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International Burkholderia cepacia Working Group
17th
Annual Meeting
April 10 – April 13, 2013
Ann Arbor, Michigan
This meeting has been possible by the generous contributions of
IBCWG 2013 Ann Arbor, MI April 2013
2
Wednesday, April 10, 2013
6:00 – 8:00 p.m.
Registration / Meet and Greet Reception – Hors d’Oeuvres
Thursday, April 11, 2013
7:30 – 8:15 a.m.
Breakfast & Registration
8:15 – 8:30 a.m. Welcome – Dr. John LiPuma, Dr. Joanna Goldberg and Dr. Amal Amer
8:30 – 9:30 a.m. KEYNOTE SPEAKER Dr. Vaughn Cooper
POPULATION GENOMICS OF EXPERIMENTALLY EVOLVED BURKHOLDERIA BIOFILMS
9:40 – 10:40 a.m. Session 1: The variation of Bcc isolates
Moderators: Joanna Goldberg and Tom Coenye
9:40 – 10:00 a.m. 1. EXPLORING THE CLINICAL IMPACT OF BURKHOLDERIA CEPACIA COMPLEX STRAINS ISOLATED FROM INDUSTRIAL PRODUCTS
José Degrossi, Agustina Lopez de Volder, Verónica Pioli, Fabricio Rugnone, Eduardo Vadell, Laura Galanternik, Sergio Teves and Mirta Franco
10:00 – 10:20 a.m. 2. PSEUDOMONAS AERUGINOSA METABOLITES INDUCE A TRANSIENT COLONY MORPHOTYPE IN SOME SPECIES OF THE BURKHOLDERIA CEPACIA COMPLEX
Steve P. Bernier, Courtney Hum, and Michael G. Surette
10:20 – 10:40 a.m. 3. COMPARATIVE PHYLOGENOMIC ANALYSIS OF THE BURKHOLDERIA MULTIVORANS ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS PATIENTS
Vijaya Brahma, Julio Diaz Caballero, Shawn Clark, Valerie Waters, Yvonne Yau, Pauline Wang, Lijie Zhang, Sylva Donaldson, D. Elizabeth Tullis, David S. Guttman and David M. Hwang.
IBCWG 2013 Ann Arbor, MI April 2013
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10:40 -11:00 a.m. Coffee break
11:00 a.m. – 12:40 p.m. Session 2. The variation of Bcc isolates II
Moderators: Annette Vergunst and Siobhán McClean
11:00 – 11:20 a.m. 4. PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF SEQUENTIAL BURKHOLDERIA MULTIVORANS ISOLATES FROM AN ADULT WITH CYSTIC FIBROSIS Shawn T. Clark, Julio Diaz Caballero, Vijaya Brahma, Pauline W. Wang, Sylva L. Donaldson, Yvonne C. Yau, Valerie J. Waters, D. Elizabeth Tullis, David S. Guttman and David M. Hwang.
11:20 – 11:40 a.m. 5. TOBRAMYCIN SUSCEPTIBILITIES OF BURKHOLDERIA CEPACIA COMPLEX ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS PATIENTS
Valerie Waters, Yvonne Yau Anina Ratjen, Jill Wettlaufer , Larissa Matukas, Felix Ratjen , Elizabeth Tullis
11:40 – 12:00 p.m. 6. EPIDEMIC B. MULTIVORANS STRAINS IN BELGIAN CF PATIENTS
C. Peeters, A. De Bel, J. Willekens, A. Malfroot, D. Pierard, P. Vandamme
12:00 – 12:20 p.m. 7. GENETIC VARIATION OF BURKHOLDERIA DOLOSA IN SINGLE CLINICAL SAMPLES PROVIDES A SIGNATURE OF SELECTIVE PRESSURES
Tami D. Lieberman, Kelly B. Flett, Thomas R. Martin, Alexander J. McAdam, Gregory P. Priebe, and Roy Kishony
12:20 – 12:40 p.m. 8. A 15 YEAR STUDY OF BURKHOLDERIA INFECTION IN PERSONS WITH CYSTIC FIBROSIS Amy A. Bumford, Theodore Spilker, Bridget Foster, Jennifer Krot, Linda M. Kalikin and John J. LiPuma
12:40 – 2:00 p.m.
Lunch
IBCWG 2013 Ann Arbor, MI April 2013
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2:00 – 3:40 p.m.
Session 3. Gene expression responses
Moderators: Vittorio Venturi and Pam Sokol
2:00 – 2:20 p.m. 9. MANNITOL ELICITS A GENOME-WIDE RESPONSE IN BURKHOLDERIA MULTIVORANS THAT IMPACTS ON NUMEROUS VIRULENCE TRAITS
Carmen C. Denman and Alan R. Brown
2:20 – 2:40 p.m. 10. LIFE AT LOW-OXYGEN TENSION IN B. CENOCEPACIA H111
Gabriella Pessi, Rubina Braunwalder, Alexander Grunau, Ulrich Omasits, Christian H. Ahrens and Leo Eberl
2:40 – 3:00 p.m. 11. GENE EXPRESSION CHANGES OF BURKHOLDERIA CENOCEPACIA ST32 WHEN CULTURED IN CF SPUTUM AND SERUM
Lucie Kalferstova, Michal Kolar, Pavel Drevinek
3:00 – 3:20 p.m. 12. HIGH THROUGHPUT SCREENING FOR GROWTH INHIBITORS OF BURKHOLDERIA CENOCEPACIA
Carrie Selin, Eric Brown and Silvia T. Cardona
3:20 – 3:40 p.m. 13. GENE-SPECIFIC SILENCING OF BURKHOLDERIA CEPACIA COMPLEX WITH PHOSPHORODIAMIDATE MORPHOLINO OLIGOMERS
Kimberly Marshall-Batty, Lacey Harbor, Bruce L. Geller and David E. Greenberg
3:40 -4:00 p.m. Coffee break
IBCWG 2013 Ann Arbor, MI April 2013
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4:00 – 5:40 p.m. Session 4. Virulence factors
Moderators: Eshwar Mahenthiralingam and Alan Brown
4:00 – 4:20 p.m. 14. COMPARATIVE GENOMIC ANALYSIS DEFINES THE BURKHOLDERIA CENOCEPACIA CORE GENOME AND CANDIDATE VIRULENCE GENES
Erick Cardenas, Linda M. Kalikin, Jean-Marie Rouillard, Jiangchao Zhao, Patrick S. Chain, Erdogan Gulari, James M. Tiedje, and John J. LiPuma
4:20 – 4:40 p.m. 15. THE ROLE OF BURKHOLDERIA DOLOSA FLAGELLA IN HOST COLONIZATION
Deborah Yoder-Himes, Damien Roux, David Skurnik, Mihaela Gadjeva, Stephen Lory
4:40 – 5:00 p.m. 16. INVESTIGATION OF GENES AND REGULATORY ELEMENTS INVOLVED IN PRODUCTION OF NOVEL ANTIFUNGAL COMPOUNDS IN BURKHOLDERIA CENOCEPACIA H111
Christian Jenul, Aurelien Carlier, Pamela A. Sokol and Leo Eberl
5:00 – 5:20 p.m.
17. GENETIC ANALYSIS OF A BROAD-HOST RANGE TAILOCIN IN BURKHOLDERIA CENOCEPACIA
Iris Duarte, Guichun W. Yao, John J. LiPuma, Ryland F. Young and Carlos F. Gonzalez
5:20 – 5:40 p.m.
6:00 p.m.
18. IDENTIFICATION OF NOVEL BURKHOLDERIA CEPACIA COMPLEX ANTIGENS BY IMMUNOPROTEOMICS AND INVESTIGATION OF THEIR ROLES IN VIRULENCE Ruth Dennehy, Cristobal Mujica, Miguel A. Valvano, Maire Callaghan and Siobhán McClean
Free time / Dinner on your own
IBCWG 2013 Ann Arbor, MI April 2013
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Friday, April 12, 2013
7:30 – 8:30 a.m.
Breakfast
8:40– 10:20 a.m. Session 5: Biofilms and quorum sensing
Moderators: Elizabeth Tullis and David Hwang
8:40 – 9:00 a.m. 19. INVOLVEMENT OF TOXIN-ANTITOXIN MODULES IN TOLERANCE OF BURKHOLDERIA CENOCEPACIA BIOFILMS
Heleen Van Acker, Hans J. Nelis and Tom Coenye
9:00 – 9:20 a.m. 20. NEW INSIGHTS INTO THE BDSF AND THE AHL-BASED QUORUM SENSING SYSTEMS OF B. CENOCEPACIA H111
Nadine Schmid, Gabriella Pessi, Yinyue Deng, Claudio Aguilar, Ulrich Omasits, Lian-Hui Zhang, Christian H. Ahrens and Leo Eberl
9:20 – 9:40 a.m. 21. ANTI-BIOFILM ROLE OF EXOPOLYSACCARIDES PRODUCED BY BURKHOLDERIA CENOCEPACIA
Elena Pellizzoni, Fabio Ravalico, Denis Scaini, Roberto Rizzo, Paola Cescutti
9:40 – 10:00 a.m. 22. AHL QUORUM SENSING IN THE ENVIRONMENTAL AND POTENTIALLY BENEFICIAL PLANT-ASSOCIATED BURKHOLDERIA CLUSTER
Bruna G. Coutinho, Birgit Mitter, Chouhra Talbi, Angela Sessitsch, Eulogio J. Bedmar, Nigel Halliday, Euan K. James, Miguel Cámara and Vittorio Venturi
10:00 – 10:20 a.m. 23. TEASING APART THE MULTIPLE ROLES OF A CzcR/CzcS-LIKE TWO-COMPONENT SYSTEM IN BURKHOLDERIA CENOCEPACIA: VIRULENCE, QUORUM SENSING AND HEAVY METAL RESISTANCE
Matthew T. Robinson and Alan R. Brown
10:20 – 10:40 a.m. Coffee Break
IBCWG 2013 Ann Arbor, MI April 2013
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10:40 a.m. – 12:20 p.m.
Session 6. Innate immunity and intracellular lifestyle
Moderators: Paola Cescutti and Leo Elberl
10:40 – 11:00 a.m. 24. PROTEINS INVOLVED IN HOST CELL ATTACHMENT ARE PROTECTIVE ANTIGENS AGAINST BCC INFECTION.
Minu Shinoy, Marc Healy, Bernard Mahon, Máire Callaghan and Siobhán McClean
11:00 – 11:20 a.m. 25. DEFECTIVE AUTOPHAGY ACTIVITY CONTRIBUTES TO INCREASED IL-1β PRODUCTION DURING BURKHOLDERIA CENOCEPACIA INFECTION
Basant A. Abdulrahman, Mia Tazi, Anwari Akhter, Kyle Caution, Hoda Hassan, Miguel A. Valvano, Benjamin Kopp, and Amal O. Amer
11:20 – 11:40 a.m. 26. AUTOPHAGY STIMULATION BY IFN-γ IMPROVES BURKHOLDERIA CENOCEPACIA CLEARANCE IN HUMAN CYSTIC FIBROSIS MACROPHAGES
Ben Kopp, Kaivon Assani, and Amal Amer
11:40 -12:00 p.m. 27. A BURKHOLDERIA CENOCEPACIA GENE ENCODING A NON-FUNCTIONAL PHOSPHOTYROSINE PHOSPHATASE IS REQUIRED FOR THE DELAYED MATURATION OF THE BACTERIA-CONTAINING VACUOLES IN MACROPHAGE
Angel Andrade and Miguel Valvano
12:00-12:20 p.m. 28. MODELING OF THE INNATE IMMUNE RESPONSE TO INFECTION WITH BACTERIA BELONGING TO THE BURKHOLDERIA CEPACIA COMPLEX USING ZEBRAFISH EMBRYOS
Jennifer Mesureur, Julien Rougeot, David O’Callaghan, Annemarie Meijer, Annette Vergunst
12:20 – 2:00 p.m. 2:00 p.m.
Lunch
Free time to visit Ann Arbor
Dinner on your own
IBCWG 2013 Ann Arbor, MI April 2013
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Saturday, April 13, 2013
7:30 – 8:30 a.m. Breakfast
8:40 – 10:20 a.m. Session 7. Modeling and biology of Bcc
Moderators: David Greenberg and Silva Cardona
8:40 – 9:00 a.m. 29. MODELLING BURKHOLDERIA CEPACIA COMPLEX ROOT COLONIZATION IN VITRO AS AN IMPORTANT ENVIRONMENTAL TRAIT
J. Cristian Vidal-Quist, Louise A. O’Sullivan, Annaëlle Desert, Amanda S. Fivian-Hughes, Angela M. Marchbank, Coralie Millet, T. Hefin Jones, Andrew J. Weightman, Hilary J. Rogers, Colin Berry and Eshwar Mahenthiralingam
9:00 – 9:20 a.m. 30. REAL TIME ANALYSIS OF BURKHOLDERIA CENOCEPACIA ST32 ISOLATES IN A ZEBRAFISH EMBRYO MODEL
Klara Vilimovska Dedeckova, Pavel Drevinek, Annette Vergunst
9:20 – 9:40 a.m. 31. EXPLORING THE TOPOLOGY OF ArnT FROM BULKHOLDERIA CENOPACIA
Faviola Tavares-Carreón and Miguel A Valvano
9:40 – 10:00 a.m. 32. STRUCTURAL DIFFERENCES BETWEEN THE GENOMES OF BURKHOLDERIA CENOCEPACIA STRAINS K56-2 AND J2315
Andrea M. Sass, Silvia Cardona, Miguel A.Valvano, Tom Coenye, Eshwar Mahenthiralingam
10:00 – 10:20 a.m. 33. COMPARATIVE FUNCTIONAL ANALYSIS OF METABOLIC ABILITIES OF BCC SPECIES USING RECONCILED GENOME-SCALE METABOLIC RECONSTRUCTIONS
Jennifer Bartell, Phillip Yen, John Varga, Joanna Goldberg and Jason Papin
10:20 – 10:40 a.m. Coffee Break
IBCWG 2013 Ann Arbor, MI April 2013
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10:40 a.m. – 12:00 p.m. Session 8. The complexity of Bcc genetics
Moderators: Peter Vandamme and Amal Amer
10:40 – 11:00 a.m. 34. INVESTIGATING FACTORS INVOLVED IN THE MAINTENANCE OF THE ‘CHROMOSOME 3’ MEGAPLASMID
Kirsty Agnoli, Carmen Frauenknecht and Leo Eberl
11:00 – 11:20 a.m. 35. GLOBAL MAPPING OF PRIMARY TRANSCRIPTION START SITES IN THE BURKHOLDERIA CENOCEPACIA J2315 GENOME BY DIFFERENTIAL RNA SEQUENCING
Andrea M Sass, Heleen van Acker, Konrad Förstner, Jörg Vogel, Tom Coenye
11:20 a.m. – 12:00 p.m. Closing remarks – Discussion about next meeting
12:00 p.m. Lunch
Adjourn
IBCWG 2013 Ann Arbor, MI April 2013
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ABSTRACTS
1
EXPLORING THE CLINICAL IMPACT OF BURKHOLDERIA CEPACIA COMPLEX
STRAINS ISOLATED FROM INDUSTRIAL PRODUCTS
José Degrossi1, Agustina Lopez de Volder
1, Verónica Pioli
1, Fabricio Rugnone
2, Eduardo
Vadell1, Laura Galanternik
3, Sergio Teves
1, 2 and Mirta Franco
1
1 Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires. Argentina
2 Laboratorio Proanalisis SA. Buenos Aires. Argentina
3 Hospital de Niños “Dr. Ricardo Gutierrez”. Buenos Aires. Argentina
Contact: [email protected]
Members of the Burkholderia cepacia complex (Bcc) are frequent contaminants of industrial
products like pharmaceuticals, cosmetics and house cleaners.
Although the transmission of Bcc from environmental sources to patients has been reported, the
establishment of industrial contaminated products as a reservoir for infections remains unclear.
In order to elucidate the potential clinical impact of Bcc isolated from industrial sources, we
studied strains of B. cepacia, B. vietnamiensis, B. contaminans and B. lata (n=15) recovered
from contaminated products and purified water employed in different industries.
A genetic comparison by RAPD analysis with Bcc isolates recovered from patients, and
pathogenicity of the industrial strains using four infection models (alfalfa, Galleria mellonella,
Drosophila melanogaster and Dictyostelium discoideum) were performed.
60 % of the industrial isolates displayed similar RAPD pattern profiles to clinical isolates.
Regarding pathogenicity, results obtained with different models were not correlative. Most
isolates displayed virulence in at least one model. B. vietnamiensis and B. lata isolates were the
less virulent studied strains. Among B. contaminans isolates, those recovered from contaminated
products in which preservative agents are present, resulted more virulent than isolates recovered
from purified water.
These results reinforce the idea that industrial products should be considered a Bcc reservoir with
potential clinical impact. On the other hand, due to the differences observed among the employed
models, a multi host infection model is suggested for pathogenicity studies of industrial isolates.
IBCWG 2013 Ann Arbor, MI April 2013
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2
PSEUDOMONAS AERUGINOSA METABOLITES INDUCE A TRANSIENT COLONY
MORPHOTYPE IN SOME SPECIES OF THE BURKHOLDERIA CEPACIA COMPLEX
Steve P. Bernier1, Courtney Hum
1, and Michael G. Surette
1,2
1
Farncombe Family Digestive Health Research Institute, Department of Medicine, Faculty of
Health Sciences, McMaster University, Hamilton, Ontario, Canada 2
Department of Biochemistry and Biological Sciences, Faculty of Health Sciences, McMaster
University, Hamilton, Ontario, Canada
Contact: Steve P. Bernier, [email protected]
Background. In multi-species communities, metabolite-mediated interactions have the potential
to trigger polymicrobial-specific behaviours that are typically cryptic in mono-species cultures.
In this study, we use the cystic fibrosis pathogens Pseudomonas aeruginosa and the
Burkholderia cepacia complex (Bcc) as an in vitro model of polymicrobial communities to
demonstrate that metabolites from P. aeruginosa trigger a biological response in some Bcc
species leading to a transient colony morphotype.
Results. Representative strains from nine species of the Bcc (B. cepacia, B. multivorans, B.
cenocepacia, B. stabilis, B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina, and B. pyrrocinia)
were initially plated onto agar medium-containing low concentrations (0.5 - 1.0) of spent
medium derived from a 24-h culture of P. aeruginosa to determine whether different colony
morphotypes would arise. Strains from B. multivorans, B. cenocepacia, and B. dolosa exhibited a
colony morphotype change upon exposure to P aeruginosa extracellular metabolites. The
observed morphotype is specific to P. aeruginosa metabolites since Bcc strains fail to respond in
a similar manner to both their own and Escherichia coli spent medium. Further, the morphotype
change is transient and not due to a mutation since wild-type colony morphotype is restored
when plated back onto regular agar medium (no spent medium). To broadly identify Bcc genes
and pathways essential for responding to P. aeruginosa metabolites, a random transposon
mutagenesis was performed in parallel on two responsive strains from the species B. multivorans
and B. dolosa. Disruption of genes coding for lipoproteins, hypothetical proteins, anti-sigma
factors, transcriptional regulators, histidine kinase, type II secretion system, and type IV pili
completely abolished the colony morphotype change in response to active P. aeruginosa spent
medium. An important overlap was observed between the mutagenesis screens of both B.
multivorans and B. dolosa with approximately 50 of the hits being located within a 16-kb
region coding for the biogenesis of type IV pili. Investigations are ongoing to identify the
bioactive molecule present in P. aeruginosa spent medium as well as the characterization of Bcc
behaviours associated with the described polymicrobial-specific colony morphotype.
Conclusions/relevance. Our study demonstrates that bacterial behaviours can emerge in
polymicrobial communities which are cryptic in mono-species cultures. Understanding
molecular mechanisms leading to polymicrobial-specific traits may reveal how multi-species
interactions collectively contribute to pathogenesis and persistence in the host.
IBCWG 2013 Ann Arbor, MI April 2013
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3
COMPARATIVE PHYLOGENOMIC ANALYSIS OF THE BURKHOLDERIA
MULTIVORANS ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS
PATIENTS
Vijaya Brahma1 , Julio Diaz Caballero2, Shawn Clark 1,3, Valerie Waters4, Yvonne Yau4 ,
Pauline Wang5, Lijie Zhang5, Sylva Donaldson5, D. Elizabeth Tullis6, David S. Guttman2,5 and
David M. Hwang.1,3
1 Latner Thoracic Surgery Research Laboratories, University Health Network, Toronto ON
2 Department of Cell & Systems Biology, University of Toronto, Toronto ON
3 Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto ON
4 Department of Infectious Diseases, Hospital for Sick Children, Toronto ON
5 Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto ON
6 Division of Respirology, St. Michael‟s Hospital, Toronto, ON
Contact: David Hwang, [email protected]
David Guttman, [email protected]
Burkholderia multivorans is an emerging opportunistic pathogen in individuals with cystic
fibrosis (CF). Current repositories are very limited with respect to B. multivorans whole genome
sequences, particularly from clinical strains. To better understand the genetic diversity of B.
multivorans infections in CF, we have sequenced and assembled the genomes of B. multivorans
isolates from pediatric and adult CF patients. Genomic DNA was sequenced with paired-end
Miseq sequencing. The resulting reads were assembled using CLC de novo assembler and
reference mapped with the Burrows Wheeler Assembler (BWA). Variant calling was performed
with Samtools and the identified variants were characterized with CLC Genomics. Initial
analysis of genome sequences from our isolates found significant differences compared to B.
multivorans ATCC 17616, an environmental isolate. Ongoing analysis will further define core
and accessory genomes in B. multivorans, delineating the diversity between isolates infecting
different patients and address the question whether or not the environmental, pediatric and adult
CF isolates follow distinct evolutionary paths.
IBCWG 2013 Ann Arbor, MI April 2013
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4
PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF SEQUENTIAL
BURKHOLDERIA MULTIVORANS ISOLATES FROM AN ADULT WITH CYSTIC
FIBROSIS
Shawn T. Clark1,2
, Julio Diaz Caballero3, Vijaya Brahma
2, Pauline W. Wang
4, Sylva L.
Donaldson4, Yvonne C. Yau
1,5, Valerie J. Waters
6, D. Elizabeth Tullis
7, David S. Guttman
3,4 and
David M. Hwang1,2
1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
2 Latner Thoracic Surgery Research Laboratories, University Health Network, Toronto, Canada
3 Department of Cell & Systems Biology, University of Toronto, Toronto, Canada
4 Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto,
Canada 5 Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Canada
6 Department of Pediatrics, Infectious Diseases, Hospital for Sick Children, Toronto, Canada
7 Respirology, University of Toronto, Toronto, Canada
Contact: Shawn Clark, [email protected]
Pulmonary infection by members of the Burkholderia cepacia complex (BCC) remains a
feared condition in the cystic fibrosis (CF) community because of their high intrinsic resistance
and association with poor patient outcome. Recent studies into the diversity and dynamics of
dominant CF pathogen populations have identified clonal-relatedness and phenotypic
heterogeneity among many chronic infection isolates. At present, similar examinations into BCC
populations are limited. The objective of this study was to survey adaptive changes in an
infecting B. multivorans population during periods of pulmonary exacerbation. We present a
preliminary analysis of the phenotypic and genotypic attributes of 20 isolates recovered from
sequential sputum samples produced by an adult CF patient chronically infected by
B. multivorans. Three non-mucoid colony morphotypes, including small colony variants, were
identified and confirmed as B. multivorans by initial 16S rDNA sequencing. Phenotypic
characterization identified that all isolates were non-motile, able to produce rhamnolipids and
later isolates demonstrated an increased ability to form biofilms in vitro. Additionally, isolates
could be grouped into two distinct phenotypic profiles based on differences in both β-lactam
resistance and growth patterns in LB. The complete genome of each isolate was sequenced by
paired-end MiSeq sequencing and comparative genomic analysis is currently being performed.
IBCWG 2013 Ann Arbor, MI April 2013
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5
TOBRAMYCIN SUSCEPTIBILITIES OF BURKHOLDERIA CEPACIA COMPLEX
ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS PATIENTS
Valerie Waters 1
, Yvonne Yau 2
, Anina Ratjen 1
, Jill Wettlaufer 1
, Larissa Matukas 3
, Felix Ratjen 4, Elizabeth Tullis
5
1Division of Infectious Diseases, Department of Pediatrics, The Hospital for Sick Children,
University of Toronto, Toronto, Canada.
2Division of Microbiology, Department of Pediatric Laboratory Medicine, The Hospital for Sick
Children, University of Toronto, Toronto, Canada. 3Division of Microbiology, St. Michael‟s Hospital, University of Toronto, Toronto, Canada.
4Division of Respirology, Department of Pediatric Laboratory Medicine, The Hospital for Sick
Children, University of Toronto, Toronto, Canada. 5Division of Respirology and Keenan Research Centre of Li Ka Shing Knowledge Institute,
Department of Medicine, St. Michael‟s Hospital, University of Toronto, Toronto, Canada.
Contact: Valerie Waters, [email protected]
Background: Pulmonary infection with Burkholderia cepacia complex (BCC) is associated with
increased lung function decline and mortality in patients with cystic fibrosis (CF) but there are
currently no effective chronic maintenance antimicrobial therapies for this condition. Although
BCC is intrinsically resistant to aminoglycosides via efflux pumps, for example, new inhaler
devices such as the podhaler can deliver very high levels of tobramycin inhalation powder (TIP)
to the lung that may be able to overcome these mechanisms of resistance. The goal of this study
was to determine the tobramycin minimum inhibitory concentrations (MICs) of a large collection
of BCC isolates in relation to the tobramycin sputum concentrations known to be achievable by
TIP (up to 1979 µg/g).
Methods: This was a 1 year (Sept 2011-Dec 2012) prospective observational study of pediatric
and adult CF patients infected with BCC attending the Hospital for Sick Children and St
Michael‟s Hospital CF clinic. BCC were isolated from sputum and genomovars were determined
using recA typing. Conventional broth microdilution susceptibility testing was done on cultures
grown planktonically by inoculating 105
CFU/ml into wells with Mueller-Hinton broth with
tobramycin at increasing concentrations: 10, 100,200, 400, 800, 1600 and 3200 µg/ml.
Results: A total of 89 BCC isolates were collected from 46 patients (8 pediatric, 38 adult). Of
the 89 BCC isolates, 64 (72%) were B. cenocepacia, 17 (19%) were B. multivorans and 8 were
unknown. The median tobramycin MIC was 200 µg/ml (range 10-3200 µg/ml) overall (median
tobramycin MIC for B. cenocepacia=200 µg/ml; for B. multivorans=100 µg/ml). When the MIC
of only the first isolate per patient was examined, the median tobramycin MIC was 100 µg/ml.
The change in tobramycin MIC in individual patients over time was examined; 21 patients had
repeated isolates (range 2-4 isolates). In 15 of the 21 patients (71%), the tobramycin MIC of the
first isolate compared to that of the last isolate was within 1 dilution. Fourteen patients had
multiple morphotypes from one sample tested; in the majority of cases (10/14, 71%) the
tobramycin MICs of different morphotypes were within 1 dilution of each other.
Conclusions: This preliminary in vitro study demonstrates that for the majority of BCC isolates
infecting both pediatric and adult CF patients, the minimum concentration required to inhibit
these organisms is 200 µg/ml. This suggests that TIP delivered via podhaler may be able to
achieve a ten-fold increase in tobramycin sputum concentration above the planktonic MIC.
Further susceptibility testing growing BCC as a biofilm is required to confirm these findings. In
addition, clinical trials are currently underway to determine therefore whether TIP administration
via podhaler can decrease the sputum bacterial density and improve lung function in CF patients
infected with BCC.
IBCWG 2013 Ann Arbor, MI April 2013
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6
EPIDEMIC B. MULTIVORANS STRAINS IN BELGIAN CF PATIENTS
C. Peeters1, A. De Bel
2, J. Willekens
3,4, A. Malfroot
3, D. Pierard
2, P. Vandamme
1
1Laboratory of Microbiology Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent,
Belgium 2Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije
Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium 3Cystic Fibrosis Clinic, Universitair Ziekenhuis Brussel, Belgium
4Department of Pediatrics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB),
Laarbeeklaan 101, B-1090 Brussels, Belgium
Contact : Charlotte Peeters, [email protected]
Burkholderia cepacia complex (Bcc) bacteria have emerged as significant respiratory pathogens
in CF patients due to the risk of cepacia syndrome (a fatal necrotizing pneumonia with
bacteraemia), the organism‟s innate multiresistance to antibiotics, and the transmissibility of
bacterial strains among patients by social contact. Of the 17 established species within the Bcc,
B. cenocepacia and B. multivorans dominate in CF. While B. cenocepacia strains have been
responsible for large epidemics among CF patients and are often extremely virulent, only a
limited number of B. multivorans outbreak strains and rare cases of cepacia syndrome caused by
B. multivorans have been reported. However, recent epidemiological surveys reveal that the
proportion of CF patients infected with B. multivorans is rising (e.g. UK and USA), or that B.
multivorans is the most prevalent Bcc CF pathogen (e.g. Belgium, France, New-Zealand).
Since January 2011 a National Reference Center is charged with the surveillance of respiratory
infections caused by non-fermenting Gram-negative bacilli in Belgian CF patients. This project
aims to identify isolates from CF patients and study the epidemiology and virulence of Bcc
bacteria in order to improve infection control guidelines.
During the two-year period 2011-2012, 88 Bcc isolates were identified using recA gene sequence
analysis and 56 isolates (64%) belonged to B. multivorans. Multilocus Sequence Typing (MLST)
was used to assess the epidemiology of the Bcc isolates and revealed the occurrence of two B.
multivorans strains among multiple Belgian CF patients. The two clusters comprised five and
twelve patients, respectively. Additionally, many unique B. multivorans strains were found,
suggesting acquisition from environmental sources.
In conclusion, this study demonstrated the presence of two epidemic B. multivorans clones in the
Belgian CF population.
This research was supported by the Special Research Council of Ghent University and National
Health Insurance (RIZIV/INAMI).
IBCWG 2013 Ann Arbor, MI April 2013
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7
GENETIC VARIATION OF BURKHOLDERIA DOLOSA IN SINGLE CLINICAL
SAMPLES PROVIDES A SIGNATURE OF SELECTIVE PRESSURES
Tami D. Lieberman1, Kelly B. Flett
2, Thomas R. Martin
3, Alexander J. McAdam
4, Gregory P.
Priebe2,5
, and Roy Kishony1,6
1Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
2Division of Infectious Diseases, Department of Medicine, Boston Children‟s Hospital; and
Harvard Medical School, Boston, MA 02115, USA. 3Division of Respiratory Diseases, Department of Medicine, Boston Children‟s Hospital and
Harvard Medical School, Boston, MA 02115, USA 4Department of Laboratory Medicine, Boston Children‟s Hospital and Harvard Medical School,
Boston, MA 02115, USA. 5Division of Critical Care Medicine, Department of Anesthesiology, Perioperative and Pain
Medicine; Boston Children‟s Hospital; and Harvard Medical School, Boston, MA 02115, USA. 6School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138,
USA.
Contact: Tami Lieberman, [email protected]
Bacterial pathogens acquire mutations during infection as they adapt to challenges posed by the
environment within the human host. Recent genomic studies tracking bacterial evolution within
individual patients have identified mutations responsible for antibiotic resistance, the rate at
which mutations accumulate, and specific bacterial genes and pathways under selective pressure.
Such studies typically use sequencing of a single colony isolate from each clinical sample, giving
only a limited picture of the diversity of the pathogen population at the time of sampling. Here,
using colony re-sequencing and deep population sequencing of sputum isolates of Burkholderia
dolosa from people with cystic fibrosis, we identify extensive intrastrain genomic diversity
within individual clinical samples. We find that mutations rarely fix within a patient's pathogen
population—instead, diversifying lineages coexist for many years. When strong selection is
acting on a gene, multiple adaptive mutations arise yet coexist, generating lasting allele diversity
that provides a signature of past selection. In the pathogen populations we studied, genes
involved in outer-membrane components, iron scavenging and antibiotic resistance all showed
signs of within-patient selection. These results offer a general approach for diagnosing selective
pressures acting on a pathogen within the human host using single clinical samples.
IBCWG 2013 Ann Arbor, MI April 2013
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8
A 15 YEAR STUDY OF BURKHOLDERIA INFECTION IN PERSONS WITH CYSTIC
FIBROSIS
Amy A. Bumford, Theodore Spilker, Bridget Foster, Jennifer Krot, Linda M. Kalikin and John J.
LiPuma
Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor,
USA
Contact: John LiPuma, [email protected]
Between 1997 and 2011, the Burkholderia cepacia Research Laboratory and Repository
(BcRLR) at the University of Michigan received 14,580 isolates that we identified as species in
the B. cepacia complex (Bcc) or B. gladioli. These isolates had been recovered from 2,670
persons with CF attending 433 CF care centers in the United States. All Bcc species, except for
B. ubonensis, were identified. Analysis also suggested the presence of several novel Bcc species.
B. cenocepacia, B. multivorans, and B. gladioli were the most commonly identified species in
each year, with the annual incidence of B. cenocepacia showing a steady decline during the
study period, and the incidence of B. gladioli increasing in recent years. Transient or persistent
co-infection with more than a single species was noted, as well as super-infection with one
infecting species being supplanted by another. Chronic infection of greater than 14 years
duration with B. cenocepacia, B. multivorans, B. dolosa, or B. arboris was observed in several
patients. Genotyping analyses on a large subset of isolates using repetitive element BOX-PCR
identified previously described „epidemic‟ strains, including B. cenocepacia PHDC, the B.
cenocepacia Midwest clone, and B. dolosa SLC6. Several novel strains shared by multiple
patients were also identified. Active surveillance genotyping is now routinely performed to
monitor for evidence of outbreaks and to support infection control efforts at referring care
centers. Strain genotyping is also a critical element contributing to our understanding of
Burkholderia global epidemiology, as well as the natural history and clinical outcomes of
infection.
IBCWG 2013 Ann Arbor, MI April 2013
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9
MANNITOL ELICITS A GENOME-WIDE RESPONSE IN BURKHOLDERIA
MULTIVORANS THAT IMPACTS ON NUMEROUS VIRULENCE TRAITS
Carmen C. Denman1 and Alan R. Brown
1
1Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter. UK.
Contact: Alan Brown, [email protected]
A dried-powder formulation of mannitol is approved as an osmolyte therapy for use in cystic
fibrosis patients. It is widely-documented that mannitol promotes exopolysaccharide (EPS)
production by members of the Burkholderia cepacia complex (BCC). In the present study we
sought to assess the wider impact that mannitol has on Burkholderia multivorans. Parallel studies
of representative wildtype B. multivorans (ATCC 17616 and C1576) and EPS-deficient isogenic
mutants of each strain enabled us to determine which components of the mannitol response were
associated with EPS production, and which were apparently independent of EPS production. In
addition, microarray (ATCC 17616) or RNA-seq (C1576) analysis was performed to characterize
the transcriptional response of the wildtype B. multivorans to mannitol. Our studies revealed a
significant whole-genome response to mannitol that impacts on a multitude of virulence-
associated traits, including biofilm formation, biotic & abiotic adherence, motility, antibiotic
resistance and epithelial cell invasion. In the majority of cases, this mannitol response was
observed equally in the wildtype and the isogenic EPS-deficient mutants, indicating it to be
largely independent of EPS production. Strain-to-strain variation was observed in the extent to
which EPS contributed to particular phenotypes, most notably adherence. Genome sequencing of
C1576 identified fimbrial and afimbrial adhesin loci that underlie the enhanced adherence
observed with this strain following growth in mannitol. In conclusion, these studies highlight the
wide-ranging effect that mannitol has on members of the BCC, and suggest that close monitoring
of BCC-infected patients receiving mannitol therapy would appear prudent.
IBCWG 2013 Ann Arbor, MI April 2013
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10
LIFE AT LOW-OXYGEN TENSION IN B. CENOCEPACIA H111
Gabriella Pessi1, Rubina Braunwalder
1, Alexander Grunau
1, Ulrich Omasits
2, Christian H.
Ahrens2 and Leo Eberl
1
1Institute of Plant Biology, Department of Microbiology, UZH, Zürich, Switzerland
2Institute of Molecular Life Sciences, UZH, Zürich, Switzerland
Contact : [email protected]
B. cenocepacia H111 is an opportunistic pathogen originally isolated from a cystic fibrosis (CF)
patient. In CF infections, bacteria grow to high densities in a mucopurulent material that is
limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to
grow and survive under oxygen-limited to anaerobic conditions by using microaerobic
respiration, denitrification and fermentative pathways. In contrast, inspection of the B.
cenocepacia H111 genome sequence suggested that it is an obligate aerobic and non-fermenting
bacterium. Accordingly, our study of growth under different oxygen concentrations
demonstrated that H111 is able to grow with as little as 0.1% O2 but not under anaerobic
conditions. A phenotypical analysis showed that H111 produced more biofilms and pellicles
under microaerobic conditions (0.5% O2, i.e. conditions that mimic infection), and was
consequently more resistant to several antibiotics. To obtain a more comprehensive view of the
molecular mechanisms underlying physiological adaptation to an oxygen-limited environment
RNA-Seq and shotgun proteomics analyses were performed on cultures of B. cenocepacia H111
grown under microaerobic (0.5% O2) and aerobic (21% O2) conditions. For further
characterization and analysis of mutant strains, we have chosen two transcriptional units that are
highly expressed and up-regulated in microaerobically growing cells: i) an FNR-like
transcriptional regulator and ii) a rpoS-related sigma factor.
IBCWG 2013 Ann Arbor, MI April 2013
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11
GENE EXPRESSION CHANGES OF BURKHOLDERIA CENOCEPACIA ST32 WHEN
CULTURED IN CF SPUTUM AND SERUM
Lucie Kalferstova1, Michal Kolar
2, Pavel Drevinek
1
1 Department of Medical Microbiology and Department of Pediatrics, 2
nd Medical School,
Charles University, Prague, Czech Republic. 2 Department of Genomics and Bioinformatics, Institute of Molecular Genetics, Academy of
Science of the Czech Republic, Prague, Czech Republic
Contact: Lucie Kalferstova, [email protected]
The aim of this study was to compare gene expression in two pairs of ST32 clinical isolates that
were subjected to cultivation in two different conditions, characteristic for chronic B.
cenocepacia infection in CF patients. ST32 strain is known to be a problematic epidemic strain,
which caused a serious outbreak at the Prague CF centre.
While one isolate per pair was recovered from blood at the time of cepacia syndrome, the other
was isolated from sputum one month prior to cepacia syndrome. Each isolate was incubated in
triplicate in: (i) CF sputum (minimal medium supplemented with 10% pooled CF sputum), (ii)
CF heat-inactivated serum and (iii) the basal salt medium (BSM) supplemented with glucose and
casein amino acids, generating in total samples for 36 microarrays: 3 growth conditions (sputum;
serum; BSM) x 4 isolates (sputum isolates from patients A and B; blood isolates from patients A
and B) x 3 replicates.
Bacterial cells were harvested after 4 hours of cultivation; their RNA was extracted and enriched
using Trizol, RiboPure and MICROBEnrich kits (Life Technologies). The conversion to cDNA,
fluorescent labeling and hybridization to the microarray was processed according to protocol for
microarrays analysis. The data mining was performed in GeneSpring v 12.0 (Agilent
Technologies).
We used a 4x44k custom array (Agilent Technologies) designed for closely related J2315 strain
since the whole genome sequence of ST32 is still unavailable. However, data from an ongoing
ST32 genome sequencing allowed to identify 5739 chip probes matching the putative CDS in
ST32 (~ 80% of all putative ST32 CDS) and 1049 probes for intergenic regions.
Numbers of genes with significantly altered expression are summarized in Tab.1. We noted that
the variable “growth condition” generated a pattern that was affected by the factor “patient” to
considerably lesser extent than the variable “origin of isolate” [an overlap between gene lists was
37% (comparisons #1 vs. #2), 51% (#3 vs. #4), 21% (#5 vs. #6) and 11% (#7 vs. #8)]. Now we
focus on the analysis of the latter variable with the ultimate goal to reveal gene expression
changes specific to origin of isolate and to septicemia, a last and critical stage of Burkholderia
infection. Tab. 1
condition compared
Comparison #
origin of isolate
growth condition
isolate from patient
origin of isolate
growth condition
isolate from patient
2-way ANOVA, p 0.05, 2-fold change up
down
growth condition
1 blood serum A
vers
us
blood sputum A 330 146 184
2 blood serum B blood sputum B 286 112 174
3 sputum serum A sputum sputum A 429 165 264
4 sputum serum B sputum sputum B 412 210 202
origin of isolate
5 blood serum A sputum serum A 135 104 31
6 blood serum B sputum serum B 127 47 80
7 blood sputum A sputum sputum A 116 79 37
8 blood sputum B sputum sputum B 140 86 54
Supported by IGA MZ NT12405-5, MSMT LD11029
IBCWG 2013 Ann Arbor, MI April 2013
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12
HIGH THROUGHPUT SCREENING FOR GROWTH INHIBITORS OF
BURKHOLDERIA CENOCEPACIA
Carrie Selin1, Eric Brown
2 and Silvia T. Cardona
1,3
1Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
2Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON,
Canada 3Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada
Contact: [email protected]
There is an urgent need for the discovery and development of new antimicrobial treatments for
Burkholderia cepacia complex infections. One approach to address this need is high-throughput
screening of small molecule libraries against whole cells to discover new, active scaffolds that
could stimulate drug discovery.
We used a whole-cell based assay to screen the Canadian Compound Collection (CCB) small
molecule library for growth inhibitory activity against B.cenocepacia K56-2. The screen used
bacterial growth in 96-well plate format, measured by optical density at 600 nm, to evaluate the
effect of 30,259 compounds. From 206 primary actives with <0.7 percent residual growth and <-
17.5 B-Score, we selected and confirmed the growth inhibitory activity of 72 compounds, named
herein Bce bioactives. The top twenty most active compounds consisted of antibiotics used in
clinics to treat Burkholderia infections, known antibiotics that are not commonly used, and
disinfectants. The remaining compounds were clustered by structure and prioritized for further
research according to novelty, growth inhibitory activity, solubility, and potential as multitarget
compounds. Thiophen, pyrazine and maleimide derivatives were among the selected small
molecules.
Our work illustrates the challenges of finding novel compounds with antibacterial activity
against B. cepacia complex and highlights the utility of whole cell-based high throughput screens
to identify bioactives as a starting point of antibacterial drug discovery.
IBCWG 2013 Ann Arbor, MI April 2013
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13
GENE-SPECIFIC SILENCING OF BURKHOLDERIA CEPACIA COMPLEX WITH
PHOSPHORODIAMIDATE MORPHOLINO OLIGOMERS
Kimberly Marshall-Batty1, Lacey Harbor
1, Bruce L. Geller
2 and David E. Greenberg
1
1Department of Internal Medicine, University of Texas Southwestern, Dallas, TX, USA
2Department of Microbiology, Oregon State University, Corvallis, OR, USA
Contact: [email protected]
Background: Members of the Burkholderia cepacia complex (Bcc) can be difficult to treat in
patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD) given their inherent
antibiotic resistance. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs)
are synthetic DNA/RNA analogues that silence expression of specific genes and prevent
translation. We studied whether PPMOs targeted to essential genes in Bcc are active in vitro and
in vivo.
Methods: A PPMO targeting the gene acpP was tested in in vitro and in vivo assays.
Bacterial strains were grown overnight and for in vitro assays, were diluted to a final
concentration of 5 x 105 CFU/mL. Blood was collected from normal donors and CF patients and
neutrophils were purified. For ex vivo studies, PPMOs were added to infected neutrophils (PMN)
one hour post-infection. For in vivo experiments, X-linked CGD mice (gp91phox
) that were 16-20
weeks old were infected intranasally with Burkholderia multivorans. Mice were treated with
various doses of PPMO intraperitoneally or through aerosol delivery. Both fixed-end point and
survival studies were undertaken and tissue burden of organism as well as pathologic findings
were determined.
Results: A PPMO targeting acpP is growth inhibitory across a variety of members of the Bcc.
This includes a pan-resistant B. cenocepacia outbreak isolate where there was a >3-log reduction
in CFU after 24 hour incubation with PPMO while incubation with peptide alone or a scrambled
sequence PPMO (Scr) had no effect on growth. The addition of the AcpP PPMO augments both
normal and CF PMN killing at both 2 and 4 hours post-infection. To better mimic the clinical
presentation and treatment of Bcc infections in humans, CGD mice were infected with B.
multivorans intranasally. Mice were infected with 6 x 105 CFU of B. multivorans IN and then
received one dose of AcpP PPMO or placebo (Scr) via an Aerogen nebulizer 6-hours post-
infection. All mice were euthanized at 24 hours and CFU/g of lung determined. A single, delayed
300 μg dose of AcpP PPMO reduced CFU by 90% compared to Scr PPMO treated mice
(p<.005). Mice were then infected with B. multivorans as above and treated with aerosolized
AcpP or Scr PPMO at 1,6 and 24 hours post-infection and then daily thereafter through day 7.
Mice received a single IP dose of PPMO at 24, 48 and 72 hours post-infection. While 50% of
AcpP PPMO treated mice survived through day 7, 100% of mice treated with Scr PPMO were
euthanized by day 5.
Conclusions: Gene-specific targeting of Bcc using PPMOs are effective in vitro and in vivo.
Targeted pulmonary delivery of PPMOs could be a viable therapeutic approach in treating
patients with Bcc infections.
IBCWG 2013 Ann Arbor, MI April 2013
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14
COMPARATIVE GENOMIC ANALYSIS DEFINES THE BURKHOLDERIA
CENOCEPACIA CORE GENOME AND CANDIDATE VIRULENCE GENES
Erick Cardenas1, Linda M. Kalikin
2, Jean-Marie Rouillard
3, Jiangchao Zhao
2, Patrick S. Chain
4,5,
Erdogan Gulari3, James M. Tiedje
1, and John J. LiPuma
2
1Center for Microbial Ecology, Michigan State University, East Lansing, Michigan, USA
2Departments of Pediatrics and , University of Michigan, Ann Arbor, Michigan, USA
3Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA
4Department of Energy Joint Genome Institute, Walnut Creek, California, USA
5Los Alamos National Laboratory, Los Alamos, New Mexico, USA
Contact: [email protected]
Burkholderia cenocepacia is a naturally occurring soil bacteria that can live in a wide range of
ecosystems. It is also an opportunistic pathogen in cystic fibrosis (CF) patients where it can lead
to chronic, and sometimes lethal respiratory infections. Our understanding of the pathogenicity
of B. cenocepacia has been shaped by epidemiological data in CF patients, as well as
experimental data using a variety of biological models of infection. However, little attention has
been given to the characterization of the intra-species genetic diversity as a way to understand its
pathogenicity. We used comparative genomic hybridization to study the distribution of over
8,000 genes in a set of 187 B. cenocepacia strains. This collection included clinical isolates,
including representatives from the ET12, PHDC, and Midwest epidemic lineages, and
environmental isolates. Hierarchical cluster analysis of array present/absent calls grouped
isolates broadly by recA lineages and in smaller clusters by epidemic lineage. Almost half
(49.1%) of the array genes belonged to the species core, defined as presence in at least 90% of all
187 strains. Gene functions associated with metabolism (COG categories C, E, F, G, H, I P, and
Q) represented the largest proportion (36.7%) of the core genome. Analysis of 165 genes
previously reported to have a potential role in Burkholderia virulence found that almost half
(48.0%) were part of the species core. While virulence genes were preferentially found in strains
from the epidemic lineages compared to non-epidemic strains, no virulence gene was exclusive
to one epidemic lineage, or to the epidemic strains at all. The lack of a strong association of
genes with the “epidemic” trait may be interpreted as a characteristic of opportunistic pathogens.
Nevertheless, we were able to find novel genes associated with epidemic strains in the collection.
Our results support the hypothesis that B. cenocepacia is a generalist, and that the versatility in
lifestyles it possesses is due to a large and adaptable genome that naturally harvests the potential
for pathogenesis.
IBCWG 2013 Ann Arbor, MI April 2013
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15
THE ROLE OF BURKHOLDERIA DOLOSA FLAGELLA IN HOST COLONIZATION
Deborah Yoder-Himes1,2
, Damien Roux3, David Skurnik
3, Mihaela Gadjeva
3, Stephen Lory
2
1 Department of Biology, University of Louisville, Louisville, Kentucky, USA
2 Department of Microbiology and Immunobiology, Harvard Medical School, Boston,
Massachusetts, USA 3
Channing Lab, Brigham and Women‟s Hospital, Boston, Massachusetts, USA
Contact: [email protected]
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. By screening a
number of B. dolosa isolates, we found that the genes encoding the lateral flagella are found in
all but one epidemic strains, sporadically in non-epidemic clinical strains, and not in the
environmental isolate. To investigate the role of the polar and lateral flagellin in a host system,
we infected mice intranasally with wild-type B. dolosa, mutant strains in either flagellar system,
wild-type Pseudomonas aeruginosa, or a P. aeruginosa flagellin mutant and tracked the
clearance of these strains, the recruitment of immune cells and the production of cytokines in
response to these strains over time. We found that B. dolosa strains persist longer in the lung
compared to P. aeruginosa strains and recruit fewer neutrophils; however, all B. dolosa strains
recruit cells that may play an active role in suppressing the immune system. We also found that
B. dolosa strains produced far fewer cytokines than P. aeruginosa strains with little difference
seen between the B. dolosa wild-type and mutant strains. We conclude that B. dolosa may be
actively avoiding or suppressing the immune system in order to proliferate and persist in a host.
IBCWG 2013 Ann Arbor, MI April 2013
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16
INVESTIGATION OF GENES AND REGULATORY ELEMENTS INVOLVED IN
PRODUCTION OF NOVEL ANTIFUNGAL COMPOUNDS IN BURKHOLDERIA
CENOCEPACIA H111
Christian Jenul1, Aurelien Carlier
1, Pamela A. Sokol
2 and Leo Eberl
1
1Department of Microbiology, Institute of Plant Biology, University of Zürich, Zürich,
Switzerland 2Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary,
Calgary, Alberta, Canada
Contact : Leo Eberl, [email protected]
Species of the Burkholderia cepacia complex (Bcc) are known for their ability to suppress fungal
pathogens by production of various antifungal compounds. Previous work has shown that in B.
cenocepacia K56-2 a large gene cluster that is present on the megaplasmid pC3 directs the
biosynthesis of an antifungal lipopeptide. Unexpectedly, we observed that a pC3-cured
derivative of B. cenocepacia H111 still displayed antifungal activity, indicating that H111
synthesizes a previously unrecognized antifungal compound. Our objective was to elucidate what
genes and regulatory elements are responsible for the different antifungal phenotypes of H111
and K56-2. Further we seeked to elucidate the nature of the bioactive compound involved in
antifungal activity of strain H111.
We found that the CepIR quorum sensing system regulates antifungal activity in H111, but not in
K56-2. Comparison of transcriptomics data of H111 and K56-2 quorum sensing mutants led to
the identification of a gene cluster comprising seven genes (H111 antifungal metabolite cluster),
that are predicted to be involved in secondary metabolism and that are differently regulated in
K56-2 and H111. In silico analysis of the gene cluster revealed that hamD encodes a non-
ribosomal peptide synthetase (NRPS). Mutation of hamD in H111 completely abrogated
antifungal activity and this phenotype could be rescued by expression of hamD from a plasmid.
This finding suggests that the ham gene cluster is involved in the biosynthesis of a so far
uncharacterized antifungal compound in H111.
IBCWG 2013 Ann Arbor, MI April 2013
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17
GENETIC ANALYSIS OF A BROAD-HOST RANGE TAILOCIN IN BURKHOLDERIA
CENOCEPACIA
Iris Duarte1, 4
, Guichun W. Yao1, 4
, John J. LiPuma2
, Ryland F. Young3,4
and Carlos F.
Gonzalez1,4
1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station,
USA 2 Department of Pediatrics and Communicable Diseases, University of Michigan Medical
School, Ann Arbor, USA
3 Department of Biochemistry and Biophysics, Texas A&M University, College Station, USA
4Center for Phage Technology, Texas A&M University, College Station, USA
Contact: Carlos F. Gonzalez, [email protected]
Members of the Burkholderia cepacia complex (Bcc) are opportunistic human pathogens that
exhibit broad-spectrum antibiotic resistance. High-molecular weight (HMW) bacteriocins, like
the well-studied R- and F-type pyocins (tailocins) from Pseudomonas aeruginosa, closely
resemble bacteriophage (phage) tails. Tailocins are attractive candidates for novel antibacterial
strategies because they can be genetically manipulated like phages and can also be deployed with
defined dosage, since they do not propagate in therapeutic use.
An 18 X 18 test matrix of B. cenocepacia clinical isolates was used to identify tailocin
Bcep0425, which exhibited broad host range activity against members of the Bcc. Electron
microscopy studies confirmed the phage tail-like structure of Bcep0425. The major tailocin
proteins were resolved using SDS-PAGE and four major protein species were subjected to N-
terminal sequence analysis. The host genome was sequenced and, using the sequence
information from the major proteins, the tailocin locus was identified and annotated in detail.
The tailocin cassette was found to be integrated into a tRNAArg
gene. The Bcep0425 integrase
gene was identified by significant sequence similarity to the integrase of the well-studied
temperate Salmonella podophage P22. Most Bcep0425 proteins showed homology to the tail
proteins of the paradigm E. coli myophage P2. A lysis cassette encoding an endolysin, i-spanin
and o-spanin was identified by sequence homology. Two genes, 26 and 27, were experimentally
confirmed as encoding the holin and antiholin, respectively, by phenotypic analysis of deletions
and complemented deletions. Bcep0425 holin contained four transmembrane domains (N-in and
C-in), defining a new holin topological class. Targeted mutagenesis of genes involved in the
biosynthesis of the bacterial lipopolysaccharide (LPS) in B. cenocepacia strain K56-2 was
conducted to determine that LPS is the putative receptor for Bcep0425. The molecular
characterization of Bcep0425 indicates that it was derived from a defective prophage.
Considering its broad host range, unprecedented for tailocins, Bcep0425 has significant potential
for antibacterial therapy.
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18
IDENTIFICATION OF NOVEL BURKHOLDERIA CEPACIA COMPLEX ANTIGENS
BY IMMUNOPROTEOMICS AND INVESTIGATION OF THEIR ROLES IN
VIRULENCE
Ruth Dennehy1, Cristobal Mujica
2, Miguel A. Valvano
2, Maire Callaghan
1 and Siobhán
McClean1
1Centre of Microbial Host Interactions, Institute of Technology Tallaght, Dublin.
2Centre of Infection and immunity, Queens University Belfast.
Contact: [email protected]
Virulence factors make direct contact with host cells and so are among the primary antigen
targets of the host immune system. Therefore, proteomic analysis using serum from
Burkholderia cepacia complex (Bcc) colonised patients can lead to identification of novel
virulence factors. The aim of this study was to identify virulence factors from two of the most
clinically relevant Bcc species, B. cenocepacia and B. multivorans, using an immunoproteomic
approach. Two-dimensional blots were probed with serum from CF patients that had been
infected with Bcc and compared to blots probed with serum from patients with no history of Bcc
infection. Twelve B. cenocepacia and 14 B. multivorans immunogenic proteins were identified
using MALDI-Tof mass spectrometry. Of the proteins identified, six were common to both
species. Two proteins were chosen for further examination: an outer membrane lipoprotein
(BCAL3204), previously shown by us to be involved in attachment to CF lung epithelial cells
(CFBEs) and a hypothetical protein (BCAS0292), located in a virulence gene cluster.
Targeted deletion mutants were constructed in the B. cenocepacia strain, K56-2, with the aim of
further examining the role of these proteins in the pathogenesis of Bcc. The mutants were
examined in vivo using the Galleria mellonella virulence model. The virulence of the
BCAL3204 mutant was significantly reduced (p < 0.02) in comparison to the wild type, while a
slight decrease in virulence was observed in the BCAS0292 mutant. Both mutants also displayed
a substantial reduction in adhesion to CF lung epithelial cells compared to the wild-type strain,
indicating that both proteins may play a role in bacterial attachment to host cells.
Further investigation into the role of these immunogenic proteins will provide a better
understanding of the role they play in the pathogenesis of Bcc, which could lead to their
development as potential vaccine antigens or as drug targets for anti-virulence therapies.
Funded by Science Foundation Ireland and EU COST Action BM1003
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19
INVOLVEMENT OF TOXIN-ANTITOXIN MODULES IN TOLERANCE OF
BURKHOLDERIA CENOCEPACIA BIOFILMS
Heleen Van Acker1, Hans J. Nelis
1& Tom Coenye
1
1
Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, België
Contact : Heleen Van Acker, [email protected]
Burkholderia cenocepacia is an opportunistic pathogen that can cause severe lung infections in
cystic fibrosis patients. Infections are often difficult to treat because of its innate resistance and
its capacity to form biofilms. Biofilms play an important role in the recalcitrance of infections
due to their tolerance. 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. The toxin (T) is a protein that inhibits an
important cellular function such as translation or replication and which can form an inactive
complex with an antitoxin (AT). 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 the present study we wanted to investigate
whether these TA modules play a role in B. cenocepacia biofilm tolerance towards different
antibiotics.
Using bioinformatics tools, 15 pairs of genes were identified as possible TA modules in the
genome of B. cenocepacia J2315 (score ≥ 60). We also found 4 other possible TA modules by
blast analysis. 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 TA
modules in treated sessile and planktonic cultures was compared to that in untreated cultures.
Mutants in which a toxin or antitoxin-coding gene was overexpressed under control of a
rhamnose inducible promoter were constructed and evaluated for growth, resistance, biofilm
formation and response to antibiotics. We constructed 14 toxin-overexpressing and 3 antitoxin
overexpressing mutants.
Generally the toxins showed a higher basal expression in sessile cells than in planktonic cells,
but the expression patterns were similar for most T-AT pairs. Four toxins were upregulated in all
conditions tested, whereas only one toxin was systematically downregulated. Operons showing
similarity with mazEF, relBE or mqsRA and five other TA modules were significantly
upregulated in biofilms and planktonic cultures after treatment with tobramycin but not after
treatment with ciprofloxacin. None of the operons was upregulated in both conditions after
treatment with ciprofloxacin. Others were only upregulated in sessile or in planktonic cultures,
which indicates that planktonic and sessile cells may have different mechanisms to regulate the
relative amounts of T/AT.
Overexpression of toxins had different effects on growth, biofilm formation and the number of
surviving cells after treatment. For example, overexpression of RelE or MazF resulted in a
prolonged lag phase and more cells surviving antibiotic treatment whereas overexpression of an
undefined antitoxin (BCAM0258) resulted in less biofilm formation. These results indicate that
various TA modules may play a role in tolerance and persistence but that their exact contribution
depends on the mode of growth and the antibiotic used.
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20
NEW INSIGHTS INTO THE BDSF AND THE AHL-BASED QUORUM SENSING
SYSTEMS OF BURKHOLDERIA CENOCEPACIA H111
Nadine Schmid1*
, Gabriella Pessi1*
, Yinyue Deng2*
, Claudio Aguilar1, Ulrich Omasits
3, Lian-Hui
Zhang2, Christian H. Ahrens
3 and Leo Eberl
1
1Department of Microbiology, Institute of Plant Biology, University of Zurich, Switzerland
2Institute of Molecular and Cell Biology, Singapore
3Institute of Molecular Life Sciences, University of Zurich, Switzerland
* These authors contributed equally to this work
Contact : [email protected]
Burkholderia cenocepacia H111, a CF isolate, employs two quorum-sensing (QS) systems to
control the expression of genes associated with virulence and biofilm formation. While the N-
acyl homoserine lactone (AHL)-based QS system has been fairly well analyzed, little is known
about the more recently discovered cis-2-dodecenoic acid (BDSF)-dependent signaling system.
Recent work showed that the regulatory effect of BDSF on gene expression is mediated through
alteration of the intracellular c-di-GMP level. RpfR, the protein responsible for this effect, is a
bifunctional enzyme with both diguanylate-cyclase and phosphodiesterase activity. Upon binding
of BDSF to its PAS-domain, the phosphodiesterase activity is stimulated and as a consequence
the cellular c-di-GMP level is lowered.
Phenotypic characterization of QS mutants and transcriptome analyses revealed that the BDSF
and AHL stimulons partially overlap. In spite of the fact that BDSF positively regulates the AHL
levels, our data suggest that both signal molecules are required for maximal expression of the
large surface protein bapA and the lectin operon bclACB. In accordance with this observation,
the defects in biofilm formation, protease activity and swarming motility of BDSF- and AHL-
deficient double mutant can only be restored to wild type level upon the addition of both signal
molecules. This suggests that the two QS systems are not hierarchically arranged but rather
operate in parallel.
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21
ANTI-BIOFILM ROLE OF EXOPOLYSACCARIDES PRODUCED BY
BURKHOLDERIA CENOCEPACIA
Elena Pellizzoni, Fabio Ravalico, Denis Scaini, Roberto Rizzo, Paola Cescutti
Dipartimento di Scienze della Vita, Università degli Studi di Trieste, via L. Giorgeri 1, Ed. C11,
34127 Trieste
Contact : Paola Cescutti, [email protected]
The preferential mode of growth of microbes in nature and disease is within a biofilm and there
are strong indications that bacteria of the BCC develop biofilms in the CF lung. The biofilm
matrix surrounds the microbial cells and confers an increased resistance to antibiotics, biocides
and host defenses. It is an active macromolecular scaffold composed mainly by proteins,
extracellular DNA and polysaccharides (PLS), but the exact composition may vary from species
to species and depends on cultivation media. We focussed our attention on B. cenocepacia
BTS2, a CF clinical isolate and an abundant biofilm producer. The aim of our investigation was
to determine the influence of growth media on the physico-chemical properties of the biofilm
matrix, with particular attention to polysaccharides (PLS), often regarded as the main matrix
components. Five different media were used: four common bacteriological media and the
synthetic cystic fibrosis medium (SCFM) which simulates the in vivo situation. Proteins and PLS
content were determined, and NMR established the structure of the latter. Biofilm morphology
was visualized by CLSM while AFM was used to investigate the thickness of the matrix. The
evaluation of PLS content in biofilms showed that media promoting abundant biofilm formation
exhibited the lowest PLS content. In particular, cepacian, the PLS synthesized by the majority of
BCC strains in YEM medium, was associated with scanty biofilm development. Interestingly, it
was recently reported that the extent of mucoidy in BCC is inversely correlated with the rate of
decline in lung function. These observations prompted an investigation of the role of the PLS
produced by B. cenocepacia strain BTS2 in biofilms. Cepacian had two main effects: when
mixed with the bacterial culture it impaired biofilm development, while its addition to mature
biofilms caused their partial detachment. These properties could be exploited for impeding
and/or removing biofilms.
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22
AHL QUORUM SENSING IN THE ENVIRONMENTAL AND POTENTIALLY
BENEFICIAL PLANT-ASSOCIATED BURKHOLDERIA CLUSTER
Bruna G. Coutinho1,2
, Birgit Mitter3, Chouhra Talbi
4, Angela Sessitsch
3, Eulogio J. Bedmar
4,
Nigel Halliday5, Euan K. James
6, Miguel Cámara
5 and Vittorio Venturi
1
1Bacteriology Group, International Centre for Genetic Engineering & Biotechnology,
Padriciano 99, 34149 Trieste, Italy 2The Capes Foundation, Ministry of Education of Brazil, Cx postal 250, Brasilia, DF 70.040-
020, Brazil 3AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-3430 Tulln, Austria
4Department of Soil Microbiology and Symbiotic Systems Estación Experimental del Zaidín,
CSIC, Apartado Postal 419, 18080-Granada, Spain 5School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of
Nottingham, Nottingham NG7 2RD, UK 6The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK
Contact: [email protected]
The genus Burkholderia is composed of functionally diverse species and it can be divided into
several clusters. One of these, designated as the plant-beneficial-environmental (PBE)
Burkholderia cluster, is formed by non-pathogenic species, which in most cases have been found
to be associated with plants. It was previously established that all members of the PBE group
share an N-acyl-homoserine lactone (AHL) quorum-sensing (QS) system designated BraI/R that
produces and responds to 3-oxo-C14-HSL. Moreover, some of them also possess a second AHL
QS system, designated XenI2/R2, producing and responding to 3-hydroxy-C8-HSL (OHC8-
HSL). We performed LC-ESI-MS/MS analysis to determine which AHL molecules are produced
by each QS system of this group of bacteria. Results showed that XenI2/R2 is mainly responsible
for the production of OHC8-HSL and that the BraI/R system is involved in the production of
several different AHLs. This analysis also revealed that B. phymatum STM815 produces greater
amounts of AHLs than the other species tested. Further studies showed that the BraR protein of
B. phymatum is more promiscuous than other BraR proteins, responding equally well to several
different AHL molecules, even at low concentrations. Transcriptome studies with B. xenovorans
LB400 and B. phymatum STM815 revealed that the BraI/R regulon is species-specific, with
exopolysaccharide production being the only common phenotype regulated by this system in the
PBE cluster. In addition, BraI/R was shown not to be important for plant nodulation by B.
phymatum spp. nor for endophytic colonization and growth promotion of maize by B.
phytofirmans PsJN.
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23
TEASING APART THE MULTIPLE ROLES OF A CzcR/CzcS-LIKE TWO-
COMPONENT SYSTEM IN BURKHOLDERIA CENOCEPACIA: VIRULENCE,
QUORUM SENSING AND HEAVY METAL RESISTANCE
Matthew T. Robinson1 and Alan R. Brown
1
1Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter. UK.
Contact: Alan Brown, [email protected]
Two-component systems (TCSs) are key regulators of bacterial adaptation to extracellular
stimuli and frequently play a pivotal role in the regulation of virulence. We have previously
reported that inactivation of a CzcR/CzcS-like TCS in Burkholderia cenocepacia results in
hypersensitivity to heavy metals and attenuated virulence (Robinson & Brown, IBCWG 2012).
Here we report further characterization of the virulence-related phenotypes associated with this
CzcR/CzcS-like TCS, and how this TCS facilitates an effective response to heavy metal stress
(using zinc as a model). We confirm that both biofilm formation and intracellular survival within
macrophages are dramatically-impaired as a consequence of czcRS deletion, phenotypes that can
be fully restored by complementation. Despite the profound attenuation observed in the ΔczcRS
mutant, transcriptional profiling suggests that only a modest set of genes are actually regulated
by the TCS in response to zinc. However, studies have revealed that in the absence of zinc, the
czcRS genes are required for the full activation of the quorum sensing network, explaining the
profound virulence attenuation observed in the ΔczcRS mutant. Whilst these observations suggest
that the CzcR transcriptional regulator possesses basal activity in the absence of TCS activation
by zinc, transcriptional profiling has also confirmed the pivotal role that this CzcR/CzcS-like
TCS plays in ensuring cellular homeostasis in the presence of zinc. We conclude that the
CzcR/CzcS-like TCS in B. cenocepacia directly and indirectly regulates diverse cellular
processes within the cell that contribute to virulence and heavy metal resistance.
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24
PROTEINS INVOLVED IN HOST CELL ATTACHMENT ARE PROTECTIVE
ANTIGENS AGAINST BCC INFECTION.
Minu Shinoy1, Marc Healy
2, Bernard Mahon
2, Máire Callaghan
1 and Siobhán McClean*
1
1Centre of Microbial Host Interactions Institute of Technology Tallaght, Dublin 24 Ireland.
2Department of Biology, National University of Ireland, Maynooth, Ireland.
Contact : [email protected]
Bcc is rarely eradicated once a patient has been colonised, therefore vaccination against this
pathogen may represent a better therapeutic option. Traditionally good vaccine candidates are
those which are involved in host cell attachment. We have developed a method to identify the
bacterial proteins that are involved in the attachment of Burkholderia multivorans and
Burkholderia cenocepacia to lung epithelial cells. Bacterial proteins were extracted from B.
multivorans strains (LMG13010 or C1962) or B. cenocepacia strains (BC-7 or C1394), separated
on 2-D gels and transferred to a membrane, before probing with lung epithelial cells (CFBE41o-
or 16HBE14o- cells). Proteins involved in attachment were identified by MALDI-ToF MS. A
total of twenty proteins were identified by 2-DE from all the four strains of Bcc. Out of these,
seven proteins were found to be common to both species, but only two were common to all four
strains.
These two proteins were selected for further study and were cloned into E. coli, expressed and
purified by chromatography. In both cases the recombinant E. coli strain showed more binding
than the wild type (12.5- and 8.7- fold), confirming that these proteins play a role in binding to
epithelial cells. Immunoproteomic analysis using serum from Bcc colonised CF patients
confirmed that both proteins are expressed in vivo and each one elicits a potent humoral
response. Groups of mice were immunized individually (i.p.) with the recombinant antigens and
challenged 5 days after the booster with either B. multivorans or B. cenocepacia. Mice
immunised with BcOMP1 were protected from B. cenocepacia infection (15 Ln reductions in
lung CFU in immunized mice) and B. multivorans infection (7 Ln reductions in lung CFU).
Immunization with BcOMP2 protected against B. cenocepacia with a reduction in lung bacterial
counts by 7 Ln (60,000 fold reduction in bacterial counts, p=0) and protected against B.
multivorans by a four-Ln reduction, (p<0.013). Both antigens induced potent antigen-specific
antibody responses (IgG titres ~ 1 x 106). The ratio of IgG2a to IgG1 antibodies in response to
BcOMP1 was indicative of a potent Th1 response, while the IgG2a: IgG1 ratio in response to
BcOMP2 was indicative of a mixed Th1/Th2 response. In conclusion, both antigens are
involved in bacterial adhesion to lung cells and are likely to be good vaccine candidates in a
multi-subunit vaccine.
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25
DEFECTIVE AUTOPHAGY ACTIVITY CONTRIBUTES TO INCREASED IL-1β
PRODUCTION DURING BURKHOLDERIA CENOCEPACIA INFECTION
Basant A. Abdulrahman1, Mia Tazi
1, Anwari Akhter
1, Kyle Caution
1, Hoda Hassan
1, Miguel A.
Valvano2, Benjamin Kopp
3, and Amal O. Amer
1
1
Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Center for Microbial Interface Biology and the Department of Internal Medicine, Ohio State University. Columbus, OH, USA. 2 Centre for Human Immunology, Department of Microbiology and Immunology, and The
Department of Medicine, University of Western Ontario, London, Ontario, N6A 5C1, Canada. 3 Division of Pediatric Pulmonology, Nationwide Children‟s Hospital, Columbus, OH 43205.
Contact: Amal Amer, [email protected]
IL-1β is expressed as a precursor inactive molecule in response to a Toll-Like Receptor-mediated
signal, and is later cleaved by caspase-1 to yield active IL-1β. Caspase-1 is activated within the
inflammasome complex in response to a Nod-Like-receptor (NLR) signal. The biological
activities of IL-1β include promoting inflammatory responses and leukocyte infiltration.
Recently, autophagy has emerged as an important innate immune response to infection and is
required to limit IL-1β production. Hence, insufficient autophagy has been implicated in disease
conditions characterized by excessive IL-1β production such as Crohn‟s disease and arthritis.
Autophagy is an evolutionary conserved, catabolic process that involves the entrapment of
cytoplasmic components within characteristic vesicles for their delivery to and degradation
within lysosomes. Autophagy is regulated via a group of genes called AuTophaGy related Genes
(ATGs) and is executed at basal levels in all cells for maintaining cellular integrity. IL-1β level is
elevated in bronchoalveolar lavages (BALs) of cystic fibrosis (CF) patients which suffer from
exacerbated inflammation leading to lung tissue damage. However, the underlying mechanism
leading to IL-1β exacerbated production is not understood. Additionally, the source of IL-1β is
not clear. Here we show that CF macrophages produce significantly high amounts of IL-1β when
infected with B. cenocepacia. IL-1β pro-form is produced downstream of TLR4 and is cleaved to
its active form via the pyrin inflammasome. We also show that IL-1β exacerbated production is
due to defective autophagy in CF macrophages. Moreover, B. cenocepacia down regulates
autophagy molecules upon infection of CF macrophages. Thus, IL-1β production is reduced in
response to autophagy stimulating agents. Interestingly, recent studies demonstrated that
epithelial cells do not release IL-1β in response to B. cenocepacia. Together, these data suggest
that IL-1β is primarily produced by activated macrophages and this production is uncontrolled in
CF macrophages due to impaired autophagy activity.
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26
AUTOPHAGY STIMULATION BY IFN-γ IMPROVES BURKHOLDERIA
CENOCEPACIA CLEARANCE IN HUMAN CYSTIC FIBROSIS MACROPHAGES
Ben Kopp1,2
, Kaivon Assani2, and Amal Amer
3
1Section of Pediatric Pulmonology, Nationwide Children‟s Hospital, Columbus, OH USA
2Center for Microbial Pathogenesis, The Research Institute at Nationwide Children‟s Hospital,
Columbus, OH USA 3Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH
Contact : Ben Kopp, [email protected]
Introduction: Burkholderia cenocepacia is the most feared bacteria for cystic fibrosis (CF)
patients to acquire due to a severe spectrum of disease with diminished long term survival due to
ineffective antibiotic treatment. Autophagy is a physiologic process that involves engulfing non-
functional organelles and delivering them to the lysosome for degradation, but also plays a role
in eliminating intracellular pathogens. Deficiencies in autophagy have been shown in CF, and a
murine CF model infected with B. cenocepacia demonstrates reduced bacterial load and
inflammation when pre-treated with the autophagy stimulator rapamycin. Therefore, we propose
that induction of autophagy in human CF macrophages infected with B. cenocepacia will
improve bacterial clearance, limiting chronic inflammatory stimulus and host injury.
Methods: Peripheral blood monocyte derived macrophages were obtained from 20 CF and 20
non-CF donors at the Nationwide Children‟s Hospital. Macrophages were infected with B.
cenocepacia clinical isolate strain k56-2 at an MOI of 10 and treated with autophagy inducers
rapamycin or IFN-γ and for 4 and 24 hours. Host-bacterial interactions were determined with
confocal microscopy. Autophagic flux was monitored through the use of autophagy inhibitors
combined with protein turnover detected on western blotting.
Results: CF macrophages have diminished IFN-γ production in supernatants after stimulation
with B. cenocepacia compared to controls. CF macrophages infected with B. cenocepacia
demonstrate reduced bacterial load with a 24 hour IFN-γ treatment, nearing non CF bacterial
load levels. Initiation of autophagy is seen in CF macrophages after 4 hours of IFN γ treatment
by the co-localization of bacteria with the autophagy marker LC3. Autophagy co-localization
was not demonstrated in untreated CF macrophages. Bacterial loads are still similar for CF
macrophages between a 4 hour treatment and no treatment, but diminish greatly by 24 hours.
Cell death is reduced by 48 hours after IFN-γ treatment. IL-1β production is significantly
reduced with rapamycin treatment, with a trend towards reduction with IFN-γ. There is a
significantly higher proportion of bacteria co-localizing with the autophagy docking molecule
P62/SQSTM1 (p62) in CF macrophages after treatment. Non CF macrophages demonstrate
decreased bacterial load compared to untreated CF macrophages, early autophagy initiation with
co-localization of bacteria to autophagosomes, and sustained clearance of bacteria through 4 or
24 hours regardless of IFN-γ treatment. Treatment with rapamycin also diminishes bacterial load
in CF macrophages. The addition of 3-MA to block autophagosome formation negates the
benefits of IFN-γ in CF macrophages.
Conclusions: Autophagy stimulation improves human CF macrophage clearance of B.
cenocepacia with subsequent reductions in host inflammation and cell death. The mechanisms
by which it enhances autophagic flux in CF patients are the focus of ongoing studies, but are
suggestive of enhanced autophagosome formation.
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27
A BURKHOLDERIA CENOCEPACIA GENE ENCODING A NON-FUNCTIONAL PHOSPHOTYROSINE PHOSPHATASE IS REQUIRED FOR THE DELAYED MATURATION OF THE BACTERIA-CONTAINING VACUOLES IN MACROPHAGES
Angel Andrade1 and Miguel Valvano
1,2
1Department of Microbiology and Immunology. Centre for Human Immunology, University of
Western Ontario, London, Ontario, Canada. N6A 5C1. 2Centre for Infection and Immunity. Queen's University of Belfast, Belfast, UK.
Contact: [email protected]
Burkholderia cepacia complex (Bcc) are a group of highly problematic opportunistic pathogens
causing chronic infections in patients with cystic fibrosis (CF) (1). The Bcc members display
high levels of intrinsic antibiotic resistance, persistence in the presence of antimicrobials, and
intracellular survival capabilities. The intracellular persistence of Burkholderia cenocepacia (a
member of the Bcc) has been associated with subverting the host cell endocytic pathway to
prevent the acidification and fusion of the phagolysosome. Previous reports showed that
intracellular B. cenocepacia arrests the assembly of the NADPH oxidase complex on the
phagocytic vacuole (BcCV) membrane, and impairs the activation of Rab7. Although delay of
NADPH oxidase assembly depends on a defect in Rac1 and Cdc42 activation, the mechanism by
which B. cenocepacia precludes fusion of the phagosome with lysosomes and persists within the
macrophage remains poorly defined. Because bacterial phosphatases have been associated with
virulence, contributing to the intracellular survival of pathogens in this work we focused on
investigate a possible role of tyrosine phosphatases in B. cenocepacia pathogenesis. Four genes
encoding predicted low molecular weight protein tyrosine phosphatases (PTP´s) were identified
in B. cenocepacia genome (BCAM0208, BCAM0628, BCAM0857 and BCAL2200). First, the four
4 ptp genes were cloned into a plasmid and expressed in B. cenocepacia to evaluate their
secretion as putative virulence factors. Secretion assays indicated that under the evaluated
conditions, BCAM0628 is the only PTP secreted by B. cenocepacia, pointing toward a potential
role of BCAM0628 interacting with a tyrosine phosphorylated host substrate. Using pulsed
macrophages with the Fluorescein Isothiocyanate-Dextran or LAMP-1 as probes for lysosomal
fusion markers showed differences between parental B. cenocepacia BCAM0628 strains.
Suggesting a contribution of BCAM0628 delaying the maturation of the BcCV´s. However,
gentamicin protection assays showed no difference in survival between wild type strain and any
ptp mutant strain. Recombinant histidine-taged versions of BCAM0628 and BCAL2200
were purified to homogeneity and phosphatase activity was evaluated using the general
chromogenic substrate pNPP. Phosphatase activity assays indicate that BCAM0628 is an inactive
enzyme most likely due to amino acid substitution in the catalytic core of the protein. However,
this is the only PTP that becomes secreted to the supernatant and is likely to have an effect on the
intracellular infection. Together, our data support a role of BCAM0628 affecting the host
machinery involved in late endosome maturation.
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28
MODELING OF THE INNATE IMMUNE RESPONSE TO INFECTION WITH
BACTERIA BELONGING TO THE BURKHOLDERIA CEPACIA COMPLEX USING
ZEBRAFISH EMBRYOS
Jennifer Mesureur1, Julien Rougeot
2, David O‟Callaghan
1, Annemarie Meijer
2, Annette
Vergunst1
1INSERM U1047. Université de Montpellier 1. UFR Médecine, Nimes, France
2Institute of Biology, Leiden University, Leiden, The Netherlands
Contact : [email protected]
Chronic respiratory infection in cystic fibrosis patients is characterized by a high level of pro-
inflammatory cytokines, leukocyte infiltration, and inflammation in the lungs due to colonization
by pathogenic bacteria. Particularly infections caused by Burkholderia cepacia complex (Bcc)
bacteria can progress into fatal respiratory inflammation and septicaemia, and infections with
these bacteria are associated with poor prognosis for patients. Several lines of evidence, using
murine and cell culture models, have shown that B. cenocepacia induces a particularly high pro-
inflammatory response (including secretion of the neutrophil chemo attractant IL-8, and IL-1β),
and that bacterial virulence factors, including LPS and flagellin, play a role in this. Importantly,
after phagocytosis, Bcc have been shown to manipulate macrophage host cell biology in vitro,
leading to bacterial survival and intracellular multiplication, and this intracellular survival
strategy has been proposed to contribute significantly to pathogenesis.
We are using zebrafish embryos to study in detail the role of host phagocytes in bacterial
virulence and the mechanism of induction of an excessive pro-inflammatory response in vivo.
We have reported that B. cenocepacia K56-2 belonging to the epidemic ET12 lineage survives
and multiplies in macrophages of zebrafish embryos prior to dissemination, and systemic fatal
infection. Our recent data show that K56-2 induces a very rapid bacterial dose-dependent strong
increase in pro-inflammatory gene expression that is much higher than the response elicited by
heat-killed K56-2 or a less pathogenic strain such as B. stabilis LMG14294, which causes
persistent infection. Using a combination of transgenic reporter fish lines, morpholino gene
knock down, deep sequencing of RNA and bacterial mutants, we are now further dissecting the
role of host phagocytes, and bacterial factors and host signaling pathways that lead to the
excessive pro-inflammatory response. We will further report on the importance of an
intramacrophage stage for progress of infection in this in vivo model, and our current analysis of
the host transcriptome in virulent versus persistent infection.
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29
MODELLING BURKHOLDERIA CEPACIA COMPLEX ROOT COLONIZATION IN
VITRO AS AN IMPORTANT ENVIRONMENTAL TRAIT
J. Cristian Vidal-Quist1, Louise A. O‟Sullivan
1, Annaëlle Desert
1, Amanda S. Fivian-Hughes
2,
Angela M. Marchbank1, Coralie Millet
1, T. Hefin Jones
1, Andrew J. Weightman
1, Hilary J.
Rogers1, Colin Berry
1 and Eshwar Mahenthiralingam
1*
1 Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University,
Cardiff, UK. 2
Imperial College, London, UK.
*Contact: [email protected]
Burkholderia cepacia complex (Bcc) bacteria occur widely in the natural environment and are
particularly dominant around the plant rhizosphere. This region around the roots is the site of
many biotechnologically useful interactions including the rhizoremediation of toxic pollutants
and the secretion of bioactive metabolites that promote the control of pathogenic fungi or
nematodes. We assessed the fitness of different Bcc species in the rhizosphere by developing an
in vitro model of root colonization using the pea (Pisum sativum) and the model plant,
Arabidopsis thaliana. A panel of 26 genetically diverse Bcc strains representing 9 different
species and different sources (clinical versus environmental) was assembled. Their rhizosphere
interactions were then systematically compared using the two species of plant. Initial
development of the A. thaliana microcosm was greatly facilitated by the use of a lux-tagged
Burkholderia vietnamiensis G4 reporter strain. Bacteria colonising the roots was determined
using viable counts and by direct observation through bioluminescence or electron microscopy.
All Bcc species colonized the roots of both plant models in vitro at levels significantly greater
than that seen for two non-rhizosphere control species, Staphylococcus aureus and Escherichia
coli K12. In general, the source of the Bcc strain, either clinical or environmental, did not
influence their root colonization phenotype. However, for species such as Burkholderia
ambifaria, strains of an environmental origin possessed greater rhizosphere colonization rates
than clinical isolates. In previous studies, the genetic basis for pea root colonization was assessed
using signature-tagged-mutagenesis of B. vietnamiensis G4. Further exploration of the genetics
of Burkholderia rhizosphere colonisation was performed with the A. thaliana model using five
mutated Bcc strains (representing 5 species) which lacked their third chromosomal replicon.
Deletion of this replicon representing greater than 1 Mb of DNA (1000 genes), did not
significantly alter the ability of these strains to colonize A. thaliana roots. Overall our data
indicate that the ability to colonize the rhizosphere is an intrinsic trait of Burkholderia cepacia
complex bacteria.
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30
REAL TIME ANALYSIS OF BURKHOLDERIA CENOCEPACIA ST32 ISOLATES IN A
ZEBRAFISH EMBRYO MODEL
Klara Vilimovska Dedeckova1, Pavel Drevinek
1, Annette Vergunst
2
1Charles University, 2
nd Faculty of Medicine,
Department of Medical Microbiology, Prague,
Czech Republic 2
INSERM U1047, Universite´ de Montpellier 1, UFR Médecine, Nimes, France
Contact: Klara Vilimovska Dedeckova, [email protected]
Burkholderia cenocepacia sequence type 32 (ST32) represents one of the globally distributed
strains from the Burkholderia cepacia complex (Bcc), which infected 30% of the Czech cystic
fibrosis (CF) patients. The aim of our study was to analyse virulence potential of several of these
ST32 isolates in an animal model, and to identify specific changes in virulence that might occur
over time.
Using a zebrafish embryo infection model, earlier we found large differences between 31
different ST32 isolates isolated from 14 different patients in their ability to cause embryo death.
Fourteen isolates were virulent (most embryos were killed between 3 and 6 dpi). Thirteen
isolates showed intermediate virulence, with between 50-80% mortality at 6 dpi. Four isolates
were not very virulent, and most embryos were still alive at 6 dpi, the end point of the infection
experiments. Interestingly, isolates originating from cepacia syndrome seemed less virulent in
the model than isolates that originated from earlier time points of chronic and/or exacerbated
stages of infection in the same patient.
The zebrafish embryo model is highly amenable to real time in vivo imaging of infection with
fluorescently labeled bacteria. Intravenous microinjection of virulent strains, such as K56-2, is
followed by rapid phagocytosis of bacteria by macrophages (Vergunst et al. 2010), bacterial
intracellular survival and multiplication from 7 hpi onwards, followed by bacterial systemic
dissemination and rapid (2 dpi) embryo death. Less virulent strains however are unable to
multiply efficiently, or unable to disseminate and do not cause fatal infection.
Based on our previous mortality assays in zebrafish embryos we analyzed some of the ST32
isolates in more detail in real time using fluorescence microscopy. We will discuss our findings
showing that a less-virulent isolate (as defined by the previous zebrafish experiment results),
originating from a patient with cepacia syndrome is unable to survive and replicate in
macrophages, whereas an isolate from preceding stages of infection of the same patient was
highly virulent in zebrafish. Further, our preliminary data suggest differences in phagocytic
behaviour by macrophages between an intermediately virulent cepacia syndrome isolate and its
virulent counterpart from the same patient. We will discuss how the zebrafish model may help us
in defining potential differences between isolates during the subsequent stages of infection, and
how this may be combined with other genomic tools to better understand the virulence potential
of ST32.
Supported by: GAUK 307311; IGA MZ NT12405-5; MS MT LD11029
IBCWG 2013 Ann Arbor, MI April 2013
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EXPLORING THE TOPOLOGY OF ArnT FROM Bulkholderia cenopacia
Faviola Tavares-Carreón1 and Miguel A Valvano
1,2.
1Department of Microbiology and Immunology, Centre for Human Immunology, University of
Western Ontario, London, Ontario, Canada. N6A 5C1 2Centre for Infection and Immunity. Queen's University of Belfast, Belfast, UK.
Contact : [email protected]
Burkholderia cenocepacia is an opportunistic pathogen displaying high resistance to
antimicrobial peptides and polymyxins. This pathogen cause severe lung infections in patients
with cystic fibrosis, which are difficult to eradicate. Lipopolysaccharide (LPS) is embedded in
the outer leaflet of the outer membrane of Gram-negative bacteria and contributes to the
structural integrity of the bacterial cell. LPS is a complex molecule consisting of lipid A, core
oligosaccharide (OS), and in some strains, O antigen and acts as endotoxin to elicit strong
inflammatory responses in mammals.
LPS may also be modified by the addition of sugars or phosphate groups, which allows
adaptation to stress condition or resistance to several antibiotics including antimicrobial
peptides. In many Gram-negative bacteria, L-Ara4N (4-amino-4-deoxy-L-arabinose) is added to
lipid A conferring increased resistance to antimicrobial peptides. In B. cenocepacia ArnT protein
modify the LPS by adding L-Ara4N at two different positions: the phosphates in the lipid A and
Ko (D-glycero-D-talo-oct-2-ulosonic acid) residue in the core OS. These modifications in B.
cenocepacia are also critical for bacterial viability. Therefore, we are interested in investigating
the structure-function of ArnT as this protein could be a possible target to develop novel
antibiotics. Understanding the structure function of ArnT, an integral membrane protein that
exerts its enzymatic function on the periplasmic side of the membrane, requires elucidation of its
topology. So far, experimentally based topological model had just been proposed for ArnT from
Salmonella enterica serovar Typhimurium. The topology of ArnT from B. cenocepacia has not
been characterized and also functional regions or residues in the protein are unknown. Therefore,
the elucidation of an accurate topological map of ArnT is essential to understand its function. In
this study, we began to characterize the topology of the ArnT protein from B. cenocepacia. The
C-terminal of ArnT is the less conserved region of this protein and we observed that is essential
for their function and to confer polymyxin B resistance. Interestingly, through LacZ and PhoA
fusions we identified that C-terminal of ArnT is located in the periplasm, which is opposite from
reported for ArnT of Salmonella. Now, we are focus on get a refined topological model of B.
cenocepacia ArnT protein by PEGylation assay. PEGylation is a method for assessing
topological accessibilities through protein modification by the covalent attachment with
monomethoxy-polyethylene glycol-maleimide (mPEG-Mal), and detection using a gel shift
assay. With this method, will be able to provide an ArnT topological map that can be extended to
other integral membrane proteins involved in Lipid A modification.
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STRUCTURAL DIFFERENCES BETWEEN THE GENOMES OF
BURKHOLDERIA CENOCEPACIA STRAINS K56-2 AND J2315
Andrea M. Sass1, Silvia Cardona
3, Miguel A.Valvano
4, Tom Coenye
1, Eshwar
Mahenthiralingam2
1Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium
2Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University,
Cardiff, Wales, UK 3Department of Microbiology, University of Manitoba, Winnipeg, Canada
4Centre for Infection and Immunity, Queen's University of Belfast, Belfast, UK
Contact : Andrea Sass, [email protected]
For molecular biological studies involving Burkholderia cenocepacia, strain K56-2 has often
been used due to its relatively high amenability for genetic manipulation compared to the
genome reference strain J2315. However, a complete genome sequence for K56-2 is not yet
available. Three datasets of high-throughput genome sequencing data are currently available at
NCBI for strain K56-2, all derived from sequencing at the Craig Venter Institute. We examined
these sequences and tested several methods for sequencing assembly. The best results, i.e. the
smallest number of contigs and the largest scaffold, were achieved with multi-span (3 kb and 8
kb) paired end Titanium 454 sequencing data, assembled with the GS assembler which uses the
Newbler algorithm. The assembly produced 798 contigs with an average size of 10580
nucleotides and an N50 contig size of 20000 nucleotides, organised in 7 large scaffolds with
together 7.25 Mb; the largest scaffold being 3.2 Mb in size.
Burkholderia genomes are large and rich in genes occurring in multiple copies, such as rRNA
genes and transposases, but these facts alone cannot account for the relatively high contig
number. To analyse the cause of the high contig number, mapping to the reference genome
sequence of B. cenocepacia J2315 was performed.
The genomes of both strains appear to be largely similar. Certain large genomic regions present
in J2315 are not present in K56-2. For example, the duplicated region on the large replicon of
J2315 (BCAL0969-1928 are identical to BCAL2845-2901) is not duplicated in K56-2. The
duplication within J2315 had let to an inversion within the large replicon. Genomic islands GI3
(BCAL0409a-0420) and GI8 (BCAL2475-2600) on the large replicon of J2315 are not present in
K56-2. A phage and adjacent genes on small replicon of J2315 are deleted from K56-2
(BCAS0440-BCAS0571). Additionally, numerous transposases and SNPs within the K56-2
genome could be mapped with relatively high probability.
Many contig breaks appear to be caused by genes that are present in K56-2, but contain regions
without mapped reads which are apparently not caused by deletion events. More often than not
these regions are correlated with an above average GC-content of >80%. This points to a locally
high-GC content impairing one or several steps within the sequencing workflow, leaving high-
GC regions un-sequenced and probably also causing locally increased number of sequencing
errors. Increased occurrence of sequencing errors can in turn lead to mis-assemblies.
In conclusion, the assembly of a high quality draft genome does not appear possible from the
currently available K56-2 sequencing data. Extensive further sequencing across contig gaps,
potential misassemblies and all transposases will need to be performed to enable accurate
assembly. Sequencing should be repeated with a method able to give reliable results for high-GC
DNA regions, ideally in combination with a method providing reads with higher average length
than transposase genes. A draft assembly, in multiple contigs, and a list of transposases and
SNPs with their location is available for basic comparisons of J2315 with K56-2.
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COMPARATIVE FUNCTIONAL ANALYSIS OF METABOLIC ABILITIES OF BCC
SPECIES USING RECONCILED GENOME-SCALE METABOLIC
RECONSTRUCTIONS
Jennifer A. Bartell*1, Phillip Yen*
1, John J. Varga
2, Joanna B. Goldberg
2 and Jason A. Papin
1
1Biomedical Engineering, University of Virginia, Charlottesville, VA, USA
2Pediatric, Emory University, Atlanta, GA, USA
Contact : [email protected]
Motivation: Cystic fibrosis patients show increased morbidity when infected with species of the
Burkholderia cepacia complex. Studies suggest B. multivorans is the most prevalent Bcc
pathogen in CF, while B. cenocepacia is considered more virulent with higher associated risk of
cepacia syndrome and death. Based on our prior work with Pseudomonas aeruginosa, we
hypothesized that unique metabolic capacities could contribute to B. cenocepacia‟s increased
virulence and sought to quantify functional differences between species using metabolic
modeling.
Methods: To investigate potential mechanisms for this differential virulence and quantitatively
compare metabolic capacity, we built genome scale metabolic reconstructions using genome
annotations of B. cenocepacia J2315 (BC) and B. multivorans ATCC17616 (BM). The
reconstructions connect reactions and metabolites using a stoichiometric matrix and link genes to
their corresponding reactions. Reaction activity is predicted using linear optimization to
maximize the flux through a Burkholderia-specific biomass synthesis reaction (representing
growth). We used a free computational resource to produce initial reconciled draft
reconstructions. By then curating the reconstructions using literature and databases (MetaCyc,
KEGG, etc), we ensured that any differences between the final models were biologically relevant
rather than artifacts of model syntax. We used Biolog and M9 minimal media growth screening
to guide our reconstruction of amino acid catabolism. We also incorporated lipid production
pathways from a previously published smaller model of B. cenocepacia, and expanded their
reconstruction of virulence-related pathways.
Results: BC model iPY1520 accounts for 1520 genes and 1512 reactions, while BM model
iJB1301 accounts for 1301 genes and 1434 reactions. The 122 unique reactions in iPY1520 and
60 unique reactions in iJB1301 are concentrated in fatty acid synthesis, galactose metabolism,
and starch metabolism, but unique reactions were spread through more than 40 canonical KEGG
pathways. iPY1520 and iJB1301 predict 74 and 80 essential genes respectively for growth on
rich media in silico, and match available growth screening data at an accuracy of 84% and
85.7%, respectively. iPY1520 shows enhanced capacity to produce an array of virulence factors,
functional differences in several amino acid catabolic pathways, and a greater number of
metabolic isozymes, suggesting potential increased metabolic adaptability. These models
account for hundreds of additional genes and reactions compared to similar curated
reconstructions. They can be integrated with transcriptomic, proteomic, and metabolomic data to
functionally characterize metabolic differences for specific growth conditions and adapted to
related Bcc strains of interest, enabling genome scale comparative study of metabolism for these
important pathogens.
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INVESTIGATING FACTORS INVOLVED IN THE MAINTENANCE OF THE
‘CHROMOSOME 3’ MEGAPLASMID
Kirsty Agnoli1, Carmen Frauenknecht
1 and Leo Eberl
1
1Department of Microbiology, Institute of Plant Biology, University of Zürich.
Contact : [email protected]
We have previously reported that Bcc chromosome 3 is a non-essential megaplasmid in
many complex members, and suggested that this element be renamed pC3. To date,
representatives of all Bcc species except B. cepacia have been cured of pC3. Amongst these
pC3-null derivatives are three B. cenocepacia strains (H111, MCO-3 and HI2424), however the
highly transmissible ET-12 lineage strain K56-2 remained resistant to such curing.
Integration of an origin of transfer into K56-2 pC3 allowed its transfer into an H111 pC3-
null derivative. K56-2 pC3 could not be cured from this strain, suggesting that the resistance of
K56-2 pC3 to curing was most likely due to the presence of efficient toxin-antitoxin systems
(TASs), rather than essential genes. A scan of putative pC3 contigs from K56-2 using the web-
based TAS finder RASTA-bacteria revealed the presence of two candidate TASs and one lone
antitoxin. Heterologous introduction of the antitoxin components allowed K56-2 to be cured of
pC3 using plasmid incompatibility. Investigations into the pathogenicity of pC3-null K56-2 are
on-going.
To investigate the stability of pC3, a derivative of B. cenocepacia H111 was constructed,
bearing a trimethoprim resistance gene controlled by a tightly regulated LacI repressable
promoter on chromosome 1, and on pC3 lacI. This strain was used to evaluate the rate of pC3
loss under nutrient-rich growth conditions. The rate of loss was calculated at 0.000147
/generation, and it was found that although the experimentally measured exponential growth
rates of wt and pC3-null H111 appeared identical, the pC3-null derivative had a slight growth
advantage over its parent strain (0.017 %).
The prevalence of pC3 was assessed by screening the 111 Bcc isolates available within
our laboratory collection for the presence of pC3 using PCR, pulsed-field gel electrophoresis and
Southern hybridisation. Only five strains were found which appeared to lack pC3, suggestive of
high selective pressures for its maintenance within the Bcc. The nature of these pressures is
currently under investigation.
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GLOBAL MAPPING OF PRIMARY TRANSCRIPTION START SITES IN THE
BURKHOLDERIA CENOCEPACIA J2315 GENOME BY DIFFERENTIAL RNA
SEQUENCING
Andrea M Sass1, Heleen van Acker
1, Konrad Förstner
2, Jörg Vogel
2, Tom Coenye
1
1Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium
2Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany
Contact: Andrea Sass, [email protected]
Since the event of transcriptomics by high-throughput sequencing, an unexpected complexity
within prokaryotic transcriptomes has been revealed. In particular, large numbers of small non-
coding RNAs have been discovered due to RNA-sequencing, and have since been found to play
an important role in regulation of gene expression in bacteria.
To analyse the transcriptome of B. cenocepacia for presence of non-coding RNAs, RNA
extracted from biofilm-grown cells was sequenced with a recently developed method aiming at
specifically identifying primary transcription start sites. RNA sequencing was performed without
fragmentation and mRNA enrichment. cDNA was generated by priming from only the 3‟-end,
and sequencing was performed from only the 5‟-end. The majority of sequencing reads located
therefore at the 5‟-end of transcripts. Prior to cDNA generation, a portion of the RNA samples
was treated with an processive exonuclease specific for 5‟-ends with a monophosphate.
Most mRNAs and non-coding RNAs carry a 5‟-triphosphate, whereas most rRNAs and tRNAs
are processed and carry a 5‟-monophosphate. Exonuclease treatment leads to an enrichment of
primary transcripts and a depletion of processed RNA. Comparison of the exonuclease-treated
sequencing libraries with untreated libraries (differential RNA sequencing) allows therefore to
precisely map the first nucleotide of a transcript, the transcription start site (TSS), by identifying
sites with peaks in read-coverage that increase in magnitude in the exonuclease-treated sample
portion. This allows more accurate analysis of 5‟-UTRs of annotated genes and of promoter
elements such as Pribnow boxes, and identification of primary transcribed non-coding RNAs.
Applied to B. cenocepacia J2315 biofilms, this method revealed high numbers of TSS in
intergenic regions, associated with adjacent genes or orphan. In most cases of intergenic regions
harbouring transcription initiation, multiple TSS occurred on both strands, regardless of the
orientation of adjacent genes. TSS also occurred in high numbers within annotated genes, in
sense and antisense direction. Antisense transcription generally occurred on a large scale.
Numerous putative non-coding RNAs could be identified, and the criteria for defining non-
coding RNAs by this method could be evaluated.
In addition to mapping TSSs and putative non-coding RNAs, the differential RNA sequencing
dataset allowed identification of genes that are expressed in a B. cenocepacia J2315 biofilm, by
evaluating reads mapping to the first 100 bases upstream of a primary TSS of annotated genes.
Numerous primary TSS were located upstream of the first base of an annotated gene, indicating
putatively misannotated genes. TSS mapping also allowed definition of genomic islands as
sequence regions with a locally increased number of intergenic TSS, and in this way additional
previously unannotated genomic islands could be identified.
1
Int'l Burkholderia cepacia Working Group Conference Roster
April 10 - April 13, 2013 University of Michigan, Ann Arbor, MI 48109
NAME TITLE ORGANIZATION E-MAIL ADDRESS ADDRESS TELEPHONE Agnoli, Kirsty
Post-doctoral researcher
University of Zurich
Zollikerstrasse 107 , Zurich, ZH CH-8008 USA
(+41) 0446348268
Amer, Amal Associate Professor
Ohio State University [email protected] 4647 Wilkin Court , New Albany, OH 43054 USA
614 247 1566
Andrade, Angel Postdoctoral Fellow
University of Western Ontario
[email protected] 1151 Richmond Street DSB, Room 3014, Department of Microbiology and Immunology, London, ON N6A 5C1 CANADA
(519) 661-3433
Bartell, Jennifer Grad Student University of Virginia [email protected] 425 Lane Rd MR5 Rm 2041, Charlottesville, VA 22908 USA
8045147986
Bernier, Steve Postdoctoral Fellow
McMaster University [email protected] 1280,Main St. West Department of Medicine, McMaster University, Rm HSC 3N4, Hamilton, ON L8S4K1 Canada
905-525-9140 ext:21905
Brahma, Vijaya Postdoctoral Researcher
University of Toronto [email protected] 101 College Street Latner Thoracic Research Laboratories,Lab 2-501, Toronto, ON M5G 1L7 CANADA
16477807123
2
Brown, Alan Dr University of Exeter [email protected] Geoffrey Pope Building, Stocker Road Biosciences, Exeter, EXETER EX4 4QD UK
01392725526
Burns, Jane Professor Seattle Children's Research Institute/Univ of Washington
[email protected] 1900 Ninth Avenue C9S8-8th Floor, Seattle, WA 98101 USA
206-987-2073
Cardenas, Erick Post Doc, Fellow Michigan State University
[email protected] Dept. of Microbiology & Immununology 2350 Health Sciences Mall, Vancouver, V6T 1Z3 Canada
809-822-5646
Cardona, Silvia Associate Professor
University of Manitoba [email protected] 45 Chancellors Circle Department of Microbiology, Winnipeg, MB R3T 2N2 Canada
1-204-474-8997
Cescutti, Paola Dr Univ Trieste [email protected] via L. Giorgieri 1, Ed C11 Dept Life Sciences, Trieste, - 34127 Italy
+39 040 5588755
Chaparro, Cecilia Respirologist University of Toronto [email protected] 485 University Av CSB - 11C-1190, Toronto, On M5M 3J6 USA
416-340-4996
Clark, Shawn Graduate Student
University of Toronto [email protected] 101 College Street TMDT-2nd Floor, Toronto, ON M5G 1L7 Canada
905-334-6101
Coenye, Tom Prof. Ghent University [email protected] Harelbekestraat 72 , Gent, - 9000 Belgium
+3292648141
3
Cooper, Vaughn Assoc Professor of Microbiology and Genetics
University of New Hampshire
[email protected] 46 College Rd 212 Rudman Hall, University of New Hampshire, Durham, NH 03824 USA
6039887590
Dedeckova, Klara Msc./PhD. student
Charles University in Prague, 2nd Faculty of Medicine
[email protected] V Úvalu 84 , Prague, CZ 150 06 Czech Republic
2 2443 2026
Degrossi, José Ph.D. Universidad de Buenos Aires
[email protected] Junín 956 , Buenos Aires, 1113 Argentina
54114964-8257
Dennehy, Ruth Postgraduate student
ITT Dublin [email protected] Old Blessington road CASH centre, Dublin, Ireland Ireland
00353857120947
Eberl, Leo Prof. University of Zurich [email protected] Zollikerstrasse 107 Dept. of Microbiology, Zurich, ZH 8008 CH
+41446348220
Goldberg, Joanna Professor Emory University School of Medicine
[email protected] 1510 Clifton Rd NE Suite 3009, Atlanta, GA 30322 USA
404-727-6760
Gonzalez, Carlos Professor Texas A&M University [email protected] 120Peterson; 2132 TAMU Department of Plant Pathology and Microbiology, College Station, TX 77843-2132 USA
979-220-6622
Greenberg, David Assistant Professor
University of Southwestern Medical Center
[email protected] 5323 Harry Hines Boulevard Y9.206C MC 9113, Dallas, TX 75390 USA
214-648-2458
4
Hwang, David Pathologist University of Toronto [email protected] 200 Elizabeth Street Department of Pathology, Toronto, ON M5G 2C4 Canada
416-340-4988
Jenul, Christian PhD student University of Zurich [email protected] Zollikerstrasse 107 , Zürich, ZH 8008 Switzerland
0041 44 63 48227
Juarez-Perez, Victor Scientific Advisor ALAXIA Company [email protected] batiment Adénine 60 avenue Rockefeller, Lyon, 69008 FRANCE
33 437 532 640
Kalferstova, Lucie MSc/PhD student
2nd Faculty of Medicine, Charles University in Prague
[email protected] V Uvalu 84 , Prague, CZ 15006 Czech Republic
+420 2 2443 2026
Kopp, Benjamin Assistant Professor
Nationwide Children's Hospital/The Ohio State University
[email protected] 700 Children's Drive , Columbus, OH 43205 USA
6142262497
Lieberman, Tami PhD Student Harvard Medical School
[email protected] 200 Longwood Avenue, WA 536 , Boston, MA 02115
732-501-6444
Lopez De Volder, Agustina
PhD Student Universidad de Buenos Aires
[email protected] Faultad de Farmacia y Bioquimica Viamonte 444, Buenos Aires CUIT, 30-54666656-1 Argentina
54 011 49648200
Mahenthiralingam, Eshwar
Professor Cardiff University [email protected] Main Building, Museum Avenue Rm. 023, Cardiff, SG CF10 3AT USA
+442920875875
McClean, Siobhan Dr ITT Dublin [email protected] Old Blessington Road Centre of Microbial Host Interactions, Tallaght, D24 Ireland
+35314042794
5
Peeters, Charlotte PhD student Ghent University [email protected] Ledeganckstraat 35 , Gent, Fl 9000 Belgium
+32.9.264.5130
Pessi Ahrens, Gabriella
Dr University of Zürich/ Institut of Plant Biology
[email protected] Winterthurerstrasse 190 Microbiology Department, Zürich, ZH 8057 Switzerland
0041446352904
Priebe, Greg Assoc. Prof. of Anesthesia
Boston Children's Hospital
[email protected] 300 Longwood Ave Bader 634, Boston, MA 02115 USA
617-794-0175
Sass, Andrea Dr. Ghent University [email protected] Harelbekestraat 72 , Gent, - 9000 Belgium
+3292648141
Schmid, Nadine MSc Institute of Plant Biology,University of Zurich
[email protected] Zollikerstrasse 107 , Zürich, ZH 8008 Switzerland
+41795626477
Sokol, Pam Professor Emeritus
University of Calgary [email protected] 3330 Hospital Dr NW Department ofMicrobiology, Immunology and Infectious Disease, Calgary, AB T2N4N1 Canada
480-982-0532
Tavares-Carreon, Faviola
Dr. University of Western Ontario
[email protected] 1151 Richmond Street DSB, Room 3014, Department of Microbiology and Immunology, London, ON N6A5C1 CANADA
(519) 661-3433
Tiedje, James M. University Distinguished Professor and Director
Michigan State University
[email protected] Michigan State University 540 Plant and Soil Science Building, East Lansing, MI 48824-1325 USA
517-353-9021
6
Tullis, Elizabeth CF Clinic Director
University of Toronto [email protected] 83 Humberview Road , Toronto, ON M6S 1W9 Canada
416 864 5406
Van Acker, Heleen PhD student Ghent University [email protected] Harelbekestraat 72 , Gent, - 9000 Belgium
+3292648141
Vandamme, Peter Professor Ghent University [email protected] Ledeganckstraat 35 , Gent, Fl 9000 Belgium
+32.9.264.5113
Venturi, Vittorio Group Leader ICGEB [email protected] Padriciano 99 Bacteriology, Trieste, I 34149 Italy
+390403757319
Vergunst, Annette Dr Universite de Montpellier/ INSERM
[email protected] 186 chemin du Carreau de lanes INSERM U1047, UFR Medecine, Nimes, 30908 France
+33466028157
Waters, Valerie Assistant Professor
Hospital for Sick Children
[email protected] 555 University Avenue , Toronto, ON M5G 1X8 Canada
416-813-7654
Yoder-Himes, Deborah
Assistant Professor
University of Louisville [email protected] 139 Life Sciences Building , Louisville, KY 40292 USA
502-852-0991