Spa secreted proteins required for invasion, escape from endocytic vacuoles, intercellular spread and pathogenesis. Antibodies to putative Bsa-secreted proteins were detected in convalescent serum from a melioidosis patient, suggesting that the system is functionally expressed in vivo. B. pseudomallei mutant strains lacking components of the Bsa secretion and translocation apparatus were constructed. The mutant strains exhibited reduced replication in J774.2 murine macrophage-like cells, an inability to escape from endocytic vacuoles and a complete absence of formation of membrane protrusions and actin tails. These findings indicate that the Bsa type III secretion system plays an essential role in modulating the intracellular behaviour of B. pseudomallei.     

Burkholderia cenocepacia lectin A binding to heptoses from the bacterial lipopolysaccharide

Bacteria from the Burkholderia cepacia complex (Bcc) cause highly contagious pneumonia among cystic fibrosis (CF) patients. Among them, Burkholderia cenocepacia is one of the most dangerous in the Bcc and is the most frequent cause of morbidity and mortality in CF patients. Indeed, it is responsible of "cepacia syndrome", a deadly exacerbation of infection, that is the main cause of poor outcomes in lung transplantation. Burkholderia cenocepacia produces several soluble lectins with specificity for fucosylated and mannosylated glycoconjugates. These lectins are present on the bacterial cell surface and it has been proposed that they bind to lipopolysaccharide epitopes. In this work, we report on the interaction of one B. cenocepacia lectin, BC2L-A, with heptose and other manno configured sugar residues. Saturation transfer difference NMR spectroscopy studies of BC2L-A with different mono- and disaccharides demonstrated the requirement of manno configuration with the hydroxyl or glycol group at C6 for the binding process. The crystal structure of BC2L-A complexed with the methyl-heptoside confirmed the location of the carbohydrate ring in the binding site and elucidated the orientation of the glycol tail, in agreement with NMR data. Titration calorimetry performed on monosaccharides, heptose disaccharides and bacterial heptose-containing oligosaccharides and polysaccharides confirmed that bacterial cell wall contains carbohydrate epitopes that can bind to BC2L-A. Additionally, the specific binding of fluorescent BC2L-A lectin on B. cenocepacia bacterial surface was demonstrated by microscopy.     

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.     

Burkholderia cepacia complex infection in a cohort of Italian patients with cystic fibrosis

The aims of this study were to detect Burkholderia cepacia complex (Bcc) strains in a cohort of Cystic Fibrosis patients (n=276) and to characterize Bcc isolates by molecular techniques. The results showed that 11.23% of patients were infected by Bcc. Burkholderia cenocepacia lineage III-A was the most prevalent species (64.3%) and, of these, 10% was cblA positive and 50% esmR positive. Less than half of the strains were sensitive to ceftazidime, meropenem, piperacillin tazobactam, and trimethoprim-sulfamethoxazole. About half of the strains (41%) had homogeneous profiles, suggesting cross-transmission. The infection by B. cenocepacia was associated to a high rate of mortality (p=0.01).     

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.