Abstract book - users.ugent.betcoenye/Abstractbook2012.pdf · ABSTRACT BOOK This meeting has been possible by the generous contributions of . IBCWG 2012 Abstract Book 2 A1 ... strains

International Burkholderia cepacia Working Group

16th Annual Meeting

April 19 – April 21, 2012

InterContinental Montreal Hotel 360 Saint-Antoine Street West

Montréal, Québec, H2Y 3X4

ABSTRACT BOOK

This meeting has been possible by the generous contributions of

IBCWG 2012 Abstract Book

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A1 COMPARATIVE GENOMICS OF BURKHOLDERIA CENOCEPACIA EPIDEMIC STRAINS J2315 AND ST32 Klara Dedeckova, Lucie Kalferstova and Pavel Drevinek Department of Pediatrics, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic Contact : Pavel Drevinek, [email protected] Burkholderia cenocepacia ET-12 is a notoriously known epidemic lineage that has been causing devastating infections in Canadian and United Kingdom cystic fibrosis (CF) patients. Strain J2315, a representative of ET-12, was the first Burkholderia cepacia complex (Bcc) isolate with fully sequenced genome. Strain ST32 is another problematic epidemic strain found in many countries around the world, associated like ET-12 with fatal necrotizing pneumonia, however its genomic data is missing. We aimed to compare genomes of these two genetically closely related strains (three locus variants by MLST) in order to provide insight into genetics of ST32 and to identify unique genetic features for each strain. Two methods were exploited to perform whole genome comparison of J2315 vs. ST32: genomic subtractive hybridization (GSH) and comparative genomic hybridization (CGH). For GSH, we used a PCR Select Bacterial Genome Subtraction kit (Clontech); a tester strain was ST32, while J2315 served as a driver. Hybridization temperature was set at 73 degC. For CGH, DNA of both strains was fluorescently labelled and co-hybridized to an 8x15k custom CGH array, containing probes for 7,049 and 6,573 annotated genetic features of J2315 and AU1054, respectively. The data were analyzed with GeneSpring v7 (Agilent). The GSH approach, intended to reveal ST32-associated sequences, led to identification of 58 DNA fragments of 62 to 756 bp in length. Although ca. 20% of them showed some sequence homology with J2315, 31 sequences were found to be unique with no similarity to any sequenced Bcc genome. Moreover, one of these sequences coding for hypothetical protein was selected for designing a PCR which proved to be specific to detect ST32. Preliminary results of CGH analysis indicated some 450 features to be specific to J2315 vs. ST32 (>2 fold change) and on contrary, 100 genes to be unique to ST32 (genes that are present within AU1054 genome). Of note, a cenocepacia island, considered to be a significant virulence factor of J2315, seems to be present also in the ST32 genome. Complementary whole genome comparative approaches were applied to describe differences between two closely related epidemic strains. Without having whole genome sequence of ST32 available, this allowed us to identify features specific to J2315 vs. ST32 (appr. 6% of its genome) and vice versa. While the latter gene list is likely far from complete, the data was sufficient to design an ST32-specific diagnostic PCR. Supported by IGA MZ NS 10543-3, GAUK 307311.


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A2 ANALYSIS OF VIRULENCE OF BURKHOLDERIA CENOCEPACIA ST32 ISOLATES FROM CYSTIC FIBROSIS PATIENTS IN ZEBRA FISH EMBRYOS Klara Dedeckova1, Annette Vergunst2, Pavel Drevinek1 1Charles University, 2nd Faculty of Medicine, Department of Paediatrics, Prague, Czech Republic

2 INSERM U1047, Universite´ de Montpellier 1, UFR Médecine, Nimes, France Contact: Klara Dedeckova, [email protected] Bacteria from Burkholderia cepacia complex (Bcc) are opportunistic pathogens which cause serious lung infections in patients with cystic fibrosis (CF). Burkholderia cenocepacia ST32 is one of recognized globally distributed strains that infected many CF patients in Canada and Europe, however little is known about its virulence potential in animal models. To evaluate ST32 virulence and differences in virulence among individual isolates in vivo, we selected a panel of clinical isolates from Prague CF Centre, where the strain infected almost 30% of the population, and analyzed their virulence in a zebra fish embryo infection model (1). The high similarity to the human immune system, transparency of fish embryos, and ease of handling make it an excellent model to study bacterial infection. We tested 31 B. cenocepacia ST32 isolates obtained at different phases of chronic infection from 14 CF patients. 28 isolates originated from sputum (22 of them were collected at stable clinical condition, 5 at the time preceding cepacia syndrome by 1 to 4 months, 1 at cepacia syndrome), and 3 isolates were from blood cultures taken from 3 patients who developed cepacia syndrome. All isolates were confirmed to be ST32 by using RAPD and/or MLST. Based on results of the zebrafish embryo survival assay, ST32 isolates were divided into three distinct virulence groups. Group 1 contained 12 isolates (out of 25 with clear result), which were able to kill 100% of infected embryos within 64 - 96 hours post infection (hpi). Group 2 was composed of 9/25 isolates with intermediate virulence that killed 40 - 75% of embryos by 120 hpi. Four isolates (Group 3) were able to kill 0 - 20% of all embryos by 120 hpi. Notably, sequential ST32 isolates from the same patient collected at different phases of a clinical state often displayed different level of virulence; for instance 2 cepacia syndrome isolates were less virulent than isolates from the stable phase of infection or before cepacia syndrome, whereas a third cepacia syndrome isolate had the same virulence as an earlier isolate. Our results indicate a high degree of variation in virulence between clonal B. cenocepacia ST32 isolates that was seen not only in isolates originating from different patients, but also in paired isolates from one patient. (1) Vergunst AC, Meijer AH, Renshaw SA, O׳Callaghan D. (2010) Burkholderia cenocepacia creates an intramacrophage replication niche in zebrafish embryos, followed by bacterial dissemination and establishment of systematic infection. Infect. Immun. 78: 1495-1508. Supported by: GAUK 307311; IGA MZ NT12405-5; MS MT LD11029.


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A3 REPORT ON DIVERSITY, POTENTIAL PATHOGENICTY OF BURKHOLDERIA CEPACIA COMPLEX FROM CHINA AND ITS SUSCEPTIBIIIY TO COPPER SURFACES AND CHITOSAN Muhammad Ibrahim, Zhu Bo, Bin Li, Xie Guan Lin, Fang Yuan, Lou Miao Miao Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, 310058 Hangzhou, China Contact : Muhammad Ibrahim, [email protected] ; Xie Guan Lin, [email protected] A collection of about 300 Burkholderia cepacia complex isolates were recovered from clinical, water, and agricultural resources in China in our previous studies. The potential pathogenicity was tested in the alternative infection models alfalfa, detached lettuce midrib, Galleria mellonella (wax moth), rat agar bead, and lettuce intact leaves. In addition, Bcc which is multidrug resistant and ubiquitous in environment, our studies reported that copper surfaces could be utilized in health care facilities to reduce the bioburdon of Bcc species which may protect susceptible member of the community from bacterial infection. Furthermore, chitosan a natural nontoxic biopolymer, a major component of the shells of crustacea and have several advantages over other type of bactericide also investigated against Bcc. The results of our studies showed that water and clinical isolates showed severe virulence and strong biofilm formation abilities compared to agricultural isolates. Copper and Chitosan possesses a higher antibacterial activity, against Bcc bacteria. Due to their commonalities, biotechnological potentials, opportunistic pathogenicity particularly recently known B. seminalis 0901 (plant pathogen, Li et al. 2010), S9 (water isolate, Fang et al. 2011), R456 (biocontrol agent Li et al., (2011) and LMG 24067T kindly provided by Dr J.J. Li Puma are under progress in genome sequencing of B. seminalis in Beijing Genomics Institute and the transcriptional analysis in our lab, could help us to understand the differentiation in adaptation of different environmental niches of this opportunistic pathogen particularly B. seminalis which is in case a new Bcc species with diverse characteristics. A4 PREVALANCE AND CHARACTERISATION OF ANTIFUNGAL ACTIVITY WITHIN THE BURKHOLDERIA CEPACIA COMPLEX Othman Boaisha, Lynne Boddy, and Eshwar Mahenthiralingam Cardiff School of Biosciences, Cardiff University, Main Building, Museum Avenue, Cardiff, CF10 3AT, UK. Contact : Eshwar Mahenthiralingam ([email protected]) Background. Burkholderia cepacia complex (Bcc) are well known for their ability to produce antifungal agents. However, the majority of past studies have screened only a limited number of isolates many of which were not taxonomically well characterized. In addition, while the activity of Bcc bacteria against plant pathogenic fungal species has been characterized, very few studies have examined the interaction between Bcc and saprotrophic wood decay basidiomycete species.


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The aim of this study was to characterize the interactions of Bcc bacteria with the model ascomycete Candida albicans and woodland basidiomycete fungi. Methods. A large collection (397 isolates) of taxonomically well identified Burkholderia species was systematically screened for antifungal activity against Candida albicans and wood decay basidiomycetes: Bjerkandera adusta, Trametes versicolor, Hypholoma fasciculare, Resinicium bicolor and Phanerochaete velutina. An agar overlay assay was used to examine the anti-candidal activity of Burkholderia isolates, while a novel overlay assay was developed using homogenised mycelial material for the basidiomycete species. Thin Layer Chromatography (TLC) bioassays were used to reveal the presence of active Burkholderia metabolites in the antimicrobial extracts. Results. The majority of Burkholderia isolates possessed the same antagonistic activity towards C. albicans as they did towards B. adusta, which was selected basidiomycete for screening the bacterial collection. Of the 397 isolates screened, 47.85% were anti-candidal (190 isolates; 89.47% of these were Bcc species and 10.53% were other Burkholderia species), while 48.10% were active against B. adusta (191 isolates; 93.19% of these were Bcc species and 6.81% were other Burkholderia). Antifungal activity was the greatest in the following Burkholderia species: B. ambifaria, B. cepacia, B. cenocepacia and B. contaminans. Interestingly, no antifungal activity was observed for B. multivorans (27 isolates) and B. stabilis (18 isolates) under the experimental conditions tested. Antifungal Burkholderia isolates produced between 1 and 10 antifungal metabolites when examined by TLC bioassay. Several antifungal agents were identified as enacyloxin, pyrrolnitrin, bactobolin and quinolines. Conclusions. Burkholderia are potentially a very large reservoir of known and novel antimicrobial agents, with nearly 50% of isolates screened exhibiting antifungal activity. Novel extraction and bioassay methods for anti-fungals were developed in this study which will provide excellent tools for the isolation and characterization of antifungal Burkholderia metabolites. B1 AZTREONAM FOR INHALATION SOLUTION (AZLI) IN CYSTIC FIBROSIS (CF) PATIENTS WITH CHRONIC BURKHOLDERIA SPECIES (BURK) INFECTION: A RANDOMIZED, PLACEBO-CONTROLLED TRIAL E. Tullis1, J.L. Burns2, G. Retsch-Bogart3, M. Bresnik4, M. McKevitt5 S. Lewis5, J.J. LiPuma6

1University of Toronto, Toronto, Canada, 2University of Washington, Seattle, WA, USA, 3University of North Carolina, Chapel Hill, NC, USA, 4Gilead Sciences, Foster City, CA, USA, 5Gilead Sciences, Seattle, WA, USA, 6University of Michigan, Ann Arbor, MI, USA Contact: Elizabeth Tullis, [email protected] A 1 yr randomized placebo (PCB)-controlled blinded 2-part trial was conducted at 35 sites in the US and Canada to evaluate the safety and efficacy of 24 and 48 weeks of continuous AZLI therapy in CF subjects with Burk infection . Results of the 24 week PCB-controlled phase are presented. Methods: CF pts ≥6 yrs old with chronic Burk infection (>50% of cultures positive in past year; confirmed Burk spp. by a reference lab) and stable pulmonary disease were enrolled. Pts were randomized to receive 24 weeks of AZLI 75 mg or PCB TID every day via a Pari Investigational eFlow® Nebulizer System, followed by 24 weeks of open label AZLI 75 mg TID daily for all pts. Efficacy endpoints included change in FEV1 and Burk sputum density, use of additional antibiotics (Abx) and hospitalizations. Results: 100 pts (AZLI: 48, placebo: 52) were randomized and treated. Mean age (yrs) AZLI: 28.0, PCB: 24.7; (83% >18 yrs). Mean baseline FEV1 57% predicted (range 16%-115%). B.


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cenocepacia (40%) and B. multivorans (27%) were the most common of the 9 Burk spp identified at baseline. Pseudomonas aeruginosa (PA) was isolated in 45% and MRSA in 39% of pts during the study. At baseline, MIC50 of aztreonam for Burk was high (AZLI: 64, PCB:128 ug/ml). There was no significant difference in primary endpoint (relative change from baseline in FEV1 % predicted, measured by the time-adjusted area under the curve through week 24) between treatment groups: AZLI: +0.16, PCB: -0.75 (p=0.66). Pts receiving additional systemic or inhaled Abx for respiratory indications: AZLI: 29; PCB: 38 (p=0.206). Median number of days to first respiratory event: AZLI: 75, PCB: 51 (p=0.27). Suppressive effects on Burk sputum density were not observed in either treatment group. Changes from baseline in the BURK and PA MIC50 of 5 Abx were similar between groups except for the BURK MIC50 of aztreonam which increased 4 fold from baseline at week 24 in AZLI subjects. Total number of pts with adverse events was similar in both groups. More wheezing was reported in the AZLI group: 21% vs. PCB: 6% (p=0.04). Conclusions: This is the largest randomized controlled trial evaluating safety and efficacy of aerosolized antibiotic treatment in Burk-infected CF pts. No difference was seen in change of FEV1% predicted for AZLI vs. PCB. Both treatment groups received substantial amounts of additional Abx for respiratory indications. AZLI pts received numerically less additional Abx for respiratory indications, and had a greater time to need for additional Abx, but the differences from PCB pts were not statistically significant. Continuous AZLI did not reduce the Burk sputum density, nor did it lead to treatment emergence of respiratory pathogens. Baseline lung function varied widely in the study pts and there was a significant amount of co-infection with other CF pathogens. Evaluation of the subset of pts exhibiting favorable FEV1 responses may identify clinical or microbiological factors predicting response to AZLI. Supported by Gilead Sciences. B2 INVESTIGATION INTO INCREASED POLYMYXIN SUSCEPTIBILITY OF BURKHOLDERIA MULTIVORANS ISOLATES DURING CHRONIC CYSTIC FIBROSIS PULMONARY INFECTION Rebecca J. Malott, Tracy D. Lee and David P. Speert Center for Understanding and Preventing Infection in Children, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada Contact: Rebecca Malott: [email protected] Chronic pulmonary infections are the major cause of morbidity and mortality in people living with cystic fibrosis (CF). Burkholderia cepacia complex (Bcc) bacteria are ominous CF pathogens due to their pathogenic potential and intrinsic resistance to multiple antibiotics. Physicians are returning to polymyxins, especially inhaled colistin for therapy of respiratory Pseudomonas aeruginosa infections. During long-term pulmonary infection, sequential Bcc isolates exhibit fluid genotypes and phenotypes. Bcc species are considered highly resistant to polymyxins, yet the dynamics of the polymyxin resistance phenotype over the course of Bcc infection has yet to be investigated. Minimum inhibitory concentrations (MICs) for colistin (polymyxin E) of RAPD-type matched early and late B. multivorans respiratory isolates from ten CF patients were evaluated by broth microdilution. Two of the late isolates had MICs to colistin of 64 µg/ml, with the remainder of the isolates having MICs greater than 512 µg/ml. The MICs


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of seven additional antimicrobials for the two early and late isolate pairs exhibiting an increase in colistin susceptibility over time were performed. Late isolate D2408 was also more susceptible to polymyxin B, gentamicin and ceftazidime compared to the matched early isolate D1285, indicating a global decrease in antimicrobial resistance over time. Conversely, late isolate D2434 was only more susceptible to the polymyxin B compared to the matched early isolate, C6398. The mechanism of specific increase in susceptibility to polymyxins observed in this pair of sequential isolates is not due to differences in permeability to 1-N-phenylnaphthylamine in the presence of polymyxin B or binding of BODIPY-conjugated polymyxin B. Investigations are ongoing to determine the mechanism of increased polymyxin susceptibility between isolate C6398 and D3434 and how this relates to the clinical course B. multivorans infection. B3 CHARACTERIZATION OF BURKHOLDERIA CONTAMINANS ISOLATED DURING THE COURSE OF THE INFECTION IN A CYSTIC FIBROSIS PATIENT José Degrossi1, Laura Galanternik2, Agustina Lopez de Volder1, Miryam Vasquez2, Miguel D´Aquino1, Alejandro Teper2, Mirta Franco1 1Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina. 2Hospital de Niños “Dr Ricardo Gutirrez”, Buenos Aires, Argentina. Contact: José Degrossi, [email protected] Burkholderia contaminans is the most frequent species of the Burkholderia cepacia complex (Bcc) isolated from cystic fibrosis (CF) patients in Argentina. There is little data about the virulence and clinical impact of this Bcc member over CF patients´ health. Many patients harboring B contaminans treated at different centres in our country are chronically infected. However during the last year, exacerbation followed by some patients´ death was observed in our centre. In order to assess changes in B. contaminans isolates during the course of the infection, we compared colonies obtained at different periods of time from a female CF chronically infected patient of 10.6 years old when she died. A total of 29 colonies were collected from respiratory secretions: 24 months (T24), 8 months (T8) and immediately before the patient´s death (T0). Isolates were genetically compared by RAPD and recA gene sequence analysis; phenotypic characterization included resistance to antibiotics, virulence on the alfalfa model, ability to form biofilms, swarming motility, haemolysis, proteases, lipases, green-yellow pigment and exopolisaccharides production. There were no differences in the recA sequences of all isolates. Among the T24 colonies, at least two groups distinguished by phenotypic characteristics such as antibiotic susceptibility, haemolytic activity, yellow green pigment production and mucoid expression were observed. Slight differences in the RAPD profiles of both groups also were detected. T8 and T0 isolates shared the same RAPD pattern and showed only slight differences in the probes assayed: all colonies were non haemolytic, non or partially mucoid, showed low motility and non pigment production, among other characteristics. Colonies collected at T0 proved to be the most virulent on the alfalfa model. These results describe some of the phenotypic variation of B. contaminans and indicate an adaptative response during the course of infection as it was described in other Bcc species.


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B4 CULTURE AND DEEP SEQUENCING ANALYSIS OF MICROBIAL COMMUNITY COMPOSITION IN THE CYSTIC FIBROSIS AIRWAYS DURING EXACERBATIONS M. M. Tunney1, A. A. Fodor2, E. Klem3, D. F. Gilpin1, J. S. Elborn1, R. C. Boucher3, and M. C. Wolfgang3,4 1CF & Airways Microbiology Research Group, Queen’s University Belfast, Belfast, UK 2University of North Carolina at Charlotte, Charlotte, USA 3Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, USA, 4Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, USA Contact: Michael Tunney, [email protected] Introduction and aims: Numerous studies have shown that CF airways are infected with complex microbial communities and that pathogens persist through multiple rounds of antibiotics. As many microbes are not detected by conventional culture methods, molecular detection techniques offer a more complete view of the nature of CF infections. The objective of this study was to characterize the CF lung microbiome and to determine the effect of antibiotic treatment on bacterial diversity and abundance during exacerbation. Methods: Sputum samples were collected from 23 CF patients at the beginning and completion of antibiotic treatment of exacerbations and from a subset of patients (n=13) when clinically stable. Samples were processed using strict anaerobic culture and 454 sequencing. Microbial community diversity was compared with markers of disease severity such as lung function (FEV1). Results: Over 400,000 sequences were generated from our study population representing nearly 170 distinct microbial taxa. Although, approximately 60% of the sequences obtained were from the dominant pathogens, Pseudomonas and Burkholderia, a range of other taxa including Prevotella, Streptococcus, Rothia and Veillonella were frequently observed. The two dominant pathogens found by culture were P. aeruginosa and B. cenocepacia and there was broad agreement between the results of 454 sequencing and culture for these bacteria. Diversity was reduced in patients predominantly infected by Bukholderia spp. (P < 0.001). Although antibiotic treatment was associated with a small, transient decrease in species richness, there was little change in the dominant members of the microbial community in response to antibiotics. Furthermore, microbial community composition was highly similar in patients during an exacerbation and when clinically stable. While the presence of Burkholderia was usually associated with a low FEV1, patients with Burkholderia that had higher overall microbial diversity tended to have a higher FEV1. Conclusions: Our results reveal that the adult CF sputum microbiome is largely stable through periods of exacerbation and antibiotic treatment. Over time a small number of taxa predominate causing more severe clinical symptoms.


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C1 THE BURKHOLDERIA GENOME DATABASE: A RESOURCE FOR COMPARING COMPLETE AND INCOMPLETE GENOME SEQUENCES AND ANNOTATIONS Geoffrey L. Winsor and Fiona S.L. Brinkman Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC. Canada. Contact: Geoff Winsor, [email protected] The Burkholderia Genome Database (http://www.burkholderia.com) is a resource for viewing and comparing Burkholderia cepacia complex genome annotations and sequences with comparative analyses available for other Burkholderia genomes. With current advances in next-generation sequencing technologies, the number of completed and ongoing Burkholderia genome projects is close to 200; however the majority is expected to remain in permanent draft status. To address this issue, the Burkholderia Genome Database now facilitates the comparison of incomplete Burkholderia genome annotations and sequences alongside those of 27 completely sequenced genomes. This framework enables the identification of putative orthologs in both complete and incomplete genomes using our high-precision method for evaluating previously-predicted orthologs (Ortholuge) as well as a clustering method for providing a more inclusive set of genes mapped to their putative orthologs and paralogs (Burkholderia Orthologous Groups). In addition to the detailed views of gene annotations, we now provide the ability to view and compare intergenic region sequences and their annotations including transcription terminator predictions and other regulatory elements. Additional analyses of interest include high quality subcellular localization and operon predictions and identification of pathogen-associated genes. With more than 1000 updates submitted by Burkholderia researchers and in-house curation efforts, and increased database curation to occur in the coming year, this database aims to provide a high quality, annotated genome resource. Funding provided by Cystic Fibrosis Foundation Therapeutics, Inc. C2 RANDOM PROMOTER REPLACEMENT OF ESSENTIAL GENES AS A TOOL FOR CHEMICAL GENOMICS STUDIES IN BURKHOLDERIA CENOCEPACIA Ruhi A.M. Bloodworth, April S. Gislason, Holly Hurst, and Silvia T. Cardona Department of Microbiology, University of Manitoba, Winnipeg, Canada Contact : Silvia T. Cardona, [email protected] Essential genes can be operationally defined as genes required for growth in rich, undefined medium and are potential antimicrobial targets. We used random transposon delivery of an outward facing rhamnose-inducible promoter to construct a genomic library of 104 Burkholderia cenocepacia essential gene conditional expression mutants. With this strategy, we identified widely conserved essential genes and Burkholderia-specific essential genes, highlighting the organismal nature of gene essentiality. Comparative expression analysis of essential genes showed that the B. cenocepacia library is representative of the whole range of expression levels


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for gene essentiality. However, the rhamnose concentrations required for exhibiting the conditional-growth phenotype were remarkable similar. Exposing the library to sub-lethal antibiotics with known target confirmed that under-expression of essential genes produced selective antibiotic hypersensitivity. Conversely, mutants were not sensitive to inhibitors of unrelated processes. This hypersensitivity was observed at various percentages of maximal growth (30-80%) requiring just 5 rhamnose concentrations for screening the entire library. This work shows that genome-wide random delivery of one inducible promoter followed by a simple screening for conditional growth, creates a cohesive tool for small-molecule target interactions given the broadly similar conditions required for modulating gene expression. C3 CENTROMERE BINDING AND EVOLUTION OF CHROMOSOMAL PARTITION SYSTEMS IN THE BURKHOLDERIALES Fanny M Passot, Virginie Calderon, Gwennaele Fichant, David Lane and Franck Pasta Laboratoire de Microbiologie et Génétique Moléculaires, UMR 5100 CNRS/Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France Contact : Dave Lane, [email protected] How split genomes emerged and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2 and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3 and pBC share many features, suggesting they arose within an extended family. Database searching revealed seven sub-families of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of Burkholderiales, which reinforces the proposal of an extended family. The sub-family of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using EMSA we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. By contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome, suggesting that we may be witnessing emergence of a third chromosome from a plasmid in this species.


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C4 REGULATION OF GENE EXPRESSION BY MAEB RNA IN BURKHOLDERIA Xiaoling Yang, Jonathan Perreault Institut national de la recherche scientifique, Institut Armand-Frappier, Québec, Canada Contact : [email protected], [email protected] The gene regulation by small RNA structures is common in cells. Recently, several novel structures of RNA in bacteria have been discovered; however, the mechanism of gene regulation for most of these RNA is unknown. The MAEB RNA (Metabolism-Associated Element in Burkholderia) is found in bacteria of the Burkholderia genus, which consists of a single hairpin structure with several conserved positions. MAEB elements are typically found upstream of a target gene in the 5 'untranslated region, and these genes are usually involved in the primary metabolism. These discoveries suggest that this element could take a role in the control of metabolic genes in Burkholderia. The first goal of our project is to identify the functional mode of MAEB motif in the metabolic genes regulation in Burkholderia. Also, the conserved bases in MAEB RNA point towards the possibility that the mechanism of MAEB to regulate gene expression could go through a RNA-binding protein action, which means that a protein could bind to MAEB structure in response to environmental changes. So we also focus on the determination of the protein partners of MAEB and the binding mechanism of these proteins to MAEB RNA in Burkholderia. C5 NON-CODING RNA IN BURKHOLDERIA Jonathan Perreault1 1Institut national de la recherche scientifique, Institut Armand-Frappier, Laval, Québec, Canada In all living organisms, a plethora of RNAs control numerous cell processes. As with proteins, these so called non-coding RNAs (ncRNAs) adopt a structure that confers function. By using this “structure signature” and the wealth of data in public sequence databases, we and others have found hundreds of novel RNAs in the recent years with comparative genomics approaches. Many ncRNAs discovered in bacteria have been characterized with microbiology, genetic and biochemical methods. Among these, riboswitches are RNAs embedded in the 5' portion of mRNAs and regulate the expression of the protein encoded downstream by binding specifically to metabolites. However, for most of the newly discovered ncRNAs no function has been identified yet. These include the hammerhead ribozyme instances that we revealed in bacteria, as well as numerous other RNAs found in many bacteria, including in the Burkholderia genus. A dozen different ncRNAs have been discovered recently in many Burkholderia species (Weinberg et al. 2007, Nucleic Acids Research; Weinberg et al. 2010, Genome Biology; Coenye et al. 2007, FEMS Microbiology Letters). These ncRNAs putatively regulate close to a hundred genes, but only a few have been experimentally deciphered, a lot of work remains to understand their roles.


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C6 CHARACTERIZATION OF THE MICROOXIC LIFESTYLE OF BURKHOLDERIA CENOCEPACIA H111 Gabriella Pessi, Rubina Braunwalder, Alexander Grunau and Leo Eberl Institute of Plant Biology, Department of Microbiology, UZH, Zürich, Switzerland Contact : Gabriella Pessi, [email protected] Burkholderia cenocepacia is an opportunistic pathogen that is especially problematic for patients with cystic fibrosis (CF). In CF infections, bacteria grow to high densities in a mucopurulent material that is limited in oxygen. Recent studies have shown that Pseudomonas aeruginosa, the dominant pathogen in CF patients, can adapt to grow and survive under oxygen-limited to anaerobic conditions by using microaerobic respiration, denitrification and fermentative pathways. Curiously, the genome of B. cenocepacia H111 lacks the genes involved in denitrification (nar, nir, nos, nor), arginine fermentation (arcABCD operon), as well as homologs of the P. aeruginosa terminal cbb3-type oxidase, which has been shown to sustain respiration when oxygen levels are low. Accordingly, our study demonstrated that H111 is not able to grow anaerobically and with less than 0.1% O2. In order to understand the molecular mechanisms underlying Bcc H111’s adaptation to microaerobic conditions, the expression of transcripts and proteins extracted from matched microaerobic (0.5% O2) and aerobic (21% O2) samples was profiled using RNA-Seq and shotgun proteomics. A preliminary phenotypical analysis showed that Bcc H111 produces more biofilm and cellulose under microaerobic conditions while the production of other virulence factors such as siderophores and autoinducers decreased. Finally, mutation of a FNR-type transcriptional regulator that is up-regulated under microaerobic conditions will hopefully help us to elucidate the importance of microaerobic adaptation of B. cenocepacia in CF infections. D1 USING TN-SEQ TO DEFINE BURKHOLDERIA DOLOSA GENES ESSENTIAL FOR COLONIZATION AND SEPTIC DISSEMINATION IN MICE, AND CELL INVASION Deborah Yoder-Himes1,2, Damien Roux3, Craig Gerard1, Stephen Lory1

1 Division of Respiratory Diseases, Children’s Hospital Boston, Boston, MA, USA 2 Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA 3 Channing Laboratory, Brigham and Women’s Hospital, Boston, MA, USA Contact: [email protected] Abstract Burkholderia dolosa is the causative agent of an epidemic at the Children’s Hospital Boston cystic fibrosis clinic that spanned from 1999 to the present day. Over 40 patients have been infected and over 7 have died from cepacia syndrome. Very little is known about the virulence of B. dolosa. The genome sequence revealed a number of loci that are not found in most Burkholderia cepacia complex members but are found in the closely related B. pseudomallei and B. mallei human and horse pathogens, including genes encoding lateral flagella and a third Type


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III secretion system (T3SS#3). We developed tools which allow us to utilize next-generation sequencing of transposon mutants (Tn-seq) to identify genes important for (i) colonization of the mouse bronchi, (ii) invasion into cultured respiratory epithelial cells, and (iii) septic dissemination in an intraperitoneal mice model of infection. The results demonstrate that hundreds of genes contribute to each model and that a different cadre of genes is required for colonization and dissemination. Of the 98 genes that are required for colonizing the lung and the peritoneum, over 10% encode transcription factors. Additionally, genes encoding proteins involved in nitrogen metabolism are also over-represented. The 213 genes essential for disseminating into the heart and spleen include those encoding some components of the T3SS#3 and Type VI secretion systems. There are many genes important for invading cultured epithelial cells and these include those encoding the inner membrane protein TonB and a Ton-dependent receptor, which are involved in iron acquisition. This work provides us with insights into multiple steps along the course of infection for B. dolosa which may help in elucidating the mechanisms that this particular pathogen uses to cause virulence and also provides a valuable new tool for Bcc research. D2 INVESTIGATION OF THE ROLE OF PC3 IN THE VIRULENCE OF BCC SPECIES Stephan Schwager, Kirsty Agnoli and Leo Eberl

Department of Microbiology, Institute of Plant Biology, University of Zurich, Zurich, Switzerland Contact: Leo Eberl, [email protected] Our group has recently reported that ‘chromosome 3’ (pC3) is not essential for growth of many Bcc strains, but is instead important for virulence. By use of a plasmid incompatibility approach, we have suceeded in removing pC3 from representatives of 16 of the 17 Bcc species characterised to-date (the exception being B. cepacia). In the majority of these strains, where the wild-type was found to be pathogenic in C. elegans or G. mellonella, the pC3 null mutant showed reduced pathogenicity. To localise genes which might play a role in pathogenicity, the sequence of pC3 from B. cenocepacia H111 was compared with those from other completed Bcc genomes. The leading half of pC3 was found to be conserved, while the remainder showed little conservation between species. This led us to construct a partial deletion of the non-conserved part of B. cenocepacia H111 pC3 by FLP/FRT recombination. This partial deletion did not affect pathogenicity in either C. elegans or G. mellonella, suggesting that the pathogenicity genes of pC3 are located on the conserved part of the replicon. Furthermore, we used a broad-host-range cosmid library to conduct a gain-of-function screen in B. cenocepacia H111Δc3. Of 2,000 mutants tested, only one was found to restore pathogenicity in C. elegans. Tests showed that this cosmid did not carry the gene encoding the nematocidal protein AidA. Investigations into the virulence factor carried on this cosmid are ongoing.


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D3 ROLE OF BUKHOLDERIA CENOCEPACIA BCAS0208 AND BCAS0201 GENES IN DETERMINING SURFACE-ASSOCIATED PHENOTYPES Sujatha Subramoni1, Kirsty Agnoli2, Leo Eberl2, Shawn Lewenza1, and Pamela A. Sokol1 1Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada 2Department of Microbiology, Institute of Plant Biology, University of Zürich, Zürich, Switzerland Contact: Pamela A. Sokol, [email protected] Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis patients. Previously we reported that mutations in shvR, a LysR-type transcriptional regulator, and the ShvR regulated genes BCAS0208 and BCAS0201 affect colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia (Bernier et al. 2008; Subramoni et al. 2011). BCAS0208 encodes an acyl-coenzyme A dehydrogenase and BCAS0201 encodes a putative FAD-dependent oxidoreductase. These genes are located in an operon adjacent to shvR and downstream of the afcAB genes previously shown to be involved in synthesis of a membrane associated antifungal lipopeptide in B. pyrrocinia (Kang et al 1998). In this study we have investigated the role of BCAS0208 and BCAS0201 in influencing colony morphotype and other cell surface associated phenotypes. As previously reported for the shvR mutant (O’Grady et al 2011), mutations in both BCAS0208 and BCAS0201 resulted in loss of antifungal activity against Fusarium and Rhizoctonia solani indicating that these genes are involved in synthesis of the antifungal lipopeptide. Mutations in BCAS0207, encoding a citrate synthase, and BCAS0204, part of an ABC transporter complex, which are in the same operon as afcAB, BCAS0208 and BCAS0201, do not alter either colony morphotype or antifungal activity suggesting that there is a correlation with the synthesis of the antifungal lipopeptide and colony morphotype. However fatty acid methyl ester analysis by MIDI-gas chromatography did not reveal any major differences in cellular fatty acid compositions of the shvR, BCAS0208 or BCAS0201 mutants. Mutations in shvR, BCAS0208 and BCAS0201 resulted in reduced swarming motility which was restored to wild type levels upon providing these genes in trans. Both the shvR and BCAS0208 mutants show increased uptake of the hydrophobic fluorescent probe N-phenyl-naphthylamine (NPN) suggesting altered outer membrane properties; however, mutations in either BCAS0208 or BCAS0201 did not affect sensitivity to polymyxin B, an antibiotic that binds and alters membrane properties. Taken together, these results suggest that BCAS0208 and BCAS0201 are involved in lipid metabolism that affects both cell surface and antifungal properties of B. cenocepacia.


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D4 A PUTATIVE CzcR-CzcS TWO-COMPONENT SYSTEM REGULATES VIRULENCE IN BURKHOLDERIA CENOCEPACIA Matthew T. Robinson & Alan R. Brown Biosciences, University of Exeter, Exeter, UK. Contact : Alan Brown, [email protected] Two-component systems (TCSs) are pivotal regulators of bacterial adaptation, facilitating the response of bacteria to extracellular stimuli. TCSs typically comprise a histidine kinase (HK) sensor protein within the cytoplasmic membrane, and a cognate response regulator (RR) protein located within the cytoplasm. In response to certain stimuli, the HK sensor controls the phosphorylation state (and thus activity) of the cognate RR, which is typically a DNA-binding protein that regulates gene expression. TCSs have received attention as antimicrobial targets, as their inhibition may block the ability of bacteria to respond to conditions encountered within the host. Preliminary studies undertaken within our laboratory have supported that viewpoint, showing that a putative TCS inhibitor can protect Galleria mellonella (wax moth larvae) from bacterial killing. Perhaps consistent with their remarkable versatility, Burkholderia species possess a large number of TCSs. In this particular study, we have characterized a TCS of B. cenocepacia that has homology to documented CzcR-CzcS TCSs which typically regulate the bacterial response to heavy metals. Deletion of the putative czcRS genes from B. cenocepacia K56-2 results in hypersusceptibility to zinc and cadmium, consistent with the proposed czc nomenclature. However, the ΔczcRS B. cenocepacia strain also exhibits profound alteration in several virulence-associated traits, including attenuation in the G. mellonella infection model, reduced biofilm formation and reduced survival within murine macrophages. Comparison of czcR and czcS single and double mutants has provided insight into the nature of the interaction between the HK and RR. Ongoing studies aim to establish the repertoire of genes that are regulated by CzcR, and how those genes explain the pleiotropic effects associated with the CzcR-CzcS TCS of B. cenocepacia. D5 A NOVEL IRON UPTAKE PATHWAY IN B. CENOCEPACIA H111 Anugraha Mathew, Aurelien Carlier and Leo Eberl Department of Microbiology, Institute of Plant Biology, University of Zurich Contact: Leo Eberl, [email protected] Iron uptake in B.cenocepacia H111 is mediated mainly by two siderophores, pyochelin and ornibactin. However, we noticed that a H111 mutant defective in the production of both siderophores was still able to grow under iron-limited conditions. This raised an intriguing question: What is the alternative, high affinity uptake strategy developed by this pathogen to scavenge iron and thrive in the absence of siderophores?


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While analysing different Burkholderia genomes to address this question, we encountered a putative iron transport locus (BCAL2298 – BCAL2301 in J2315), which is conserved across all sequenced Burkholderia species. An iron permease protein which is homologous to the high affinity ferric permease (ftr1p) found in Saccharomyces cerevisiae, clustered with other ORFs encoding ferredoxin and putative iron transporters implied the possible involvement of this locus in iron uptake. A conditional mutation was introduced in this target locus in a siderophore mutant of H111 to investigate its role in iron uptake. This mutant failed to grow in the absence of siderophores under iron limited conditions, indicating its role in iron acquisition. In addition, radiolabeled iron transport assays showed that these mutants were highly impaired in iron acquisition when compared to the siderophore mutants. This provides initial evidence of a novel iron uptake pathway in Burkholderia. D6 ORNIBACTIN-LIKE SIDEROPHORE ARE PRODUCED BY BURKHOLDERIA THAILANDENSIS Sok Gheck Tan, François Lépine and Eric Déziel INRS-Institut Armand-Frappier, Université du Québec, Laval, Canada

Contact : Eric Deziel, [email protected] Iron, one of the most abundant elements on Earth, is required for the survival of all microorganisms. In most environments, whether biological or not, the concentration of available iron is very low due to the insolubility of the ionic forms of iron and due to the sequestration of the metal by specialised proteins like transferrin and lactoferrin in a mammalian host. This results in a low bioavailability of iron for the survival of the microorganisms. To bypass this obstacle, they have developed different strategies, direct or indirect, to capture iron. A siderophore is a chelator that has a high-affinity for ferric iron, the most common ion form of iron. These molecules are produced and secreted by many bacteria, fungi and even some plants under iron-deficient conditions. Siderophores are considered to be virulence factors because of their capacity to compete against specialised proteins for the iron acquisition allowing a pathogen to survive in a host. This project aims to study the siderophores produced by Burkholderia pseudomallei, a Gram-negative bacillus which is the causative agent of meliodosis, an infectious disease endemic in parts of Southeast Asia and Northern Australia. For this matter, Burkholderia thailandensis, an avirulent species which is phylogenetically and phenotypically similar to B. pseudomallei, is used as an alternative model. It is already confirmed that B. pseudomallei produces siderophores. However, little is known as to what types of siderophores are produced and which genes are involved. Therefore, the primary objectives set for this project were to characterize the siderophores produced by B. thailandensis and to identify the genes involved in the biosynthesis and the regulation on these molecules. A screening by random transposon mutagenesis has allowed the identification of several genes implicated in the regulation and biosynthesis of siderophores similar to ornibactin, a siderophore belonging to the hydroxamate family produced by some members of the Burkholderia cepacia complex. Analysis by liquid chromatography–mass spectrometry (LC-MS) has shown that these ornibactin-like siderophores are the main ones produced by B. thailandensis and that many forms, like ornibactin which exists in three differents forms, appear to be present.


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D7 BCAL1491, A POTENTIAL PERIPLASMIC PHOSPHATASE THAT CONTROLS MULTIPLE CELL ENVELOPE COMPONENTS IN BURKHOLDERIA CENOCEPACIA Andrea D. Valderrey1, Daniel Aubert2, Roberto Rosales-Reyes2, Rafael Rotger1, Miguel A. Valvano2 1Department of Microbiology II Ramón y Cajal and Institute for Health Research (IRYCIS), Faculty of Pharmacy, Universidad Complutense de Madrid. 2Center of Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada. Contact: Andrea D. Valderrey, [email protected] Burkholderia cenocepacia is an opportunistic pathogen in humans, able to produce a serious infection in patients with cystic fibrosis. A proportion of infected patients develop a fulminant form known as "cepacia syndrome". Although the incidence varies by geographic area, B. cenocepacia is the species of the B. cepacia complex most commonly involved. The bases of the virulence B. cenocepacia are currently under study. The ability to survive intracellularly in macrophages and also a type VI secretion system (T6SS) are required for virulence. The T6SS is a recently discovered secretion system associated with bacterial virulence, but the factors secreted by T6SS in B. cenocepacia are still unknown. Bioinformatic analyses of the B. cenocepacia genome revealed that the gene BCAL1491 encodes a predicted phosphatase with homology to SopB from Salmonella. BCAL1491is located between genes encoding a tRNA and a possible transposase, suggesting it may have arisen from horizontal transfer and could be an effector of the T6SS. To examine the role of BCAL1491 we constructed a deletion mutant in B. cenocepacia K56-2. Macrophages infected with K56-2ΔBCAL1491 did not form the characteristic cytoplasmic protrusions attributed to a functional T6SS, similar to macrophages infected with a Δhcp mutant (T6SS functionally inactive). Lack of protrusions was not due to differences in macrophage internalization, since both K56-2ΔBCAL1491 and the parental strain were internalized at similar levels. Co-localization assays with dextran and bacteria expressing red fluorescent protein showed that the K56-2ΔBCAL1491 bacteria were located in lysosomes less frequently than the wild type strain, and this co-location was further decreased in the mutant Δhcp. Moreover, K56-2ΔBCAL1491 fixed more Congo red on the bacterial surface, showed a morphological arrangement in pairs, and lost swimming and swarming, suggesting the absence of a functional BCAL1491 is associated with defects in the bacterial cell envelope. An analysis of conserved domains revealed the presence of a TAT site (twin arginine translocation) at the amino-terminal end of the putative phosphatase. Therefore, we conclude that the BCAL1491 is a putative periplasmic phosphatase that is required for the homeostasis of the bacterial cell envelope, including functionality of the T6SS.


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E1 QUORUM SENSING STUDIES IN BURKHOLDERIA Bruna G. Coutinho1, Zulma Rocio Suarez-Moreno1, K. Sonal Choudhary1, Sanjarbek Hudaiberdiev1, Daniel Passos da Silva1, Eulogio Bedmar2, Birgit Mitter3, Angela Sessitsch3, Geoff Elliott4, Euan James5, Sándor Pongor1 and Vittorio Venturi1 1International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149, Trieste, Italy 2Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Apartado Postal 419, 18080-Granada, Spain 3AIT Austrian Institute of Technology GMBH, Konrad-Lorenz-Strasse 24, 3430 Tulln an der Donau, Austria 4The James Hutton Institute, Craigiebuckler, Aberdeen AB15 8QH, Scotland, UK 5The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK Contacts : Vittorio Venturi, [email protected] and Sandor Pongor, [email protected] The BraI/R quorum sensing system in plant-associated Burkholderia cluster is highly conserved in all the species, thus being a common feature however its role in plant-bacteria interaction is poorly understood. It is therefore of interest to establish the role of BraI/R in this group of species; we have investigated this in nodulating B. phymatum, in the B. phytofirmans endophyte and in the biodegrader B. xenovorans. Results will be presented indicating a species-specific role for BraI/R. On a more general note, we have surveyed the topological arrangement of genes involved in N-acyl homoserine lactone (AHL) quorum sensing in 27 complete and 43 draft Burkholderia genomes (23,431 contigs). Patterns of quorum sensing genes are variable throughout Burkholderia genomes, although there are unifying themes that are common among the various topological arrangements. The gene neighborhoods of the quorum sensing circuits and solo luxR genes have also been analyzed. Results of this in silico study will also be presented. E2 PSEUDOMONAS AERUGINOSA MEDIATES ANTAGONISTIC INTERACTIONS WITH THE BCC VIA THE RELEASE OF SMALL BIOACTIVE MOLECULES. Steve P. Bernier and Michael G. Surette Department of Medicine, McMaster University, Farncombe Family Digestive Health Research Institute, Hamilton, Ontario, Canada Contact: Steve P. Bernier, [email protected] Brief Introduction: The co-infection of the cystic fibrosis (CF) lung by different pathogens is a common occurrence, for which Pseudomonas aeruginosa still remains the most prevalent one. When sharing the same microenvironment, dynamic and competitive interactions will likely occur between these organisms. For instance, a number of P. aeruginosa extracellular metabolites were shown to exhibit antibiotic-like activities against the CF pathogens Staphylococcus aureus and Candida albicans. A few reports have previously described that


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unidentified small molecule(s) of P. aeruginosa could mediate killing of some Burkholderia cepacia complex (Bcc) species. Here, we hypothesized that P. aeruginosa may prevent other CF pathogens including the Bcc to share a polymicrobial community by releasing toxic bioactive small molecules(s). Aims: To determine the susceptibility of various Bcc species to P. aeruginosa spent medium and to identify the bioactive small molecules(s) having antibiotic-like activity against the Bcc. Results summary: In total, 32 strains representing nine Bcc species, namely B. cepacia, B. multivorans, B. cenocepacia, B. stabilis, B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina, and B. pyrrocinia were assessed for their susceptibility to P. aeruginosa spent media isolated 8, 16, 24, or 48 hours of growth. For this study, four P. aeruginosa strains were used including the non-mucoid laboratory strains PA14 and PAO1 as well as the mucoid PA2192 and the mall colony variant C3719, two typical features expressed by P. aeruginosa in the CF lung. The killing efficiency was enhanced in 24- and 48-h spent media compared to those isolated at earlier time points suggesting that accumulation of active metabolites was critical. In general, most tested strains were totally eradicated upon exposure to 24-h spent media from strain PA14, PAO1, and PA2192 with only two strains being totally resistant. Although C3719 spent medium could kill a subset of strains, it was generally less toxic than the other three. However, the metabolite-mediated killing was dependent of various environmental conditions such as pH, temperature, nutrients, oxygen, and movement. Briefly, modification of these factors could make an active supernatant inactive or vice versa. Further, we demonstrated that the loss-of-function adaptive mutations of the quorum sensing regulator genes lasR and/or rhlR could allow most tested strains, but not all, to survive in their respective spent medium. Lastly, analyses of various active and inactive spent media (lasR/rhlR mutants and pH shifts) by mass spectrometry are currently in progress as a combined approach to specifically identify the various bioactive molecules described above. Relevance of this study: Competitive interactions between CF pathogens are still poorly understood and the possible identification of new bioactive molecules may lead to the design of new drugs against Bcc infections. Further, the demonstration that adaptive mutations of P. aeruginosa may allow a greater microbial diversity is of importance to understand the evolution of CF lung infections in a context of polymicrobial interactions. E3 BIOFILM PRODUCED BY BURKHOLDERIA SPP: INFLUENCE OF SOLID SUPPORTS ON MATRIX COMPOSITION Paola Cescutti, Bruno Cuzzi and Roberto Rizzo Dipartimento di Scienze della Vita, Università di Trieste, Trieste, Italy Contact : Paola Cescutti, [email protected] The biofilm matrix1 is an active macromolecular scaffold composed mainly by proteins, extracellular DNA and polysaccharides (PLS), and the latter are generally the main biofilm component. In spite of their abundance in the biofilm matrix, little is known about their primary structure and their role in biofilm architecture. In fact, most of the studies involving PLS resort to growth of bacteria on agar plates, or in liquid media, although both these methods do not follow the definition of biofilm, as clearly reported by Donlan and Costerton2:” ...These ‘nonbiofilm’ population, which include colonies of bacteria growing on the surface of agar, behave like


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planktonic cells stranded on a surface and exhibit none of the inherent resistance characteristics of true biofilms…”. An investigation on the PLS present in biofilm matrix was recently undertaken by our group. A CF clinical isolate of B. cenocepacia strain BTS2, a good biofilm producer on microtiter plates, was compared with the reference strain C1576 of B. multivorans, which did not produce biofilm on the same support. When grown on YEM agar plates, BTS2 produced a mixture of cepacian and galactan-Kdo, while C1576 produced only cepacian. Recovery of matrix from microtiter plates was unsuccessful. Therefore, two others supports were chosen: a cellulose porous membrane layered on agar dish and a glass slide half dipped in culture broth. Both strains grew on both solid supports. Surprisingly, also C1576 stick to the glass and formed some slime. The matrices were recovered and proteins and PLS content were determined. Preliminary data showed that the PLS produced were the same as those obtained in non-biofilm mode of growth, although BTS2 produced the two PLS in different ratios and with different acetylation degree. 1) Flemming HC, Wingender J. (2010) The biofilm matrix. Nat. Rev. Microbiol. 8, 623-633. 2) Donlan RM, Costerton J.W. (2002) Biofilms: survival mechanisms of clinically relevant microorganisms Clin. Microbiol. Rev. 15, 167-193. E4 REGULATION OF RHAMNOLIPIDS PRODUCTION BY BURKHOLDERIA THAILANDENSIS Adeline Humery and Eric Déziel INRS-Institut Armand-Frappier, Laval, Québec, H7V 1B7, Canada Contact : [email protected] Rhamnolipids are amphiphilic molecules with excellent surface active properties. They have been exhaustively studied in Pseudomonas aeruginosa due to their roles in multicellular behaviours of this bacterium and to their commercial potential as alternative surfactants. In Burkholderia thailandensis, Burkholderia glumae, Burkholderia ambifaria and Burkholderia cenocepacia, we have discovered ortholog genes to rhlA, rhlB and rhlC which are responsible for rhamnolipid production in P. aeruginosa. Unlike this latter bacterium, B. thailandensis contains the three genes inside one operon that exists in two paralog and functional copies (even if one copy seems to contribute more strongly to global rhamnolipid production). The aim of my project is to investigate the factors that regulate the transcription of the rhl operons in B.

β-D-Galp-(1→2)-α-D-Rhap 1 ↓ 4 [3)-β-D-Glcp-(1→3)-α-D-GlcpA-(1→3)-α-D-Manp-(1→]n 2 6 ↑ ↑ 1 1

α β

[3)-β-D-Galp2Ac-(1→4)-α-D-Galp-(1→3)-β-D-Galp-(1→5)-β-Kdo-(2→ ]n

cepacian

galactan-Kdo


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thailandensis. Since cell-to-cell communication (quorum sensing or QS) activates rhamnolipid production in P. aeruginosa, we verified if the same is true in B. thailandensis by testing various QS mutants for their rhamnolipid production (characterised and quantified by LC/MS). Unexpectedly, QS in B. thailandensis appears to repress rhamnolipid production because some QS mutants achieve a ten-fold increase in production compared to wild type strain. However, this regulation does not seem to be direct. Using constructions of different transcriptional reporter genes (lacZ fusion type) transferred into wild type strain and QS mutants, we investigated the regulation of both operon from rhlA1 and rhlA2 promoters, and their specific implication in total rhamnolipid production. Our results confirm that one of the operon is expressed at a higher level than the other. In order to identify direct regulator(s), two lines of research are pursued: (1) random transposon mutagenesis of strains containing rhlA reporter genes, followed by mutant screening, (2) pull-down approach using the targeted promoters (identified by 5’RACE method) to capture possible regulatory molecules. 5’RACE experiments have revealed promoters much shorter than expected that does not explain the differential transcriptional regulation of the two operons. This work will help us understand rhamnolipid production in other Burkholderia species. E5 THE IMPORTANCE OF QUORUM SENSING IN THE VIRULENCE OF BURKHOLDERIA CENOCEPACIA J2315 Claudia Udine1, Gilles Brackman2, Silvia Bazzini1, Maria Rosalia Pasca1, Tom Coenye2 and Giovanna Riccardi1 1Dipartimento di Biologia e Biotecnologie, Università degli Studi di Pavia, Pavia, Italy 2Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium Contact: Giovanna Riccardi ([email protected]), Tom Coenye ([email protected]) Burkholderia cenocepacia J2315 is a Gram-negative opportunistic pathogen, which is able to cause serious infections in immune-compromised individuals, such as cystic fibrosis patients. In the virulence of this pathogen, the cell–cell communication mechanism, known as quorum sensing (QS), has an important role to coordinate gene expression and biofilm formation. B. cenocepacia possesses two QS systems (CepIR and CciIR), which operate using acyl homoserine lactones (AHL) as signaling molecules. In addition, a more recently identified system is based on a diffusible fatty acid signal molecule (cis-2-dodecenoic acid, also called “Burkholderia diffusible signal factor”, BDSF). The aim of the present study was to analyze the phenotype of deletion mutants in which BCAM1870 (CepI), BCAM0239a (CciI) or BCAM0581 (BDSF synthase) were inactivated. Three single (ΔcepI, ΔcciI and ΔBCAM0581), a double (ΔcepI-ΔBCAM0581) and a triple (ΔcepI-ΔcciI-ΔBCAM0581) mutants were tested to assess their virulence and ability to form biofilm, using different in vitro and in vivo model systems. β-lactam susceptibility of planktonic cells of the ΔBCAM0581 mutant, as well as of the double and triple mutant, was increased (for example, for meropenem MIC = 8 µg/ml for the mutants vs. 32 µg/ml for WT and the ΔcepI and ΔcciI single mutants). Susceptibility of planktonic cells to ciprofloxacin or tobramycin was not altered. In vitro biofilm formation was impaired in all mutants, as demonstrated by crystal violet and resazurine staining, as well as by CLSM. Addition of BDSF restored biofilm formation of the ΔBCAM0581 mutant to WT levels. We showed that all mutants formed biofilms that were more susceptible to tobramycin than the WT. This effect


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was most prounounced for the double (ΔcepI-ΔBCAM0581) and triple (ΔcepI-ΔcciI-ΔBCAM0581) mutants. Production of AHL QS-molecules was drastically reduced in all mutants and no AHLs were detected in the triple (ΔcepI-ΔcciI-ΔBCAM0581) mutant. Finally, the ΔBCAM0581 and triple (ΔcepI-ΔcciI-ΔBCAM0581) mutants were less virulent in a Caenorhabditis elegans model system. E6 CYTOTOXIC EFFECTS OF 4-HYDROXY-3-METHYL-2-ALKYLQUINOLINES (HMAQ) PRODUCED BY BURKHOLDERIA Annelise Chapalain, Sylvain Milot, Anastasia Nikolakakis, Christine Matte, François Lépine, Albert Descôteaux & Eric Déziel INRS-Institut Armand-Frappier (Laval) Qc Canada Contact : [email protected] The human opportunistic pathogen Pseudomonas aeruginosa is the microorganism most encountered in patients suffering from Cystic Fibrosis (CF). This bacterium expresses numerous virulence factors, mostly controlled by quorum sensing (QS) regulatory networks. QS in P. aeruginosa relies on three main parts: the two first, LasRI and RhlRI, are based on acyl-homoserinelactone (AHL) signal molecules, whereas the third part relies on 4-hydroxy-2-alkylquinolines (HAQ). These molecules, in addition to their signaling role, have more recently been shown to have a direct virulence potential, by acting as immunomodulatory or cytotoxic agents. We have reported that at least three Burkholderia species, namely Burkholderia pseudomallei, Burkholderia thailandensis and Burkholderia ambifaria produce molecules similar to HAQ, bearing a methyl group at the 3’ position and thus named 4-hydroxy-3-methyl-2-alkylquinolines (HMAQ). More recently, another team has shown that several Burkholderia cepacia strains were also able to produce HMAQ, which displayed antifungal properties. Given that several Burkholderia species, including but not limited to those belonging to the Burkholderia cepacia complex, can also be isolated from CF patient, our hypothesis is that, similarly to the HAQ from P. aeruginosa, HMAQ from Burkholderia interact with eukaryotic cells, and participate to the virulence of Burkholderia species. We are comparing the cytotoxicity on murine macrophages of the two main HMAQ molecules (HMAQ(C7:2’) and HMAQ(C9:2’)) produced by Burkholderia with the two main HAQ of P. aeruginosa (Pseudomonas Quinolone Signal (PQS) and 4-hydroxy-2-heptylquinoline (HHQ)) . Our results indicate that HMAQ(C9:2’) and HHQ display an important cytotoxic effect, whereas HMAQ (C7:2’) and PQS are less cytotoxic, but induce cellular changes after 24h of exposure. Experiments are currently underway to determine which kind of cellular death is triggered by each molecule, and if the observed effects are limited to macrophages.


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F1 CHARACTERIZATION OF BURKHOLDERIA CENOCEPACIA-INDUCED NEUTROPHIL DEATH IN CHRONIC GRANULOMATOUS DISEASE Allison M. McDonald and David P. Speert Centre for Understanding and Preventing Infections in Children, Department of Pediatrics, Faculty of Medicine, University of British Columbia, Vancouver, Canada Contact: David Speert, [email protected]

The phagocyte NADPH oxidase is responsible for producing toxic reactive oxygen species (ROS) upon interaction with pathogens. Patients with chronic granulomatous disease (CGD) have loss-of-function mutations in the NADPH oxidase complex and thus suffer from chronic, recurrent infections. In CGD, the Burkholderia cepacia complex can cause necrotizing pneumonia and sepsis and is the leading bacterial cause of death. The pathology is characterized by infiltrating neutrophils and B. cenocepacia induces enhanced neutrophil death in CGD compared to controls in vitro. This study was driven by the hypothesis that differences in neutrophil death in normal and CGD cells upon challenge with B. cenocepacia are due to an inability to control the replication of ingested bacteria or to activate specific pathways involved in physiological cell death. The molecular mechanisms leading to neutrophil death were investigated in primary neutrophils pretreated with the potent NADPH oxidase inhibitor diphenyleneiodonium (DPI) to mimic CGD and compared to untreated neutrophils. Neutrophil necrosis was enhanced in ROS-deficient cells compared to normal cells following challenge with a panel of clinical B. cenocepacia isolates. In contrast to the rapid killing of B. cenocepacia by normal neutrophils, the bacteria were able to survive in the absence of ROS. This increased persistence was associated with a heightened, prolonged neutrophil caspase-3 response. Caspase activation was not the basis for the observed differences in neutrophil necrosis. Current studies are focused on investigating the contribution of apoptosis, programmed necrosis, and autophagy on B. cenocepacia-induced neutrophil necrosis. F2 REAL TIME ANALYIS OF THE ROLE OF ZEBRAFISH PHAGOCYTES TO INFECTION WITH BURKHOLDERIA CENOCEPACIA Jennifer Mesureur, Nelly Wagner, David O’Callaghan and Annette Vergunst

INSERM U1047. Université de Montpellier 1. UFR Médecine, Nimes, France Contact : Annette Vergunst, [email protected] Bacteria belonging to the ‘Burkholderia cepacia complex’ (Bcc) have emerged as opportunistic pathogens in immunocompromised patients, particularly those with cystic fibrosis (CF) and chronic granulomatous disease. After an unpredictable period of colonisation, the bacteria can cause acute, fatal necrotizing pneumonia and septicaemia, known as ‘cepacia syndrome’. These Gram negative bacteria can adopt an intracellular life style, which is important for disease


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development. We use the zebrafish embryo model to study the early innate immune response and the role of host phagocytes to infection with Bcc. Earlier, we have shown that B. cenocepacia isolates belonging to the epidemic ET12 lineage are highly virulent for zebrafish embryos, although young embryos can control infection with for instance B. vietnamiensis FC441 and B. stabilis LMG14294. Using real time analysis with DSRed expressing bacteria we have shown that Bcc bacteria are mainly phagocytosed by, and survive and multiply in macrophages of zebrafish embryos. After intracellular replication, bacteria escape from infected macrophages by non-lytic release resulting first in local dissemination, followed by systemic infection. Using live imaging, we studied the role of phagocytes during the different steps of the infection process. We will report on the role of macrophages for progression of disease, and we will show how neutrophil-macrophage interactions may be important for the outcome of infection. F3 THE INTRAMACROPHAGE LIFESTYLE OF BURKHOLDERIA MULTIVORANS Crystal L. Schmerk and Miguel A. Valvano

Department of Microbiology and Immunology, and Center for Human Immunology, University of Western Ontario, London, Ontario, N6A 5C1, Canada Contact : Crystal Schmerk, [email protected] The Burkholderia cepacia complex (Bcc) consists of 17 closely related species of opportunistic bacterial pathogens, which can cause lethal infections in cystic fibrosis patients and immune-compromised individuals. Together, B. multivorans and B. cenocepacia account for the majority of Bcc infections. Recent research efforts have concentrated on elucidating the infection mechanisms of the more virulent B. cenocepacia with little focus on B. multivorans. However, while current infection control measures are reducing the number of B. cenocepacia infections, B. multivorans infection levels remain steady. To gain a better understanding of B. multivorans infection, RAW264.7 murine macrophages were infected with 3 B. multivorans strains: ATCC17616, C5568, and C0514. The infection characteristics of these strains were compared to each other as well as the B. cenocepacia strain K56-2. The B. multivorans strains ATCC17616 and C5568 infected macrophages at a significantly higher level than C0514 and K56-2. This is likely a result of increased phagocytosis due to differences in LPS O-antigen chain length. Unlike B. cenocepacia, all B. multivorans strains were able to survive but unable to replicate within murine macrophages. Previous research has shown that B. cenocepacia is able to interfere with endocytic trafficking and delay fusion of the bacteria-containing phagosome with lysosomes. There was no lag in the fusion of B. multivorans containing vacuoles (BmCV) with lysosomes, as the majority of BmCVs were positive for LysoTracker Green and Fluorescein Dextran 2 hrs post-infection. Autophagy does not appear to play a significant role in the control of these strains as BmCV co-localization with the autophagic marker LC3B remained at low levels throughout 24 hrs of infection. Hyper-vacuolarization of macrophages could be observed after infection with both ATCC17616 and C5568 strains. This vacuolar phenotype does not appear to be attributed to differences in the production of exoenzymes and may be attributed to their heightened level of infection. This research demonstrates that Bcc species can differ greatly in their ability to invade and manipulate the intramacrophage environment.


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F4 THE BURKHOLDERIA CENOCEPACIA TYPE VI SECRETION SYSTEM MEDIATES ESCAPE OF TYPE II SECRETED PROTEINS INTO THE CYTOPLASM OF INFECTED MACROPHAGES Roberto Rosales-Reyes1,2, Daniel F. Aubert1, Jennifer S. Tolman1, Amal O. Amer3, and Miguel A. Valvano1 1 Centre for Human Immunology, Department of Microbiology and Immunology, The University of Western Ontario, London ON, N6A 5C1, Canada. 2 Laboratorio de Infectología, Microbiología e Inmunología Clínicas, Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, C.P. 06726. México. 3 Centre for Microbial Interface Biology, Department of Microbial Infection and Immunity and the Department of Internal Medicine, Ohio State University. Columbus, OH 43210. USA. Contact: Roberto Rosales-Reyes; [email protected] Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in a bacteria-containing membrane vacuole (BcCV) with features of an arrested autophagosome. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and cause dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with the adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. Interestingly, ZmpA and ZmpB are required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results reveal a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.


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F5 HOW IS BURKHOLDERIA CENOCEPACIA RECOGNIZED BY THE AUTOPHAGY MACHINERY Basant A. Abdulrahman1, Arwa Abu Khweek1, Kyle J. Caution1, Anwari Akhter1, Mia Tazi 1, Benjamin Kopp2, Karen McCoy2, Miguel A. Valvano3 and Amal O. Amer1 1 Centre for Microbial Interface Biology, Department of Microbial Infection and Immunity and the Department of Internal Medicine, Ohio State University. Columbus, OH 43210. USA. 2 Division of Pediatric Pulmonology, Nationwide Children’s Hospital, Columbus, OH 43205. 3 Centre for Human Immunology, Department of Microbiology and Immunology, The University of Western Ontario, London ON, N6A 5C1, Canada. Contact: [email protected] Burkholderia cenocepacia can survive within a variety of eukaryotic cells such as amoebae, epithelial cells and human macrophages. B. cepacia lingers in macrophages harboring the CFTR mutation CFTR-ΔF508, and evades lysosomal degradation. In wild-type but not in CFTR-ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Autophagy is a cell survival mechanism in which multilamellar compartments called autophagosomes engulf non-functional organelles during periods of stress or starvation. Then, these autophagosomes deliver their contents to the lysosomes for degradation and production of nutrients. Autophagy markers such as LC3 were acquired by the B. cepacia vacuole in wild-type macrophages but not in CFTR-ΔF508 macrophages. The B. cepacia enclosing vacuole depicted a typical multilamellar characteristic of autophagosomes in wild-type macrophages. In CFTR-ΔF508 macrophages, the B. cepacia vacuole did not acquire multiple membranes nor did it acquire LC3. The ability of the autophagy machinery to recognize B. cepacia vacuole in wild-type macrophages leads to containment of B. cepacia infection. However, it is not clear how this recognition occurs in wild-type macrophages. Yet, the determination of this recognition mechanism will allow the design of new approaches to help clear infection. Typically, cargo destined for autophagy is ubiquitinated then, labeled with p62, NBR1 or NDP52. Here we show that B. cepacia vacuole is equally ubiquitinated in wild-type and CFTR-ΔF508 macrophages. We found that in wild-type macrophages, the ubiquitinated vacuole acquired p62 but did not do so in their CFTR-ΔF508 counterparts. Depletion of wild-type macrophages from p62 by specific SiRNA prevented B. cepacia vacuole from acquiring LC3 and fusing with the lysosome. The depletion of p62 rendered wild-type macrophages as permissive as CFTR-ΔF508 macrophages to B. cepacia infection. Thus, we have identified p62 as an efficacious autophagy molecule that labels the B. cepacia vacuole for autophagy. We consider p62 as a novel drug target for ameliorating the clearance of B. cepacia by the autophagy machinery in infected cystic fibrosis patients.


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G1 HETERORESISTANCE OF BURKHOLDERIA CENOCEPACIA TO ANTIMICROBIAL PEPTIDES Omar M. El-Halfawy1 and Miguel A. Valvano1,2 1 Department of Microbiology and Immunology and 2 Department of Medicine, Infectious Diseases Research Group, Siebens Drake Medical Research Institute, University of Western Ontario, London, Ontario, Canada Contact : Miguel A. Valvano, [email protected] Burkholderia cenocepacia, a Gram-negative opportunistic pathogen, is a significant cause of death in cystic fibrosis patients. These bacteria are extremely resistant to antimicrobial peptides (APs). Moreover, we detected that the resistance of B. cenocepacia to APs is heterogeneous across an isogenic population, whereby higher levels of resistance are expressed by a subset of the microbial population. Heteroresistance may aggravate the clinical situation via selection for the more resistant cells by antibiotic therapy. Furthermore, the high level of resistance could be transferred to the remainder of the less resistant population, hence protecting them. Therefore, our objectives are to test the mechanism of increased resistance of the more resistant subpopulation, and to determine how they protect the less resistant cells from APs. We compared the proteins secreted by normal and the more resistant cells. Amino acids (lysine-arginine-ornithine) transporters were more expressed in the more resistant cells in response to APs. These amino acids are involved in the biosynthesis of polyamines (PAs) such as spermidine. Thus, we hypothesized that the more resistant cells secrete excess PAs resulting in increased resistance and its transfer to the less resistant cells. Spermidine increased the resistance of normal cells to APs, whereas inhibitors of spermidine synthase lowered the resistance of the more resistant cells to APs. Deletion of genes encoding the enzymes synthesizing PAs also resulted in higher susceptibility to APs. The significance of this study is to increase our understanding of the mechanism of intrinsic resistance of B. cenocepacia to APs and to provide potential therapeutic solutions to its severe infections. G2 AMINOARABINOSE MODIFICATION OF THE BURKHOLDERIA CENOCEPACIA LIPOPOLYSACCHARIDE (LPS) IS ESSENTIAL FOR EXTREME ANTIMICROBIAL PEPTIDE RESISTANCE AND LPS EXPORT TO THE OUTER MEMBRANE. Mohamad A. Hamad1, Flaviana Di Lorenzo2, Antonio Molinaro2, and Miguel A. Valvano1

1Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada, N6A 5C1 2Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Naples, Italy Contact : Mohamad A. Hamad, [email protected] One common mechanism for Gram-negative bacteria to resist killing by antimicrobial peptides (APs) is the modification of lipid A with the sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N). Burkholderia cenocepacia is a Gram-negative bacterium that causes opportunistic infections in


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cystic fibrosis and other immunocompromised patients. B. cenocepacia is extraordinary resistant to APs as well as most clinically useful antibiotics. The “intrinsic” resistance of B. cenocepacia to APs is attributed to outer membrane integrity however the molecular basis of “intrinsic” resistance are not well understood. We have previously discovered that the synthesis of L-Ara4N is essential for the viability of B. cenocepacia. This is unique to B. cenocepacia (and likely all Burkholderia) since in other Gram-negative bacteria L-Ara4N synthesis is dispensable for growth. In this study, we describe the isolation of B. cenocepacia suppressor mutants that lack L-Ara4N synthesis genes and the L-Ara4N transferase gene arnT but remain viable. Chemical characterization of the lipopolysaccharide (LPS) from these mutants revealed the lack of L-Ara4N modification. The L-Ara4N-deficient mutants were highly sensitive to polymyxin B and melittin, two different classes of APs indicating that L-Ara4N modification of LPS is critical for the intrinsic resistance of B. cenocepacia to APs. We also determined that the suppressor phenotype was a result of an aspartic acid to a histidine replacement of residue 31 of LptG, a protein component of the multi-protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that L-Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly of the outer membrane of B. cenocepacia. G3 INVOLVEMENT OF TOXIN-ANTITOXIN MODULES IN BURKHOLDERIA CEPACIA COMPLEX PERSISTENCE Heleen Van Acker, Hans J. Nelis & Tom Coenye

Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, België Contact : Tom Coenye, [email protected] Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens which can cause severe lung infections in immunocomprimised people, like cystic fibrosis patients. Infections are often difficult to treat because of the innate resistance of these species and their capacity to form biofilms. Biofilms play an important role in the recalcitrance of infections due to their tolerance against many antibiotics. Tolerance is an ability to survive antibiotic treatment without expressing a resistance mechanism. Although the molecular basis of tolerance is still largely unknown, toxin-antitoxin modules (TA) are thought to play a role in it. They were first identified on plasmids and play a role in plasmid maintenance. TA modules consist of two genes in an operon, encoding a stable toxin and a labile antitoxin. The toxin is a protein that inhibits an important cellular function such as translation or replication and which can form an inactive complex with an antitoxin. Bactericidal antibiotics kill cells by corrupting the target functions. Inhibition of these functions would prevent antibiotics from corrupting their targets and would give rise to tolerant persister cells. In this study we investigated whether these TA modules play a role in the tolerance of Bcc biofilms and planktonic cultures against different antibiotics. Using the RASTA-Bacteria software (http://genoweb1.irisa.fr/duals/RASTA-Bacteria/) 17 pairs of genes were identified as possible TA modules in the genome of Burkholderia cenocepacia J2315 (score ≥ 60). Based on microarray data which indicated that these genes were upregulated in biofilms treated with tobramycin, RNA of treated and untreated B. cenocepacia J2315 biofilms and planktonic cultures was extracted and analyzed by qPCR. The expression of these


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TA modules in tobramycin and ciprofloxacin treated sessile and planktonic cells was compared to those in untreated cells. Generally the toxins showed a higher basal expression in biofilm grown cells than in planktonic cultures. Only one toxin was upregulated in all conditions tested whereas two toxins were systematically downregulated. Operons showing similarity with mazEF, relBE or mqsRA and three other operons were significantly upregulated in biofilms and planktonic cultures after treatment with tobramycin but not after treatment with ciprofloxacin. Others were only upregulated in sessile or planktonic cells treated with tobramycin but none of the operons was only influenced by treatment with ciprofloxacin. These preliminary results indicate that various TA modules are present in the Bcc and may play a role in persistence. G4 BCAL1938 AND BCAL2128 TWO ENZYMES REQUIRED FOR CELL WALL REMODELING IN BURKHOLDERIA CENOCEPACIA Cristóbal Mujica Troncoso, Chelsea Clarke and Miguel Valvano Department of Microbiology & Immunology, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada. Contact: Miguel Valvano, [email protected] The Burkholderia cepacia complex (Bcc) is a group of opportunistic pathogens associated with cystic fibrosis patients. B. cenocepacia and other Burkholderia display a remarkable ability to colonize diverse niches, leading to various lifestyles. One characteristic feature of Bcc species is that they have evolved a diversity of resistance mechanism against several antimicrobial agents, as well as the ability to interact with multiple hosts, including the ability to survive intracellularly in amoebae, macrophages, and epithelial cells. Like most opportunistic pathogens, B. cenocepacia must adapt to environmental stresses and have the ability to persist in the infected host. Persistence is in part associated to the bacterial ability to control cell division. We have identified two cell wall hydrolases, BCAL1938 and BCAL2128, with high sequence similarity to a Mycobacterium tuberculosis protein known to be essential for later stages of cell division. These proteins belong to the NlpC/P60 family of endopeptidases that chiefly function to modify peptidoglycan during growth, but are often required for cell wall turnover, separation of daughter cells during cell division, biofilm formation, bacterial adhesion, and motility. Our results show that these proteins are able to degrade peptidoglycan in vitro and B. cenocepacia mutant strains not expressing BCAL1938 have a defect in O antigen production and are sensitive to polymyxin. BCAL2128 mutant strains are sensitive to cell-wall acting antibiotics. And double mutants strains show growth defects characterized by the formation of long chains. Hence, our result suggests the necessity of these proteins in B. cenocepacia during adaptive responses makes them novel putative targets for antimicrobial drug development.


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H1 EXPERIMENTAL THERAPEUTIC TREATMENT OF THE BURKHOLDERIA CEPACIA COMPLEX WITH BACTERIOPHAGES Diana D. Semler, Amanda D. Goudie and Jonathan J. Dennis Centennial Centre for Interdisciplinary Science, Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada Contact : Jon Dennis, [email protected] Recently, in situations where a bacterial pathogen is also highly antibiotic resistant, the efficacy of bacteriophage therapy has come under increasing examination. In this regard, bacteriophage therapy may be especially applicable to infections caused by the Burkholderia cepacia complex (Bcc) since members of the Bcc are highly antibiotic resistant. Bcc infections can significantly reduce the life expectancy of CF patients and in severe cases can lead to “cepacia syndrome”, which is characterized by a rapid deterioration of pulmonary function. A therapy that utilizes bacteriophages (or phages) to specifically target Bcc species would complement current chemical antibiotic therapeutic regimens. Phages are bacterial viruses that exclusively attack and lyse particular species or strains of bacteria. Although there have been reports of successful phage treatment, not all phage therapy testing has been effective. We demonstrate effective phage therapy for different strains of Burkholderia cenocepacia in an aerosol mouse model, including important improvements over previous descriptions of Bcc phage therapy. In addition, we observe in vitro that some Bcc bacteriophage can provide a dual role in combating the growth of Pseudomonas aeruginosa. Finally, we examine the possibility of phage loss from the lungs by diffusion, which could result in a reduction of phage therapy effectiveness. Our results suggest that Bcc phage therapy can effectively clear murine lungs (by a magnitude of 3-4 log units) with a treatment delay of up to 48 hours. H2 MICROBICIDAL NATURAL KILER CELLS HAVE ANTI-BURKHOLDERIA ACTIVITY Marwah Saleh1, Shaunna M. Huston1, Danuta Stack1, Eoin O’Grady1, Pamela A. Sokol1 and Christopher H. Mody1,2 1Department Microbiology, Immunology and Infectious Diseases and the 2Department of Internal Medicine, Snyder Institute for Chronic Diseases, University of Calgary, Calgary Alberta Contact : Chris Mody, [email protected] Burkholderia cenocepacia and B. multivorans pose a considerable risk to patients with cystic fibrosis. The organisms are intrinsically resistant to multiple antibiotics, which makes antimicrobial therapy particularly challenging. Thus, there is an important need for novel therapeutic approaches. Recently, cytotoxic lymphocytes that bind to organisms directly, and deploy their cytotoxic payload, resulting in the destruction of the microbe have been described. Both NK cells and T lymphocytes have the ability to directly kill microbes using either perforin or granulysin as the effector molecules. Consequently, we hypothesized that NK cells would


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have anti-Burkholderia activity. By co-culturing B. multivorans, strain LMG 3010 and B. cenocepacia, strain J2315 with YT cells (a human NK cell line) or primary NK cells from healthy adults, we were able to determine the anti-Burkholderia activity by measuring colony forming units at the beginning of culture and at various times afterward. YT cells were able to inhibit the growth of B. cenocepacia to a maximum of 294 ± 15% at 2 hours (n=4, similar results obtained in a second experiment). There was a dose-dependent reduction in anti-Burkholderia activity as the effector to target (E:T) ratio was reduced from 100:1 to 6.25:1, but even at the lowest E:T ratio, there was over 90% inhibition of growth. YT cells also had significant anti-B. multivorans activity (62±1% inhibition, n=4, similar results obtained in a second experiment) although the activity was less than for B. cenocepacia. Anti-Burkholderia activity was also observed in primary NK cells. Thus, NK cells have significant anti-Burkholderia activity that results in killing of the organism (reduction in CFU below the starting inoculum). We speculate that the anti-Burkholderia activity of NK cells may provide novel insights into the mechanism of lymphocyte-mediated microbicidal activity, and may motivate studies of this novel killing mechanism as a potential therapeutic approach. This work was supported by a grant from Cystic Fibrosis Canada. H3 SINGLE PARTICLE TRACKING IN BURKHOLDERIA CEPACIA COMPLEX BIOFILMS AS A TOOL TO STUDY TRANSPORT OF DRUG-LOADED LIPOSOMES Anne-Sophie Messiaen1, Katrien Forier2,3, Hans Nelis1, Kevin Braeckmans2,3 and Tom Coenye1 1Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium 2Laboratorium voor Algemene Biochemie en Fysische Farmacie, Universiteit Gent, Gent, Belgium 3Center for Nano- and Biophotonics, Ghent University, Ghent, Belgium Contact: Tom Coenye, [email protected] In cystic fibrosis patients acute lung infections or exacerbations by Pseudomonas aeruginosa or Burkholderia cepacia complex are often treated with high doses of intravenously administered antibiotics combined with the daily use of nebulized tobramycin to prevent chronic lung infections. Once chronic lung infections are established, eradication of these pathogens is very difficult. This is due to the biofilm mode of growth of certain pathogenic P. aeruginosa and B. cepacia complex strains, as sessile cells are resistant to most antibiotics, partly, because of the latter’s limited diffusion into the biofilm. In the present study we investigated whether a liposomal tobramycin formulation can deliver a high dose of antibiotics closely to the bacterial cells and increase the success rate of the treatment. In order to optimize the liposomal formulation, we used a model system based on single particle tracking to study the transport of positive and negative 200 nm fluorescent polystyrene nanospheres in Burkholderia cepacia complex biofilms. Negative particles penetrated into the biofilm and were then immobilized in cell clusters while positively charged particles hardly penetrated into the biofilm but adhered to threadlike structures in the extracellular matrix. These threadlike structures are believed to consist of DNA as they disappeared when biofilms were grown in the presence of recombinant human DNase. Positively charged particles added to biofilms grown in the presence of rhDNase showed higher


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mobility. Some of these particles were able to penetrate deeply into the biofilm where they were immobilized in cell clusters. Our data indicate that negative particles are directed towards cell clusters, which means that negatively charged nanomedicines could be suitable candidates to deliver antibiotics to the bacterial clusters. Positively charged nanomedicines can only be promising when they are administered in combination with rhDNase therapy. H4 IMMUNOPROTEOMIC ANALYSIS OF BURKHOLDERIA MULTIVORANS AND BURKHOLDERIA CENOCEPACIA M. Shinoy1, K. Schaffer3, S. Doyle2, M. Callaghan1, S. McClean1 1Centre of Microbial Host Interactions, Institute of Technology Tallaght Dublin, Ireland 2 Dept of Biology, NUI, Maynooth, Co Kildare. 3 St Vincent’s University Hospital, Dublin Ireland. Contact : [email protected] The antimicrobial resistance of Burkholderia cepacia complex (Bcc) is a major barrier to effective treatment and once acquired it is rarely eradicated. Prevention of colonization of CF patients should have major impact on their mortality and quality of life. The aim of this study was to identify potential immunogens which could be developed as vaccine antigens to protect against both B. cenocepacia, the most virulent species and B. multivorans, the most frequently isolated species. We have used an immunoproteomics approach to investigate the immunogens expressed by 4 Bcc strains: two B. cenocepacia strains (BC-7 and C1394 which are piliated and non-piliated, respectively) and two B. multivorans strains (LMG 13010 and C1962) in order to identify antigens that would protect against both key species. We focused on the outer membrane proteins (OMPs) as these are exposed directly to host cells and are therefore among the primary targets of the host immune response. The OMPs were separated by 2-DE, transferred and probed with serum from CF patients colonised with Bcc (and compared to blots probed with serum from patients with no history of Bcc). Immunogenic proteins were excised from parallel Coomassie blue stained gels and analysed by MALDI-Tof MS. A total of 55 proteins were reproducibly identified across the four strains examined. Of these, only 6 immunogens were common to all four strains, including known immunogenic proteins such as chaperonin GroEL and an efflux protein. Other intracellular proteins were also shown to be strongly immunogenic on the OMP blots including, DNA-directed RNA polymerase subunit alpha and a 38kDa aldo/keto reductase. Fifteen identified immunogens were exclusive to B. cenocepacia, of which ten proteins identified were exclusive to C1394 immunoblots. Eight were exclusive to B. multivorans. Several proteins which have been shown to be virulent in other pathogens were also identified amongst the immunogens in three of the four strains. Further study will evaluate the potential of the common immunogens as vaccine candidates and the potential virulence of the identified immunogenic proteins.

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