Synthesis and evaluation of aromatic BDSF bioisosteres on biofilm formation and colistin sensitivity in pathogenic bacteria.

The diffusible signal factor family (DSF) of molecules play an important role in regulating intercellular communication, or quorum sensing, in several disease-causing bacteria. These messenger molecules, which are comprised of cis-unsaturated fatty acids, are involved in the regulation of biofilm formation, antibiotic tolerance, virulence and the control of bacterial resistance. We have previously demonstrated how olefinic N-acyl sulfonamide bioisosteric analogues of diffusible signal factor can reduce biofilm formation or enhance antibiotic sensitivity in a number of bacterial strains. This work describes the design and synthesis of a second generation of aromatic N-acyl sulfonamide bioisosteres. The impact of these compounds on biofilm production in Acinetobacter baumannii, Escherichia coli, Burkholderia multivorans, Burkholderia cepacia, Burkholderia cenocepacia, Pseudomonas aeruginosa and Stenotrophomonas maltophilia is evaluated, in addition to their effects on antibiotic tolerance. The ability of these molecules to increase survival rates on co-administration with colistin is also investigated using the Galleria infection model.

Identification of Key Factors for Anoxic Survival of B. cenocepacia H111.

Burkholderia cenocepacia is an opportunistic pathogen that can lead to severe infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Being an obligate aerobe, B. cenocepacia is unable to grow in the absence of oxygen. In this study, we show that the CF isolate B. cenocepacia H111 can survive in the absence of oxygen. Using a transposon sequencing (Tn-seq) approach, we identified 71 fitness determinants involved in anoxic survival, including a Crp-Fnr family transcriptional regulatory gene (anr2), genes coding for the sensor kinase RoxS and its response regulator RoxR, the sigma factor for flagella biosynthesis (FliA) and subunits of a cytochrome bd oxidase (CydA, CydB and the potentially novel subunit CydP). Individual knockouts of these fitness determinants significantly reduced anoxic survival, and inactivation of both anr copies is shown to be lethal under anoxic conditions. We also show that the two-component system RoxS/RoxR and FliA are important for virulence and swarming/swimming, respectively.

Protein with negative surface charge distribution, Bnr1, shows characteristics of a DNA-mimic protein and may be involved in the adaptation of Burkholderia cenocepacia.

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5-fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5-fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host-cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three-dimensional structure revealed BCAS0292 presents a dimeric ß-structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull-downs with FLAG-tagged BCAS0292, including the Histone H1-like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface-charged regulatory protein 1 (Bnr1), acts as a DNA-mimic and binds to DNA-binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

Biofilm formation by strains of Burkholderia cenocepacia lineages IIIA and IIIB and B. gladioli pv. alliicola associated with onion bacterial scale rot.

The Burkholderia genus has high ecological and nutritional versatility, having species capable of causing diseases in animals, humans, and plants. During chronic infections in humans, biofilm formation is a characteristic often associated with strains from different species of this genus. However, there is still no information on the formation of biofilms by plant pathogenic strains of B. cenocepacia (Bce) lineages IIIA and IIIB and B. gladioli pv. alliicola (Bga), which are associated with onion bacterial scale rot in the semi-arid region of northeast Brazil. In this study, we performed an in vitro characterization of biofilm formation ability in different culture media by the phytopathogenic strains of Bce and Bga and investigated its relationship with swarming motility. Our results indicated the existence of an intraspecific variation in biofilm formation capacity in vitro by these bacteria and the existence of a negative correlation between swarming motility and biofilm formation for strains of Bce lineage IIIB. In addition, histopathological analyses performed using optical microscopy and scanning electron microscopy revealed the formation of biofilm in vivo by Bce strains in onion tissues.

A LysR Family Transcriptional Regulator Modulates Burkholderia cenocepacia Biofilm Formation and Protease Production.

Quorum-sensing (QS) signals are widely employed by bacteria to regulate biological functions in response to cell densities. Previous studies showed that Burkholderia cenocepacia mostly utilizes two types of QS systems, including the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) systems, to regulate biological functions. We demonstrated here that a LysR family transcriptional regulator, Bcal3178, controls the QS-regulated phenotypes, including biofilm formation and protease production, in B. cenocepacia H111. Expression of Bcal3178 at the transcriptional level was obviously downregulated in both the AHL-deficient and BDSF-deficient mutant strains compared to the wild-type H111 strain. It was further identified that Bcal3178 regulated target gene expression by directly binding to the promoter DNA regions. We also revealed that Bcal3178 was directly controlled by the AHL system regulator CepR. These results show that Bcal3178 is a new downstream component of the QS signaling network that modulates a subset of genes and functions coregulated by the AHL and BDSF QS systems in B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important opportunistic pathogen in humans that utilizes the BDSF and AHL quorum-sensing (QS) systems to regulate biological functions and virulence. We demonstrated here that a new downstream regulator, Bcal3178 of the QS signaling network, controls biofilm formation and protease production. Bcal3178 is a LysR family transcriptional regulator modulated by both the BDSF and AHL QS systems. Furthermore, Bcal3178 controls many target genes, which are regulated by the QS systems in B. cenocepacia. Collectively, our findings depict a novel molecular mechanism with which QS systems regulate some target gene expression and biological functions by modulating the expression level of a LysR family transcriptional regulator in B. cenocepacia.

Antagonistic interactions subdue inter-species green-beard cooperation in bacteria.

Cooperation can be favoured through the green-beard mechanism, where a set of linked genes encodes both a cooperative trait and a phenotypic marker (green beard), which allows carriers of the trait to selectively direct cooperative acts to other carriers. In theory, the green-beard mechanism should favour cooperation even when interacting partners are totally unrelated at the genome level. Here, we explore such an extreme green-beard scenario between two unrelated bacterial species-Pseudomonas aeruginosa and Burkholderia cenocepacia, which share a cooperative locus encoding the public good pyochelin (an iron-scavenging siderophore) and its cognate receptor (green beard) required for iron-pyochelin uptake. We show that pyochelin, when provided in cell-free supernatants, can be mutually exchanged between species and provide fitness benefits under iron limitation. However, in co-culture we observed that these cooperative benefits vanished and communities were dominated by P. aeruginosa, regardless of strain background and species starting frequencies. Our results further suggest that P. aeruginosa engages in interference competition to suppress B. cenocepacia, indicating that inter-species conflict arising from dissimilarities at the genome level overrule the aligned cooperative interests at the pyochelin locus. Thus, green-beard cooperation is subdued by competition, indicating that interspecific siderophore cooperation is difficult to evolve and to be maintained.

DNA Methylation Epigenetically Regulates Gene Expression in Burkholderia cenocepacia and Controls Biofilm Formation, Cell Aggregation, and Motility.

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation is an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2 and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, while ΔBCAM0992 was less motile. B. cenocepacia wild-type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-molecule real-time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified, and qPCR experiments showed an upregulation of several genes, including biofilm- and motility-related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation, cell aggregation, and motility.IMPORTANCE CF patients diagnosed with Burkholderia cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multireplicon genome, and much remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to advance the biological understanding of B. cenocepacia cell adaptation via epigenetic regulation. In time, this might lead to novel approaches to tackle B. cenocepacia infections in CF patients.

Inactivation of Rho GTPases by Burkholderia cenocepacia Induces a WASH-Mediated Actin Polymerization that Delays Phagosome Maturation.

Burkholderia cenocepacia is an opportunistic bacterial pathogen that causes severe pulmonary infections in cystic fibrosis and chronic granulomatous disease patients. B. cenocepacia can survive inside infected macrophages within the B. cenocepacia-containing vacuole (BcCV) and to elicit a severe inflammatory response. By inactivating the host macrophage Rho GTPases, the bacterial effector TecA causes depolymerization of the cortical actin cytoskeleton. In this study, we find that B. cenocepacia induces the formation of large cytosolic F-actin clusters in infected macrophages. Cluster formation requires the nucleation-promoting factor WASH, the Arp2/3 complex, and TecA. Inactivation of Rho GTPases by bacterial toxins is necessary and sufficient to induce the formation of the cytosolic actin clusters. By hijacking WASH and Arp2/3 activity, B. cenocepacia disrupts interactions with the endolysosomal system, thereby delaying the maturation of the BcCV.

Burkholderia cenocepacia H111 Produces a Water-Insoluble Exopolysaccharide in Biofilm: Structural Determination and Molecular Modelling.

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.

Negative frequency-dependent selection maintains coexisting genotypes during fluctuating selection.

Natural environments are rarely static; rather selection can fluctuate on timescales ranging from hours to centuries. However, it is unclear how adaptation to fluctuating environments differs from adaptation to constant environments at the genetic level. For bacteria, one key axis of environmental variation is selection for planktonic or biofilm modes of growth. We conducted an evolution experiment with Burkholderia cenocepacia, comparing the evolutionary dynamics of populations evolving under constant selection for either biofilm formation or planktonic growth with populations in which selection fluctuated between the two environments on a weekly basis. Populations evolved in the fluctuating environment shared many of the same genetic targets of selection as those evolved in constant biofilm selection, but were genetically distinct from the constant planktonic populations. In the fluctuating environment, mutations in the biofilm-regulating genes wspA and rpfR rose to high frequency in all replicate populations. A mutation in wspA first rose rapidly and nearly fixed during the initial biofilm phase but was subsequently displaced by a collection of rpfR mutants upon the shift to the planktonic phase. The wspA and rpfR genotypes coexisted via negative frequency-dependent selection around an equilibrium frequency that shifted between the environments. The maintenance of coexisting genotypes in the fluctuating environment was unexpected. Under temporally fluctuating environments, coexistence of two genotypes is only predicted under a narrow range of conditions, but the frequency-dependent interactions we observed provide a mechanism that can increase the likelihood of coexistence in fluctuating environments.

Variation of Burkholderia cenocepacia cell wall morphology and mechanical properties during cystic fibrosis lung infection, assessed by atomic force microscopy.

The influence that Burkholderia cenocepacia adaptive evolution during long-term infection in cystic fibrosis (CF) patients has on cell wall morphology and mechanical properties is poorly understood despite their crucial role in cell physiology, persistent infection and pathogenesis. Cell wall morphology and physical properties of three B. cenocepacia isolates collected from a CF patient over a period of 3.5 years were compared using atomic force microscopy (AFM). These serial clonal variants include the first isolate retrieved from the patient and two late isolates obtained after three years of infection and before the patient's death with cepacia syndrome. A consistent and progressive decrease of cell height and a cell shape evolution during infection, from the typical rods to morphology closer to cocci, were observed. The images of cells grown in biofilms showed an identical cell size reduction pattern. Additionally, the apparent elasticity modulus significantly decreases from the early isolate to the last clonal variant retrieved from the patient but the intermediary highly antibiotic resistant clonal isolate showed the highest elasticity values. Concerning the adhesion of bacteria surface to the AFM tip, the first isolate was found to adhere better than the late isolates whose lipopolysaccharide (LPS) structure loss the O-antigen (OAg) during CF infection. The OAg is known to influence Gram-negative bacteria adhesion and be an important factor in B. cenocepacia adaptation to chronic infection. Results reinforce the concept of the occurrence of phenotypic heterogeneity and adaptive evolution, also at the level of cell size, form, envelope topography and physical properties during long-term infection.

Genomic analyses of Burkholderia cenocepacia reveal multiple species with differential host-adaptation to plants and humans.

BACKGROUND: Burkholderia cenocepacia is a human opportunistic pathogen causing devastating symptoms in patients suffering from immunodeficiency and cystic fibrosis. Out of the 303 B. cenocepacia strains with available genomes, the large majority were isolated from a clinical context. However, several isolates originate from other environmental sources ranging from aerosols to plant endosphere. Plants can represent reservoirs for human infections as some pathogens can survive and sometimes proliferate in the rhizosphere. We therefore investigated if B. cenocepacia had the same potential. RESULTS: We selected genome sequences from 31 different strains, representative of the diversity of ecological niches of B. cenocepacia, and conducted comparative genomic analyses in the aim of finding specific niche or host-related genetic determinants. Phylogenetic analyses and whole genome average nucleotide identity suggest that strains, registered as B. cenocepacia, belong to at least two different species. Core-genome analyses show that the clade enriched in environmental isolates lacks multiple key virulence factors, which are conserved in the sister clade where most clinical isolates fall, including the highly virulent ET12 lineage. Similarly, several plant associated genes display an opposite distribution between the two clades. Finally, we suggest that B. cenocepacia underwent a host jump from plants/environment to animals, as supported by the phylogenetic analysis. We eventually propose a name for the new species that lacks several genetic traits involved in human virulence. CONCLUSION: Regardless of the method used, our studies resulted in a disunited perspective of the B. cenocepacia species. Strains currently affiliated to this taxon belong to at least two distinct species, one having lost several determining animal virulence factors.

Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

Burkholderia cenocepacia Induces Macropinocytosis to Enter Macrophages.

Burkholderia cenocepacia is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. B. cenocepacia survives in macrophages in membrane-bound vacuoles; however, the mechanism by which B. cenocepacia gains entry into macrophages remains unknown. After macrophage internalization, survival of B. cenocepacia within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that B. cenocepacia induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of B. cenocepacia contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of Burkholderia to enter macrophages through the induction of macropinocytosis.

Environmental Burkholderia cenocepacia Strain Enhances Fitness by Serial Passages during Long-Term Chronic Airways Infection in Mice.

Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656T. Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts.

Effects of Extended Storage of Chlorhexidine Gluconate and Benzalkonium Chloride Solutions on the Viability of Burkholderia cenocepacia.

Chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) formulations are frequently used as antiseptics in healthcare and consumer products. Burkholderia cepacia complex (BCC) contamination of pharmaceutical products could be due to the use of contaminated water in the manufacturing process, over-diluted antiseptic solutions in the product, and the use of outdated products, which in turn reduces the antimicrobial activity of CHX and BZK. To establish a "safe use" period following opening containers of CHX and BZK, we measured the antimicrobial effects of CHX (2-10 µg/ml) and BZK (10-50 µg/ml) at sublethal concentrations on six strains of Burkholderia cenocepacia using chemical and microbiological assays. CHX (2, 4, and 10 µg/ml) and BZK (10, 20, and 50 µg/ml) stored for 42 days at 23°C showed almost the same concentration and toxicity compared with freshly prepared CHX and BZK on B. cenocepacia strains. When 5 µg/ml CHX and 20 µg/ml BZK were spiked to six B. cenocepacia strains with different inoculum sizes (10° -105 CFU/ml), their toxic effects were not changed for 28 days. B. cenocepacia strains in diluted CHX and BZK were detectable at concentrations up to 10² CFU/ml after incubation for 28 days at 23°C. Although abiotic and biotic changes in the toxicity of both antiseptics were not observed, our results indicate that B. cenocepacia strains could remain viable in CHX and BZK for 28 days, which in turn, indicates the importance of control measures to monitor BCC contamination in pharmaceutical products.

Evaluation of the electron transfer flavoprotein as an antibacterial target in Burkholderia cenocepacia.

There are hundreds of essential genes in multidrug-resistant bacterial genomes, but only a few of their products are exploited as antibacterial targets. An example is the electron transfer flavoprotein (ETF), which is required for growth and viability in Burkholderia cenocepacia. Here, we evaluated ETF as an antibiotic target for Burkholderia cepacia complex (Bcc). Depletion of the bacterial ETF during infection of Caenorhabditis elegans significantly extended survival of the nematodes, proving that ETF is essential for survival of B. cenocepacia in this host model. In spite of the arrest in respiration in ETF mutants, the inhibition of etf expression did not increase the formation of persister cells, when treated with high doses of ciprofloxacin or meropenem. To test if etf translation could be inhibited by RNA interference, antisense oligonucleotides that target the etfBA operon were synthesized. One antisense oligonucleotide was effective in inhibiting etfB translation in vitro but not in vivo, highlighting the challenge of reduced membrane permeability for the design of drugs against B. cenocepacia. This work contributes to the validation of ETF of B. cenocepacia as a target for antibacterial therapy and demonstrates the utility of a C. elegans liquid killing assay to validate gene essentiality in an in vivo infection model.

NtrC-dependent control of exopolysaccharide synthesis and motility in Burkholderia cenocepacia H111.

Burkholderia cenocepacia is a versatile opportunistic pathogen that survives in a wide variety of environments, which can be limited in nutrients such as nitrogen. We have previously shown that the sigma factor σ54 is involved in the control of nitrogen assimilation and virulence in B. cenocepacia H111. In this work, we investigated the role of the σ54 enhancer binding protein NtrC in response to nitrogen limitation and in the pathogenicity of H111. Of 95 alternative nitrogen sources tested the ntrC showed defects in the utilisation of nitrate, urea, L-citrulline, acetamide, DL-lactamide, allantoin and parabanic acid. RNA-Seq and phenotypic analyses of an ntrC mutant strain showed that NtrC positively regulates two important phenotypic traits: exopolysaccharide (EPS) production and motility. However, the ntrC mutant was not attenuated in C. elegans virulence.

Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c-di-GMP effector BerB.

Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm-control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c-di-GMP) is a positive regulator of biofilm formation in many bacteria, but more knowledge about c-di-GMP effectors is needed. We provide evidence that c-di-GMP, the alternative sigma factor RpoN (σ54), and the enhancer-binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm-stabilizing exopolysaccharide. Our findings suggest that BerB binds c-di-GMP, and activates RpoN-dependent transcription of the berA gene coding for a c-di-GMP-responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm-stabilizing exopolysaccharide in response to high c-di-GMP levels. Our findings imply that the production of biofilm exopolysaccharide in B. cenocepacia is regulated through a cascade involving two consecutive transcription events that are both activated by c-di-GMP. This type of regulation may allow tight control of the expenditure of cellular resources.

Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models.

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.