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Isolement Et Screening Des Actinobactéries À Partir Des Écosystèmes Extrêmement Salins

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Isolement Et Screening Des Actinobactéries À Partir Des Écosystèmes Extrêmement Salins REPUBLIQUE ALGERIENNE DEMOCRATIQUE ET POPULAIRE MINISTERE DE L’ENSEIGNEMENT SUPERIEURE ET DE LA RECHERCHE SCIENTIFIQUE Université Larbi Ben M'hidi Oum El Bouaghi Faculté Des Sciences Exactes et des Sciences de la Nature et de la Vie Département des Sciences de la Nature et de la Vie N ° d’ordre…… N ° de série…... Mémoire Présenté pour l’obtention du diplôme de MASTER Filière : Sciences biologiques OPTION : Microbiologie Appliquée Thème Isolement et screening des actinobactéries à partir des écosystèmes extrêmement salins Présenté par: MENASRIA Nour El Houda MEZIANE Amira Randa Rimas Devant le jury Président : DEROUICHE K. M.C.A Univ. Oum EL Bouaghi. Rapporteur : DJABALLAH C. M.A.A Univ. Oum EL Bouaghi. Examinateur : HAMAMES M. M.A.A Univ. Oum EL Bouaghi. Année universitaire : 2019-2020 Remerciements L’encadrement scientifique de ce travail a été assuré par Mr. DJABALLAH Chamsseddine, Maître-Assistant à l’université Larbi Ben M’Hidi Oum El Bouaghi. Nous tenons vivement à lui exprimer notre profonde reconnaissance et gratitude pour le temps qu’il a consacré pour sa patience, son suivi, sa compréhension, ses écoutes, ses qualités humaines et encore pour les précieuses informations qu’il nous a prodiguées avec intérêt. Nous tenons à remercier également les membres du jury Mr. DEROUICHE K. et Mr. HAMAMES M. pour avoir accepté d’examiner et d’évaluer ce travail. Nos remerciements les plus respectueux s’adressent aussi à Mr.ARABI Abed, Mr.Rouar Salim qui n’ont pas hésité à tout moment d’offrir leurs aides scientifiques, encouragements et fructueux conseils. Nous remercions tous nos chers professeurs qu’on a pu rencontrer pendant notre parcours universitaire. Nos vifs remerciements vont à tous les membres de BIO-ART CLUB pour leur soutien et encouragements Dédicace A la mémoire de ma chére grand-mère maternelle « Fatiha » tu reste toujours présente dans mon cœur, aucun hommage ni remerciement ne saurait être suffisant A mon très cher grand père maternel « Ahmed »que dieu t’accorde une longue vie pleine de bonheur et de santé A mes très chers parents, pour leurs sacrifices, leur amour, leur soutien et leurs prières tout au long de mes études A mon petit frère Mohamed Amir Arseléne que dieu te bénisse A mes chères tantes Leila et Nora A ma petite cousine Mimicha Ainsi qu’a toute ma famille A mes amies : Doucha, Hadjer, Aida, Rania Noussaiba, Kenza et Karima A toute l’équipe BioArt je dédie ce mémoire Randa Dédicace À l’être le plus cher de ma vie, à la femme qui a souffert sans me laisser souffrir qui n’a épargné aucun effort pour me rendre heureuse : Ma mère. Quoi que je fasse ou que je dise, je ne saurai point te remercier comme il se doit. À la mémoire de mon père disparu trop tôt Toi qui n’est plus de ce monde tu demeure à jamais dans mon cœur À mon très cher oncle ‶ Mohamed ″ Tu as toujours été à mes coté pour me soutenir et m’encourager À mon très cher ‶Houssem″ À toute ma famille ; ma grand-mère et mon petit frère adorable‶ Hichem″. À mon Enseignant ‶Amine HIMEUR″ Il m’a toujours encouragé, stimulé et me guidé Ton appui est inestimable À tous mes amis qui m’ont toujours encouragé, et a qui je souhaite plus de succès ‶Amira, Noussaiba, Arselane, Akram, Oussama et Islem″ À toute l’équipe de Bio-Art surtout ‶Marou, Jouhaina, Dalal et Maria″ Je dédie ce mémoire Houda LISTE DES ABREVIATIONS LB : Luria–Bertani ARDRA : L’analyse amplifiée des restrictions de l’ADNr. MB : Méga Base ATCC : American Type Culture Collection MG : mannosylglycérate Mod-Actino: Actinobactéries modernes CCA : Colloidal chitin agar CCMS : Cellulose-caséine-multisels ISP : Milieu International Streptomyces Project. CE : Conductivité Electrique CM : Complexe Medium CMB : Concentration minimale bactéricide OTR : Oxygene transfert Rate CMI : Concentration minimale inhibitrice P/V : poids par volume PHB : Dépolymérase extracellulaire de Polyhydroxybutyrate ELL : Extraction liquide-liquide RC : Réhydration et centrifugation SARM : Staphylococcus aureus résistant à la méthicilline GTY : Glucose Tryptone Yeast Extract agar GW1 : Mannitol- casein acid hydrolysis SCA : Starch -Casein Agar GYM : Glucose-yeast extract-malt extract agar LISTE DES ABREVIATIONS SDS : Sodium Dodécyl Sulfate HV : Humic acid Vitamine agar SHF : Super haute fréquence HV : Vitamine et acide humique SMF : Submerged fermetation Smf : Fermentation submergée SSC : Sodium saline citraté SSF : Solide state fermentation SSF : Fermentation de substrats solides SW 10%: 10% milieu d’eau saline w/v : weight / volume LISTE DES FIGURES Page Figure 1: Système de classification phylogénétique pour les Actinobacteria 7 propos par Stackebrandt et al. (1997) (Lawson, 2018). Figure 2: Les étapes impliquées dans le cycle de vie d’une actinobactérie 9 filamenteuse (Trujillo, 2016). Figure 3: Écologie des actinomycètes dans la nature (van der Meij et al., 2017). 12 Figure 4: Méthode d’isolation du filtre à membrane pour les actinomycètes 36 (Subhashini 2018). Figure N° 5: Recherche de l’activité antimicrobienne par la technique de stries 55 croisées (Benouagueni., 2015). Figure N° 6 : dessin schématique d’une ampoule de décantation avec deux 62 couches distinctes de liquides (Moldoveanu et David, 2015). LISTE DES TABLEAUX Page Tableaux 1 : Diversité taxonomique des actinomycètes halophiles 15 (Hozzein, 2015). Tableaux 2 : Récapitulatif des différents métabolites bioactifs produits par les 22 actinomycètes halophiles et halotolérants Tableau 3 : Pourcentages d’utilisation des milieux de culture dans les protocoles 40 d’isolement des actinomycètes halophiles et halotolérants dans les articles scientifiques. Tableau 4 : Les milieux de culture et les conditions d’incubation nécessaire à 42 l’isolement des actinomycètes halophiles et halotolérant appartenant au genre Nocardiopsis. Tableau 5 : Les milieux de culture et les conditions d’incubation nécessaire à 43 l’isolement des actinomycètes halophiles et halotolérant appartenant aux genres Actinopolyspora et Nesterenkonia. Tableau 6 : les milieux de culture et les conditions d’incubation nécessaire à 45 l’isolement des actinomycètes halophiles et halotolérant appartenant aux genres Streptomonospora, Saccharopolyspora, Prauserella et Amycolatopsis. Tableau 7 : Les milieux de culture et les conditions d’incubation nécessaire à 46 l’isolement des actinomycètes halophiles et halotolérant appartenant aux genres Glycomyces et Streptomyces. Tableau 8 : Les sources de carbone et d’azote utilisés dans l’isolement des 49 actinobactéries halophiles et halotolérants. Tableau 9 : Comparaison entre la fermentation solide et liquide (Manpreet et al., 60 2005) . REMERCIEMENTS DÉDICACES LISTE DES ABRÉVIATIONS LISTE DES FIGURES LISTE DES TABLEAUX Table de matières Pages INTRODUCTION 2 Chapitre 01 : les actinomycètes 1. Généralités 5 2. Classification des actinobactéries 6 3. Morphologie des actinomycètes 8 4. Ecologie et distribution dans la nature 10 5. Les actinobactéries halophiles 12 5-1-Halophilie et halotolérance 13 5-2-Diversité taxonomique des actinomycètes halophiles 14 5-3-Distribution des actinomycètes halophiles et halotolérants dans 17 l’environnement 5-4-Facteurs influençant la distribution des actinomycètes dans le sol 18 salin 5-4-1-NaCl 19 5-4-2—Humidité 5-4-3-pH 5-5-Mécanisme d'adaptation en milieu salin 19 5-6-Biotechnologie des actinobactéries halophiles et halotolérantes 21 Chapitre 02 : Isolement des actinomycètes 1-Collecte des échantillons 27 1-1-À partir de l’eau 27 1-2- À partir du sol 28 2-Caractéristiques physico-chimiques des échantillons 29 2-1-Propriétés physico-chimiques du sol 29 2-2-Propriété physicochimique de l’eau 30 3-Principes de base pour l'isolement des actinomycètes 30 4-Les approches d’isolement des actinomycètes 31 4-1-Prétraitement physique et chimique de l’échantillon 32 4-2-Inhibition sélective 34 4-3-Sélection nutritionnelle 35 4-4- Isolement sur les membranes à filtres 35 4-5-Les méthodes d’enrichissement 36 4-6-Méthode intégrée 37 5-Conception des milieux de culture utilisés pour l’isolement des 37 actinomycètes 6-Isolement des actinomycètes halophiles 39 Chapitre 03 : Criblage, Fermentation et Extraction I-Criblage et évaluation de l’activité antibactérienne des actinomycètes 53 1-Méthodes par diffusion en milieu gélosé 53 1-1-Principe de la méthode 53 1-2-Méthode de diffusion sur disque / méthode Kirby-Bauer 53 1-3-Méthode de diffusion des puits d’agar 54 1-3-1-Technique des cylindres d’agar 54 1-3-2-La méthode à stries croisées 54 II-Production de biomolécules par les techniques de fermentation 56 1-Fermentation solide 57 1-2- Principe 57 1-3- Types de la fermentation solide 57 1-4- Avantages et limites 57 2-Fermentation liquide 58 2-1-Définition 58 2-2- Principe 58 2-3-Influence de la morphologie sur la capacité des actinomycètes à 59 produire des antibiotiques et d'autres produits en culture submergée 2-4-Avantages et limites 59 3-Les conditions optimales de la fermentation 61 III-Extraction liquide-liquide 64 1-Procédures courantes d’extraction liquide-liquide 64 2-Sélection des solvants 65 3- Les facteurs qui influencent l’extraction 65 3-1-L’influence du pH sur l’extraction 65 3-2-Modifications chimiques qui affectent l’extraction 65 3-3-Facteurs non chimiques affectant l’extraction 65 4-Avantages et inconvénients de l’extraction liquide-liquide 66 CONCLUSION GÉNÉRALE ET PERSPECTIVES 68 RÉFÉRENCES BIBLIOGRAPHIQUES ANNEXES RÉSUMÉ Introduction INTRODUCTION La résistance aux antibiotiques est un aspect particulier de l'évolution bactérienne. L'apparition d'une résistance peut être un événement rare, voire transitoire, si elle n'offre pas un avantage sélectif contre une molécule présente dans l'environnement (Courvalin, 2008). L'évolution des bactéries vers la résistance résulte de deux étapes indépendantes - l'émergence et la dissémination - bien que, comme nous le verrons, le mécanisme de la première étape puisse largement influer sur le succès de la deuxième étape (Courvalin, 2008). La perception commune, cependant, est que le problème est considéré comme exclusivement associé à la surutilisation / mauvaise utilisation d'antibiotiques chez l'homme et les animaux.
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