Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome

Background  

Burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND) efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance.     

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 cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life‐threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin‐binding adaptors p62 and NDP52 and the autophagosome membrane‐associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre‐infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.     

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Background  

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.     

Metabolic Profiling of Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia Isolates from Maize Rhizosphere

Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia are the Burkholderia cepacia complex (Bcc) species most frequently associated with roots of crop plants. To investigate the ecophysiological diversity of these species, metabolic profiling of maize rhizosphere isolates was carried out by means of the Biolog system, using GN2 and SFN2 plates and different parameters related to optical density (OD). The metabolic profiles produced by the SFN2 and GN2 plates were identical, but the SFN2's narrower range of OD values and significantly longer reaction times made these plates less suitable for differentiation of isolates. Principal component analysis of maximum OD (OD_M) and maximum substrate oxidation rate (μ_M) data generated by GN2 plates allowed the selection of a reduced number of carbon sources. Statistical analysis of OD_M values highlighted marked differences between the metabolic profiles of B. cenocepacia and B. ambifaria, whereas metabolic profiles of B. pyrrocinia clustered very often with those of B. cenocepacia. Analysis of the μ_M parameter resulted in a slightly lower differentiation among the three Bcc species and a higher metabolic diversity within the single species, in particular within B. cenocepacia. Finally, B. cenocepacia and B. pyrrocinia showed generally higher oxidation rates than B. ambifaria on those GN2 substrates that commonly occur in maize root exudates.     

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.     

Differences in strategies to combat osmotic stress in Burkholderia cenocepacia elucidated by NMR-based metabolic profiling

Aims: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance. Methods and Results: We employed NMR‐based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo‐protective metabolites glycine‐betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites. Conclusions: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress. Significance and impact of the study: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.     

A functional phenylacetic acid catabolic pathway is required for full pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans host model

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of metabolically versatile bacteria that have emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Previously a screen of transposon mutants in a rat pulmonary infection model identified an attenuated mutant with an insertion in paaE, a gene related to the phenylacetic acid (PA) catabolic pathway. In this study, we characterized gene clusters involved in the PA degradation pathway of B. cenocepacia K56-2 in relation to its pathogenicity in the Caenorhabditis elegans model of infection. We demonstrated that targeted-insertion mutagenesis of paaA and paaE, which encode part of the putative PA-coenzyme A (CoA) ring hydroxylation system, paaZ, coding for a putative ring opening enzyme, and paaF, encoding part of the putative beta-oxidation system, severely reduces growth on PA as a sole carbon source. paaA and paaE insertional mutants were attenuated for virulence, and expression of paaE in trans restored pathogenicity of the paaE mutant to wild-type levels. Interruption of paaZ and paaF slightly increased virulence. Using gene interference by ingested double-stranded RNA, we showed that the attenuated phenotype of the paaA and paaE mutants is dependent on a functional p38 mitogen-activated protein kinase pathway in C. elegans. Taken together, our results demonstrate that B. cenocepacia possesses a functional PA degradation pathway and that the putative PA-CoA ring hydroxylation system is required for full pathogenicity in C. elegans.     

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.     

Polyphasic characterisation of Burkholderia cepacia complex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.     

Phenotypic characterization of trimeric autotransporter adhesin-defective bcaC mutant of Burkholderia cenocepacia: cross-talk towards the histidine kinase BCAM0218

Burkholderia cenocepacia is a virulent species belonging to the Burkholderia cepacia complex (Bcc) and one of the most problematic agents of chronic lung infection in cystic fibrosis patients. B. cenocepacia possesses a large panel of virulence traits that include trimeric autotransporter adhesins (TAAs). Such proteins are obligate homotrimeric anchored in the outer membrane. They are players in the adhesion events that occur between bacteria and biotic/abiotic surfaces. In this study, we constructed two insertional-mutants for TAA bcaC and Histidine kinase (HK) BCAM0218 genes, which are clustered together within the B. cenocepacia K56-2 TAA cluster. The bcaC-mutant affects B. cenocepacia adhesion to extracellular matrix proteins and red blood cells hemagglutination. BcaC contributes to enhancing B. cenocepacia K56-2 adhesion to bronchial epithelial cells. The expression of bcaC seems to affect biofilm formation negatively. Due to a BCAM0218 disruption, the bcaC expression increases significantly, indicating that they are functionally linked. The overexpression of bcaC in the BCAM0218-mutant background rescues at least part of the BcaC functions. Altogether, these findings reveal the multifunctionality of BcaC as a novel B. cenocepacia K56-2 virulence factor and postulate the involvement of a sensor HK (BCAM0218) in the control of this TAA gene.     

Improved Electrotransformation and Decreased Antibiotic Resistance of the Cystic Fibrosis Pathogen Burkholderia cenocepacia Strain J2315

The bacterium Burkholderia cenocepacia is pathogenic for sufferers from cystic fibrosis (CF) and certain immunocompromised conditions. The B. cenocepacia strain most frequently isolated from CF patients, and which serves as the reference for CF epidemiology, is J2315. The J2315 genome is split into three chromosomes and one plasmid. The strain was sequenced several years ago, and its annotation has been released recently. This information should allow genetic experimentation with J2315, but two major impediments appear: the poor potential of J2315 to act as a recipient in transformation and conjugation and the high level of resistance it mounts to nearly all antibiotics. Here, we describe modifications to the standard electroporation procedure that allow routine transformation of J2315 by DNA. In addition, we show that deletion of an efflux pump gene and addition of spermine to the medium enhance the sensitivity of J2315 to certain commonly used antibiotics and so allow a wider range of antibiotic resistance genes to be used for selection.Burkholderia cenocepacia is part of the Burkholderia cepacia complex (Bcc), a group of closely related bacteria of soil, water, and roots (41) recently updated to at least 15 related species (42). Bcc displays many interesting features (see reference 27 for a review). Originally discovered as responsible for soft onion rot (3), Bcc species also interact beneficently with plants (see reference 34 for a review) and may degrade pollutants such as phthalate or the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5,-T) (25, 33). But it is the emergence of Bcc as an opportunistic pathogen of people suffering from cystic fibrosis (CF) (19) and immunocompromizing conditions that has drawn most attention to these bacteria. Among Bcc species, Burkholderia multivorans and B. cenocepacia are the most prevalent in the epidemiology of CF. In particular, strains of the ET12 lineage of B. cenocepacia were responsible for a major transcontinental epidemic among CF patients in the 1990s (20), an outbreak aggravated by the high levels of resistance to nearly all antibiotics that characterizes Bcc. Species of the Bcc have large genomes (7 to 9 Mb) composed of two or three chromosomes and one or more plasmids, an unusual genomic organization among bacteria. The first Bcc genome to be sequenced was that of B. cenocepacia J2315 (also known as LMG16656), the type strain of the ET12 lineage and the reference strain for CF epidemiology; the sequence was completed and made available by the Wellcome Trust Sanger Institute in 2003. It revealed three chromosomes of 3.9, 3.2, and 0.9 Mb and a plasmid of 93 kb. The annotation of this genome was released recently (15).The pathogenicity and multipartite genome of B. cenocepacia make it an important subject for both practical and fundamental study. Genetic modification is essential to the success of many such investigations. Unfortunately, J2315 throws up major barriers to genetic manipulation. Standard electrotransformation techniques are ineffective with this strain, as also found elsewhere (26). Conjugal introduction of DNA has proved unreliable despite adaptations (7) that have enabled occasional successes with B. cenocepacia species (9, 40) including J2315 (39) (see also Results below). Besides, the natural resistance of J2315 to antibiotics, high even on the scale of the generally extensive resistance of B. cenocepacia species (31), severely restricts the use of antibiotic resistance in genetic selections. Circumventing these problems by resorting to a proxy strain, B. cenocepacia K56-2, that has not been sequenced and is more permissive to gene transfer (26, 17, 32, 9) runs the risk that results will be of uncertain relevance to J2315.In the context of our general aim to decipher the role of the four replicon-specific ParABS systems of J2315 (6), we have sought to overcome these obstacles. We report here the reproducible electrotransformation of J2315, and we analyze factors that improve its efficiency. We report also our isolation of a J2315 derivative with reduced antibiotic resistance and the broadened selection possibilities this offers. Detailed protocols are provided which should facilitate studies of this pathogen.     

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.     

The Third Replicon of Members of the Burkholderia cepacia Complex,Plasmid pC3, Plays a Role in Stress Tolerance

The metabolically versatile Burkholderia cepacia complex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined. B. cenocepacia strains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and the Caenorhabditis elegans model of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance of B. cenocepacia H111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc.     

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome

Background  

The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs.     

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.     

In vivo fluorescence imaging of bacteriogenic cyanide in the lungs of live mice infected with cystic fibrosis pathogens

Background

Pseudomonas aeruginosa (PA) and Burkholderia cepacia complex (Bcc), commonly found in the lungs of cystic fibrosis (CF) patients, often produce cyanide (CN), which inhibits cellular respiration. CN in sputa is a potential biomarker for lung infection by CF pathogens. However, its actual concentration in the infected lungs is unknown.

Methods and Findings

This work reports observation of CN in the lungs of mice infected with cyanogenic PA or Bcc strains using a CN fluorescent chemosensor (4′,5′-fluorescein dicarboxaldehyde) with a whole animal imaging system. When the CN chemosensor was injected into the lungs of mice intratracheally infected with either PA or B. cepacia strains embedded in agar beads, CN was detected in the millimolar range (1.8 to 4 mM) in the infected lungs. CN concentration in PA-infected lungs rapidly increased within 24 hours but gradually decreased over the following days, while CN concentration in B. cepacia-infected lungs slowly increased, reaching a maximum at 5 days. CN concentrations correlated with the bacterial loads in the lungs. In vivo efficacy of antimicrobial treatments was tested in live mice by monitoring bacteriogenic CN in the lungs.

Conclusions

The in vivo imaging method was also found suitable for minimally invasive testing the efficacy of antibiotic compounds as well as for aiding the understanding of bacterial cyanogenesis in CF lungs.     

Burkholderia cenocepacia K56‐2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation

Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF‐associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56‐2, is a tumor necrosis factor receptor 1‐interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL‐8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.