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7th INTERNATIONAL CLOSTRIDIUM DIFFICILE SYMPOSIUM VIRTUAL MEETING 2020 Á Á C. RODRIGUEZ* 1,2 , B. T AMINIAU 3 , F. MARTIN-REYES 1,2 , A. HO-PLÁGARO 1,2 , P. BLANC 4 , G. ALCAÍN-MARTINEZ 1,2 , G. DAUBE 4 , E. GARCÍA-FUENTES 1,2 1 UGC Aparato Digestivo, Hospital Universitario Virgen de la Victoria, Málaga, Spain 2 Instituto de Investigación Biomédica de Málaga, Málaga, Spain 3 University of Liege, Faculty of Veterinary Medicine, Department of Food Science & FARAH, Liège, Belgium 4 UGC-Microbiología, Hospital Universitario Virgen de la Victoria, Málaga, Spain C. RODRIGUEZ* 1,2 , B. T AMINIAU 3 , F. MARTIN-REYES 1,2 , A. HO-PLÁGARO 1,2 , P. BLANC 4 , G. ALCAÍN-MARTINEZ 1,2 , G. DAUBE 4 , E. GARCÍA-FUENTES 1,2 1 UGC Aparato Digestivo, Hospital Universitario Virgen de la Victoria, Málaga, Spain 2 Instituto de Investigación Biomédica de Málaga, Málaga, Spain 3 University of Liege, Faculty of Veterinary Medicine, Department of Food Science & FARAH, Liège, Belgium 4 UGC-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 microbial diversity of feces and secreted EMV in healthy patients, vesicles (EMV) with important biological functions, including immune‐response regulation, long distance transport of virulence factors, lateral transfer of antibiotic resistance genes, or RNA transfer agents, among others. For Clostridium difficile (C. difficile), these vesicles have been associated with the infection (CDI), since they can induce the expression of pro‐inflammatory genes and epithelial cells cytotoxicity diarrheic patients and patients with CDI. The link between microbiota composition and their derived EMV could reveal newt insights into the microbial activities in the host. Furthermore, the identification of these changes opens up new possibilities of disease diagnostic and assessment induce the expression of pro inflammatory genes and epithelial cells cytotoxicity . new possibilities of disease diagnostic and assessment. EMV ISOLATION C DIFFICILE DETECTION METAGENETIC ANALYSIS EMV 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 patients with diarrhea (c) Vesicles (V) composition versus faecal microbiota (F) composition at genus level in a group of 10 healthy patients and 10 patients with diarrhea (d) Statistical differences using 2‐way ANOVA and Sidak multiple comparisons test at genus level between faecal microbiota composition and vesicles composition of diarrheic patients (e) Multiple comparisons between 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 samples Genus level (a) (b) (c) (e) Statistical differences were found in taxa Alistipes, Bacteroides, Holdemia, Barnesiella, and Veillonellaceae (difference in mean proportions %). D1_F D2_F D3_F D4_MF D5_F D6_MF D7_F D8_F D9_F D10_F D1_V D2_V D3_V D4_V D5_V D6_V D7_V D8_V D9_V D10_V 0 5000 Pacients and type of sample Relative abundance Lachnospiraceae_ge Blautia Prevotella Ruminiclostridium_6 D1_F D1_V D2_F D2_V D3_F D3_V D4_F D4_V D5_F D5_V D6_F D6_V D7_F D7_V D8_F D8_V D10_F D10_V D11_F D11_V 0 5000 Pacients and type of sample Relative abundance Prevotella Ruminiclostridium 6 High throughput sequencing results: diarrhea vs C. difficile diarrhea F l t ll b t il 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 Subdoligranulum Ruminococcaceae_ge Blautia Citrobacter _ Lactococcus Agathobacter Lachnoclostridium Ruminococcus_1 Terrisporobacter Others Romboutsia a l ib a c te r i u m e p t o c o c c u s o s t r id iu m _ 5 t e r o c o c c u s 0 1000 2000 3000 Statistical differences genus level Mean cumulative relative abundance Vesicles composition Feces composition (d) 6000 8000 10000 ive relative abundance Feacal extracelular bacterial vesicules: presence of Clostridioides 4000 6000 8000 10000 Relative abundance 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) F a e c a l i S t r e p L a c h n o c l o s E n t e (C) Vesicules_Clostridioides Vesicles_without Clostridioides 0 2000 4000 Type of samples Mean cumulat Parasutterella Others NC_v1 NC_v3 NC_v4 NC_v5 NC_v6 NC_v13 NC_v14 NC_v3.1 NC_v3.2 NC_v3.3 NC_v3.5 NC_v3.6 NC_v3.14 NC_v3.18 NC_v3.22 NC_v3.28 NC_v3.30 C_v3.13 C_v3.15 C_v3.16 C_v3.17 C_v3.25 C_v3.26 C_v3.31 C_v3.32 C_v3.33 C_v3.34 C_v3.35 C_v3.36 C_v3.37 C_v3.38 C_v3.42 C_v3.43 C_v3.44 0 2000 Type of sample NC: without Clostridioides genus; C: with Clostridioides genus R Cutibacterium Others 1000 2000 ean cumulative relative abundance 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 a e c a lib a c t e r i u m C lo s tr i d io i d e s S t a p h y l o c o c c u s B a c t e r o i d e s E n t e r o c o c c u s 0 Genus bacteria level M Figure Cell Figure 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 of Liège (Microbiology Unit, Prof. G. Daube). C. Rodriguez is supported by a grant from the ISCIII (Spain) (CD18/00188) This work has been performed in part under the ECVPH resident 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 suggest that the increased production of EMV by these taxa could be associated with the dysbiosis establishment, and therefore with the development of the infectious disease. More extensive research to investigate the specific role of the identified EMV in the CDI is now warranted.
