UNIVERSITÉ DE LILLE1-SCIENCES ET TECHNOLOGIES École doctorale de Science de la Matière, de Rayonnement et de l’Environnement THÈSE DE DOCTORAT Spécialité : Ingénierie des Fonctions Biologiques Présentée par ALAA ABDULHUSSAIN AL-SERAIH Pour l’obtention du grade de DOCTEUR DE L’UNIVERSITÉ DE LILLE 1 Propriétés Antagonistes et Probiotiques de Nouvelles Bactéries Lactiques et Levures Isolées des Matières Fécales Humaine et Animale No Ordre 42128 Préparée à l’institut Charles Viollette (ICV)- EA7394 Présentée, le 10 Novembre 2016 devant le jury composé de : Véronique Delcenserie Professeur Université de Liège, Belgique Nathalie Connil Maître de conférence-HDR. Université de Rouen-Haute Normandie Djamel Drider Professeur Université de Lille 1 John Baah Directeur de Recherche et développement Best Environmental Technologies,Canada Benoit Cudennec Maître de conférence. Université de Lille 1 François Krier Maître de conférence. Université de Lille1 Acknowledgements I would like to convey my heartfelt gratitude and sincere appreciation to all the people who have helped and inspired me during my doctoral study. This thesis would not have been possible without the support of many people. First of all, I would like to express my deep appreciation and gratitude to my honorable advisor, Professor Dr. Djamel Drider, for the patient guidance and mentorship he provided to me throughout my PhD study. Professor Drider’s intellectual heft is matched only by his genuine good nature and humility, and I am truly fortunate to have had the opportunity to work with him. Also, with a deep sense of honor, I would like to express my sincere gratitude to Professor Dr. Pascal Dhulster, the director of Charles Viollette Institute, for his unceasing and encouraging support. I would like to thank all of the thesis committee, Professor Dr. Véronique Delcenserie, Dr. Nathalie Connil, Dr. John Baah, Dr. Benoit Cudennec, and Dr François Krier, for accepting the evaluation of my PhD work, and for their insightful comments and encouragement. My sincere thanks also go to Dr. Rabah Boukherroub (IEMN, CNRS) and Professor Dr. Sabine Szunerits (IMEN, Lille 1) for their collaboration and SEM assistance. I would like to extend thanks to Dr. Annee Vachee (Roubaix hospital, France) for her cooperation in the antibiotic resistance analysis. I would also like to express my heartfelt gratitude to Dr. Yanath Belguesmia, Dr. Max Béchet, and Dr. Gabrielle Chataigné for their help, support, and encouragement throughout my PhD work. Similar profound gratitude goes to my friends Ahmed, Ameen, Yazen, Hamza, Mahammed, Qassim, Michael, Debarun, Alexandre, Delphine, Juliette and all other members of the QSA and ProBioGEM teams who provided me a family environment for more than four years. II I would like to extend thanks to the Iraqi Ministry of Higher Education and Scientific Research for giving me the opportunity to do my PhD by awarding the doctoral scholarship. Similar deep gratitude goes to Campus France Organization, especially to Mr. Adrien Chalancon, who successfully managed the dossiers of Iraqi students. Also with deep sense of honor, I wish to express my heartily gratitude to the Best Environmental Technologies Company (Alberta, Canada), especially to Mr. Jim G. Watson and Dr. John Baah for all their support of my PhD project. Finally, I have no words to express my profound gratitude and appreciation to my family. My loving mother, father and brothers Bahaa, Osama, Ahmed, and Amir always gave me unconditional love, support, and encouragement during all the stages of my life. To each of them, I would like to say thank you from the bottom of my heart. No words can either express the deepest gratitude and appreciation I have for my loving wife Hiba, who has always supported me and helped me overcome all difficulties during my studies. She has been there for me every minute of every year of my studies and supported me and my work without complaint. I would like to extend my warmest thanks to my dear son, Murtadha. If this work has sometimes prevented us from sharing important moments of life, know that I never stopped thinking about you. You are the most important people in my life, and I dedicate this work to all of you. III Scientific valorization Publications in peer reviewed journals 1- Al-Seraih A, Flahaut C, Krier F, Cudennec B, Drider D., 2015. Characterization of Candida famata Isolated from Poultry Feces for Possible Probiotic Applications. Probiotics and antimicrobial proteins. 7, 233-41. 2- Al-Seraih A, Belguesmiaa Y, Baah J, Szuneritsc S, Boukherroubc R, Drider D., 2016. Enterocin B3A-B3B produced by LAB collected from infant feces: potential utilization in the food industry for Listeria monocytogenes biofilm management. In Antonie van Leeuwenhoek Journal of Microbiology (Under review). 3- Al-Seraih, Kergourlay G, Cudennec B, Baah J, Belguesmia Y, Drider D., 2016. Enterococcus faecalis B3A-B3B: Genome analysis and experimental evidences to claim its beneficial attributes. (Manuscript in preparation). Posters and workshops 1- Al-Seraih A, Flahaut C, Krier F, Cudennec B, Drider D. Characterization of Candida famata Isolated from Poultry Feces for Possible Probiotic Applications. Annual Conference of Microbiology Society. 21-24 March 2016, Liverpool, UK. 2- Al-Seraih A, Belguesmia Y, Desriac F, Drider D. Enterocin B3A-B3B from infant origin helps nisin to control growth of the foodborne pathogen Listeria monocytogenes. AMP. 6-8 June 2016. Montpellier, France. 3- 2nd International workshop on (Bioinformatics Tools for PKS and NRPS Discovery from Genomic Data for the Product), University of Lille1. 28-30 October, 2015. Lille, France. IV ABSTRACT In this study, we isolated probiotic yeasts and lactic acid bacteria (LAB) from different microbial sources. Eighty-one (81) yeasts and seventy (70) LAB isolates were randomly selected and identified from fecal samples of poultry feces and healthy Iraqi infants, respectively. The yeast strains were obtained from a farm of broiler chickens located in the city of Lille. They were clustered into 22 groups by GTG5-rep PCR technique, then identified as Debaryomyces hansenii, (teleomorph of Candida famata) species using the biochemical ID-32C system and molecular sequencing of 26S rDNA and ITS1-5.8-ITS2 rDNA region methods. Only one yeast strain, designated as Candida famata Y.5 (C. famata Y.5), exhibited antimicrobial activity against Listeria innocua. For more accurate discrimination, the antagonistic strain C. famata Y.5 was identified by MALDI-TOF-MS technology. Further characterization of this anti-Listeria strain, permitted to unveil its probiotic potential. Thus, C. famata Y.5 appeared to be a non-hemolytic strain. In vitro tests of cytotoxicity and adhesion on human Caco-2 epithelial cells confirmed the safety traits of this strain. C. famata Y.5 displayed good surface properties, especially auto-aggregation, in addition to high survival ability under harsh conditions mimicking those of the gastrointestinal tract (GIT). The LAB strains were isolated from fecal samples of a group of Iraqi children living in the north of France. LAB strains were obtained from six blind donors and then identified as 41 cocci and 29 bacilli. Two strains displayed antagonistic activities against Gram-positive bacteria (GPB) including: Listeria monocytogenes, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Clostridium perfringens but not against fungi or Gram-negative bacteria (GNB), except for Salmonella Newport. The biochemical, MALDI-TOF-MS, and molecular (16S rDNA sequencing) methods identified these two strains as Enterococcus faecalis B3A-B3B and B20A-B20B . Bacteriocin produced by strain B3A-B3B, designed as V enterocin B3A-B3B, was purified by a simplified two-step procedure including a liquid-liquid phase extraction and reverse phase high-performance liquid chromatography (RP-HPLC). The predicted molecular mass of this enterocin consists of two peptides of 5,176.31 Da (B3A) and 5,182.21 Da (B3B). Notably, B3A-B3B hampered the biofilm installation of L. monocytogenes strain grown on AISI 304 stainless steel slides. The treatment of stainless steel with nisin (1 mg. ml-1 or 16 mg. ml-1) diminished the cell numbers by about 2 logs CFU. ml-1, preventing therefore the biofilm formation by L. monocytogenes 162 or by its nisin-resistant variant L. monocytogenes 162R. Further combination of nisin and B3A-B3B enterocin reduced the MIC value needed to inhibit this pathogen about 2 logs CFU. ml-1. To gain insights on the probiotic profile of the E. faecalis B3A-B3B strain, the whole genome was sequenced and in silico analysis was performed and compared with those of clinical strains as E. faecalis MMH594, E. faecalis V583, and E. faecalis OG1RF from humans, and also compared to that of the well-known probiotic E. faecalis Symbioflor1 strain. Even harboring gelE, cpd, efaAfm, ccf, agg, and cob coding for virulence factors, the B3A-B3B strain resulted to be sensitive to most antibiotics tested here, non-cytotoxic, non-hemolytic, and devoid of inflammatory effects. Moreover, B3A-B3B strain showed remarkable hydrophobicity, auto- aggregation, adhesion to human Caco-2 cells, viability in simulated GIT conditions, and cholesterol assimilation. These features together introduce the E. faecalis B3A- B3B strain as an interesting probiotic candidate. VI RESUME Dans cette étude, nous avons isolé des levures et des bactéries lactiques (BL) potentiellement probiotiques, à partir de différents écosystèmes microbiens. Quatre- vingt-une (81) levures, et soixante-dix (70) BL, ont été isolées et identifiées à partir de matières fécales animales (poulet) et humaines (enfants Irakiens en bonne santé). Ainsi, les souches de levures ont été isolées à partir de matières fécales de poulets, dans une ferme située dans la région de Lille (France). Elles ont été regroupées en 22 groupes par la technique de Rep-PCR utilisant une amorce unique 5'- GTG5-3', puis identifiées comme appartenant à l’espèce Debaryomyces hansenii (téléomorphe de Candida famata) en utilisant des méthodes biochimique (système ID- 32C) et moléculaire (séquençage de l'ADNr 26S et les régions ITS1-5.8-ITS2 de l’ADNr).