Rôle des probiotiques lors d'infections entériques d'origine bactérienne et virale : analyses in vitro et études in vivo chez des modèles murins

Liste des illustrations

• Figure 1.1 : Souche de bifidobactérie. Microscopie électronique à balayage. Tiré de Rouvet (2004).
• Figure 1.2 : Changement du microbiote intestinal en fonction de l’âge.Tiré de Ballongue (1998).
• Figure 1.3 :Guide pour l’évaluation des probiotiques en utilisation alimentaire.Tiré et traduit de FAO/WHO (2002).
• Figure 1.4 : Mécanismes d’action proposés des micro-organismes probiotiques dans le traitement des infections entériques. Adapté de Calder et Kew (2002); Kaur et al. (2002).
• Figure 1.5 : Mécanisme d’inhibition de l’adhésion des pathogènes par un effet barrière dû à l’adhésion spécifique (a) et non spécifique (b) des probiotiques.
• Figure 1.6 : Mode d’action des acides organiques produits par les probiotiques contre les pathogènes bactériens. *X-COOH = CH₃-CHOH-COOH dans le cas de l’acide lactique ou CH₃COOH dans le cas de l’acide acétique.
• Figure 1.7 : E. coli O157:H7. Microscopie électronique à balayage. Tiré de Centers for Disease Control and Prevention (2006).
• Figure 1.8 : Pathogénie de la souche entérohémorragique E. coli O157:H7 chez l’humain. Adapté de Nataro et Kaper (1998); EcL (2004).
• Figure 1.9 : Particules du rotavirus. Microscopie électronique à transmission. Tiré de Centers for Disease Control and Prevention (1981).
• Figure 1.10 : Pathogénie des rotavirus chez l’humain. Adapté de Levy et al. (1994); Clarin (2007).
• Figure 1.11 : Défenses antimicrobiennes de l’hôte face à l’intrusion de micro-organismes pathogènes au niveau du tractus intestinal. Tiré de Liévin-Le Moal et Servin (2006).
• Figure 1.12 : Colonisation microbienne du tractus gastrointestinal humain. Adapté de Holzapfel et al. (1998).
• Figure 1.13 : Le système immunitaire intestinal. Tiré et traduit de Magalhaes et al. (2007).
• Figure 2.1 : Agarose gel electrophoresis of PCR products from infant bifidobacterial isolates used in this study. PCR products were amplified with Bifidobacterium genus-specific primers lm26-f and lm3-r. Lane 1: size markers generated by HindIII-digested DNA. Lanes 2-6: infant bifidobacterial isolates RBL71; RBL81; RBL82; RBL460 and RBL461, respectively. Lane 7: B. longum ATCC 15707.
• Figure 2.2 : Agar spot test showing antagonistic activity of infant bifidobacterial isolates and B. breve ATCC 15700 against E. coli O157:H7. Clear area is zone of inhibition.
• Figure 2.3 : Bacterial adhesion to the human intestinal cell-line Caco-2. (A) Gram staining of E. coli O157:H7 adhesion (magnification ×630), (B) confocal laser-scanning micrograph of E. coli O157:H7 detected with ALEXA 488-labeled antibodies (magnification ×1000), and (C) simultaneous double labeling of B. longum ATCC 15707 (green) and E. coli O157:H7 (red) with ALEXA 488 and 568-labeled antibodies respectively, by immunofluorescence (magnification ×1000).
• Figure 2.4 : Inhibition of enterohemorrhagic E. coli O157:H7 adhesion to Caco-2 cells by infant bifdobacterial isolates,(a) B. bifidum RBL71 and (b) B. bifidum RBL460. To each well of growing Caco-2 cells, 2.5 × 10⁷ cfu of E. coli O157:H7 were added simultaneously with increasing concentrations of bifidobacteria. Values are means ± SE of triplicate analyses. Asterik indicates significant differences (P < 0.05) from trials with E. coli O157:H7 alone (  ).
• Figure 3.1 : Fecal B. thermacidophilum RBL71 (A) and E. coli O157:H7 (B) counts in group mice during the experiment (means ± standard deviation, n = 3).(*) Significant difference between pre- or post-infection group and infected group (P < 0.05);(**) Significant difference between pre- or post-infection group and infected group (P < 0.01).
• Figure 3.2 : Body weights of the five groups of mice during the week following challenge with E. coli O157:H7 (means ± SEM,n = 5).(*)Significant difference between pre-infection feeding group and infected group (P < 0.05);(**)Significant difference between control and infected groups (P < 0.01).
• Figure 3.3 : Hematoxylin and eosin stained cross-sections of mouse intestines at day 7. (A) jejunum of a control group mouse; (B) jejunum of an E. coli O157:H7-infected mouse; (C) ileum of a control group mouse; (D) ileum of an E. coli O157:H7-infected mouse; (E) colon of a control group mouse; (F) colon of an E. coli O157:H7-infected mouse; (G) ileum of mouse fed B. thermacidophilum before E. coli O157:H7 infection; (H) ileum of mouse fed B. thermacidophilum after E. coli O157:H7 infection. Black arrows: panel F, edematouscolonic epithelia; panels G and H, lymphocyte infiltration (magnification, × 100).
• Figure 3.4 : Periodic acid-Schiff-stained cross-sections of mouse colons at day 7. (A) control group; (B) E. coli O157:H7-infected; (C and D) given B. thermacidophilum for seven days respectively before and after infection (magnification, × 400).
• Figure 3.5 : Levels of E. coli O157:H7-specific IgA in feces (A) and IgG+IgM in serum (B) after E. coli O157:H7 challenge in non infected + feeding, infected and in pre- and post-infection feeding groups. Each column shows the mean ± SEM of three and five mice for IgA and IgG+IgM, respectively. (, P < 0.01).
• Figure 4.1 : Visualization of detection methods used for evaluating viral concentration. (A) Plaque assay of serial 10-fold dilution of FCV starting at 1×10⁷ PFU/ml in CrFK cells stained with crystal violet two days post-infection. Control well is non infected CrFK cells. (B) Indirect immunofluorescence of rotavirus at concentration of 1×10⁶ PFU/ml in MA-104 cells. The inset shows control non infected MA-104 cells (magnification × 200).
• Figure 4.2 : Attachment of enteric viruses at concentration of 1×10⁶ PFU/well to intestinal epithelial cells at different times post-infection.The results shown represent the means ± SEM of three replicate infected cultures.
• Figure 4.3 : Replication of enteric viruses in intestinal epithelial cells. Viruses were added at a concentration of 1×10⁶ PFU/well on human intestinal cells. Culture supernatants were collected at various times post-infection and virus titers measured as described in Materials and Methods.
• Figure 5.1 : Fluorescent micrographs of MA-104 cells used for the determination of rotavirus titer. (A) Inoculum rotavirus (5×10⁶ PFU/ml) used for exclusion, competition and displacement assays on HT-29 cells; (B and C) Rotavirus at 5×10⁶ PFU/ml applied to HT-29 cells 30 min after (exclusion assay) and simultaneously (competition assay) with B. thermophilum RBL67 at 5×10⁹ CFU/ml, respectively; (D) Controls non infected cells.
• Figure 6.1 : Body weights of the five groups of mice during the week following challenge with rotavirus (means ± SEM, n > 30).
• Figure 6.2 : Viable bifidobacteria in the intestines of suckling CD-1 mice fed B. thermophilum for seven days (means ± SEM, n = 3).
• Figure 6.3 : Content of infectious rotavirus SA-11 in the intestines of suckling CD-1 mice after challenge. Titers of virus in the combined small and large intestine were determined at various times post-infection by plaque assay. Data are means ± SEM for three mice per group. (*) P< 0.05 vs infected group; (**) P < 0.01 vs infected group.
• Figure 6.4 : Hematoxylin and eosin-stained cross-sections of the colons of suckling CD-1 mice at different times post-infection (PI) with rotavirus SA-11 (magnification, ×40).
• Figure 6.5 : Specific immunoglobulin production in suckling CD-1 mice after challenge with rotavirus SA-11. (A) IgA in the intestinal homogenate; (B) Serum IgG and IgM (means ± SEM, n = 3; ** significantly different, P < 0.01).