1

THEODOR ESCHERICH SYMPOSIUM 2015: ABSTRACTS

SHORT TALKS

SHORT TALK 1

"Biosynthesis and degradation of Klebsiella oxytoca enterotoxin tilivalline”

Dornisch E.1, , Pletz J.

2, Schneditz G.

1, Högenauer C.

3, Kienesberger S.

1, Breinbauer R,

2, Zechner E.

1

1University of Graz,

2Graz University of Technology,

3Medical University of Graz, Austria

Antibiotic-associated hemorrhagic colitis (AAHC) develops during antibiotic-driven intestinal dysbiosis and is

caused by the bacterial pyrrolobenzodiazepine tilivalline produced by the pathobiont Klebsiella oxytoca.[1,2].

To understand its’ function during health and disease we study the regulation of tilivalline biosynthesis.

Essential biosynthetic genes in the clinical isolate AHC-6 – aroX, aroB and two non-ribosomal peptide synthases

(npsA and npsB) – are located on a pathogenicity island present in all toxin positive Klebsiella isolates.[2].

Under laboratory conditions tilivalline is detected in conditioned medium of bacterial cultures. Cytotoxicity

towards HeLa cells appears at the end of the exponential phase, maximizes during stationary phase and

decreases to undetectable levels at 48 h. This loss indicates a growth-dependent regulation of biosynthesis

and/or inactivation of tilivalline. To test the latter hypothesis we butanol-extracted culture supernatants

obtained following 0-52h of growth. The presence of tilivalline, or a modified product, was analyzed by HPLC-

MS and NMR. Loss of tilivalline over the time course was observed as well as the appearance of a small

molecule of previously unknown structure. Our data indicate that tilivalline is detoxified enyzmatically.

Remarkably, however, the new nonribosomal peptide is not the result of tilivalline processing but is formed by

an additional branch of the biosynthetic pathway. Under laboratory conditions tilivalline is detected in

conditioned medium of bacterial cultures. Cytotoxicity towards HeLa cells appears at the end of the

exponential phase, maximizes during stationary phase and decreases to undetectable levels at 48 h. This loss

indicates a growth-dependent regulation of biosynthesis and/or inactivation of tilivalline. To test the latter

hypothesis we butanol-extracted culture supernatants obtained following 0-52h of growth. The presence of

tilivalline, or a modified product, was analyzed by HPLC-MS and NMR. Loss of tilivalline over the time course

was observed as well as the appearance of a small molecule of previously unknown structure. Our data indicate

that tilivalline is detoxified enyzmatically. Remarkably, however, the new nonribosomal peptide is not the

result of tilivalline processing but is formed by an additional branch of the biosynthetic pathway.Moreover,

qRT-PCR transcripts of npsA and npsB were downregulated in a growth dependent manner in bacterial

monoculture as well as during cocultivation with human epithelial cells.

In summary, K. oxytoca inactivates tilivalline in vitro and instead synthesizes a non-cytotoxic substance. Current

studies focus on the characterization of the new product’s bioactivities and on host and microbial impact on

tilivalline.

[1] Högenauer et al. (2006): Klebsiella oxytoca as a causative organism of antibiotic associated hemorrhagic

colitis. N Engl J Med 355(23):2418–2426.

[2] Schneditz et al. (2014): Enterotoxicity of a nonribosomal peptide causes antibiotic-associated colitis. Proc

Natl Acad Sci USA 111(36):13181-13186."

2

SHORT TALK 2

“Assessment of Illumina-Nanopore hybrid assembly performance for bacterial genomes”

Pamela Ferretti1, Mattia Bolzan

1, Edoardo Pasolli

1, Slvia Fillo

2, Fabrizio Anniballi

3, Florigio Lista

2, Adrian Tett1,

Nicola Segata1

1 Centre for Integrative Biology, University of Trento, Italy

2 Army Medical and Veterinary Research Center, Rome, Italy

3National Reference Center for Botulism, Dept. of Veterinary Public Health and Food Safety, Istituto Superiore

di Sanità (ISS), Rome, Italy.

Recent advances in sequencing technologies are making microbial genome sequencing a routine and cost-

effective task. However, producing high-quality complete assemblies is still extremely challenging when using

Illumina as short reads cannot resolve repetitive regions of the genome. Recently, a new promising sequencing

platform, Nanopore MinIONTM, has been released. This technology generates longer reads (tens of kilobases),

but with higher error rates than Illumina. Combining Illumina and Nanopore reads in the assembly process has

been shown to produce very high-quality ungapped genome reconstruction, as long Nanopore reads can guide

the scaffolding process of the contigs assembled with short high-quality reads. In our work we comprehensively

evaluated the hybrid assembly performance on several real datasets using the SPAdes assembler. We first

considered two available datasets of Salmonella typhi strains, subsampling both Illumina (up to 24X) and

MinIONTM reads (up to 15X) to identify the right combination that maximizes the assembly performance,

minimizing costs and run length. Several QC filters were also tested. Hybrid assembly significantly decreased

the number of contigs (-54%) and increased the N50 (+48%) compared to single-technology assembly. We then

applied the hybrid assembly approach to two isolates of Clostridium butyricum and Pseudomonas aeruginosa.

For both organisms we obtained almost complete consensus sequences (9 and 3 scaffolds respectively). The

optimal coverage thread-off resulted to be 8X for MinIONTM and 24X for Illumina. Our work suggests that, if

the cost per base of MinIONTM sequencing will be competitive, the hybrid approach can become the standard

for very high-quality microbial genome reconstruction.

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SHORT TALK 3

"Improving the detection of Archaea in human samples

Michael Beck1, Alexandra Perras

1, Kaisa Koskinen

1, Maximilian Mora

1, Lisa Wink

1 and Christine Moissl-

Eichinger1,2

1 Medical University Graz, Austria

2 BioTechMed Graz, Austria

Archaea were discovered more than 40 years ago representing a microbial kingdom separate from Bacteria.

Their wide-spread presence has been shown in numerous extreme and moderate environments, including the

human body. Although they are recognized as members of the microbiome, the question whether archaeal

pathogens exist or not remains unanswered.

Archaea are different from Bacteria in many ways: In particular their cell walls, metabolism and molecular

machineries can clearly be distinguished from bacterial and eukaryotic systems. They possess a 16S rRNA gene,

but it is highly different in sequence and three-dimensional structure, making it less accessible for standard,

bacteria-focused microbiome analyses. Moreover, generally used analysis pipelines remove archaeal reads, and

these microorganisms remain hidden once more. However, even without NGS-based analysis methods, the

presence of Archaea has been linked to e.g. periodontal disease and vaginal infections, and their immunogenic

properties have been shown. In order to understand their role in human microbiome and health and disease,

current methods have to be improved and adapted to state-of-the-art technologies.

Here, we describe improvements in the area of quantitative PCR and next generation sequencing for enhanced

detection of Archaea in human skin, tissue, gut, nose and lung samples. Using these specialized protocols, we

were able to detect Archaea in all tested clinical sample types, and substantially more archaeal taxa compared

to standard procedures.

These improvements will help us and other researchers in a broad field to better detect Archaea in a variety of

samples and estimate their richness, distribution and quantity.

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SHORT TALK 4

MICROBIOMES OF THE BUILT ENVIRONMENT ARE SHAPED BY OUR ROOM MAINTENANCE

Alexander MAHNERT1*

, Christine MOISSL-EICHINGER2,3

, Parag VAISHAMPAYAN4, Thomas RATTEI

5, Henry

MÜLLER1, Alexander J. PROBST

6, Lisa OBERAUNER-WAPPIS

7, Roscel Amor ORTEGA

1, Kasthuri

VENKATESWARAN4, and Gabriele BERG

1

1Institute of Environmental Biotechnology, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria

2Medical University Graz, Department of Internal Medicine, Auenbruggerplatz 15, 8036 Graz, Austria

3BioTechMed Graz, Krenngasse 37, 8010 Graz, Austria

4Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, 4800 Oak Grove Drive, Pasadena,

CA 91109, USA 5Department of Microbiology and Ecosystem Science, University of Vienna, Althanstraße 14, 1090 Wien,

Austria. 6Lawrence Berkeley National Laboratory, Earth Sciences Division, 1 Cyclotron Rd., Berkeley, CA 94720, USA

7Medical University Graz, Institute of Pathology, Auenbruggerplatz 15, 8036 Graz, Austria

*Corresponding email: a.mahnert@tugraz.at

Keywords: Viable controlled and uncontrolled indoor microbiomes, cleanrooms, intensive care units, wildlife

park, indoor plants, greenhouse

The excessive removal of microbes from controlled built environments like intensive care units, operation

theatres and especially cleanrooms is daily routine. However effects of these stringent maintenance

procedures on our surrounding microbiomes in indoor environments are unknown.

Since recent studies showed the enormous potential of the human microbiome towards our health, we aim to

resolve the interactive interference of all microbiomes in the built environment and determine its potential for

human well-being indoors.

High throughput deep sequencing technologies were applied for shotgun metagenomes and 16S rRNA gene

and ITS region amplicons of Archaea, Bacteria, and fungi. Viability assays using ATP (adenosine tri-phosphate)

and PMA (propidium monoazide) were deployed and supported by qPCR to investigate viable microbial

abundance. The biotechnological potential was further characterized by VOC’s (volatile organic compounds)

assays of culturable indoor bacteria.

These assays revealed a high reduction of viable microbial abundance in cleanrooms, higher similarities of

viable microbiomes between controlled and uncontrolled areas compared to the total microbial fraction,

distinct profiles of microbial communities from floors, medical devices and workplaces in an intensive care unit,

and for all controlled built environments not only an overlap with the human, but also the plant microbiome.

Hence plant leaves could be identified as a main source for microbial dispersal on the surrounding indoor

environment.

5

The knowledge of certain key species and their ecological key functions in controlled as well as uncontrolled

built environments will help to control indoor environments in a “bioinformed” way for our health inside

buildings in the future.

Berg G, Mahnert A, Moissl-Eichinger C. (2014). Beneficial effects of plant-associated microbes on

indoor microbiomes and human health? Front Microbiol 5:1–5.

Mahnert A, Moissl-Eichinger C, Berg G. (2015). Microbiome interplay: plants alter microbial abundance

and diversity within the built environment. Front Microbiol 6:1–11.

Mahnert A, Vaishampayan P, Probst AJ, Auerbach A, Moissl-Eichinger C, Venkateswaran K, et al.

(2015). Cleanroom Maintenance Significantly Reduces Abundance but Not Diversity of Indoor

Microbiomes. PLoS One 10:e0134848.

Moissl-Eichinger C, Auerbach AK, Probst AJ, Mahnert A, Tom L, Piceno Y, et al. (2015). Quo vadis?

Microbial profiling revealed strong effects of cleanroom maintenance and routes of contamination in

indoor environments. Sci Rep 5:9156.

Oberauner L, Mahnert A, Bragina A, Berg G. (2014). Complex indoor bacterial communities.

In:Encyclopedia of Metagenomics, Nelson, KE (ed), Springer Berlin / Heidelberg: Berlin Heidelberg, pp.

1–7.

Oberauner L, Zachow C, Lackner S, Högenauer C, Smolle K-H, Berg G. (2013). The ignored diversity:

complex bacterial communities in intensive care units revealed by 16S pyrosequencing. Sci Rep

3:1413.

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SHORT TALK 5

PLANHAB: INSIGHTS INTO HUMAN INTESTINAL MICROFLORA DYNAMICS OF NORMOXIC AND HYPOXIC

BEDREST STUDIES

Robert Šket1, Nicole Treichel

2, Tadej Debevec

3, Ola Eiken

4, Igor Mekjavic

3, Michael Schloter

2, Marius Vital

5,

Jenna Chandler5, James M. Tiedje

5, Blaž Stres

1*

1Department of Animal Science, University of Ljubljana, Ljubljana, Slovenia

2German Research Center for Environmental Health, Neuherberg, Germany

3Department of Automation, Biocybernetics and Robotics, Jozef Stefan Institute, Ljubljana, Slovenia

4Department of Environmental Physiology, Swedish Aerospace Physiology Centre, Royal Institute of

Technology, Stockholm, Sweden 5Michigan State University, Center for Microbial Ecology, East Lansing, Michigan, USA

*Corresponding author: blaz.stres@bf.uni-lj.si

The impact of physical inactivity and hypoxia on human intestinal microflora was explored. Human intestinal

tract microbiome samples were collected within Planetary Habitat Simulation FP7-SPACE project. For analysis

of bacterial microbial community structure, stool samples during run-in period (days -5 and -1 before the onset

of experiments) and days 3, 10, 18 and 21 of the three experimental settings (normoxic bedrest, hypoxic

bedrest, hypoxic ambulatory) were analyzed using paired-end MiSeq approach. Existing human physiological

data were compiled for the same days of experiments. Changes in microbial communities occurred in response

to hypoxia, subject variability and gut-metabolites. Differences in intestinal microbial communities between

test subjects observed at the beginning of experiments increased through time in response to test environment

and were additionally exacerbated by prolonged hypoxia. The results suggest that microbial communities

respond to changes in human physiology that was changed by environmental setting (hypoxia, bedrest). The

analyses of butyrate synthase genes by QPCR and sequencing showed that subjects had relatively stable,

abundant functional communities throughout the study. Metabolic networks showed significant differences in

interactions between cornerstone species and microbial metabolites present in intestinal tract, suggesting that

functional aspects of microbiomes play significant role in shaping the observed changes in host metabolism

recorded within PlanHab project. "

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SHORT TALK 6

Enterotoxin tilivalline is a mitotic poison that stabilizes microtubules

Unterhauser Katrin1, Georg Schneditz

1, Jakob Pletz

2, Christoph Högenauer

3, Rolf Breinbauer

2 and Ellen L.

Zechner1

1University of Graz,

2Graz University of Technology,

3Medical University of Graz, Austria

Klebsiella oxytoca is a resident of the human gut microbiota. During antibiotic-induced intestinal dysbiosis,

colonic overgrowth of this bacterium in some patients rapidly leads to antibiotic-associated hemorrhagic colitis

(AAHC). We have shown that bacterial production of the secondary metabolite tilivalline, which is structurally

related to pyrrolobenzodiazepines, induces apoptosis of epithelial cells, disrupts their barrier function and is

key to the pathogenesis of colitis in an animal model of AAHC (1). The impact of tilivalline on host physiology in

carriers of low (commensal) levels of Klebsiella oxytoca and the molecular mechanism of cytotoxicity are

unknown. We synthesized tilivalline chemically to investigate the human cellular processes altered by this

enterotoxin in vitro.

Different human cancer cell lines were treated with a range of tilivalline concentrations and cellular stress

responses were monitored with proliferation assay, through microscopic imaging and FACS, as well as by

biochemical analyses. Apoptosis is induced by high doses of tilivalline. Low concentrations of the cytotoxin

inhibit cell proliferation and induce a G2/M cell cycle arrest. We have applied a variety of approaches to

identify tubulin as the molecular target of tilivalline. Microscopy of tilivalline treated cells revealed disruption

of the interphase microtubuli and dysfunctional spindle apparatus formation blocking mitosis. In vitro

microtubule polymerization assays and fluorescence based binding studies confirm that tilivalline - tubulin

interaction stabilizes the polymer form. These findings determine the mechanism underlying tilivalline toxicity

in AAHC to be disruption of host microtubule dependent processes. The current challenge is to understand the

impact of tilivalline on host physiology in carriers of low (commensal) levels of Klebsiella oxytoca.

(1) Schneditz, G., Rentner, J., Roier, S., Pletz, J., Herzog, K.A.T., Bücker, R., Troeger, H., Schild, S., Weber, H.,

Breinbauer, R., Gorkiewicz, G., Högenauer, C., Zechner, E.L. (2014) PNAS 111, 13181-13186"

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SHORT TALK 7

How to compare subgingival microbiome profiles of healthy children: a case control study

Elisabeth Santigli

Medical University Graz

9

POSTERS

POSTER 1

Diverse ecological adaptation of Prevotella species, a major group in mammalian gut, as revealed by

comparative genomic analysis of glycan binding and degrading protein repertoires.

Tomaž Accetto* and Gorazd Avguštin

University of Ljubljana, Biotechnical faculty, Animal science department, Groblje3, 1230 Domžale, Slovenia

*Corresponding author: tomaz.accetto@bf.uni-lj.si

The metagenomic studies have established that the bacteria of the genus Prevotella are one of the major

groups found in the oral cavity and large intestine of man and also dominate the cow rumen. They belong to

the Bacteroidetes, a large bacterial phylum, well known for the polysaccharide degrading potential of its

members. This potential stems from the outer membrane localized enzyme/binding protein complexes coded

in polysaccharide utilization loci (PULs). While dozens of Prevotella species, primarily from oral cavity, have

been described and many can occur simultaneously at the same sites, there is little research on their ecological

adaptation. Here we analyze the repertoires of PULs and carbohydrate acting enzymes (CAZYmes) found in

Prevotella genomes. The polysaccharide binding SusD-like proteins detected in fifty Prevotella genomes were

used as PULs indicators. Almost 1200 SusD-like proteins were detected in Prevotella genomes and for half thus

identified PULs a target substrate was predicted. The CAZYme repertoires were in the agreement with the

SusD-like protein data. Using the approach outlined in methods, we have found out that Prevotella species are

heterogeneous and display several distinct adaptations with regard to the number, source and nature of

substrates most likely preferred for growth. Although the genus Prevotella currently contains mostly species

isolated from mammal oral cavity, the ecological adaptations are diverse: on one hand there is a profound

reduction of glycan utilization apparatus in some periodonto-pathogenic species yet on the other hand some

oral species seem proficient in degrading both plant and host secreted glycans.

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POSTER 2

Fic proteins of the niche-adapted pathogen Campylobacter fetus subsp. venerealis form cis- and trans-acting

toxin-antitoxin systems.

Priya Bhutada

Karl-Franzens Universität Graz

Key words: bacterial pathogen, protein toxin, posttranslational regulation, adenylylation, filamentation.

Background: Campylobacter fetus cause human infection and are important veterinary pathogens. Recent

comparative genomics of C. fetus subspecies revealed subspecies-specific fic loci encoding Fido superfamily

proteins, which may contribute to niche adaptation and pathogenicity.

Objectives: We analyzed function of 4 fic loci organized as toxin–antitoxin (TA) modules on the chromosome

and ICE of C. fetus subsp. venerealis 84-112.

Conclusions: We show that Fic proteins are cytotoxic to human cells but not S. cerevisiae. The fic loci are

organized as TA modules on the chromosome and ICE of C. fetus subsp. venerealis 84-112. Expression in E. coli

validated the cytotoxic and neutralizing activities of the proteins, providing the first functional evidence for TA

systems in Campylobacter. Reversal of fic-mediated filamentation and growth inhibition in E. coli also revealed

antitoxin crossreactivity between loci. Key active site residues involved in adenylylation by Fic proteins are

conserved in Fic1, Fic3 and Fic4, but degenerated in Fic2. We show the non-canonical Fic2 disrupts assembly of

E. coli ribosomes. fic genes are prevalent in C. fetus subsp. venerealis but not generally conserved among

Campylobacters. Strikingly, homologous genes are found in some Campylobacters and unrelated pathogens

adapted to the human and animal urogenital tract. C. fetus fic genes thus appear to be important to adaptation

and virulence in this niche.

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POSTER 3

MICROBIOMES OF INTESTINAL MUCOSA OF CHILDREN AND ADOLESCENTS WITH IBD UNDERGONE

DIFFERENT THERAPIES

Matijašić BB1, Blaž Stres T

2, Kamhi Trop T

3, Obermajer T

1, Mohar Lorbeg P

1, Orel R

3, Rogelj I

1

bojana.bogovic@bf.uni-lj.si, blaz.stres@bf.uni-lj.si, tina.kamhi@kclj.si, tanja.obermajer@bf.uni-lj.si,

petra.mohar@bf.uni-lj.si, rok.orel@kclj.si, irena.rogelj@bf.uni-lj.si

1Institute of Dairy Science and Probiotics, Department of Animal Science, Biotechnical Faculty, University of

Ljubljana, Slovenia,

2Chair for Microbiology and Microbial Biotechnology, Department of Animal Science, Biotechnical Faculty,

University of Ljubljana, Slovenia,

3Department of Gastroenterology, Hepatology and Nutrition, University Childrens’ Hospital, University Medical

Centre Ljubljana, Slovenia

Microbiota composition of intestinal mucosa (ileum, colon) of children and adolescents (aged ≤18 years) with

Crohn’s disease (CD, n=25), ulcerative colitis (UC, n=19) or controls (functional gastrointestinal disorders, n=39)

was first assessed before therapy. Selected groups were quantified by plate counting and/or qPCR of total

bacterial DNA. 44 IBD patients (average age 13.9 years, 21 females, 23 males) subjected to treatment with

enteral nutrition, steroids or biological drugs were selected for next generation sequencing (NGS, Illumina

MiSeq, 2x300 bp) of 16S rDNA amplicons. Plate counting revealed lower count (CFU/mg) of coliforms in ileum

of CD patients and lower counts of bifidobacteria in colon of CD patients compared to controls. qPCR analysis

didn’t show significant differences among the groups. NGS resulted in 44.040.416 reads. After assembly in

mother, 72.2% of reads passed QC and the signal to noise ratio between the shallowest sequenced sample

(n=23.766). Sequences were analysed within 97% OTU assignment in mother using Human Microbiome

standard approach. Statistical analysis relating microbial community assembly to available study parameters

(patient, location, time (pre-post treatment), diagnose, sex, age, or medical treatment) was conducted. The

larges part of variability in microbial community data was explained by medical treatment type suggesting that

it had higher impact on the microbiomes of intestinal mucosa than the other factors. One interesting aspect

was the interaction of the parameters analysed as some of them were orthogonal and consequently produced

mixed effects due to their interaction. Additional study parameters are currently being collated from available

medical databases and will be included in future in-depth analyses.

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POSTER 4

Thyme and olive extracts are subject to efflux and show anti-adhesive effects against Campylobacter jejuni

on polystyrene and intestine epithelial cells

Maja Šikić Pogačar 1,2

, Anja Klančnik1, Franz Bucar

3, Tomaž Langerholc

4, Sonja Smole Možina

1

1Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia

2Medical Faculty, University of Maribor, Maribor, Slovenia

3Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria

4Department of Microbiology, Biochemistry, Molecular Biology and Biotechnology, Faculty of Agriculture and

Life Science, University of Maribor, Hoče, Slovenia

Campylobacter is the most frequent cause of food-borne gastrointestinal bacterial infections in the EU. New

strategies targeting early stage infection have thus become crucial to control the prevalence of this bacterial

pathogen. Here we investigated agro-food waste material from thyme (Thymus vulgaris) and olive leaves (Olea

europaea).

Phytochemical analysis of thyme ethanolic extract (TE), thyme post-hydrodistillation residue (TE-R) and olive

leaf extract (OE) was done by HPLC-PDA analysis.

Using gene-specific knock-out mutants and an efflux-pump inhibitor, we show that for the efflux pump system,

CmeABC is the most active pump in extrusion across the outer membrane and is even more active in its biofilm

form. To avoid selection pressure for the emergence of antimicrobial resistance, we thus present an alternative

strategy that targets Campylobacter adhesion, the first step in biofilm formation and colonization. TE and TE-R

reduced C. jejuni adhesion to abiotic surfaces by less than 30% with concentrations of 0.2 to 12.5 mg/L.

Concentrations of OE from 3.125 mg/L to 200 mg/L reduced bacterial adhesion to polystyrene of 10% to 23%.

On the other hand, C. jejuni adhesion to PSI cl1 cells was inhibited by almost 30% over a large concentration

range of these extracts. Our findings suggest that TE or TE-R as agro-food waste material and OE as by products

represent a source of bioactive phytochemicals which could be utilized further as agents that prevent bacterial

adhesion.

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POSTER 5

Effect of hydrolysable tannins on gut microbiota of male pigs

Lijana Fanedl*, Luka Lipoglavšek*, Marjeta Čandek-Potokar**, Martin Škrlep**, Nina Batorek-Lukač**, Gorazd

Avguštin*

*University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Groblje 3, 1230 Domžale, Slovenia

**Agricultural Institute of Slovenia, Department of Animal Science, Hacquetova ulica 17, 1000 Ljubljana,

Slovenia

Boar taint is one of the problems occurring in meat production of pigs, namely of entire males when they reach

sexual maturity. Two substances with interconnected metabolism are held responsible, androstenone and

skatole. The accumulation of the skatole in adipose tissues depends on microbial metabolism of L-tryptophane

in the colon. The production of skatole depends on the activity of intestinal microbiota and the availability of

the substrate, which may be altered by dietary means.

The aim of our study was to investigate the effect of the supplementation of hydrolysable tannin-rich extract

from chestnut on intestinal microbiota and consequently the formation of skatole in intestinal content.

Twenty-four Landrace × Large White boars were assigned within a litter to four treatment groups: control (fed

mixture with 13.2 MJ/kg, 17.5% crude proteins) and three experimental diets for which the control diet was

supplemented with 1%, 2% and 3% of hydrolysable tannin-rich extract. Pigs were kept individually with ad

libitum access to feed and water and slaughtered at 193 days of age and 122 ±10 kg live weight. Total microbial

DNA from caecum and distal part of large intestine was extracted and gut microbiota composition was

evaluated by the DGGE analysis of 16S rRNA.

Next generation sequencing (NGS) method employing the Illumina MiSeq platform was used for further

characterization of gut microbiota composition. The findings are in agreement with the DGGE results and

confirm the effect of tannins on gut microbiota. Correlations between abundant bacterial populations and

tannin supplementation and other variables were discovered and will be discussed.

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POSTER 6

Visualization-Combined Microbiome Detection (VCMD) method enable deep analyses of Central Venous

Catheter Infections

Alexander Fuchs, Anastasia Bragina, Maria Sensen, Robert Krause & Gabriele Berg

Biofilms, bacteria-protective exopolysaccharide layers, are ubiquitous in environment and can also be found in

different medical indwelling devices. Biofilm formation on central venous catheters (CVCs) in many cases leads

to CVC-related blood stream infections (CRBSI), which results in higher mortality rate and increased treatment

costs.

The aim of our study was to investigate the CVC colonization patterns of removed catheters from patients who

developed infection by applying Visualization-Combined Microbiome Detection (VCMD) method. VCDM

includes fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscope

(CLSM) as well as sequencing based microbiome analyses.

In summary:

1. By applying FISH we were able to visualize the inner biofilm structure of CVC.

2. We could detect a two-layer structure in relation to the inner surface of the catheter.

3. The bacterial communities tend to localize both in the soft layer close to the lumen of the catheter and in

between the two layers.

4. The microbiome analyses of the community shows three distinct origins of bacteria:

a) environmental species e.g. Herbaspirillum seropaedicae, Brucella sp;

b) human/hospital-related species, e.g. Staphylococcus aureus;

c) commensal human flora-related species, e.g. Staphylococcus epidermitis, Enterococcus faecium.

15

POSTER 7

Deriving patterns of gastrointestinal dysbiosis

Bettina Halwachs

Medical University Graz

The human gut microbiota is a complex community of microbes with enormous implications for health and

disease. Microbial marker gene based amplicon sequencing studies (e.g. 16S rDNA) have shown a high degree

of interindividual variation of the microbiota, even in absence of disease, making the definition of a healthy vs a

diseased microbial community cumbersome. Indeed, this high degree of interindividual variation succumbs

possible disease specific microbial community patterns. Nevertheless, several diseases showed alterations of

the microbiota in previous investigations. Hence these changes are under suspect to be important drivers of

diseases, such as inflammatory bowel disease (IBD). Therefore, the term “dysbiosis”, describing an altered

microbiota, is widely used in the biomedical literature, despite its exact definition.

Here we introduce an effort to better define dysbiosis, by focusing on 16S amplicon data to derive signatures of

dysbiotic microbial community profiles.

16

POSTER 8

Campylobacter bacteriophage genome analysis paves the way to simple tools for classifying bacteriophages

in metagenomic studies.

Nika Janež1, Matjaž Peterka

1, Tomaž Accetto

2

1 Center of Excellence for Biosensors, Instrumentation and Process Control, Center for Biotechnology,

Ajdovščina,Slovenia

2 University of Ljubljana, Biotechnical Faculty, Animal sciences department, Domžale, Slovenia

The viral microbiome of humans and animals is dominated by bacterial viruses -bacteriophages (Paepae et al.,

2014). Despite their abundance, the role of bacteriophages, the bacterial predators, in shaping the microbiota

remains to be elucidated. Here, we present bacteriophages specific for Campylobacter, the leading cause of

human bacterial gastroenteritis in the EU since 2005 (EFSA and EDCD, 2014). We isolated several

Campylobacter specific bacteriophages from poultry (PC5-48, PM1-5) and pigs (PK80-83), because they have

been recognized as the major reservoirs for this pathogen. All isolated bacteriophages belong to Myoviridae

family and have 150 kb large genomes. Though they all rely on the presence of capsule for infection, they are

able to lyse different sets of human and poultry C. jejuni isolates. Campylobacter specific bacteriophages were

historically divided into three groups of which only representatives of groups II and III have been sequenced.

Resemblance within one group is large in terms of genome content, organisation and tetranucleotide usage,

and differs significantly from other group. Results of tetranucleotide statistics calculations combined with

average nucleotide identity analysis suggest existence of bacteriophage populations capable of recombining

over most of their genomes during host coinfection, but are at the same time very removed from other lytic

Myoviridae infecting the same host. This was also observed in Myoviridae bacteriophages of enterobacterial

genera where more than 35 groups were defined by the same criteria. This approach could be used to rapidly

classify the bacteriophage genomes obtained in metagenomic studies.

17

POSTER 9

FECAL MICROBIOMES OF CHILDREN AND ADOLESCENTS WITH INFLAMMATORY BOWEL DISEASE (IBD)

Kamhi Trop T1, Matijašić BB

2, Obermajer T

2, Treven P

2, Rogelj I

2, Orel R

1

tina.kamhi@kclj.si, bojana.bogovic@bf.uni-lj.si, tanja.obermajer@bf.uni-lj.si, primoz.treven@bf.uni-lj.si,

irena.rogelj@bf.uni-lj.si, rok.orel@kclj.si

1Department of Gastroenterology, Hepatology and Nutrition, University Childrens’ Hospital, University Medical

Centre Ljubljana, Slovenia

2Institute of Dairy Science and Probiotics, Department of Animal Science, Biotechnical Faculty, University of

Ljubljana, Slovenia

The microbiota composition of stool samples of 79 children and adolescents (aged ≤18 years) with active

Crohn’s disease (CD, n=24) or ulcerative colitis (UC, n=16), and controls (functional gastrointestinal disorders,

n=39), was assessed to determine the main differences in microbial composition between inflammatory bowel

disease patients and controls. Selected bacterial groups in stool samples were quantified by plate counting

and/or by qPCR of total bacterial DNA. The plate counting revealed higher count (CFU/g) of Enterococcus in

Crohn’s disease patients compared to controls and ulcerative colitis. Differences in CFU/g of

Enterobacteriaceae, Lactobacillus, Bifidobacterium, E. coli, coliforms, Enterobacteriaceae, or Staphylococcus

were not significant. qPCR analysis showed significantly higher amount of DNA specific for all bacteria, Cl.

coccoides and F. prausnitzii in controls compared to UC. On the contrary, the abundance of Enterobacteriaceae

and E. faecalis was lower in controls compared to UC. It also showed significantly higher concentrations and

ration of Clostridium leptum in control samples as compared to CD. The amounts of Bacteroides/Prevotella or

Bifidobacterium determined by qPCR did not differ among the three groups. These results indicate that fecal

microbiomes’ composition of young IBD patients (up to 18 years) are altered.

18

POSTER 10

Fecal Microbiota Transplantation (FMT)

Markus Koglmann

Technical University Graz

Fecal Microbiota Transplantation (FMT) has been applied successfully to cure severe Clostridium

difficile infections. In our preliminary study we have found that standard aerobic preparation procedure

of donor stool only minimally affects the bacterial composition of fecal transplants.

POSTER 11

Polycystic ovary syndrome (PCOS)

Lisa Lindheim

Medical University Graz

Background: Polycystic ovary syndrome (PCOS) is a complex female endocrinopathy characterized by

hyperandrogenism, oligo- or anovulation, and ovarian cysts, frequently associated with obesity and insulin

resistance. PCOS has a multifactorial etiology with environmental and genetic components. To date, no studies

on the gut microbiome in PCOS have been published, despite the fact that the gut represents an important

interface between the body and the environment and may thereby have a role in PCOS development.

Methods: Stool and saliva was obtained from 24 PCOS patients and 19 healthy controls. The 16S rRNA gene

(V1-2) was sequenced using Illumina’s MiSeq System. Reads were processed in mothur and microbiome

analyses performed in QIIME and R. Logistic regression models and correlation analyses were used to relate

microbiome parameters with clinical metadata.

Results: PCOS patients exhibited a reduced stool phylogenetic diversity compared to controls, with no obvious

clustering in beta diversity analyses. Saliva samples did not differ significantly between patients and controls. In

stool, taxa from several low-abundance taxa differed significantly between patients and controls, with patients

rarely harboring these taxa. Absence of these taxa was associated with unfavorable hormonal and metabolic

characteristics.

Conclusion: PCOS patients have an altered stool, but not saliva, microbiome compared to healthy controls. This

difference is due to a reduced alpha diversity and the absence of certain low-abundance taxa, which need to be

further investigated as to their effect on metabolic and hormone homeostasis.

19

POSTER 12

Comparative microbiota profiling of cutaneous tumors identifies Staphylococcus aureus as a possible driver

of neoplasia development

Madhusudhan, Nandhitha1; Becker C, Jürgen

2 ; Gorkiewicz Gregor

1

1Medical University of Graz, Austria

2 DKTK, Essen, Germany

Malignant epithelial skin tumors (squamous cell carcinoma-SCC) arise from specific precursors (actinic

keratosis-AK). The development of the tumor alters the host-microbiome equilibrium and an altered pro-

inflammatory microbiome might drive cancer progression. Thus, we aimed to assess the microbial communities

during cutaneous neoplasia development of pre-invasive AK and invasive SCC & BCC, correlate these findings

with the respective immunophenotypes and further identify the molecular basis of microbiome-driven

neoplasia development.

Microbial community profiling using Miseq 16S rDNA sequencing showed that AK and SCC have a higher

microbial richness but lower evenness and diversity compared to BCC suggesting the presence of certain

dominant bacteria in these tumors. A significantly increased relative abundance of Staphylococcus was

observed in AK & SCC whereas a higher abundance of Streptococcus was seen in BCC compared to SCC. qPCR

confirmed these findings and hints to a direct involvement of S. aureus in neoplasia development of the

squamous epithelium. Further, AK and SCC showed a significantly increased expression of specific AMPs, HBD-2

& 3, know to cause hyperproliferation of squamous epithelia in comparison to healthy skin and BCC.

Noteworthy, the severity of inflammation significantly correlated with increased species richness and

Staphylococcus overabundance in AK but not in SCC and BCC, suggestive of a direct involvement of

Staphylococcus in skin tumor development.

Our data identifies S. aureus as a potential driver of cutaneous neoplasia development. Since, S. aureus is

known to provoke specific immune-responses, analyses of the interplay between microbe, immune-system and

the host epithelium will deepen our understanding of processes which govern skin tumor development.

20

POSTER 13

The microbiome of the International Space Station

Authors: Maximilian Mora, Christine Moissl-Eichinger

Medical University Graz

Almost complete isolation from the outside world and extreme environmental conditions, such as microgravity

and enhanced background radiation, define the International Space Station ISS as an unique biotope which is

now continuously inhabited by humans for 15 years. For future long term space flight missions it is critical to

assess the dynamics and eventual development of resistances of the microbial population in such a special

closed system. This is important because these microbes might pose a threat to humans onboard a spacecraft

or even the integrity of the spacecraft itself. Therefore it is mandatory to investigate the ISS´ microbial

population beyond the current regular monitoring. The ARBEX project (ARchaeal and Bacterial EXtremophiles

onboard the ISS), is designed to do exactly that. A broad assembly of cultivation based and molecular assays

focuses not only on possibly pathogenic but also yet undetected microbes onboard the ISS which might

influence the crew´s health in different ways. Isolates will also be compared to isolates of ground controls to

evaluate if the microbes adapted to a life in space. This poster will present first data obtained from the recent

analysis of ISS indoor dust samples. Furthermore data of the analysis of the first ground control, a clean-room

in Kourou, French Guiana will be presented. Additionally, the progress made in defining the sampling spots for

the ARBEX sampling in the ISS, which was done during a visit of the ISS mock-up model at the European

Astronaut Training Centre (EAC) at DLR in Cologne will be reported.

21

POSTER 14

Is life out there?

Alexandra Perras

Medical University Graz

Is life out there? Microorganisms are the key players in all conceivable habitats and can be even detected in

environments regarded hostile for life. Assessing the habitability of the most Earth-like planet, the Mars,

demands a deep understanding of the adaption and occurrence of microbial life thriving in similar conditions.

So-called “Mars analogues sites” – habitats on Earth, which resemble Mars environments–can be found all over

Europe (e.g. brine ponds, acidic lakes, sulfur springs) and share life-hostile conditions such as low nutrients

availability and the absence of oxygen.

Since 2014, the MASE (Mars Analogues for Space Exploration) team, a joint collaboration of researchers from

Europe, aims i) the identification of the living proportion of the microbial community thriving in selected Mars

analogues sites , ii) the cultivation and characterization of novel anaerobic microorganisms and iii) the testing

of their capacity to survive when exposed to Mars similar stresses e.g. desiccation or high doses of radiation.

Until now, several microorganisms were isolated, cultivated under anaerobic conditions and identified.

Interestingly, the majority of cultivated microorganisms is rather known to be associated with diseases in

human beings (e.g. pathogen related species of the genera Yersinia and Clostridium). In future studies, we

intend to understand the niche differentiation of pathogen-related microorganisms by revealing their genomic

inventories with the key factors and the phylogeny of pathogenicity triggering genes. By facing the knowledge

of in particular poorly understood anaerobic pathogen-related microbes, we can develop further defeating

strategies for pathogenic microbes.

22

POSTER 15

TRACING THE SYSTEMATIC ERRORS IN PRIMER-GUIDED AMPLICON SEQUENCING

Blaz Stres1, Bostjan Murovec

2

University of Ljubljana,

1 Biotechnical Faculty, Group for Microbiology and Microbial Biotechnology, Groblje 3, 1230 Domzale, Slovenia;

2 Faculty of Electrical Engineering, Group for Systems, Control and Cybernetics, Trzaska 25, 1000 Ljubljana,

Slovenia

Amplicon sequencing represents a backbone for analyses of microbial communities via deep-sequencing of

phylogenetic, house-keeping and functional genes. Despite its wide adoption it suffers from inconsistent use of

various primer sets, unequal sampling efficiencies, specificity and combinatorial exclusion of sequences due to

sequence mismatch. The limitations in the relationship between the short reads and full range genes further

complicates elucidation of biological meaning due to comparison of various stretches of targeted genes with

unique evolutionary paths. In this study the sampling capacity of various primer combinations (n>2000) at full

stringency (0 mismatches allowed) was tested on high-quality full length 16S rRNA genes collected from Silva

and RDP II databases. Each primer pair generated its own virtual microbial community. Contrary to previous

studies, the detected sequences were not clipped to remove stretches outside primer binding sites, instead,

full-length sequences were retained in order to compare unbiased signal of full length datasets. The shift in

community structure arising from the use of different primer pairs (in deep-sequencing low-cycle-number PCR

amplification) was identified by mapping the signals of virtual microbial communities (obtained as a function of

primer-region constellations) to ground truth – the original database. Significant systematic error was

introduced by various primer sets that effectively guided sequence selection and resulted in significant

undersampling. In conclusion, various primer sets distorted the obtained picture of ground-truth microbial

community beyond recognition, making possible only the comparisons of datasets generated within one study

only and making cross-study comparisons and data agglomeration a difficult task.