Download Vaneechoutte. 2016. Research at the LBR at the dawn of

The LBR @ the dawn of 2016
I. Overview
People
Logistic tasks and challenges
Research areas
Techniques
Collaborations
Publications
II. A more detailed look at some of the past, present and future research
I. Phage Therapy/Immunomodulation by phages
II. Microbiome research
III. Biofilm Research
IV. VMF Research
V. Detection, quantification, identification
VI. Fungi
VII. Genotyping
III. Future Research Possibilites
The LBR @ the dawn of 2016. People
UGent
Mario Vaneechoutte, Principal Investigator
Muco
Leen Van Simaey, Technician 1: Pseudomonas aeruginosa – Cystic fibrosis - McRAPD
[Min. Defense] Maya Merabishvili, PostDoc 1: Bacteriophage therapy
UGent
IWT
PWO
VLIR-UOS
VLIR-UOS
[ITM]
FWO
OPATHY
Piet Cools, PhD 1: Streptococcus agalactiae/E. coli – Africa – MALDI-TOF typing
Jonas Van Belleghem, PhD 2: Bacteriophage immunomodulation - FISH
Hans Duyvejonck, (PhD 3?): Cytokine quantification – Candida detection – HRM
Abel Abera Negash, PhD 4: Streptococcus pneumoniae – Ethiopia (Adis Ababa)
Simon Masha, PhD 5: Trichomonas vaginalis and STI – Kenia (Kilifi)
Liselotte Hardy, PhD 6: Vaginal biofilm – FISH – Gardnerella vaginalis
Tessa Gryp, PhD 7: Intestinal microbiome of dialysis patients - Culture
To be selected, PhD 8: Candida vaginitis metagenomics – Host/Pathogen interaction
Student
Leander Jonckheere (Leen), Master Thesis Med 1: McRAPD typing P. aeruginosa CF
Student
Sophie Decuypere (Piet), Master Thesis BioMed 2: MALDI-TOF typing
Student/IWT Cara De Galan (Jonas), Master Thesis BioMed 3, (PhD 9): Bacteriophage phagocytosis
Student
Meryam Ahalli (Tania/Mario), Master Thesis BioMed 4: Vaginal biofilm - FISH
Student
Elke Maquestiau (Leen), Master stage Biotech 1: Gardnerella vaginalis sialidase
Student
Liesl Phlypo (Jonas), Master stage BioMed 2: Bacteriophage phagocytosis
Student
Maholo Pascaline (Tessa), Master stage BioMed 3: Intestinal microbiome
Student
Jarne Van Belle, Trainee Technician Vesalius (Hans)
Visitor
Visitor
Cedric Valcke (Jonas/Maia), Scheppers Wetteren
Guy Mulinganya (MD) (Simon), one month visit?
via Daniel De Vos and Steven Callens
Visiting postdoc Guido Lopes [Postdoc 2]
Visiting postdoc Nabil El Aila [Postdoc 3]
Logistic tasks and challenges of the LBR
New funding
Gates: Phage immunomodulation. Submitted
IWT Cara: Phage phagocytosis?
Vesalius PWO: Phage therapy?
Logistics-organisation
Move to MRB2: 18-22 april!!!
Freezer min 80 (new one has been ordered)
Collection files (Excel)
Folder organisation and clearance
EasyMag problems: new extractor (Isogen)?
Transport of materials from and to Africa
Internet access
BioNumerics software
Practical courses biomedical sciences: bachelors 29feb/2mar; 7mar/9 mar
masters: 17-19 February
New technical research possibilities
MALDI + BioNumerics
Phagocytosis techniques
Microscopy:
Use of our own light microscope
Electron microscopy (phages, phagocytosis, biofilms)
FISH optimization for samples: currently too dehydratated
Binocular microscope: colony types, culture of anaerobe intestinal microbes
Minion Deep Sequencing?
Research areas @ the LBR
Taxa
Viruses
Bacteriophages: Maya, Jonas, Cara
Eukaryote viruses: Trichomonas vaginalis virus (TVV): Masha
Influenza: Tessa (Gryp = flew)
Prokaryotes (Bacteria)
Gardnerella vaginalis: Guido, Leen, Liselotte, Meryam
Pseudomonas aeruginosa: Leen, Leander, Piet
Streptococcus agalactiae (GBS): Piet
Streptococcus pneumoniae: Abel
Intestinal microflora (IMF): Tessa
Naso-oropharyngeal microflora (OMF): saliva: Guido, middle ear: Leen
Vaginal microflora (VMF): Piet, Masha, Liselotte
Nosocomial bacteria (Enterobacter cloacae): Piet, Sophie
Urinary tract: Piet, Leen
Mycobacteria: Maia
Acinetobacter: Maia, Mario
Eukaryotes
Yeasts: Hans, Tessa
Trichomonas vaginalis: Masha
Humans: PBMCs: Jonas, Cara, OPATHY project
Research areas @ the LBR
Research Areas
Identification: 16S rRNA gene sequencing, [cpn60], MALDI, species specific qPCR, ITS2-HRM
Detection/quantification: yeasts in blood, uropathogens in urine,
uncultivable bacteria in faeces, T. vaginalis & STIs in vaginal swabs
Typing: MALDI & McRAPD  hospital epidemiology, G. vaginalis, P. aeruginosa, GBS
Host/Pathogen interaction: yeasts (OPATHY), sialidase-G. vaginalis-BV, biofilms
Microbiome characterization: OMF (Brusselle), IMF (Glorieux), VMF (Opathy)
Immunomodulation: phages, bacteria
Antibiotic resistance: biofilm research (EVSM), phage therapy
Microbiomes/types of infection
OMF: adenoid & middle ear - nasopharynx
VMF: microbiome, Gardnerella, Atopobium, biofilm, Trichomonas, GBS, STIs
yeasts: Candida, host-pathogen (transcriptomics, OPATHY)
Airways: CF: Pseudomonas, (Burkholderia phages) – Mycobacterium (phages)
Urinary Tract infections
Immune system (blood): phage immunomodulation
IMF: gut microbiome of hemodialysis patients
Hospital infections (nosocomial infections): MALDI, yeasts
Blood infection: Candidemia (upcoming study Hans)
Water contamination (PIDPA)
Horse shit: Acinetobacter gandensis
Techniques @ the LBR (and collaborating centers)
Culture: optimization of urine culture in UTI
uncultivable (intestinal) organisms – anaerobic culture (BugBox)
biofilm in ex vivo sputum model, middle ear (COME)
phage propagation
Nucleotide extraction:
Qiazol - EasyMag – Phenol - QiaGen – [(PMA): Isogen – Molysis]
protease – DNAse – mutanolysin – lyticase – alkaline extraction – boiling/freezing
Amplification:
(PMA: propidium monoazide) qPCR: specific (primers, probes), universal (ITS2, 16S, cpn60)
[TaqMan array] icw AZ St. Jan Bruges, VMF
RT-qPCR: rRNA (viability PCR), mRNA (gene expression PCR), dsRNA viruses (TVV)
Characterization of amplification products:
Agarose gel electrophoresis
Melting curve analysis: McRAPD, ITS2-HRM, specificity control of species specific PCRs
Sequencing: GATC outsourcing
Deep sequencing: Roche Junior 454 – BGI outsourcing – Barcelona OPATHY outsourcing
MALDI-TOF & BioNumerics: identification – typing
FISH + Microscopy: CLSM:
P. aeruginosa in CF sputum biofilm,
H. influenzae in middle ear fluid
G. vaginalis on vaginal rings
Immunity testing:
Phagocytosis (phages)
PBMC platform: immunomodulation by phages and P. aeruginosa/S. aureus
Collaborations of the LBR (1)
DEPARTMENT (Clinical Chemistry - GE06)
Bacteriology Routine Laboratory (2P8): Geert Claeys, Jerina Boelens, Liza Padalko
Hospital Hygiene: Isabelle Leroux-Roels
Toxicology: Alain Verstraete, Veronique Stove, Katrien Forier
CEVAC: Frédéric Clement, (Philip Meuleman): PBMC stimulation
AIDS Reference Center: Chris Verhofstede, Kenny Dauwe
HOSPITAL (GUH)
UroGynaecology: Hans Verstraelen: Candida OPATHY, Review HIV-GBS Piet
General Practitioners: Stefan Heytens, Urinary Tract Infection, Candida OPATHY
ENT: Inge Dhooge, Helen Van Hoecke, Edward Lambert, Ann-Sophie De Paepe: COME
ENT Research: Claus Bachert, Nan Zhang, Feng Lan, Gaby Holtappels: S. aureus in nasal polyps
Nephrology: Griet Glorieux, (Geert Huys): IMF
MucoCenter: Frans De Baets, Sabine Van daele, Petra Schelstraete: CF
[Medical Genetics: Paul Coucke: primer development cytokines?]
Asthma Research Center: Guy Brusselle: Azithromycine in asthma, OMF
UNIVERSITY (Ugent)
VIB Protein Core Facility: Jurgen Haustraete: LPS quantification
LEGEST: Kathleen Vancraenenbroeck, Karen Heyninck: cell microscopy
Electron Microscopy: Riet De Rycke (transmission); Wim Bert (Marjolein Couvreur) (scanning)
Department of Plant Systems Biology (VIB): Dries Vaneechoutte: useful macros
Collaborations of the LBR (2)
BELGIUM
BWC, Queen Astrid Military Hospital, Nederoverheembeek,
Jean-Paul Pirnay, Daniel De Vos, Gilbert Verbeken
Bacteriophage therapy
Vesalius Hogeschool: Els Van Mechelen, Stefan Vermeulen Interleukines qPCR (PERPLEX)
New phage project?
Rehabilitation Centre De Haan: Bettina Wurth:
AUH (UZA): Stijn Verhulst, Erwin Ho:
Pseudomonas aeruginosa
Achromobacter xylosoxidans
KULeuven: Rob Lavigne:
Phages
KULeuven: Jeroen Raes, Marie Joossens:
Deep sequencing IMF
Institute Tropical Medicine: Tania Crucitti, Vicky Jespers:
VMF
Trichomonas vaginalis
St. Jan Bruges: Patrick Descheemaecker, Marijke Reynders: TaqMan DNA array for STI
Royal Belgian Institute of Natural Sciences, Ostend: Jan Haelters: marine biology
Collaborations of the LBR (3)
ABROAD
Amsterdam, the Netherlands: Janneke Van de Wijgert: vaginal microflora
Rome, Italy: Renato Fani:
Acinetobacter
Institut Pasteur Lille, France: Laurence Delhaes:
Fungi
KEMRI, Kilifi, Kenya: Eduard Sanders:
Trichomonas vaginalis
Adis Ababa University, Ethiopia: Daniel Asrath:
Streptococcus pneumoniae
Saskatchewan, Canada: Janet Hill, John Schellenberg:
Gardnerella vaginalis
Kiev, Ukrain: Larisa Churkina:
Batumin,
a staphylococcal antibiotic, produced by Pseudomonas batumici
Baylor College of Medicine, Houston, TX: Kevin Riehle, Matthew Roth: AZISAST
OPATHY Consortium: Spain, Germany, Austria, Ireland, …
Fungi
EDCTP consortium (Kenya, Rwanda, South-Africa)
VMF
[Bukavu, Congo]: Guy Mulinganya
VMF
COMPANIES
Applied Maths (St. Martens-Latem, Belgium): Katleen Vranckx: BioNumerics
Pfizer (Brussels, Belgium): Patricia Slachmuylders:
MAMBO-study (OMF)
Lysando (Germany): Kristin Neumann:
Artilysin
PIDPA (Antwerp, Belgium): Katrien De Maeyer: pollution with Enterobacter amnigenus
Published/Submitted this year (1)
1. Duyvejonck, H., P. Cools, J. Decruyenaere, K. Roelens, L. Noens, S. Vermeulen, G. Claeys, E. Decat, E.
Van Mechelen, and M. Vaneechoutte. 2015. Comparison between culture and High Resolution Melting
analysis (HRM) of the amplified ITS2 region for the detection and identification of yeasts from clinical
samples. PLOS ONE. http://pone.edmgr.com/ Accepted 1506115.
2. Cools, P., E. Ho, K. Vranckx, P. Schelstraete, B. Wurth, H. Franckx, G. Ieven, L. Van Simaey, S. Van
daele, S. Verhulst, F. De Baets, and M. Vaneechoutte. 2016. Epidemic Achromobacter xylosoxidans
strain among Belgian cystic fibrosis patients and review of literature.
J Cystic Fibrosis. Rejected without review. BMC Microbiol. Resubmitted 151202.
3. Van Hoecke, H., A.-S. De Paepe, E. Lambert, J. D. Van Belleghem, P. Cools, L. Van Simaey, P.
Deschaght, M. Vaneechoutte, and I. Dhooge. Haemophilus influenzae biofilm formation in chronic otitis
media with effusion. Submitted Int. J. ORL. December 2015
4. Merabishvili, M., M. Vaneechoutte, D. De Vos, R. Monserez, J.-P. Pirnay. Stability of bacteriophages in
burn wound care products.
Submitted to The JEAN-PAUL Int J for Delaying Publication of Interesting Results
Published/Submitted this year (2)
5. Hardy, L., V. Jespers, N. Dahchour, L. Mwambarangwe, V. Musengamana, M. Vaneechoutte, and T.
Crucitti. 2015. Unravelling the bacterial vaginosis-associated biofilm: a multiplex Gardnerella vaginalis
and Atopobium vaginae Fluorescence In Situ Hybridization assay using peptide nucleic acid probes.
PLoS ONE 10(8): e0136658. doi:10.1371/journal.pone.0136658.
6. Hardy, L., V. Jespers, S. Abdellati, I. De Baetselier, L. Mwambarangwe, V. Musengamana, J. van de
Wijgert, M. Vaneechoutte, and T. Crucitti. 2015. Atopobium vaginae in bacterial vaginosis-associated
biofilm. Submitted PlosOne: August 2015.
7. Hardy, L., V. Jespers, S. Abdellati, I. De Baetselier, L. Mwambarangwe, V. Musengamana, J. van de
Wijgert, M. Vaneechoutte, and T. Crucitti. A fruitful alliance: the synergy between Atopobium vaginae
and Gardnerella vaginalis in bacterial vaginosis-associated biofilm. Sex Transm Infections.
8. Hardy, L., V. Jespers, S. Abdellati, I. De Baetselier, L. Mwambarangwe, V. Musengamana, J. van de
Wijgert, M. Vaneechoutte, T. Crucitti. A fruitful alliance: the synergy between Atopobium vaginae and
Gardnerella vaginalis in bacterial vaginosis-associated biofilm. Submitted to Sex Transm Infections.
9. Jespers V., T. Crucitti, J. van de Wijgert, M. Vaneechoutte, S. Delany-Moretlwe, M. Mwaura, S.
Agaba, J. Menten. 2015. A DNA tool for early detection of vaginal dysbiosis in African women. Res.
Microbiol. doi:10.1016/j.resmic.
10. Schellenberg, J.J., T. P Jayaprakash, N. Withana Gamage, M.H. Patterson, M.H., M. Vaneechoutte,
and J.E. Hill. 2015. Gardnerella vaginalis subgroups defined by cpn60 sequencing and sialidase activity
in 112 isolates from Canada, Belgium and Kenya. PlosOne Accepted.
Published/Submitted this year (3)
11. Cools, P., V. Jespers, L. Hardy, T. Crucitti, S. Delany-Moretlwe, M. Mwaura, G.F. Ndayisaba, J.H.H.M.
van de Wijgert, and M. Vaneechoutte. A multi-country cross-sectional study of vaginal carriage of
group B streptococci (GBS) and Escherichia coli in resource-poor settings: prevalences and risk factors.
Plos One Re-re-submitted 151206.
12. Jespers V, van de Wijgert J, Cools P, Verhelst R, Verstraelen H, Delany-Moretlwe S, Mwaura M,
Ndayisaba GF, Mandaliya K, Menten J, Hardy L, Crucitti T; Vaginal Biomarkers Study Group. 2015. The
significance of Lactobacillus crispatus and L. vaginalis for vaginal health and the negative effect of
recent sex: a cross-sectional descriptive study across groups of African women. BMC Infect Dis 15: 115.
doi: 10.1186/s12879-015-0825-z.
13. Kyongo JK, Crucitti T, Menten J, Hardy L, Cools P, Michiels J, Delany-Moretlwe S, Mwaura M,
Ndayisaba G, Joseph S, Fichorova R, van de Wijgert J, Vanham G, Ariën KK, Jespers V. 2015. Crosssectional analysis of selected genital tract immunological markers and molecular vaginal microbiota in
Sub-Saharan African women, with relevance to HIV risk and prevention. Clin Vaccine Immunol 22:526538. doi: 10.1128/CVI.00762-14.
Published/Submitted this year (4)
14. Churkina, L., M. Vaneechoutte, E. Kiprianova, N. Perunova, L. Avdeeva, and O. Bukharin. 2015.
Batumin - a selective inhibitor of staphylococci - reduces biofilm formation in methicillin resistant
Staphylococcus aureus. Open J Med Microbiol 5: 193-201.
15. Perrin, E., M. Fondi, I. Maida, A. Mengoni, C. Chiellini, S. Mocali, P. Cocchi, S. Campana, G.
Taccetti, M. Vaneechoutte, and R. Fani. 2015. Genomes analysis and bacteria identification: the use
of overlapping genes as molecular markers. J. Microbiol. Methods. Accepted August 1st 2015.
16. Fondi, M., I. Maida, E. Perrin, V. Orlandini, L. La Torre, A. Negroni, G. Zanaroli, F. Fava, F. Decorosi,
L. Giovannetti, C. Viti, M. Vaneechoutte, L. Dijkshoorn, and R. Fani. 2015. Genomic and phenotypic
characterization of the Acinetobacter venetianus species. Submitted 151004: Nature Scientific
Reports.
17. Nguyen, L. D. N., P. Deschaght, S. Merlin, A. Loywick, C. Audebert, S. Van daele, E. Viscogliosi, M.
Vaneechoutte, and L. Delhaes. Effects of propidium monoazide (PMA) treatment on cystic fibrosis
airway mycobiome and bacteriome analysis. SREP-15-33879. Nature Scientific Reports. Submitted
151118.
18. Pirnay, J.-P., B. G. Blasdel, L. Bretaudeau, A. Buckling, N. Chanishvili, J. R. Clark, S. Corte-Real, L.
Debarbieux, A. Dublanchet, D. De Vos, J. Gabard, M. Garcia, M. Goderdzishvili, A. Górski, J.
Hardcastle, I. Huys, E. Kutter, R. Lavigne, M. Merabishvili, E. Olchawa, K. J. Parikka, O. Patey, F.
Pouilot, G. Resch, C. Rohde, J. Scheres, M. Skurnik, M. Vaneechoutte, L. Van Parys, G. Verbeken, M.
Zizi, and G. Van den Eede. 2015. Quality and safety requirements for sustainable phage therapy
products. Pharm. Res. 32: 2173-2179. doi: 10.1007/s11095-014-1617-7.
19. Pirnay, J.-P., G. Verbeken, M. Vaneechoutte, S. Jennes, M. Merabishvili, I. Serrano, and D. De Vos.
2015. Bacteriophages as antibacterial agents: Why are we facing an antibiotic crisis and how could
bacteriophages be of help? Frontiers Antimicrob Agents 1: 16-38.
A more detailed look at some of the
past, present and future research
I. Phage Therapy/Immunomodulation by phages
Maia, Jonas, Cara, Hans, (Liesl, Jarne)
II. Microbiome research
Guido, Tessa, Pascaline, Opathy
III. Biofilm Research
Liselotte, Jonas, Leen, Meryam
IV. VMF Research
Piet, Liselotte, Masha, Guido, Meryam
V. Detection, quantification, identification
Piet, Leen, Hans, Liselotte, Abel, Masha, Tessa
VI. Fungi
Hans, Leen, Opathy
VII. Genotyping
Piet, Leen, Sophie, Guido, Elke, Leander
I. Phage Therapy/Immunomodulation by phages
Maya Merabishvili (Ministry of Defense)
Icw Jean-Paul Pirnay, Daniel De Vos, Gilbert Verbeken
New project: bacteriophages for Mycobacterium tuberculosis
rapidly growing mycobacteria
Looking for phages against
Burkholderia multivorans/Pseudomonas aerigunosa (CF, burns)
Acinetobacter baumannii
Phage therapy issues/ NATO and phage therapy/ Phage stability study (submitted)
NATO: was member of HFM-RTG 194 :
Development of Evidence-based Guidelines for the Management of Severely Burnt Patients
and introduced discussion on the phage therapy option.
Assisting Jonas, Cara, and many other phage enthusiasts
Phage therapy remains a promessing option for the future and is constantly in the news.
I. Phage Therapy/Immunomodulation by phages
Maya Merabishvili: phage stability in burn wound products
1E+10
1E+10
1E+10
P.O.H.
Colisitin
Flammazin
1E+09
1E+09
1E+09
100000000
100000000
100000000
10000000
10000000
10000000
1000000
1000000
ISP
ISP
ISP
PNM
PNM
PNM
14.1
14.1
14.1
Acibel004
Acibel004
Acibel004
Acibel007
Acibel007
Acibel007
100000
100000
10000
10000
1000
1000
1000
100
100100
10
10 10
1
1 1
Control
Control
Control
0
0 0
2h
2h2h
4h
4h4h
24h
24h
24h
I. Phage Therapy/Immunomodulation by phages
Jonas D. Van Belleghem
PBMC stimulation with phage PNM and its host P. aeruginosa
RNAseq results
RT-qPCR results for 7 donors, including duplicate stimulations
Research: Host-Pathogen Interaction: Immunomodulation by bacteriophages
Background/SetUp: Do phages have an (anti-)inflammatory effect?
 additional curative effect in bacterial infection
 phage products useful as new anti-inflammatory products?
Funding: IWT, PhD. Bench fee: 4000 €
Gates foundation submitted
Techniques:
PBMC stimulation, RNA-extraction, RNA-seq, RT-qPCR,
ELISA, In situ cytokine staining (ICS), protein-protein interaction
@ LBR: JVB
Collaboration with:
Philip Meuleman-Frédéric Clement (CEVAC, GEO6),
Rob Lavigne (KULeuven, copromoter)
Results: Research ongoing: LPS problems!
Publication Status: ?
I. Phage Therapy/Immunomodulation by phages
Experimental setup
RT-qPCR
RNA
RNA-Seq
I. Phage Therapy/Immunomodulation by phages
Whole transcriptome analysis
Result:
DiffGene (Up)
Phage PNM lysate
P. aeruginosa strain PA573
704
996
DiffGene (Down) 392
1377
Total
2373
1096
 Selection for RT-qPCR
Anti-inflammatory
↑
↓
IL10
IL6
SOCS3
TGFBI
IL1RN
CD14
LYZ
Pro-inflammatory
↑
TNFα
IL1A
IL1B
CXCL1
CXCL5
Differential gene expression of selected genes: RT-qPCR
for 5 different bacteriophages, 2 purifications, 2 bacterial hosts
2 purification strategies:
LPS purified phage lysate
LPS and CsCl purified
LPS purification only in theory!!!
Still large amounts of LPS are present!
5 different phages:
P. aeruginosa phage PNM
P. aeruginosa phage LUZ19
P. aeruginosa phage GE_vB_Pae_Kakheti25
P. aeruginosa phage 14-1
S. aureus phage ISP
2 bacterial hosts:
P. aeruginosa strain PA573
S. aureus strain 6538
6 PBMC donors
I. Phage Therapy/Immunomodulation by phages
Cara De Galan
Functional assays for immune stimulation by phages:
Macrophage adherence – phagocytosis of phages
Electron microscopy: uptake of phages?
Fluorescence microscopy
qPCR for phage quantification: contamination??
Funding: New PhD? Via IWT
I. Phage Therapy/Immunomodulation by phages
Hans Duyvejonck. Development of cytokine qPCR
Research: PERPLEX
Background/SetUp: Optimization of qPCR for different cytokines
Funding: Association UGent: Vesalius: PWO (Project Wetenschappelijk Onderzoek)
Techniques: PBMC, RNA-extraction, RT-qPCR
@ LBR: HD, JVB
Collaboration with:
Els Van Mechelen, Stefan Vermeulen (Vesalius)
Chris Verhofstede (ARL)
[Paul Coucke (Medical Genetics)]
Results: First optimizations done
Hampered by teaching obligations …
Publication Status:
A more detailed look at some of the
past, present and future research
I. Phage Therapy/Immunomodulation by phages
Maia, Jonas, Cara, Liesl, Hans, Jarne
II. Microbiome research
Guido, Tessa, Pascaline, Opathy
III. Biofilm Research
Liselotte, Jonas, Leen, Meryam
IV. VMF Research
Piet, Liselotte, Masha, Guido, Meryam
V. Detection, quantification, identification
Piet, Leen, Hans, Liselotte, Abel, Masha, Tessa
VI. Fungi
Hans, Leen, Opathy
VII. Genotyping
Piet, Leen, Sophie, Guido, Elke, Leander
II. Microbiome research
1. AZISAST (OMF)
Guido Lopes dos Santos Santiago
Background/Setup: deep sequencing of oronasopharyngeal microflora
of asthma patients
treated/nontreated with azithromycin
incl. responders and non-responders.
Funding: Guy Brusselle, 5000 Euro
@ LBR: [GLS]
Collaboration with: Guy Brusselle (GE01)
Kevin Riehle (GenBoree, Baylor College of Medicine, Houston, Texas.
Publication status: first draft sent to GenBoree
II. Microbiome research
1. AZISAST (OMF)
Guido Lopes dos Santos Santiago
What is the influence of long term azithromycin treatment
on the microbial species richness and species diversity of the oropharynx ?
Do there exist differences in the oropharyngeal microbiome between individuals
which could be predictive for the therapeutic response to azithromycin?
Set up:
Microbiome analysis of the oropharynx of three groups of asthmatic patients,
azithromycin (AZ) responders
AZ non-responders
patients receiving placebo
II. Microbiome research
1. AZISAST (OMF)
Guido Lopes dos Santos Santiago
A total of 1429 OTUs were recognized.
Of these, only 59 (= 0.04% of the number of OTUs)
were represented by 0.2% or more of the reads
These 59 OTUs accounted for 89.7% of all the reads.
Moreover, only 13 OTUs accounted for 64.0% of the reads:
Prevotella melaninogenica (14.57% of all reads)
Streptococcus mitis (10.68%)
Streptococcus parasanguinis (6.54%)
Veillonella atypica (5.41%)
Streptococcus salivarius (4.13%)
Granulicatella adiacens/G. para-adiacens (3.85%)
Leptotrichia wadei (4.16%)
Fusobacterium periodonticum (3.01%)
Neisseria flavescens (2.94%)
Gemella sanguinis (2.60%)
Actinomyces graevenitzii (2.45%)
Terrahaemophilus aromaticivorans (2.11%)
Prevotella pallens (1.99%).
II. Microbiome research
1. AZISAST (OMF)
Guido Lopes dos Santos Santiago
Moreover, some of these 59 most prevalent species
were only abundant in one or two subjects:
e.g. Abiotrophia defectiva (2 patients accounted for 86% of the reads),
Actinomyces sp. clone CT047 (2 patients = 62%),
Actinomyces graevenitzii (one patient = 59%),
Alloprevotella tannerae (1 patient = 63%),
Fusobacterium nucleatum subsp. vincentii (1 patient = 60%),
Mycoplasma salivarium (1 patient = 94%),
Important message from an old man with limited statistical skills:
study your raw data before starting with blind statistical analyses!!
Of the 59 abundantly present OTUs (Supplementary Table 1),
Streptococcus salivarius (the fifth most abundant species) increased in all 9
azithromycin treated patients, between 1.5 and 79.8 fold (average 16-fold change) in
treatment samples (V3 samples), when compared to pretreatment samples (V2).
To the contrary, the OTU with highest similarity to Leptotrichia wadei (97-98%)
decreased on average 33-fold from an average of 504 reads (SD = 878) per
pretreatment sample (V2) to 16 reads (SD = 38) per treatment sample (V3), although
for patient 115 a slight increase was observed (from 39 to 111 reads). ***
II. Microbiome research
1. AZISAST (OMF) Guido Lopes dos Santos Santiago
AZ responders
AZ nonresponders
Remark: also S. salivarius increases, but this can’t be seen at the genus level
 Analyses at the genus level (as most deep sequencing studies): not very useful
II. Microbiome research
2. Toxin producing gut microbiome
of hemodialysis patients
Tessa Gryp
Cardiovascular problems for hemodialysis patients
These have been linked to toxins produced by gut microbiome
 Deep sequencing of GMB for different nephropathology
(incl. hemodialysis) patient groups: Marie Joossens (KULeuven):
Are there differences between gut microbiomes?
Research Day 2016 (Wetenschapsdag) op 16 maart 2016.
Individual variation of hippuric acid and p-cresyl sulfate plasma levels
correlate with variation of intestinal microbial phylotypes
in hemodialysis patients.
 Culture efforts to pinpoint/isolate the bacterial species responsible
for this toxin production: Tessa.
II. Microbiome research
2. Toxin producing microflora of hemodialysis patients
Tessa Gryp
Challenge: culture of not yet cultured bacteria
“Too boldly culture what no woman has cultured before”
II. Microbiome research
2. Toxin producing microflora of hemodialysis patients
Tessa Gryp
Applications
Up to date CellCrown™ cell culture inserts have been used in cell culturing
experiments in the research of biomaterials, tissue engineering, regenerative
medicine, pharmaceuticals, cancer models, and skin substitutes for animal free
testing. The studied materials have been varying from biological tissue samples to
completely synthetic ones, these studies are listed in Scaffdex web site.
II. Microbiome research
2. Toxin producing microflora of hemodialysis patients
Tessa Gryp
High diluate of faeces
(= few cells)
Plain agar
Isolated unknown strain
Plain agar + enrichment
(amino acids)
Filter: 0.2 or 0.4 µm
Autoclaved faecal suspension
= complex nutrients
Or: broth with specific enrichments
II. Microbiome research
3. Characterization of VMF with emphasis on transcriptomics
Host pathogen interaction during Candida vaginitis
See VI. Fungi. OPATHY
A more detailed look at some of the
past, present and future research
I. Phage Therapy/Immunomodulation by phages
Maia, Jonas, Cara, Hans, (Liesl, Jarne)
II. Microbiome research
Guido, Tessa, [Opathy], (Pascaline)
III. Biofilm Research
Liselotte, Jonas, Leen, Meryam
IV. VMF Research
Piet, Liselotte, Masha, Guido, Meryam
V. Detection, quantification, identification
Piet, Leen, Hans, Liselotte, Abel, Masha, Tessa
VI. Fungi
Hans, Leen, Opathy
VII. Genotyping
Piet, Leen, Sophie, Guido, Elke, Leander
III. Biofilm Research: 1. COME
Research: Resistance-Biofilms: Chronic otitis media with effusion (COME)
Background/SetUp: Is the adenoid a reservoir for chronic infection in COME?
Can biofilm of Haemophilus influenzae be shown in middle ear fluid (MEF)?
Funding: None
Techniques: FISH, qPCR, culture, genotyping with McRAPD
@ LBR: LVS, JVB (FISH for biofilm!)
Collaboration with: Inge Dhooge-Helen Van Hoecke (ENT)
Ann-Sophie De Paepe & Edward Lambert (master students medicine)
Results: Same species isolates can be cultured from ears and adenoid in 84.6% of the patients
Strong correspondence between genotypes of isolates from same patient: 81.6%
Excellent FISH CLSM biofilm images
FISH confirms presence of H. influenzae biofilms in MEF of children with COME.
Publication Status:
Submitted to European Archives of Oto-Rhino-Laryngology and Head & Neck, 151215.
Not yet rejected 151224.
III. Biofilm Research: 2. CF sputum
Research: Resistance-Biofilms: P. aeruginosa biofilms in airways of CF patients
Background/SetUp:
Which treatment is capable of eradicating P. aeruginosa biofilms from patient sputum?
Funding: Belgian Cystic Fibrosis Association
Techniques: ex vivo sputum model: add treatment directly to P. aeruginosa positive CF sputum
analyse effect with culture, FISH, qPCR
@ LBR: LVS, JVB (FISH for biofilm!)
Collaboration with: CF pediatricians, CF physiotherapists
Results: Ongoing since more than 5 years. Current focus on azithromycin, tobramycin,
artilysin and EDTA
Artilysin kills strains immediately in broth: LINK, not in sputum
EDTA: 0-32 mM: kills in sputum at 4 mM, not in broth!
Further work:
Malic acid – Citric acid
Phage (+ EDTA + Citric acid + Azithro?)
Publication Status: Negative artilysin results: difficult to publish
Should publish EDTA results!
III. Biofilm Research: 2. CF sputum
Research: Resistance-Biofilms: P. aeruginosa biofilms in airways of CF patients
III. Biofilm Research:
3. BV in vaginal smears
see IV. VMF: Liselotte Hardy
A more detailed look at some of the
past, present and future research
I. Phage Therapy/Immunomodulation by phages
Maia, Jonas, Cara, Hans, (Liesl, Jarne)
II. Microbiome research
Guido, Tessa, [Opathy], (Pascaline)
III. Biofilm Research
Liselotte, Jonas, Leen, Meryam
IV. VMF Research
Piet, Liselotte, Masha, Guido, Meryam
V. Detection, quantification, identification
Piet, Leen, Hans, Liselotte, Abel, Masha, Tessa
VI. Fungi
Hans, Leen, Opathy
VII. Genotyping
Piet, Leen, Sophie, Guido, Elke, Leander
IV. VMF Research
Liselotte Hardy: vaginal biofilms
FISH to visualize G. vaginalis and A. vaginae biofilm on vaginal rings
FISH of G. vaginalis and sialidase to distinguish BV and normal VMF
Research: Microbiome studies: Vaginal microflora characterization with FISH
Background/SetUp:
FISH for Gardnerella vaginalis & Atopobium vaginae to differentiate healthy VMF from
bacterial vaginosis.
Role of sialidase positive and negative G. vaginalis in BV biofilm.
Funding: Institute Tropical Medicine, PhD  None at present
Techniques: FISH, qPCR
@ LBR: PC, JVB
Collaboration with: Tania Crucitti, Vicky Jespers, ITM, Antwerp.
Status:
1. Excellent biofilm images, confirming role of G. vaginalis and A. vaginae in BV biofilm.
2. Link between sialidase positivity and G. vaginalis biofilm activity.
Meryam heeft nu signaal met 3 probes, nu is het probleem dat ook de GV die PCR neg zijn
voor sialidase ook een FISH signaal geven. Vreemd omdat 2 van de 3 probes in feite de primer
sequenties zijn, ik vermoed dat het een concentratie probleem is .
Update 160112
SEM ringen (bij)
Maandag 11/1: eerste try-out met 3 ringen (2 van gezonde, 1 van BV) -- ik
ben zelf jammer genoeg niet kunnen gaan kijken, omdat ik gisteren een
opleiding had en de foto's zijn volgende week pas klaar (dat is nog een
analoog fototoestel), maar Marjolein liet me wel net weten dat het gelukt
was, dat er een verschil in structuur was tussen de twee types stalen (gezond
vs. BV)
Papers
1) FISH methodology paper: published PLoS One
2) FISH AV paper: rejected mBio, resubmitted STI 18/11/15
3) Sialidase paper: finalising - stats done on all samples (527 vs. 120):
sialidase is still associated with BV and Biofilm
4) Ring biofilm paper: finalising - FISH done, SEM ongoing
IV. VMF Research
Liselotte Hardy: vaginal biofilms
IV. VMF Research
Liselotte Hardy: vaginal biofilms
A fruitful alliance: the synergy between Atopobium vaginae and Gardnerella
vaginalis in bacterial vaginosis-associated biofilm.
Liselotte Hardy, Vicky Jespers, Said Abdellati, Irith De Baetselier, Lambert
Mwambarangwe, Viateur Musengamana, Janneke van de Wijgert, Mario
Vaneechoutte, Tania Crucitti.
Submitted to Sexually Transmitted Infections.
A better understanding of vaginal biofilm with emphasis
on Gardnerella vaginalis and Atopobium vaginae
may contribute to a better diagnosis and treatment of BV.
Evaluation of the association between the presence of both bacteria and BV
by FISH and
by Nugent scoring
in 463 vaginal slides of 120 participants from a clinical trial in Rwanda.
Bacterial biofilm was detected using the universal bacteria probe in half of the samples.
A. vaginae and G. vaginalis were part of this biofilm in resp. 54.1% and 82.0%
A. vaginae was almost always accompanied by G. vaginalis.
Condition
OR for Nugent score > 4
dispersed G. vaginalis and/or A. vaginae
4.5
adherent G. vaginalis and dispersed A. vaginae 75.6
adherent G. vaginalis and adherent A. vaginae 119
IV. VMF Research
Liselotte Hardy: vaginal biofilms
IV. VMF Research
Liselotte Hardy: vaginal biofilms
IV. VMF Research
Piet Cools: VMF and GBS
1. Original PhD subject: Helminths in Africa: but too low prevalence
In the meantime: Help in setting up molecular laboratory in ICRH Mombasa
2. New PhD subject: Biomarker study (EDCTP): additional studies regarding
Streptococcus agalactiae (group B streptococci, GBS) and Escherichia coli
1. A multi-country cross-sectional study of vaginal carriage of group B streptococci (GBS)
and Escherichia coli in resource-poor settings: prevalences and risk factors.
PLOS ONE. Resubmitted 2nd time December 2015
2. GBS, vaginal microbiome and cervicovaginal immune system. In preparation
3. Influence of HIV on (recto)vaginal GBS colonization and GBS neonatal sepsis.
Systematic review and meta-analysis. In preparation
3. Logistics
 Support in practical courses of the department
 Sequencing: GATC outsourcing
 BioNumerics software
 BioStatistics
IV. VMF Research
Piet Cools: VMF and GBS.
Paper 1: carriage of GBS and E. coli; serotype distribution of GBS
Background
One million neonates die each year in low- and middle-income countries because of neonatal sepsis
Group B Streptococcus (GBS) and Escherichia coli are the leading causes.
In sub-Saharan Africa, epidemiological data on vaginal GBS and E. coli carriage are scarce
but necessary to design and implement prevention strategies.
Therefore, we assessed vaginal GBS and E. coli carriage rates and risk factors
and the GBS serotype distribution in three sub-Saharan countries.
Setup
A total of 430 women from Kenya, Rwanda and South Africa were studied crosssectionally.
Vaginal carriage of GBS and E. coli, and GBS serotype were assessed using molecular techniques.
Risk factors for carriage were identified using multivariable logistic regression analysis.
Results
1. Vaginal carriage rates in reference groups from Kenya and South Africa:
for GBS 20.2%
23.1%
E. coli 25.0%
27.1%
2. GBS serotypes Ia (36.8%), V (26.3%) and III (14.0%) were most prevalent.
IV. VMF Research
Piet Cools: VMF and GBS.
Paper 1: carriage of GBS and E. coli; serotype distribution of GBS
Results
3. Factors independently associated with GBS and E. coli carriage:
Candida albicans
an intermediate vaginal microbiome
bacterial vaginosis
recent vaginal intercourse
vaginal washing
cervical ectopy
working as a sex worker.
4. GBS and E. coli carriage (and C. albicans carriage) were positively associated!
Conclusions
Reduced vaginal GBS carriage rates might be accomplished
by advocating behavioral changes
such as abstinence from sexual intercourse
and avoidance of vaginal washing
during late pregnancy.
It might be advisable to include vaginal carriage of C. albicans, GBS and E. coli
and the presence of cervical ectopy
in a riskand/ or screening-based administration of antibiotic prophylaxis.
Current phase II GBS vaccines (a trivalent vaccine targeting serotypes Ia, Ib, and III, and a
conjugate vaccine targeting serotype III) would not protect the majority of women against
carriage in our study population.
IV. VMF Research
Simon Masha Chengo: TV, TVV and STI in Kilifi, Kenya
Goals only achievable when
collaborating closely with
colleague Abel Abera Negash
from Ethiopia
IV. VMF Research
Simon Masha Chengo: TV, TVV and STI in Kilifi, Kenya
Research: Epidemiology and pathogenicity of Trichomonas vaginalis in Kenya
Background/SetUp:
Epidemiology of TV and STI in Kilifi, Kenya
Relation of TV with TVV and mycoplasms: symbionts? Virulence enhancers?
Funding: VLIR-UOS. 6000 € bench fee per year
@LBR: SMC, PC
Collaboration: Eduard Sanders, KIMRI, Kilifi, Kenya
Tania Crucitti, ITM, Antwerp, Belgium
Patrick Descheemacker, Marijke Reynders, Bruges General Hospital St. Jan
Results: sample collection
qPCRs, culture, TaqMan array: TV positive samples + controls (x 2) to Bruges
Publication Status:
1. TV and STIs in Kenya, quantified by PCR and TAC
2. Epidemiology of TV and STIs in Kenya
3. Systematic review on co-occurrence of TV and mycoplasms
IV. VMF Research
Simon Masha Chengo: TV, TVV and STI in Kilifi, Kenya
TaqMan array
Principle
Range of TAC for STI (AZ St. Jan Bruges, Patrick Descheemaecker)
TV TV
Btub 2kb
TV
repea
t rev MG
CT
NG
TP
MH
UP
UU
HD
HSV1 HSV2 Adeno CMV
IV. VMF Research
Simon Masha Chengo: TV, TVV and STI in Kilifi, Kenya
Results TaqManAssay Card (1): TV culture and PCR positives
Original Sample New sample TV results culture TV results STI TAC
ID
ID
PCR
TV Btub
TV022
TV042
TV050
TV066
TV075
TV116
TV131
TV140
TV156
TV176
TV185
TV188
TV190
TV203
TV207
TV210
TV224
TV234
TV238
TV279
TV299
TV307
TV323
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
26,7
28,16
26,4
20,4
21,78
29,25
22,96
22,33
25,79
22,99
32,09
27,15
26,28
25,19
28,87
25,54
23,7
28,69
22,92
28,92
19,1
22,17
32,09
TV 2kb
TV repeat
rev
24,35
25,11
25,93
25,61
24,51
24,58
17,78
17,7
18,5
17,72
25,67
24,73
19,55
19,12
18,69
18,61
20,67
22,03
18,2
18,84
27,78
28,46
24,1
25,16
21,18
22,4
21
21,53
24,88
25,73
19,55
20,73
19,3
20,7
22,1
23,45
17,11
17,87
23,55
23,74
16,42
16,64
17,13
18
27,47
27,88
IV. VMF Research
TV044
40 TV054
41 TV056
42 TV059
43 TV063
44 TV069
45 TV073
46 TV076
47 TV077
Original
New 48 -TV results
TV079
Sample ID sample 49
ID - culture
TV091
50 TV094
51 TV099
52 TV109
53 TV113
54 TV117
55 TV118
56 TV126
57 TV134
58 TV152
59 TV153
60 TV154
61 TV163
62 TV164
63 TV173
64 TV174
65 TV175
66 TV178
67 TV179
68 TV180
69 TV182
70 TV184
71 TV206
72 TV209
73 TV229
74 TV231
75 TV232
76 TV233
77 TV243
78 TV256
79 TV268
80 -
- TV
STI TAC
-results
TV
- PCR
repeat
TV Btub TV 2kb rev
34,74
39,71
38,38
39,32
33,87
33,74
34,19
35,21
33,41
32,04
33,37
34,8
36,04
32,61
38
35,56
36,77
33,04
34,08
-
Simon Masha Chengo: TV, TVV and STI in Kilifi, Kenya
Results TaqManAssay Card (2): TV culture and PCR negatives
Original
New
TV results
Sample ID sample ID culture
TV002
TV003
TV004
TV005
TV009
TV011
TV014
TV018
TV019
TV020
TV023
TV030
TV031
TV032
TV033
TV037
TV044
TV054
TV056
TV059
TV063
TV069
TV073
TV076
TV077
TV079
TV091
TV094
TV099
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 -
TV
STI TAC
results
PCR
TV
repeat
TV Btub TV 2kb rev
+
-
25,4
22,62
23,22
IV. VMF Research
Vicky Jespers
This paper is now on line as in press and has been on twitter! Captured by a microbiome researcher. Nice.
Thank you all for the contributions and for the nice collaboration especially Joris for his amazing analysis.
If you know how to tweet, please do so. Vicky
https://twitter.com/MicrobiomDigest/status/666495730958209025
http://www.sciencedirect.com/science/article/pii/S0923250815001801
IV. VMF Research
Vicky Jespers
A next-generation diagnostic tool for bacterial vaginosis
consisting of quantitative and/or qualitative molecular criteria
The optimal diagnostic tool should not only diagnose bacterial vaginosis in diverse populations,
but should also detect early signs of transition to dysbiosis.
A tool based on log10-transformed qPCR data for
Lactobacillus crispatus, L. iners, L. jensenii, L. gasseri, L. vaginalis, Lactobacillus genus/
Atopobium vaginae and Gardnerella vaginalis
in vaginal specimens of 426 African women to detect dysbiosis
and predict transition to dysbiosis.
G. vaginalis (p=0.204) and A. vaginae (p=0.001) were more commonly present in women who evolved
to an intermediate (Nugent 4-6) or bacterial vaginosis score (Nugent 7-10) compared to women who
continued to have a normal Nugent score.
The combination of G. vaginalis, A. vaginae and Lactobacillus genus counts performed best
for diagnostic accuracy for bacterial vaginosis: sensitivity 93.4% and specificity 83.6%; and
for predictive accuracy for bacterial vaginosis--sensitivity 79% and specificity 52%.
We conclude that a triple-G. vaginalis-A. vaginae-Lactobacillus genus-qPCR tool holds promise for
research in sub-Saharan Africa or when developed as a next-generation clinical diagnostic modality for
bacterial vaginosis, ideally engineered as a rapid assay.
IV. VMF Research
John Schellenberg & Janet Hill
A more detailed look at some of the
past, present and future research
I. Phage Therapy/Immunomodulation
Maia, Jonas, Cara, Liesl, Hans, Jarne
II. Microbiome research
Guido, Tessa, Pascaline, Opathy
III. Biofilm Research
Jonas, Leen
IV. VMF Research
Piet, Liselotte, Masha, Guido, Meryam
V. Detection, quantification, identification
Piet, Leen, Hans, Abel, Masha, Tessa
VI. Fungi
Hans, Leen, Opathy
VII. Genotyping
Piet, Leen, Sophie, Guido, Elke, Leander
V. Detection, quantification, identification
Resistance-Biofilms: MucoDrug (CF)
Background/Setup: Monitoring of antibiotics in sputum of CF patients.
Funding: Forton, Boudewijnstichting & MucoVereniging
Period: 1 September 2014 - 31 August 2016.
@ LBR: MV, LVS
Collaboration with:
Alain Verstraete – Veronique Stove – Katrien Forier (GE06)
Frans De Baets (GE02)
Piet Cools
 MALDI for
Identification: identifying all kinds of isolates, incl. from whales: Edwardsiella tarda
Theofiel de Potvis
V. Detection, quantification, identification
Piet Cools
Research: Detection/Quantification: Aerobic vs anaerobic culture vs qPCRof urine samples
Background/SetUp:
Can differences previously (NVDE) found between aerobic and anaerobic culture of urine
samples be confirmed?
How do culture and qPCR for E. coli compare?
Healthy subjects: negative for E. coli culture and qPCR? And for other pathogens?
Funding: None
@ LBR: PC, LVS, (NVDE, GW)
Collaboration with: Geert Claeys (GE06)
Gwenn Waerlop (master student biomed)
Stefan Heytens (GE21)
Techniques:
Culture aerobic and anaerobic & identification with MALDI-TOF
qPCR for E. coli and 10 other possible uropathogens-STI germs
Populations:
100 hospital patients: midstream urine, catheters
250 women with UTI symptoms
100 controls (87 women) without UTI symptoms
V. Detection, quantification, identification
Can differences previously (NVDE) found between aerobic and anaerobic culture of urine
samples be confirmed?
Test: New series of 100 urine samples (hospital) was cultured by 7 different conditions
Result: Few differences between culture conditions
Answer: NO
How do culture and qPCR for E. coli compare?
Test: Extended culture and qPCR for E. coli was carried out on urines from UTI patients
Result: PCR is 100-1000 x more sensitive than culture!
All UTI from community are E. coli positive with qPCR: closing the culture-negative gap?
Answer: good correspondence, but qPCR much more sensitive.
Is this higher sensitivity false, due to qPCR picking up minor contaminations?
Test: Control population without symptoms was tested for E. coli by culture and qPCR
Result: perfect correspondence between culture & qPCR:
most samples are culture and qPCR negative
Answer: NO
How is this higher sensitivity explained?
Due to biofilm? qPCR picks up dormant cells
Due to culture bias: one CFU > one cell?
Test: Known number of living cells was added to urine and qPCR and culture were carried out.
Result: PCR is again 100-1000 x more sensitive
Answer: difference is not due to biofilm (because these were all freshly cultured cells)
V. Detection, quantification, identification
Spiked C. albicans, quantified by culture and qPCR
(comparable results for E. coli spiked in urine)
Conclusion: qPCR is 100 times more sensitive for quantification of freshly cultured cells
V. Detection, quantification, identification
of Fungi in clinical samples, in blood: see VI. Fungi
A more detailed look at some of the
past, present and future research
I. Phage Therapy/Immunomodulation
Maia, Jonas, Cara, Liesl, Hans, Jarne
II. Microbiome research
Guido, Tessa, Pascaline, Opathy
III. Biofilm Research
Leen, Jonas
IV. VMF Research
Piet, Liselotte, Masha, Guido, Meryam
V. Detection, quantification, identification
Piet, Leen, Hans, Abel, Masha, Tessa
VI. Fungi
Hans, Leen, Tessa, Opathy
VII. Genotyping
Piet, Leen, Sophie, Guido, Elke, Leander
VI. Fungi
1. Duyvejonck. Direct detection of Candida in clinical samples
DNA amplification method for direct detection of Candida spp. from clinical samples
easyMAG DNA extraction, directly on the clinical samples
+ ITS2-High Resolution Melting Analysis (direct method), compared with
culture and MALDI-TOF (indirect method)
to establish the presence of Candida species in
347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs
Complete concordance: 83.9% of the samples
i.e. the same Candida species were detected in 31.1% of the samples
no Candida species were detected in 52.8% of the samples.
Discrepant results in 16.1% of the clinical samples
only 6.01%were considered as major discrepancies.
Discrepancies occurred mostly when overall numbers of Candida cells in the samples
were low and/or when multiple species were present in the sample.
Most of the discrepancies could be decided in the advantage of the direct method:
a) Samples culture negative but with low amounts with the direct method
b) Samples with high quantities of Candida robusta according to ITS2-HRM were missed by
culture on Candida ID agar.
VI. Fungi
1. Duyvejonck. Direct detection of Candida in clinical samples
Tm range of ITS2 for different Candida species
C. famata
C. tropicalis
C. guilliermondii
C. parapsilosis
Tm range minor peak
C. glabrata
Tm range major peak
C. dubliniensis
C. albicans
C. kefyr
C. lusitaniae
C. inconspicua
C. krusei
C. norvegensis
C. robusta
79
81
83
85
87
Melting temperature (°C)
89
91
VI. Fungi
2. Species specific qPCR: Duyvejonck & Gryp.
Hans:
Development of species specific PCR for direct detection of
Candida albicans
Candida glabrata
Candida dublinienis
Tessa:
Candida robusta = Saccharomyces cerevisiae
 role in hemodialysis patients?
Advantage over ITS2-qPCR:
no interference: straightforward quantification.
can also be combined into one reaction.
VI. Fungi (+ II. Microbiome Research)
3. Antonio José Rodriguez Sanchez. OPATHY
Research: Detection/Quantification: Opathy
Background/Setup: Diagnostics of fungal infections
Funding: Horizon 2020 (H2020-MSCA-ITN-2014)
Techniques: qPCR – Transcriptomics
OPATHY: Omics of PATHogenic Yeasts
AntonioJoseRodriguezSanchez
LeentjeMiekeProsperVanSimaey
GuidoLopesdosSantosSantiago
MarioGilbertKarelCorneliusVaneechoutte
OPATHY Partner 9: LBR. Early Scientific Researcher (ESR)12:
Clinical molecular ecology and microbiology of Candida vaginitis
This ESR's goal will be to identify virulence factors up-regulated in C. albicans and the host,
as compared to uninfected women with normal vaginal microflora.
Candidate genes will be developed into prototype diagnostic tools.
A more detailed look at some of the
past, present and future research
I. Phage Therapy/Immunomodulation
Maia, Jonas, Cara, Liesl, Hans, Jarne
II. Microbiome research
Guido, Tessa, Pascaline, Opathy
III. Biofilm Research
Jonas, Leen
IV. VMF Research
Piet, Liselotte, Masha, Guido, Meryam
V. Detection, quantification, identification
Piet, Leen, Hans, Abel, Masha, Tessa
VI. Fungi
Hans, Leen, Opathy
VII. (Geno)typing
Piet, Leen, Sophie, Guido, Elke, Leander
VII. (Geno)typing
1. Epidemiology:
Achromobacter xylosoxidans epidemiology in CF
Enterobacter amnigenus in water supplies: PIDPA
Gardnerella vaginalis subtypes  taxonomy, new species description?
2. Predicting chronic colonisation in CF: Pseudomonas aeruginos (Leander)
3. Hospital hygiene: GUH, Hospital Hygiene: E. cloacae  all hospital isolates?
What if MALDI could predict the antibiotic resistance, virulence of individual isolates?
This would drastically change the impact of microbiology on hospital infection control
VII. (Geno)typing
1. Epidemiology: Achromobacter xylosoxidans (CF)
Background/Setup:
Is there an epidemic strain of AX in Belgium?
Already 15 years ago we showed a large cluster in the Rehabilitation Centre, De Haan
Funding: None
LBR: LVS, PC
Collaboration:
Frans De Baets - Sabine Van daele - Petra Schelstraete (GE02, GUH, MucoCenter)
Stijn Verhulst (UZA), Erwin Ho (AZ Turnhout)
Bettina Wurth, Hilde Franckx (Rehabilitation Center De Haan)
Techniques: McRAPD & MALDI-TOF
Results
First application of MALDI-TOF for typing, combined with BioNumerics software
Strong correspondence with McRAPD: confirmation that McRAPD is useful
Indeed: one large cluster of AX isolates among CF patients.
Publication status
Submitted December: JCF: rejected without review!
Submitted December: BMC Microbiol: not yet rejected! (@ 151224)
VII. (Geno)typing
1. Epidemiology. Achromobacter xylosoxidans (CF)
A
Clusters I and II
Pairs P1 and P2
B
Singletons S1, S2 and S3
McRAPD
VII. (Geno)typing
1. Epidemiology: Achromobacter xylosoxidans (CF)
Comparison of MALDI-TOF patterns for two isolates of genotypes A and B
A
B
C
VII. (Geno)typing
1.
Epidemiology: Achromobacter xylosoxidans (CF)
Bionumerics gives epidemiologically more relevant interpretation than
human interpreters
VII. (Geno)typing
1. Epidemiology: Enterobacter cloacae & hospital hygiene
Piet Cools, Sophie Decuypere
Prototype study to establish whether MALDI-typing
can predict antibiotic susceptibility, epidemiological relationship
Data have been gathered.
Are being introduced into BioNumerics.
Waiting for license upgrade.
VII. (Geno)typing
2. Predicting chronic colonisation in CF: Pseudomonas aeruginos (Leander Jonckheere)
CF patients: how to decide upon chronic colonization?
Why is it important to know?
Current guidelines (ECC): 3 PA positive samples in a period of 6 months.
We studied x patients thus far.
E.g. patient with 3 PA+ samples within 6 months. Since than: no more isolations, but patient
is classified as chronically colonized. Genotyping: 3 different types.
E.g. patient with 3 PA+ samples, but over a period of one year
 ECC: not chronically colonized.
(Geno)typing: all PA strains have same genotype.
Patient was chronically colonized since first time, but still not according to current guidelines.
VII. (Geno)typing
3. Genotyping/Detection: Streptococcus pneumoniae in Ethiopia
Abel Abera Negash
Background/Setup:
How is the prevalence of different capsular types influenced by vaccination programmes?
Funding: VLIR-UOS: Bench Fee – Travel Costs
@LBR: PC, AAN
Collaboration with: Geert Claeys (GE06) – Daniel Asrat (Addis Abeba, Ethiopia)
Results: collection samples started. Products sent?
Publication status: none
III. NEW RESEARCH OPPORTUNITIES
TaqMan Array (Geert Lion ThermoFisher) (Masha – Bruges)
Minion deep sequencing technology (Guido)
MinIon sequencer: 1000 $:
the MinION MkI (sequencer) and your starter pack.
A starter pack includes 3 MkI compatible flow cells,
1 Nanopore Sequencing kit, 1 Wash kit and ongoing intermittent free supplies.
In addition, this also covers access to the MinION Community,
where you can learn how to operate the system and collaborate with other members of the community.
The fee also includes use of the MinION operating software, MinKNOW,
and some analyses using Metrichor.
New DNA extractor: Iris Van Winge (Isogen).
Molysis? Hans?
Digital PCR: Geert Lion (ThermoFisher): for Microbiome research (Tessa?)
QuantStudio 3D: chips with 20.000 wells to make the partitions for digital PCR.
= no emulsion PCR, which has advantages, such as
reproducable manner for same numbero f partitions
simpler workflow, closed chip system
1 chip = 1 sample.
De QS 3D is eigenlijk een platform en bestaat uit een PCR toestel met vlak blok (om de chips te PCRen)
een loader om semi-manueel de chips te laden met sample en mastermix,
en de reader (de eigenlijke QuantStudio 3D).
De reader is een dual channel systeem dus kan je (naast een 3de passieve referentiedye)
2 targets detecteren per sample. Voor kleine tot middelgrote doorvoer is dit systeem zeer geschikt.
Hans: problemen met specifieke qPCR voor C. albicans – C. dubliniensis – C. glabrata
Ann Van Roost (Roche) experimenten: ander probe PCR mix: 1523 voor kleine? mixen ipv Roche
ProbeMasterMix.
Besteld door Hans. Nog vergelijkend experiment uitvoeren
Ann Van Roost: Voor wat de mix betreft:
dit is een mix die inderdaad speciaal ontwikkeld is om in zeer kleine volumes te werken (vanaf 2 µl totaal volume) en
wordt o.a. gebruikt op ons LightCycler 1536 toestel.
De compositie van de mix of de verschillen tussen beide mixen is spijtig genoeg confidentieel (ook ik heb hier geen
weet van). We gaan ervan uit dat er iets meer additieven (en/of iets meer enzyme) inzit.
Als jullie alle reacties met deze mix willen doen, kan je zeker altijd proberen te optimaliseren in een totaal volume
van 5µl. In 384 platen kan je dit zeker testen. Bij lagere volumes dan 10 µl wordt wel meestal in de labo's met
pipetteerrobotten gewerkt. Het is ook belangrijk dat de plaat zeer goed geseald wordt.
Bij het testen van de nieuwe mix is het aangeraden om telkens (voor elke target) verdunningsreeksen uit te testen
(huidig volume/mix versus nieuw verlaagd volume/mix) om na te gaan of jullie niet moeten inboeten op
gevoeligheid. Daarnaast zou ik ook altijd testen op specificiteit.
Ik had je de prijs doorgegeven van de dienst bacteriologie maar blijkbaar bestellen jullie onder virologie.
Jullie netto prijs is dan nog lager: nl 343 eur (ipv de vorige nettoprijs van 648,90 euro) .
Aangezien jullie in 10 µl werken en het een 5x mix is, kunnen jullie 2500 reacties uitvoeren met één kit "LC1536 DNA
Probe Master" => dit wil zeggen dat jullie 0,14 eur per reactie betalen (met de LC480 probe master kit was dit 0,13
eur per reactie). De prijs per reactie is dus identiek.
Als jullie bijkomende informatie nodig hebben: mijn collega Ann Crosiers komt vrijdag bij jullie langs voor de
sequencing.
Others?
Research: Microbiome studies: [CCPTB]
Background/Setup: Case-control study of preterm births
Funding: [GOA]
LBR: [GLS]
Collaboration with: Hans Verstraelen - [Inge Tency](GE04)
Detection/Quantification: Mambo-study
Background/Setup: Quantitation of Streptococcus pneumoniae and Haemophilus influenzae by qPCR in
samples from ears and throats after vaccination. LINK
Funding: Pfizer. X € per analysis
@ LBR: HD
Collaboration with: Patricia Slachmuylders (Pfizer)
Research: Microbiome studies: Vaginal microflora of ruminants. LINK
Background/Setup: Vaginal pH in humans is acid (4.5), but this is not the case in any other animal. Can
this be due to the absence of lactobacilli. Later on: different perinatal health problems between meat
and milk cows. Is this due to different VMF?
Funding: Honours Programme and Master Thesis. Bench fee.
@LBR: PC
Collaboration with: Geert Opsomer - Jolien Marga (master thesis) (DI08)
Jonas: CMAH gene of marine mammals: selectie gestuurd naar Jauniaux. Primers voor CMAH (gevraagd
aan Chak: 131113) en FoxP2 en breinneuronen zoeken. Chimp en gorilla DNA (John) zoeken (min 20).
6. CCPTB
Extra qPCRs werden uitgevoerd voor Gardnerella, Atopobium, Prevotella, Lactobacillus, maar kwaliteit
zwak (door nieuw toestel?). Nog eens data presenteren.