Physiology of EHEC carriage by ruminants Antagonist properties of probiotics

1. Physiology of EHEC carriage by ruminants Antagonist properties of probiotics

2. Objectives Limiting the risk of food-borne infections Limiting EHEC shedding by ruminants: reduction of EHEC carriage Knowledge of physiology and ecology of EHEC in the bovine gastro-intestinal tract Selection and evaluation of probiotic strains

3. Effects of probiotics on EHEC survival in ruminant digestive contents • Probiotics can positively modulate the microbial balance of the animal gut and thereby reduce its colonisation by foreign bacteria • Some of them may also exert direct inhibitory effects on foreign bacteria (competition for adhesion/nutrients, bacteriocins, inhibitory compounds…)

4. Method 3 sheep fed hay + wheat + 5 x 109 CFU Lactobacillus acidophilus BT-1386 3 sheep fed hay + wheat 3 sheep fed hay + wheat + 4 x 109 CFU Saccharomyces cerevisiae I-1077 Rumen contents through canula (pH 5.7) Abomasum contents after feeding (pH 2.5) at slaughter Jejunal and caecal contents at slaughter (pH 7.8 and 7.7 respectively) Incubation of STEC strains at 39°C EDL933 (O157:H7), 4 acid-resistant, 4 acid-sensitive strains (diverse serotypes)

5. Consequences of acid-resistance in STEC Adaptation to acid stress Induction of acid-resistance mechanisms - to acid stress in the bovine abomasum (pH 2.5) - to food processing - to acid stress in the human stomach Resistance Increases the risk of food poisoning in humans

6. STEC survival after 24 h in the rumen fluid Anaerobiosis In the presence of S. cerevisiae or L. acidophilus (3 x 105 CFU/ml), the survival rates dropped below 1.48%. Mortality occurred earlier in the presence of L. acidophilus EDL933 persists 12 h in the rumen fluid, then declines. Mortality is observed at 18 h. Chaucheyras Durand et al. 2010, AEM

7. STEC survival after 2 h in the abomasal fluid Static culture EDL933 and acid-resistant strains survive in the abomasal contents at variable levels. Acid-sensitive strains show high mortality rates Chaucheyras Durand et al. 2010, AEM

8. Induction of acid-resistance in the rumen contents STEC 106 cfu/ml 6 h Rumen fluid Abomasal fluid Survival Acid-sensitive strains become acid-resistant after transit in the rumen fluid whatever the diet Transit of STEC strains in the cattle gastro-intestinal tract may increase the risk of human infection Chaucheyras Durand et al. 2010, AEM

9. t = 0 h t = 8 h t = 0 h t = 2 h STEC growth after 2 h in jejunal contents Static cultures Significant growth of STEC strains in both intestinal contents Probiotics did not limit STEC growth in these compartments STEC growth after 8 h in caecal contents Chaucheyras Durand et al. 2010, AEM

10. Control without L.A. L.A. EHEC O157:H7 growth after 24 h in faecal suspension • (CFU t24-t0) Log10.ml-1 + L.A. 3.4 x 105.ml-1 + L.A. 3.2 x 106.ml-1 + L.A. 8.5 x 107.ml-1 Dose-dependent inhibition of STEC growth by L. acidophilus Chaucheyras Durand et al. 2006, AEM

11. Acid-resistance confers an ecological advantage to STEC strains for their persistence in the rumen Acid sensitive strains can become resistant after a short transit in rumen contents The rumen is a critical compartment controlling STEC development STEC are able to grow significantly in intestinal and fecal contents Probiotics used in this in vitro study induce delayed STEC mortality but only in rumen contents, or high concentrations are required Summary Need for selection of more efficient probiotic strains: • Acting more quickly in the rumen • Efficient in the hindgut at lower concentration • Not inducing acid resistance mechanisms • Targeting STEC strains Deep knowledge of ecology and physiology of STEC in the bovine digestive tract is required

12. pH Acetate concentration Hay 6.65 48.25 mM Hay+Wheat 5.37 71.80 mM Combination of low pH and high VFA concentration in the rumen lead to EHEC mortality Faecal contents, static cultures Rumen contents Initial inoculum The faecal flora does not exert a barrier effect The faecal environment promotes EHEC growth The ruminal flora exerts a strong barrier effect EHEC growth is oxygen-dependent Effect of the diet, autochtonous flora, and oxygen on EHEC growth Rumen contents, anaerobiosis Growth when animals were fed hay Mortality when animals were fed a more acidic diet EHEC O157:H7 Chaucheyras Durand et al. 2006, AEM

13. Physiology of EHEC in the bovine gastro-intestinal tract Metabolic pathways Stress responses Virulence factors In the digestive content of ruminants To investigate expression of genes involved in Transcriptomic profiling using microarrays

14. Methods Microarray analysis cDNAs

15. What is the carbon source used by EHEC in the digestive content? Mucus-derived monosaccharides, fermentation products of the resident microbiota, and compounds released from degradation of intestinal epithelial cells and from the diet, are potential carbon sources for EHEC in the bovine gut.

16. eut gene cluster Fold increase in cattle gut content / minimal medium What is the nitrogen source used by EHEC in the digestive content? • Amino acid degradation • Ethanolamine degradation Bertin et al., 2011, Environ. Microbiol.

17. Phosphatidylethanolamine (PE) What is the nitrogen source used by EHEC in the digestive content? EDL933 uses ethanolamine as a nitrogen source but not as a carbon source The ability to metabolize ethanolamine confers a growth advantage to EDL933 in the bovine intestinal content Bertin et al., 2011, Environ. Microbiol.

18. Only 3.7% of bacterial sequenced genomes possess the eut operon. In particular, bacteria of the Bacteroidetes and Firmicutes phyla, representing about 99% of the autochthonous digestive microbiota species, do not possess the eut operon. The eut operon of commensal E. coli is poorly expressed. EDL933 may persist in the digestive tract of ruminants by taking advantage of nutrients that are not consumed by commensal E. coli and by the normal microbiota Ethanolamine utilisation as a nitrogen source represents an ecological niche that confers a competitive advantage for EHEC strains to persist and develop in the bovine digestive content Bertin et al., 2011, Environ. Microbiol.

19. Stress responses Alteration of the cell wall and cell surface structures Alteration of cell division Alteration of cell shape

20. Stress responses Multi drug resistance acrA acrB acrR tolC RND ydhC bcrC emrD yidY MFS (major facilitator superfamily) - Solvents, dyes, detergents, antibiotics (novobiocin, erythromycin, fusidic acid, bacitracin, sulfathiazole, chloramphenicol, ethidium bromide, biliary acids), uncouplers of oxidative phosphorylation, - - quorum sensing signals ybjG, mdaA, yciD (ompW) Chromium, drugs (adriamycin, tetracyclin, ampicillin) www.nature.com

21. Stress responses AFI Acid-Resistance Most of the genes involved in acid-resistance are down-regulated in BIC. Phage induction A number of BP-933W phage genes, including stx2, are down –regulated in BIC in comparison to M9 medium. Expression of most of other prophages harbored by EDL933 is also down-regulated

22. Host cell Bacterial cell Virulence factors Attaching/effacing lesions Stevens et al., IAI, 2002

23. Locus of enterocyte effacement 1.4 1.6 4.9 4.3 4.7 5.5 1.2 1.9 1.6 3.6 10.4 3.3 2.1 11.3 1.5 2.8 9.2 4.4 3.3 3.3 8.1 5.7 9.6 2.2 1.3 1.6 3.5 6.4 1.6 5.6 2.8 2.3 3 1.8 6.8 3.2 0.4

24. Virulence factors Adhesive fimbriae: adhesion to epithelial cells Up-regulated • F9 involved in colonisation of the bovine gut at other sites than the recto-anal junction • Sfa-like, not yet characterized Down-regulated • Lpf Involvement in gut persistence in adult ruminant not clear • Curli Involvement in gut persistence not investigated Involved in biofilm formation, invasion of epithelial cell lines

25. Virulence factors Grys et al., IAI, 2005 Up-regulation of the StcE protease that cleaves mucus glycoproteins, facilitating intimate adherence of EHEC to epithelial cells.

26. Mucus Fermentation metabolites Fucose arabinose mannose galactose Lactate Acetate Epithelial cells, diet Glycerol Type 3 secretion system Summary Sfa-like F9 Indole Quorum sensing signals Antimicrobials StcE Ethanolamine Carbon source Nitrogen source Intimin Lipid A-LPS Multi-drug efflux pumps Effector proteins

27. Conclusion Dramatic changes in gene expression allow EHEC to adapt its physiology to survive in the highly competitive environment of the cattle digestive content Competitive probiotics could be selected to target specifically one or more of the metabolic or stress pathways promoting EHEC survival in cattle

28. Future work • Screening of a panel of probiotic strains (lactic acid bacteria, yeasts) for their antagonist properties • on diverse EHEC serotypes • in cattle digestive contents • using semi-continuous fermentors • Effect of the selected probiotic strains • on crucial pathways previously identified • on virulence properties (acid resistance, stx-phage release)

29. Frédérique CHAUCHEYRAS-DURAND Aurélie AMEILBONNE Jordan MADIC Yolande BERTIN Jean-Pierre GIRARDEAU Alexandra DURAND Annie GARRIVIER Fahima FAQIR Josée HAREL Estelle PUJOS Bernard LYAN Christine Rozand