Host Adaptation Predisposes Pseudomonas aeruginosa to Type VI Secretion System-Mediated Predation by the Burkholderia cepacia Complex.

Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species are opportunistic lung pathogens of cystic fibrosis (CF) patients. While P. aeruginosa can initiate long-term infections in younger CF patients, Bcc infections only arise in teenagers and adults. Both P. aeruginosa and Bcc use type VI secretion systems (T6SSs) to mediate interbacterial competition. Here, we show P. aeruginosa isolates from teenage and adult CF patients, but not those from young CF patients, are outcompeted by the epidemic Bcc isolate Burkholderia cenocepacia strain AU1054 in a T6SS-dependent manner. The genomes of susceptible P. aeruginosa isolates harbor T6SS-abrogating mutations, the repair of which, in some cases, rendered the isolates resistant. Moreover, seven of eight Bcc strains outcompeted P. aeruginosa strains isolated from the same patients. Our findings suggest certain mutations that arise as P. aeruginosa adapts to the CF lung abrogate T6SS activity, making P. aeruginosa and its human host susceptible to potentially fatal Bcc superinfection.

Burkholderia cepacia Complex Bacteria: a Feared Contamination Risk in Water-Based Pharmaceutical Products.

Burkholderia cepacia (formerly Pseudomonas cepacia) was once thought to be a single bacterial species but has expanded to the Burkholderia cepacia complex (Bcc), comprising 24 closely related opportunistic pathogenic species. These bacteria have a widespread environmental distribution, an extraordinary metabolic versatility, a complex genome with three chromosomes, and a high capacity for rapid mutation and adaptation. Additionally, they present an inherent resistance to antibiotics and antiseptics, as well as the abilities to survive under nutrient-limited conditions and to metabolize the organic matter present in oligotrophic aquatic environments, even using certain antimicrobials as carbon sources. These traits constitute the reason that Bcc bacteria are considered feared contaminants of aqueous pharmaceutical and personal care products and the frequent reason behind nonsterile product recalls. Contamination with Bcc has caused numerous nosocomial outbreaks in health care facilities, presenting a health threat, particularly for patients with cystic fibrosis and chronic granulomatous disease and for immunocompromised individuals. This review addresses the role of Bcc bacteria as a potential public health problem, the mechanisms behind their success as contaminants of pharmaceutical products, particularly in the presence of biocides, the difficulties encountered in their detection, and the preventive measures applied during manufacturing processes to control contamination with these objectionable microorganisms. A summary of Bcc-related outbreaks in different clinical settings, due to contamination of diverse types of pharmaceutical products, is provided.

Regulation of Virulence by Two-Component Systems in Pathogenic Burkholderia.

The regulation and timely expression of bacterial genes during infection is critical for a pathogen to cause an infection. Bacteria have multiple mechanisms to regulate gene expression in response to their environment, one of which is two-component systems (TCS). TCS have two components. One component is a sensory histidine kinase (HK) that autophosphorylates when activated by a signal. The activated sensory histidine kinase then transfers the phosphoryl group to the second component, the response regulator, which activates transcription of target genes. The genus Burkholderia contains members that cause human disease and are often extensively resistant to many antibiotics. The Burkholderia cepacia complex (BCC) can cause severe lung infections in patients with cystic fibrosis (CF) or chronic granulomatous disease (CGD). BCC members have also recently been associated with several outbreaks of bacteremia from contaminated pharmaceutical products. Separate from the BCC is Burkholderia pseudomallei, which is the causative agent of melioidosis, a serious disease that occurs in the tropics, and a potential bioterrorism weapon. Bioinformatic analysis of sequenced Burkholderia isolates predicts that most strains have at least 40 TCS. The vast majority of these TCS are uncharacterized both in terms of the signals that activate them and the genes that are regulated by them. This review will highlight TCS that have been described to play a role in virulence in either the BCC or B. pseudomallei Since many of these TCS are involved in virulence, TCS are potential novel therapeutic targets, and elucidating their function is critical for understanding Burkholderia pathogenesis.

Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition.

Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens.IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

[The controversial Burkholderia cepacia complex, a group of plant growth promoting species and plant, animals and human pathogens]. / El controvertido complejo Burkholderia cepacia, un grupo de especies promotoras del crecimiento vegetal y patógenas de plantas, animales y humanos.

The Burkholderia cepacia complex is a group of 22 species, which are known as opportunistic pathogens in immunocompromised people, especially those suffering from cystic fibrosis. It is also found in nosocomial infections and is difficult to eradicate due to intrinsic resistance to several antibiotics. The species have large genomes (up to 9 Mbp), distributed into 2-5 replicons. These features significantly contribute to genome plasticity, which makes them thrive in different environments like soil, water, plants or even producing nodules in legume plants. Some B. cepacia complex species are beneficial in bioremediation, biocontrol and plant-growth promotion. However, because the B. cepacia complex is involved in human infection, its use in agriculture is restricted. B. cepacia complex is being constantly studied due to the health problems that it causes and because of its agricultural potential. In this review, the history of B. cepacia complex and the most recently published information related to this complex are revised.

Effectiveness of multi-trait Burkholderia contaminans KNU17BI1 in growth promotion and management of banded leaf and sheath blight in maize seedling.

Plant growth promoting (PGP) bacteria enhance plant growth and are a green alternative to chemical fertilizers. In our study, an effective plant growth promoting rhizobacteria (PGPR) strain, KNU17BI1, was isolated from rhizospheric soil of maize, South Korea. The strain was tested in vitro for specific PGP and antifungal traits, such as phosphate solubilization, zinc solubilization, indole acetic acid (IAA) production, ammonia production, nitrogen fixation, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, siderophore hydrogen cyanide production (HCN) and hydrolytic enzyme activity. Furthermore, in viro antifungal activity was done in a laboratory and in vivo effect of KNU17BI1 on banded leaf and sheath blight intensity as well as plant growth promotion on maize seedling were conducted under greenhouse conditions. The strain was found to be highly effective toward all the parameters except HCN production. The strain KNU17BI1 was identified on the basis of 16S RNA and multilocus sequence analysis (MLSA) and confirmed as Burkholderia contaminans. This study for the first time demonstrated potent in vitro antifungal activity of B. contaminans against Rhizoctonia solani AG-1(IA), Pythium graminicola, Fusarium moniliforme, Alternaria alternata, Alternaria solani, Fusarium graminearum, Stemphylium botryosum Wallr, Colletotrichum dematium, Stemphylium lycopersici and Fusarium oxysporum f.sp. melonis. Furthermore, in this study, for the first time, the potential of B. contaminans stain KNU17BI1 in controlling banded leaf and sheath blight of maize caused by R. solani AG-1(IA) was reported. Therefore, further studies are warranted on the structural identification of actual compounds behind such activities that would be exploited further for biocontrol as well as plant growth promotion.

Use of antibiotic disks to evolve drug-resistant bacteria.

The methods used to generate antibiotic-resistant bacterial strains can be labour-intensive, costly, lengthy and/or prone to plate-to-plate variation. We propose a simple, inexpensive and easily replicated method to expose bacteria to a continuous gradient of antibiotic concentration, providing an environment of positive selective pressure for evolution of antibiotic-resistant strains.

An outbreak of Burkholderia cepacia complex in the paediatric unit of a tertiary care hospital.

INTRODUCTION: Burkholderia cepacia complex (Bcc) has emerged as a serious nosocomial pathogen worldwide especially in patients with indwelling catheters and cystic fibrosis. Bcc is a common contaminant of pharmaceutical products. We describe an outbreak of Bcc bacteraemia amongst children admitted in Paediatric Intensive Care Unit (PICU) and paediatric ward at a tertiary care hospital, Mumbai, in Western India. MATERIALS AND METHODS: Blood culture samples from paediatric patients yielded growth of non-fermenting, oxidase positive, motile, Gram negative bacilli (NFGNB) (76/909) over a period of 8 months. Based on conventional biochemical tests and antimicrobial susceptibility testing, these isolates were provisionally identified as Bcc. The increased, repeated and continued isolation of Bcc alerted the possibility of an outbreak confined to PICU and paediatric ward. Active surveillance was undertaken to trace the source and contain the outbreak. Isolates were subjected to recA polymerase chain reaction (PCR) and Expanded multilocus sequence typing (EMLST). RESULTS: Surveillance revealed the presence of Bcc on the upper surface of rubber stopper of sealed multidose amikacin vials. Isolates from blood culture and rubber stoppers were confirmed as Bcc by recA PCR. EMLST revealed that these isolates shared an identical novel sequence type 824 proving clonality. Timely interventions instituted led to control of the outbreak. CONCLUSION: This study highlights the importance of identification and molecular characterization of Bcc to establish its role in infection and outbreak.

Pseudomonas aeruginosa-Derived Rhamnolipids and Other Detergents Modulate Colony Morphotype and Motility in the Burkholderia cepacia Complex.

Competitive interactions mediated by released chemicals (e.g., toxins) are prominent in multispecies communities, but the effects of these chemicals at subinhibitory concentrations on susceptible bacteria are poorly understood. Although Pseudomonas aeruginosa and species of the Burkholderia cepacia complex (Bcc) can exist together as a coinfection in cystic fibrosis airways, P. aeruginosa toxins can kill Bcc species in vitro Consequently, these bacteria become an ideal in vitro model system to study the impact of sublethal levels of toxins on the biology of typical susceptible bacteria, such as the Bcc, when exposed to P. aeruginosa toxins. Using P. aeruginosa spent medium as a source of toxins, we showed that a small window of subinhibitory concentrations modulated the colony morphotype and swarming motility of some but not all tested Bcc strains, for which rhamnolipids were identified as the active molecule. Using a random transposon mutagenesis approach, we identified several genes required by the Bcc to respond to low concentrations of rhamnolipids and consequently affect the ability of this microbe to change its morphotype and swarm over surfaces. Among those genes identified were those coding for type IVb-Tad pili, which are often required for virulence in various bacterial pathogens. Our study demonstrates that manipulating chemical gradients in vitro can lead to the identification of bacterial behaviors relevant to polymicrobial infections.IMPORTANCE Interspecies interactions can have profound effects on the development and outcomes of polymicrobial infections. Consequently, improving the molecular understanding of these interactions could provide us with new insights on the possible long-term consequences of these chronic infections. In this study, we show that P. aeruginosa-derived rhamnolipids, which participate in Bcc killing at high concentrations, can also trigger biological responses in Burkholderia spp. at low concentrations. The modulation of potential virulence phenotypes in the Bcc by P. aeruginosa suggests that these interactions contribute to pathogenesis and disease severity in the context of polymicrobial infections.

Preliminary data on antibacterial activity of Echinacea purpurea-associated bacterial communities against Burkholderia cepacia complex strains, opportunistic pathogens of Cystic Fibrosis patients.

Burkholderia cepacia complex bacteria (Bcc) represent a serious threat for immune-compromised patient affected by Cystic Fibrosis (CF) since they are resistant to many substances and to most antibiotics. For this reason, the research of new natural compounds able to inhibit the growth of Bcc strains has raised new interest during the last years. A source of such natural compounds is represented by medicinal plants and, in particular, by bacterial communities associated with these plants able to produce molecules with antimicrobial activity. In this work, a panel of 151 (endophytic) bacteria isolated from three different compartments (rhizospheric soil, roots, and stem/leaves) of the medicinal plant Echinacea purpurea were tested (using the cross-streak method) for their ability to inhibit the growth of 10 Bcc strains. Data obtained revealed that bacteria isolated from the roots of E. purpurea are the most active in the inhibition of Bcc strains, followed by bacteria isolated from the rhizospheric soil, and endophytes from stem/leaf compartment. At the same time, Bcc strains of environmental origin showed a higher resistance toward inhibition than the Bcc strains with clinical (i.e. CF patients) origin. Differences in the inhibition activity of E. purpurea-associated bacteria are mainly linked to the environment -the plant compartment- rather than to their taxonomical position.

Bacterial proteases and haemostasis dysregulation in the CF lung.

BACKGROUND: Pathogenic bacteria which chronically colonise the cystic fibrosis (CF) lung produce a number of virulence determinants, including distinct proteolytic activities. The potential role bacterial proteases play on haemostatic dysregulation within the CF lung is, however, poorly defined, despite haemoptysis being a common complication in CF. METHODS: The potential impact of known CF pathogens (Pseudomonas aeruginosa and Burkholderia cepacia complex spp.) on haemostasis was examined for their ability to degrade fibrinogen and dysregulate fibrin clot formation and platelet aggregation. RESULTS: Results demonstrate that key CF pathogens growing as a biofilm on mucin exhibit considerable fibrinogenolytic activity, resulting in fibrinogen breakdown, impaired clot formation, and modulation of platelet aggregation. Human neutrophil elastase may also contribute to fibrinogen breakdown and dysregulated clot formation at high concentration. CONCLUSION: Bacterial-derived proteases may play an important role in the dysregulation of airway haemostasis, and potentially contribute to episodes of haemoptysis within the CF lung.

Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread. IMPORTANCE: Benzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.

Screening Complex Biological Samples with Peptide Microarrays: The Favorable Impact of Probe Orientation via Chemoselective Immobilization Strategies on Clickable Polymeric Coatings.

The generation of robust analytical data using microarray platforms strictly relies on optimal ligand-target interaction at the sensor surface, which, in turn, is inherently bound to the correct immobilization scheme of the interrogated bioprobes. In the present work, we performed a rigorous comparative analysis of the impact of peptide ligands immobilization strategy in the screening of Burkholderia cepacia complex (BCC) infections in patients affected by cystic fibrosis (CF). We generated arrays of previously validated Burkholderia derived peptide probes that were selectively oriented on polymeric coatings by means of different click-type reactions including thiol maleimide, copper-catalyzed azide-alkyne cycloaddition (CuAAC), and strain-promoted azide-alkyne cycloaddition (SPAAC). We compared immobilization efficiency among the different chemoselective reactions, and we evaluated diagnostic performances at a statistically significant level, also in contrast to random immobilization strategies. Our findings clearly support the favorable role of correct bioprobe orientation in discriminating seronegative from infected individuals and, in the last analysis, in generating more-reliable and more-reproducible data. Spacing biomolecules from the sensor surface by means of small hydrophilic linkers also positively affects the analytical performance and leads to increased statistical significance of data. Overall, all of the click immobilization strategies that were considered displayed a good efficiency. Interestingly, SPAAC-mediated conjugation using DBCO cyclooctyne for some peptides resulted in sequence-dependent autofluorescence in the Cy5 emission range wavelength, which could be circumvented by using a different fluorescence detection channel. On the basis of our results, we critically discuss the immobilization parameters that need to be carefully considered for peptide ligand immobilization purposes.

Linocin and OmpW Are Involved in Attachment of the Cystic Fibrosis-Associated Pathogen Burkholderia cepacia Complex to Lung Epithelial Cells and Protect Mice against Infection.

Members of the Burkholderia cepacia complex (Bcc) cause chronic opportunistic lung infections in people with cystic fibrosis (CF), resulting in a gradual lung function decline and, ultimately, patient death. The Bcc is a complex of 20 species and is rarely eradicated once a patient is colonized; therefore, vaccination may represent a better therapeutic option. We developed a new proteomics approach to identify bacterial proteins that are involved in the attachment of Bcc bacteria to lung epithelial cells. Fourteen proteins were reproducibly identified by two-dimensional gel electrophoresis from four Bcc strains representative of two Bcc species: Burkholderia cenocepacia, the most virulent, and B. multivorans, the most frequently acquired. Seven proteins were identified in both species, but only two were common to all four strains, linocin and OmpW. Both proteins were selected based on previously reported data on these proteins in other species. Escherichia coli strains expressing recombinant linocin and OmpW showed enhanced attachment (4.2- and 3.9-fold) to lung cells compared to the control, confirming that both proteins are involved in host cell attachment. Immunoproteomic analysis using serum from Bcc-colonized CF patients confirmed that both proteins elicit potent humoral responses in vivo Mice immunized with either recombinant linocin or OmpW were protected from B. cenocepacia and B. multivorans challenge. Both antigens induced potent antigen-specific antibody responses and stimulated strong cytokine responses. In conclusion, our approach identified adhesins that induced excellent protection against two Bcc species and are promising vaccine candidates for a multisubunit vaccine. Furthermore, this study highlights the potential of our proteomics approach to identify potent antigens against other difficult pathogens.

Evidence for the widespread production of DSF family signal molecules by members of the genus Burkholderia by the aid of novel biosensors.

Many bacteria employ cis-2-unsaturated fatty acids, referred to as DSF (diffusible signal factor) family signals, to communicate with each other. Such systems have been shown to control biofilm formation, motility, production of hydrolytic enzymes and expression of virulence factors. We report the construction of novel biosensors on the basis of components of the Burkholderia-DSF (BDSF) dependent circuitry of Burkholderia cenocepacia H111 and evaluated their utility for detecting the production of DSF family signal molecules. We show that a luxAB-based biosensor responds to nM levels of synthetic BDSF and is suitable to detect a wide range of cis-2 fatty acid molecules. Using this biosensor we show that the production of DSF family molecules is widespread among members of the B. cepacia complex and demonstrate for the first time that DSF-based molecules are also produced by plant-associated Burkholderia species.

Residence in biofilms allows Burkholderia cepacia complex (Bcc) bacteria to evade the antimicrobial activities of neutrophil-like dHL60 cells.

Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics.

Swimming motility in a longitudinal collection of clinical isolates of Burkholderia cepacia complex bacteria from people with cystic fibrosis.

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.

Investigation of aminoglycoside resistance inducing conditions and a putative AmrAB-OprM efflux system in Burkholderia vietnamiensis.

BACKGROUND: Burkholderia cepacia complex (BCC) bacteria are highly virulent, typically multidrug-resistant, opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. B. vietnamiensis is more often susceptible to aminoglycosides than other BCC species, and strains acquire aminoglycoside resistance during chronic CF infection and under tobramycin and azithromycin exposure in vitro, apparently from gain of antimicrobial efflux as determined through pump inhibition. The aims of the present study were to determine if oxidative stress could also induce aminoglycoside resistance and provide further observations in support of a role for antimicrobial efflux in aminoglycoside resistance in B. vietnamiensis. FINDINGS: Here we identified hydrogen peroxide as an additional aminoglycoside resistance inducing agent in B. vietnamiensis. After antibiotic and hydrogen peroxide exposure, isolates accumulated significantly less [3H] gentamicin than the susceptible isolate from which they were derived. Strains that acquired aminoglycoside resistance during infection and after exposure to tobramycin or azithromycin overexpressed a putative resistance-nodulation-division (RND) transporter gene, amrB. Missense mutations in the repressor of amrB, amrR, were identified in isolates that acquired resistance during infection, and not in those generated in vitro. CONCLUSIONS: These data identify oxidative stress as an inducer of aminoglycoside resistance in B. vietnamiensis and further suggest that active efflux via a RND efflux system impairs aminoglycoside accumulation in clinical B. vietnamiensis strains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but not hydrogen peroxide, in vitro. Furthermore, the repressor AmrR is likely just one regulator of the putative AmrAB-OprM efflux system in B. vietnamiensis.

Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner.

In common with other members of the Burkholderia cepacia complex (BCC), Burkholderia multivorans is capable of producing exopolysaccharide (EPS) when grown on certain mannitol-rich media. The significance of the resulting mucoid phenotype and the genome-wide response to mannitol has never been characterized despite its clinical relevance following the approval of a dried-powder preparation of mannitol as an inhaled osmolyte therapy for cystic fibrosis (CF) patients. In the present study we defined the transcriptional response of B. multivorans ATCC 17616, a model genome-sequenced strain of environmental origin, to growth on mannitol-rich yeast extract media (MYEM). EPS-dependent and -independent impact of MYEM on virulence-associated traits was assessed in both strain ATCC 17616 and the CF isolate B. multivorans C1576. Our studies revealed a significant transcriptional response to MYEM encompassing approximately 23 % of predicted genes within the genome. Strikingly, this transcriptional response identified that EPS induction occurs in ATCC 17616 without the upregulation of the bce-I and bce-II EPS gene clusters, despite their pivotal role in EPS biosynthesis. Of approximately 20 differentially expressed putative virulence factors, 16 exhibited upregulation including flagella, ornibactin, oxidative stress proteins and phospholipases. MYEM-grown B. multivorans also exhibited enhanced motility, biofilm formation and epithelial cell invasion. In contrast to these potential virulence enhancements, MYEM-grown B. multivorans C1576 showed attenuated virulence in the Galleria mellonella infection model. All of the observed phenotypic responses occurred independently of EPS production, highlighting the profound impact that mannitol-based growth has on the physiology and virulence of B. multivorans.

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.