FEMS Online Conference on Microbiology 2020 Belgrade, Serbia 28‐31 October 2020

FEMS Online Conference on Microbiology 2020 Belgrade, Serbia 28‐31 October 2020

7th INTERNATIONAL CLOSTRIDIUM DIFFICILE SYMPOSIUMVIRTUAL MEETING 2020

ÁÁC. RODRIGUEZ*1,2, B. TAMINIAU 3, F. MARTIN-REYES 1,2, A. HO-PLÁGARO1,2, P. BLANC4, G. ALCAÍN-MARTINEZ 1,2, G. DAUBE 4, E. GARCÍA-FUENTES1,2

1 UGC Aparato Digestivo, Hospital Universitario Virgen de la Victoria, Málaga, Spain2 Instituto de Investigación Biomédica de Málaga, Málaga, Spain

3University of Liege, Faculty of Veterinary Medicine, Department of Food Science & FARAH, Liège, Belgium4UGC-Microbiología, Hospital Universitario Virgen de la Victoria, Málaga, Spain

C. RODRIGUEZ*1,2, B. TAMINIAU 3, F. MARTIN-REYES 1,2, A. HO-PLÁGARO1,2, P. BLANC4, G. ALCAÍN-MARTINEZ 1,2, G. DAUBE 4, E. GARCÍA-FUENTES1,2

1 UGC Aparato Digestivo, Hospital Universitario Virgen de la Victoria, Málaga, Spain2 Instituto de Investigación Biomédica de Málaga, Málaga, Spain

3University of Liege, Faculty of Veterinary Medicine, Department of Food Science & FARAH, Liège, Belgium4UGC-Microbiología, Hospital Universitario Virgen de la Victoria, Málaga, Spain *presenting author: cris.rdrz@gmail.com

The release of membrane‐bound vesicles is a conserved cellular process. Gram‐positive and Gram‐negative secrete nanometer‐scale extracellular membrane

l ( ) h b l l f l

The aim of this study was to evaluate the microbialdiversity of feces and secreted EMV in healthy patients,

vesicles (EMV) with important biological functions, including immune‐responseregulation, long distance transport of virulence factors, lateral transfer of antibioticresistance genes, or RNA transfer agents, among others. For Clostridium difficile (C.difficile), these vesicles have been associated with the infection (CDI), since they caninduce the expression of pro‐inflammatory genes and epithelial cells cytotoxicity

diarrheic patients and patients with CDI. The link betweenmicrobiota composition and their derived EMV could revealnewt insights into the microbial activities in the host.Furthermore, the identification of these changes opens upnew possibilities of disease diagnostic and assessmentinduce the expression of pro inflammatory genes and epithelial cells cytotoxicity. new possibilities of disease diagnostic and assessment.

EMV ISOLATIONC DIFFICILE DETECTION METAGENETIC ANALYSISEMV ISOLATION

Fresh faecal samples of all patients 

were tested for the presence of C. 

difficile using:

C. DIFFICILE DETECTION

Vesicles isolation was performed by

faecal dilution and 

t if ti (5000 5600

METAGENETIC ANALYSIS

difficile using:

chromatographic immunoassay 

rapid test

rapid PCR 

classical microbiological culture

centrifugations (5000‐5600 rpm 

30‐40 min 4ºC x 3 times)

supernatant filtrations (filters of 

0.45 μm and 0.22 μm)

ultracentrifugations (130 000g

Figure 1. Metagenetic approach to determine the

proportions and absolute counts of microorganisms.

Work plan includes sample preparation, DNA

extraction, bench metagenetic analysis, sequence

analysis and interpretation of the results. 16RNA

classical microbiological culture

Further confirmation and 

characterisation of the isolates was 

performed by classical PCR with the 

detection of the tpi gene and the

ultracentrifugations (130.000g 

180 min 4ºC)

The vesicles concentrations and sizes 

were analysed using the Nanosight 

technique with the technology of sequence is a universal highly conserved bacterial

genetic marker. In this study bacterial 16S rRNA gene

amplification and barcoded pyrosequencing was

performed by targeting the V1‐V3 hypervariable region

detection of the tpi gene, and the 

toxin genes tcdA, tcdB and cdtA.technique, with  the technology of 

nanoparticle tracking analysis (NAT).

High throughput sequencing results: healthy vs diarrhea  Figure 2. (a, b) Vesicles (V) composition versus faecal microbiota (F) composition at genus level in a group of 10 patientswith diarrhea (c) Vesicles (V) composition versus faecal microbiota (F) composition at genus level in a group of 10 healthypatients and 10 patients with diarrhea (d) Statistical differences using 2‐way ANOVA and Sidak multiple comparisons test atgenus level between faecal microbiota composition and vesicles composition of diarrheic patients (e) Multiple comparisonsbetween the different groups at genus level (i) feces healthy (ii) feces diarrhea (iii) vesicles diarrhea (iv) vesicles healthy. 10000

e

Vesicles and feces composition in diarrheic patients Genus level

10000

Diarrhea feacal samples vs diarrhea vesicules samplesGenus level

(a) (b) (c) (e)

Statistical differences were found in taxa Alistipes, Bacteroides, Holdemia, Barnesiella, and Veillonellaceae (difference inmean proportions %).

D1

_FD

2_F

D3

_FD

4_M

FD

5_F

D6_

MF

D7

_FD

8_F

D9

_FD

10_F

D1_

VD

2_V

D3_

VD

4_V

D5_

VD

6_V

D7_

VD

8_V

D9_

VD

10_

V

0

5000

Pacients and type of sample

Rel

ativ

e ab

un

dan

ce

Lachnospiraceae_geBlautia

Prevotella Ruminiclostridium_6

D1_

FD

1_V

D2_

FD

2_V

D3_

FD

3_V

D4_

FD

4_V

D5_

FD

5_V

D6_

FD

6_V

D7_

FD

7_V

D8_

FD

8_V

D10

_FD

10_V

D11

_FD

11_V

0

5000

Pacients and type of sample

Rel

ativ

e ab

un

dan

ce

Prevotella Ruminiclostridium 6

High throughput sequencing results: diarrhea  vs C. difficile diarrhea  

F l t l l b t i l i l f Cl t idi id C ti f i l l ith d Cl t idi id Statistical differences Clostridioides vesicles composition( ) (b) ( )

Faecalibacterium

Prevotella_9

Fusobacterium

Clostridioides

Staphylococcus

Escherichia-Shigella

Streptococcus

Flavonifractor

Lachnoclostridium_5

Enterococcus

Bacteroides

Erysipelatoclostridium

Romboutsia

Clostridium_sensu_stricto_1

Subdoligranulum

Ruminococcaceae_ge

Citrobacter

Lactococcus

Agathobacter

Lachnoclostridium

Ruminococcus_1

Terrisporobacter

Others

Faecalibacterium

Prevotella_9

Fusobacterium

Clostridioides

Staphylococcus

Escherichia-Shigella

Streptococcus

Flavonifractor

Lachnoclostridium_5

Enterococcus

Bacteroides

Erysipelatoclostridium

Clostridium_sensu_stricto_1

Lachnospiraceae_ge

SubdoligranulumRuminococcaceae_ge

Blautia

Citrobacter

_

Lactococcus

Agathobacter

Lachnoclostridium

Ruminococcus_1

Terrisporobacter

Others

Romboutsia

alib

acte

rium

epto

cocc

us

ostrid

ium

_5

tero

cocc

us0

1000

2000

3000

Statistical differences genus level

Mea

n c

um

ula

tive

rel

ativ

e ab

undan

ce

Vesicles composition

Feces composition

(d)

6000

8000

10000

ive

rela

tive

abundan

ce

Feacal extracelular bacterial vesicules: presence of Clostridioides

4000

6000

8000

10000

Rel

ativ

e ab

un

dan

ce

Comparation of vesicules samples with and genus Clostridioides

3000

Statistical differences Clostridioides vesicles composition

Vesicles with Clostridioides

Vesicles without Clostridioides

(a) (b) (c)

Characterization of the isolated EMV

(A) (B)

Faeca

li

S

trep

L

achnocl

os

E

nte

(C)

Vesicules_Clostridioides Vesicles_without Clostridioides0

2000

4000

Type of samples

Mea

n c

um

ula

t

ParasutterellaOthers

NC

_v1

NC

_v3

NC

_v4

NC

_v5

NC

_v6

NC

_v13

NC

_v14

NC

_v3

.1N

C_v3

.2N

C_v3

.3N

C_v3

.5N

C_v3

.6N

C_v

3.14

NC

_v3.

18N

C_v

3.22

NC

_v3.

28N

C_v

3.30

C_v

3.13

C_v

3.15

C_v

3.16

C_v

3.17

C_v

3.25

C_v

3.26

C_v

3.31

C_v

3.32

C_v

3.33

C_v

3.34

C_v

3.35

C_v

3.36

C_v

3.37

C_v

3.38

C_v

3.42

C_v

3.43

C_v

3.440

2000

Type of sampleNC: without Clostridioides genus; C: with Clostridioides genus

R

CutibacteriumOthers

1000

2000

ean

cu

mu

lati

ve r

elat

ive

abu

nd

ance

Faecalibacterium

Clostridioides

Staphylococcus

Bacteroides

Flavonifractor

Prevotella_9

Enterococcus

Ruminococcaceae_UCG-013

Escherichia-Shigella

Romboutsia

Subdoligranulum

Clostridium_sensu_stricto_1

Cutibacterium

Flavobacterium

Hafnia-Obesumbacterium

Turicibacter

Ruminococcaceae_ge

Pseudomonas

Lachnospiraceae_ge

Alistipes

Fusobacterium

Agathobacter

Peptostreptococcaceae_ge

Blautia

Prevotella

Faecalibacterium

Clostridioides

Staphylococcus

Bacteroides

Flavonifractor

Prevotella_9

Enterococcus

Ruminococcaceae_UCG-013

Escherichia-Shigella

Romboutsia

Subdoligranulum

Clostridium_sensu_stricto_1

Parasutterella

Flavobacterium

Hafnia-Obesumbacterium

Turicibacter

Ruminococcaceae_ge

Pseudomonas

Lachnospiraceae_ge

Alistipes

Fusobacterium

Agathobacter

Peptostreptococcaceae_ge

Blautia

Prevotella

F

aeca

libac

teriu

m

C

lost

ridio

ides

S

taphyl

ococc

us

B

acte

roid

es

Entero

cocc

us

0

Genus bacteria level

M

Figure CellFigure 4. A) Electron microscope image of EMVs B) Fluorescence microscope photography. Cell

nucleus (blue) and bacterial vesicles (red) C) Characterization and quantification of EMVs by

Nanosight technology

Figure 3. (a) Mean cumulative relative abundance at genus level. Vesicles with Clostridioides versus Vesicles without Clostridioides

(n=17) (b) Microbiota composition at genus level in a group of 17 patients. Vesicles with Clostridioides versus vesicles without

Clostridioides (c) Statistical differences using 2‐way ANOVA and multiple T test comparisons were found at genus level when we

compare vesicles composition with and without Clostridioides from diarrheic patients. Faecalibacterium, and Bacteroides are

reduced when proportions of Clostridioides, Staphylococcus and Enterococcus are high

Study financially supported by ISCIII (Spain) (E. García‐Fuentes) and the University ofLiège (Microbiology Unit, Prof. G. Daube). C. Rodriguez is supported by a grant from theISCIII (Spain) (CD18/00188) This work has been performed in part under the ECVPHresident program (C. Rodriguez, G. Daube)

EMV were enriched in the 3 groups of patients, but their composition differed significantly between them. Regarding global differences between feces and EMV,Lachnoclostridium and Streptococcus were more abundant in feces, but their vesicles production was limited and dominated by Faecalibacterium. At genus level,proportions of Clostridioides, Staphylococcus and Enterococcus were significantly higher in vesicles from CDI patients than in the other groups. These findings suggestthat the increased production of EMV by these taxa could be associated with the dysbiosis establishment, and therefore with the development of the infectiousdisease. More extensive research to investigate the specific role of the identified EMV in the CDI is now warranted.