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Isolement Et Caractérisation De Bactéries Cadmium-Résistantes De Sols Des Sites Pollués : Etude De L’Accumulation Des Ions Métalliques

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Isolement Et Caractérisation De Bactéries Cadmium-Résistantes De Sols Des Sites Pollués : Etude De L’Accumulation Des Ions Métalliques N° d’ordre 05/2019-D/S.B UNIVERSITE DES SCIENCES ET DE LA TECHNOLOGIE HOUARI BOUMEDIENE USTHB/ALGER FACULTE DES SCIENCES BIOLOGIQUES THESE de Doctorat en Sciences Présentée pour l’obtention du grade de Docteur en Sciences Biologiques Spécialité : Ecologie Microbienne de la Rhizosphère Par Yakoubi Lila SUJET Isolement et caractérisation de bactéries cadmium-résistantes de sols des sites pollués : Etude de l’accumulation des ions métalliques Soutenue publiquement le 07 / 02 /2019 devant le jury composé de : Mr Hacene H. Professeur à l‘ USTHB /FSB Président Mme Benmalek Y. Professeur à l’ USTHB /FSB Directrice de thèse Mme Abrous O. Professeur à l’ USTHB /FSB Examinatrice Mme Gana-Kebbouche S . Professeur à l’ UMBB Examinatrice Mr Jaouadi B. Maître de Conférences A au CBS /Tunisie Examinateur Mme Bouanane A. Professeur à l’ USTHB/FSB Examinatrice Mme Zermane N. Professeur à l’ Université d’Alger Invité d’honneur Résumé Au cours de cette étude, 103 bactéries cadmium -résistantes, ont été isolées de 9 sols provenant de différentes régions d'Algérie et pollués par les métaux lourds. 94% des isolats se sont révélés à Gram négatif, appartenant principalement aux genres Pseudomonas, Burkholderia, Aeromonas et à la famille des Enterobacteriaceae. Les Gram positifs (4%), appartiennent principalement aux genres Bacillus. Toutes les souches cadmium-résistantes ont affiché des CMIs très importantes, qui oscillent entre 400 et 2000 µg mL-1, et une multirésistance vis-à-vis de 12 antibiotiques testés. Quatre souches hautement résistantes au cadmium, ont été caractérisées au niveau moléculaire, par séquençage de l'ARNr 16S, étaient affiliées aux espèces Raoultella ornithinolytica (souche YL-ML1), Klebsiella pneumoniae (souche YL-SS2) ,Pseudomonas fluorescens (souche YL-SS3) et Bacillus infantis (souche YL-SS8). Ces souches ont accumulé de très grandes quantités de cadmium au cours des 24 premières heures d’incubation, malgré l’effet toxique du Cd, exprimé par une inhibition de leur croissance. L’accumulation du cadmium est légèrement meilleure lorsque les cellules sont immobilisées dans des billes d’alginate que lorsqu’elles sont libres en suspension. Le cadmium provoque une chute du contenu en protéines totale, mais induit en revanche, une surproduction d’Exopolysaccharides (EPS). Par ailleurs, le cadmium stimule les activités enzymatiques, de la catalase, de la superoxyde dismutase et du glutathion chez les trois Protéobactéries YL-ML1, YL-SS2 et YL-SS3. Cependant, la souche YL-SS8 (Gram-Positif) a affiché une réduction de ces trois paramètres. Nous avons noté une différence dans le profil protéique des souches testées en l'absence et en présence de cadmium. Ce changement s’est matérialisé par une surexpression, induction et/ou répression de protéines de différentes masses moléculaires. Une PCR nous a permis de détecter le gène cadA dans les souches sélectionnées, ce gène confère une résistance au cadmium en codant une protéine d'efflux. Ce gène a été confirmé par séquençage après clonage, au niveau de la souche YL-ML1. Ce résultat original, révèle le rôle du transfert latéral des gènes dans l’acquisition de la résistance de cette entérobactérie dans un sol pollué par le cadmium. Les résultats de la présente étude montrent que les souches répondent au stress généré par le cadmium, par une surproduction d’EPS, activation du système antioxydant et induction de certaines protéines de stress, ainsi qu’une possible extrusion des ions métalliques par des pompes d’efflux codées par le gène cadA. Mots clés : Pollution des sols, cadmium, toxicité, mécanismes de résistance , bactéries. Remerciements La présente étude a été réalisée au laboratoire de Microbiologie de la FSB/USTHB, sous la direction de Madame Benmalek Y. Au terme de ce travail, je tiens à exprimer ma reconnaissance et ma gratitude à ma directrice de thèse Mme Benmalek Y. professeur à l’ISN/USTHB, d’avoir accepté de diriger ce travail , je la remercie vivement pour ses conseils et ses orientations précieuses, qu’elle trouve ici l’expression de mon profond respect. J’adresse mes plus grands remerciements à Monsieur Hacène H. de m’avoir accueilli dans son laboratoire et de m’offrir une paillasse de travail et aussi pour l’honneur qu’il m’accorde en présidant ce jury ,et ce, en dépit de ses charges. Mes remerciements les plus profonds à mon enseignante Mme Abrous O., professeur à la FSB/USTHB de m’avoir accordé de son temps si précieux, j’imagine, pour évaluer mon travail .Je tiens très sincèrement à lui exprimer mon profond respect. Mon immense reconnaissance va à Monsieur Jaouadi B. pour avoir accepté de faire partie du jury ,mais aussi pour ses conseils et ses orientations. Il m’est spécialement agréable que mon travail soit évalué par Madame Gana S., laquelle j’ai eu l’honneur d’apprécier sa compétence et son sérieux . Madame Zermane , a qui j’exprime toute ma gratitude et mon respect pour le modèle de l’enseignant très soucieux de l’intérêt des étudiants ,qu’elle représente .Je tiens à la remercier vivement pour sa présence . Et en fin Madame Bouanane, que je ne remercierai jamais assez, pour l’âme qu’elle a insufflée à mon travail, c’est un grand plaisir pour moi de vous compter parmi le jury de ma soutenance.Merci pour votre soutien et votre agréable moralité ! Je remercie très chaleureusement l’ensemble des membres du laboratoire de Microbiologie, de la FSB spécialement Lydia,Khelifa, Fawzi,Nawel ,Okba ,et tout le reste. Afin de n’’oublier personne, je tiens à remercier tous ceux qui ont participé, à titre professionnel ou personnel, à la réalisation de cette thèse. Je pense particulièrement à Mme Rahmania F. ,Professeur à l’USTHB/FSB , Mr Benayad T.,directeur du laboratoire de la police scientifique. , Mme Berka S . Mme Djebari K . , enseignante à l’ENSA , Monsieur Djili ,chef de département de Biologie des sols à l’ENSA (El-Harrach). Et en fin, j’adresse mes plus grands remerciements à mon mari, qui a non seulement soutenu tant financièrement que moralement une éternelle étudiante !, mais qui, en plus a été présent et patient depuis déjà 10 ans. J’adresse une tendresse toute particulière à mes enfants, mes parents et toute ma famille et ma belle famille, pour leurs encouragements tout au long de mes études et vis à vis de mes choix. Je leur dédie ce travail. Liste des abréviations ADN acide désoxyrionucléique AFNOR association française de normalisation ATB antibiotique ARN acide ribonucléique ARN 16S Sous unité 16S de l’ARN ribosomal BSA bovine serum albumine BN Bouillon nutritif CAT Catalase CMI : concentration minimale inhibitrice Ca Calcium Cd Cadmium CaCl2 Calcium chloride Cr Chrome CMI Concentration minimale inhibitrice Cu cuivre DO : densité optique EPS Exopolysaccharides ERO espèces réactives d’oxygéne ETM Eléments Trace Métallique GN gélose Nutritive GSH glutathion réduit h heure K Potassium KDa kilo dalton mg milligramme mL Millilitre Mm Masse molaire PAGE Protein Analysis Gel Electrophoresis Pb plomb pb paire de base PCR abréviation anglaise de réaction en chaîne par polymérase ppm particule par million rpm r otation par minute SAA spectrophotométrie à absorption atomique SDS sodium dodecyl Sulfate T témoin Zn zinc Liste des tableaux Tableau I. Propriétés physico-chimiques du cadmium ………………………………………………………………………………….. ………….7 Tableau II. Protéines de transport des éléments métalliques chez les microorganismes ……………………………………………………………………………………………….19 Tableau III : Principaux groupements fonctionnels et constituants membranaires chez les bactéries…………………………………………………………………… ………………21 Tableau IV : Nomenclature des ATPases-P selon la classification d’Alxelsen et al., (1998) et correspondance entre les classes d’ATPases-P avec la classifcation de Lutsenko et al . , (1995)…………………………………………………………………………………………29 Tableau V. Classification et structure des protéines RND…………………………………..37 Tableau VI. Sites et souches isolées pour chaque prélèvement…………….. 44 Tableau VII .Les antibiotiques testés (Molécules, codes et charge des disques) …… 53 Tableau VIII. Résultats des analyses physico-chimiques des sols étudiés………… 63 Tableau IX .Classification des sols selon leur conductivité électrique……………….. 64 Tableau X .Distribution des genres bactériens cadmium-résistants en fonction de leur origine et les valeurs des CMI ………………………………………………………………. 70 Tableau XI. Profil de résistance des bactéries cadmium-résistantes vis-à-vis des antibiotiques testés…………………………………………………………………………………………..73 Tableau XII. Résultats de l’identification moléculaire des souches cadmium-résistantes…77 Tableau XIII. Effet de différentes concentrations de Cd sur les teneurs en protéines totales chez la souche test YL-ML.......................................................................................................92 Tableau XIV. Effet de différentes concentrations de Cd sur les teneurs en protéines totales chez la souche test YL-SS8………………………………………………………………….96 Tableau XV. Effet de différentes concentrations de Cd sur les teneurs en protéines totales chez la souche YL-SS3……………………………………………………………………..93 Tableau XVI. Effet de différentes concentrations de Cd sur les teneurs en protéines totales chez la souche test YL-SS2…………………………………………………………………94 Tableau XVII. Effet de différentes concentrations de Cd sur la production d’EPS chez la souche test YL-ML1.. …………………………………………………………….96 Tableau XVIII. Effet de différentes concentrations de Cd sur la production d’EPS chez la souche test YL-SS8………………………………………………………………………….97 Tableau XIX. Effet de différentes concentrations de Cd sur la production d’EPS chez la souche test
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