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The Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a polycationic, amphiphilic and helical neuropeptide, is well known for its neuroprotective actions and cell penetrating properties. In the present study, we evaluated the potent antibacterial property of PACAP38 and related analogs against various bacterial strains. Interestingly, PACAP38 and related analogs can inhibit the growth of various bacteria including Escherichia coli (JM109), Bacillus subtilis (PY79), and the pathogenic Burkholderia cenocepacia (J2315). Investigation of the mechanism of action suggested that a PACAP metabolite, identified as PACAP(9-38), might indeed be responsible for the observed PACAP38 antibacterial action. Surprisingly, PACAP(9-38), which does not induce haemolysis, exhibits an increased specificity toward Burkholderia cenocepacia J2315 compared to other tested bacteria. Finally, the predisposition of PACAP(9-38) to adopt a π-helix conformation rather than an α-helical conformation like PACAP38 could explain this gain in specificity. Overall, this study has revealed a new function for PACAP38 and related derivatives that can be added to its pleiotropic biological activities. This innovative study could therefore pave the way toward the development of new therapeutic agents against multiresistant bacteria, and more specifically the Burkholderia cenocepacia complex.

It was recently proposed that bactericidal antibiotics, besides through specific drug-target interactions, kill bacteria by a common mechanism involving the production of reactive oxygen species (ROS). However, this mechanism involving the production of hydroxyl radicals has become the subject of a lot of debate. Since the contribution of ROS to antibiotic mediated killing most likely depends on the conditions, differences in experimental procedures are expected to be at the basis of the conflicting results. In the present study different methods (ROS specific stainings, gene-expression analyses, electron paramagnetic resonance, genetic and phenotypic experiments, detection of protein carbonylation and DNA oxidation) to measure the production of ROS upon antibiotic treatment in Burkholderia cepacia complex (Bcc) bacteria were compared. Different classes of antibiotics (tobramycin, ciprofloxacin, meropenem) were included, and both planktonic and biofilm cultures were studied. Our results indicate that some of the methods investigated were not sensitive enough to measure antibiotic induced production of ROS, including the spectrophotometric detection of protein carbonylation. Secondly, other methods were found to be useful only in specific conditions. For example, an increase in the expression of OxyR was measured in Burkholderia cenocepacia K56-2 after treatment with ciprofloxacin or meropenem (both in biofilms and planktonic cultures) but not after treatment with tobramycin. In addition results vary with the experimental conditions and the species tested. Nevertheless our data strongly suggest that ROS contribute to antibiotic mediated killing in Bcc species and that enhancing ROS production or interfering with the protection against ROS may form a novel strategy to improve antibiotic treatment.

Determining how an organism responds to its environment by altering gene expression is key to understanding its ecology. Here, we used RNA-seq to comprehensively and quantitatively assess the transcriptional response of the bacterial opportunistic cystic fibrosis (CF) pathogen and endemic soil dweller, Burkholderia cenocepacia, in conditions mimicking these 2 environments. By sequencing 762 million bases of cDNA from 2 closely related B. cenocepacia strains (one isolated from a CF patient and one from soil), we identified a number of potential virulence factors expressed under CF-like conditions, whereas genes whose protein products are involved in nitrogen scavenging and 2-component sensing were among those induced under soil-like conditions. Interestingly, 13 new putative noncoding RNAs were discovered using this technique, 12 of which are preferentially induced in the soil environment, suggesting that ncRNAs play an important role in survival in the soil. In addition, we detected a surprisingly large number of regulatory differences between the 2 strains, which may represent specific adaptations to the niches from which each strain was isolated, despite their high degree of DNA sequence similarity. Compared with the CF strain, the soil strain shows a stronger global gene expression response to its environment, which is consistent with the need for a more dynamic reaction to the heterogeneous conditions of soil.

The Burkholderia cepacia Complex assembles at least eighteen closely related species that are ubiquitous in nature. Some isolates show beneficial potential for biocontrol, bioremediation and plant growth promotion. On the contrary, other strains are pathogens for plants and immunocompromised individuals, like cystic fibrosis patients. In these subjects, they can cause respiratory tract infections sometimes characterised by fatal outcome. Most of the Burkholderia cepacia Complex species are mucoid when grown on a mannitol rich medium and they also form biofilms, two related characteristics, since polysaccharides are important component of biofilm matrices. Moreover, polysaccharides contribute to bacterial survival in a hostile environment by inhibiting both neutrophils chemotaxis and antimicrobial peptides activity, and by scavenging reactive oxygen species. The ability of these microorganisms to produce exopolysaccharides with different structures is testified by numerous articles in the literature. However, little is known about the type of polysaccharides produced in biofilms and their relationship with those obtained in non-biofilm conditions. The aim of this study was to define the type of exopolysaccharides produced by nine species of the Burkholderia cepacia Complex. Two isolates were then selected to compare the polysaccharides produced on agar plates with those formed in biofilms developed on cellulose membranes. The investigation was conducted using NMR spectroscopy, high performance size exclusion chromatography, and gas chromatography coupled to mass spectrometry. The results showed that the Complex is capable of producing a variety of exopolysaccharides, most often in mixture, and that the most common exopolysaccharide is always cepacian. In addition, two novel polysaccharide structures were determined: one composed of mannose and rhamnose and another containing galactose and glucuronic acid. Comparison of exopolysaccharides obtained from cultures on agar plates with those extracted from biofilms on cellulose membranes showed important differences, thus suggesting that extrapolating data from non-biofilm conditions might not always be applicable.

Burkholderia cepacia complex (Bcc) is a group of bacteria with conflicting biological characteristics, which make them simultaneously beneficial and harmful to humans. They have been exploited for biocontrol, bioremediation, and plant growth promotion. However, their capacity as opportunistic bacteria that infect humans restricts their biotechnological applications. Therefore, the risks of using these bacteria should be assessed. In this study, Burkholderia multivorans WS-FJ9 originally isolated from pine rhizosphere, which was shown to be efficient in solubilizing phosphate, was evaluated with respect to its biosafety, colonization in poplar rhizosphere, and growth-promoting effects on poplar seedlings. Pathogenicity of B. multivorans WS-FJ9 on plants was determined experimentally using onion and tobacco as model plants. Onion bulb inoculated with B. multivorans WS-FJ9 showed slight hypersensitive responses around the inoculation points, but effects were not detectable based on the inner color and odor of the onion. Tobacco leaves inoculated with B. multivorans WS-FJ9 exhibited slightly water-soaked spots around the inoculation points, which did not expand or develop into lesions even with repeated incubation. Pathogenicity of the strain in alfalfa, which has been suggested as an alternative Bcc model for mice, was not detectable. Results from gene-specific polymerase chain reactions showed that the tested B. multivorans WS-FJ9 strain did not possess the BCESM and cblA virulence genes. Scanning electron microscopy revealed that the colonization of the WS-FJ9 strain reached 1.4 × 10⁴ colony forming units (cfu) g⁻¹ rhizosphere soil on day 77 post-inoculation. The B. multivorans WS-FJ9 strain could colonize the rhizosphere as well as the root tissues and cells of poplars. Greenhouse evaluations in both sterilized and non-sterilized soils indicated that B. multivorans WS-FJ9 significantly promoted growth in height, root collar diameter, and plant biomass of inoculated poplar seedlings compared with controls. Phosphorus contents of roots and stems of treated seedlings were 0.57 and 0.55 mg g⁻¹ higher than those of the controls, respectively. Phosphorus content was lower in the rhizosphere soils by an average of 1.03 mg g⁻¹ compared with controls. The results demonstrated that B. multivorans WS-FJ9 is a nonpathogenic strain that could colonize the roots and significantly promote the growth of poplar seedlings.

Bacteria of the Burkholderia cepacia complex (BCC) are associated with severe infection in cystic fibrosis. Recent evidence shows that the mucoid phenotype is common in BCC bacteria; however, during chronic infection, transitions from the mucoid to nonmucoid morphology have been shown to take place. Here we use RNA microarray and proteomic isobaric tagging relative and absolute quantitation technologies to gain insight into a pair of mucoid and nonmucoid isolates of B. cenocepacia obtained from a chronically infected patient with cystic fibrosis in the year prior to her death. During chronic infection, the mucoid isolate lost the B. cepacia epidemic strain marker and acquired a mutation in the cepR gene. In the nonmucoid isolate, we observed overexpression at both the RNA and protein level of several described putative virulence factors, including a nematocidal protein AidA and the oxidative stress response protein AhpC. We show that this translates into increased resistance to oxidative stress in the nonmucoid isolate, a key microbial determinant for resistance against phagocytic cell killing. These data illuminate the biological differences between mucoid and nonmucoid BCC bacteria, provide targets for elucidating the genetic control of exopolysaccharide production in the BCC, and highlight that chronic infection can produce both genetically and phenotypically distinct microbial variants in the cystic fibrosis lung.

Burkholderia cepacia complex bacteria are opportunistic pathogens that cause respiratory tract infections in susceptible patients, mainly people with cystic fibrosis. There is convincing evidence that B. cepacia complex bacteria can form biofilms, not only on abiotic surfaces (e.g., glass and plastics), but also on biotic surfaces such as epithelial cells, leading to the suggestion that biofilm formation plays a key role in persistent infection of cystic fibrosis lungs. This article presents an overview of the molecular mechanisms involved in B. cepacia complex biofilm formation, the increased resistance of sessile B. cepacia complex cells and the role of quorum sensing in B. cepacia complex biofilm formation.

This study determined the antibiotic susceptibility of planktonic and biofilm cultures of Burkholderia cepacia complex organisms, a group of highly problematic pathogens associated with cystic fibrosis patients. The biofilm inhibitory concentrations were considerably higher than the corresponding minimum inhibitory concentrations for meropenem and piperacillin-tazobactam. However, tobramycin and amikacin were efficacious against both biofilm and planktonic cultures. Overall this study showed that biofilm susceptibility testing might be more clinically appropriate for determining antibiotic therapy for Burkholderia cepacia complex infections in cystic fibrosis patients.

Despite the characterization of some Burkholderia cepacia complex exopolysaccharides (EPSs), little is known about the role of EPSs in the pathogenicity of B. cepacia complex organisms. We describe 2 Burkholderia cenocepacia (genomovar III) isolates obtained from a patient with cystic fibrosis (CF): the nonmucoid isolate C8963 and the mucoid isolate C9343. Both isolates had identical random amplified polymorphic DNA patterns. C9343 produced a capsule composed of the EPSs PS-I and PS-II, as well as α-1,6-glucan. These isolates exhibited several phenotypic differences: C8963 synthesized octanoyl-homoserine lactone and produced biofilms, but C9343 did not; in a mouse model of pulmonary infection, C8963 was cleared more rapidly than was C9343; and C9343 interacted poorly with macrophages and neutrophils, compared with C8963, suggesting that the C9343 capsule interfered with cell-surface interactions. Overproduction of EPS by C9343 resulted in a mucoid appearance and interfered with cell-surface interactions and clearance in an animal model. This mucoid colonial appearance could enhance the persistence and virulence of this important CF-related pathogen.

Members of the Burkholderia cepacia complex (Bcc), such as B. ambifaria , are effective biocontrol strains, for instance, as plant growth-promoting bacteria; however, Bcc isolates can also cause severe respiratory infections in people suffering from cystic fibrosis (CF). No distinction is known between isolates from environmental and human origins, suggesting that the natural environment is a potential source of infectious Bcc species. While investigating the presence and role of phase variation in B. ambifaria HSJ1, an isolate recovered from a CF patient, we identified stable variants that arose spontaneously irrespective of the culture conditions. Phenotypic and proteomic approaches revealed that the transition from wild-type to variant types affects the expression of several putative virulence factors. By using four different infection models ( Drosophila melanogaster , Galleria mellonella , macrophages and Dictyostelium discoideum ), we showed that the wild-type was more virulent than the variant. It may be noted that the variant showed reduced replication in a human monocyte cell line when compared with the wild-type. On the other hand, the variant of isolate HSJ1 was more competitive in colonizing plant roots than the wild-type. Furthermore, we observed that only clinical B. ambifaria isolates generated phase variants, and that these variants showed the same phenotypes as observed with the HSJ1 variant. Finally, we determined that environmental B. ambifaria isolates showed traits that were characteristic of variants derived from clinical isolates. Our study therefore suggest that B. ambifaria uses phase variation to adapt to drastically different environments: the lung of patients with CF or the rhizosphere.