ICDS conference 2020 ves
Transcript of ICDS conference 2020 ves
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: [email protected]
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.