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Thesis Presented by Selma HOUCHI Submitted for the Fulfillment of the Requirements for the Degree of Doctorate of Sciences in Biology

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Thesis Presented by Selma HOUCHI Submitted for the Fulfillment of the Requirements for the Degree of Doctorate of Sciences in Biology الجمهورية الجزائرية الديمقراطية الشعبية وزارة التعليم العالي و البحث العلمي جامعة فرحات عباس، سطيف University of Ferhat Abbas Setif 1 1 كلية علوم الطبيعة و الحياة Faculty of Life and Nature Sciences DEPARTMENT OF BIOCHEMISTRY N°………………………………………….…………..…….……/SNV/2019 Thesis Presented by Selma HOUCHI Submitted for the fulfillment of the requirements for the degree of Doctorate of Sciences In Biology OPTION: Biochemistry Theme Characterization of ß-lactamase genes in clinical isolates and determination of the inhibitory effects of Algerian seaweeds extracts on recombinant ß-lactamases GES-22 and OXA-1 Discussed on 22/ 07/ 2019 Board of Examiners Chairman Abdelhalim KHENCHOUCHE Associated professor (A). UFA Setif-1 Supervisor Rachid MAHDADI Pr. University Ferhat Abbas Setif-1 Examiner Karim HOUALI Pr.Univ. Mouloud Mammeri Tizi Ouzou Examiner El-Hafidh NABTI Pr. Univ. Abderrahmane Mira Bejaia Examiner Kamel AISSET Pr. Univ. Hadj Lakhdar Batna-1 Examiner Widad SOBHI Associated professor (A). UFA-Setif-1 Invited member Cemal SANDALLI Pr. Recep Tayyip Erdogan University, Turkey Laboratory of Applied Biochemistry In the name of Allah, the Beneficent, the Merciful Dedication I am dedicating this thesis, First and foremost, to my loving parents my father, Ahmed, May Allah grant him Jannah Firdaws. and My mother MAIZA Hafsa for their love, endless support and encouragement To the people who helped me a lot and for their support moral and their sacrifices along my PhD; My dear brothers Abdelkarim, Faycel and Mounir To my dear cousin Khaoula Acknowledgements First and foremost, I would like to thank ALLAH Almighty for giving me the strength, ability and opportunity to undertake this modest research study. There are many people who have helped me during this study. There is not enough space, nor words, to acknowledge everyone to the extent they deserve. I hope people understand the brevity of this part. First of all, I would like to thank my doctorate advisor and research guide at Ferhat Abbas SETIF-1 University, (ALGERIA) Professor Rachid MAHDADI. Under his guidance, I was given the freedom to pursue my research in my own way and I greatly appreciated that freedom. It was a great pleasure to work with him. Thanks for supporting and encouraging me through these years. Additionally, I would like to thank the jury members for agreeing to judge this work and to be the examiners of this thesis: Dr. Abdelhalim KHENCHOUCHE, for agreeing to judge this work and to do me the honor of chairing this thesis jury. Pr. Karim HOUALI, Pr. El-Hafidh NABTI and Pr. Kamel AISSET, Dr. Widad SOBHI to have accepted to judge this work. I am deeply grateful to all members of the jury for agreeing to read the manuscript and to participate in the defense of this thesis. The realization of this work was only possible due to the several people's collaboration, to which desire to express my gratefulness: To Pr. Cemal sandalli at Recep tayyib Erdogan University, (TURKEY) and to all his team at the Microbiology and Molecular Biology Research Laboratory. With deep sense of gratitude, I owe sincere thanks to him for agreeing to participate in the defense of this thesis as invited member. Pr. Guanhua DUGH and Pr. ZHANG Li at Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (CHINA), and all the staff of the Laboratory of Bioactive Substances and Functions of Natural Medicines. Pr. Hassiba TALI-MAAMAR at the Medical bacteriology laboratory in Pasteur institute, Dely Brahim (ALGERIA) and all members of this Laboratory Dr. Wahid REFES. Associated Professor at the National Higher School of Marine Sciences and Coastal Management (ENSSMAL). The Marine and Littoral Ecosystems Laboratory (ECOSYSMarL), (ALGERIA) and to his student my dear colleague BAHRI Nabila. Dr. Moussa MENNAD. Elected representative of the research staff of the National Center for Research and Development of Fisheries and Aquaculture (CNRDPA), Bou Ismail, Tipaza (ALGERIA). I also take a real pleasure to thank Dr. Abdelhakim AOUF at Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, University of Science and Technology Houari Boumediene, Algiers, (ALGERIA). I would also like to thank all the members of the Laboratory of Applied Biochemistry (Ferhat Abbas SETIF-1 University, ALGERIA). Particularly: Pr.Lekhmici ARRAR and Pr.Noureddine CHAREF. Besides this, I would like to thank Dr. Azeddine TEMAMNA may ALLAH grant him vast paradise; Pr. Abdelouahab BOUZIDI; Pr.Rachid BELHATTAB; Pr. Hammama BOURICHE and Pr. M. Mihoub ZERROUG. ملخص هدفت هذه الدراسة الى تحديد نوع الجينات المشفرة لبناء اﻻنزيمات المسماة بيتاﻻكتماز و كذا شيوعها في عزﻻت بكتيرية سالبة الغرام جمعت من خارج و داخل مستشفيات جزائرية. هدفت أيضا الى التحقق من قدرة مستخلصات ميثانولية لسبعة طحالب نامية على الساحل الجزائري ,Ulva intestinalis, Codium tomentosum, Bryopsis pulmosa] [Sargassum vulgare, Dictyota dichtoma, Halopteris scoparia, Cystoseira compressa على تثبيط نوعين من بيتاﻻكتماز و هما GES-22 و OXA-1. بينت النتائج أن العزﻻت تحوي على اﻷقل جينا واحدا مشفرا لبناء بيتاﻻكتماز ((blaTEM, blaSHV, blaCTX-M1-M2, blaGES, blaPER-2, blaNDM, bla blaOXA-M (23,24,51,58) و ﻻ تحوي أي من الجينات (blaKPC, blaVIM, blaIMP) لكنها بالمقابل تحوي اﻷجزاء المحفوظة بالعليبات الجينية لﻻنتجرونات قسم I و II منفصلة او مجتمعة. تختلف الطحالب السبعة من حيث محتواها من عديدات الفينول الذي تتغير قيمته من 0.65 ± 0.93 و 0.91 ± 3.22 mgGAEs/g DW .تفوق هذه القيمة عند الطحالب الخضراء بمرتين تلك للطحالب البنية. في حين، كان محتوى هذه اﻷخيرة من فيتامين E أعلى من الطحالب الخضراء. بينت الدراسة التحليلية وجود مركبات مشتركة بين المستخلصات المثانولية للطحالب السبعة و هي حمض الفا-لينولينيك (C18: 3 ω3) ، حمض اللينولييك )C18: 2 ω 6( ، حمض اﻷولييك )C18: 1 ω9( ، وحمض اﻷراكيدونيك C20: 4) ω6)(. عﻻوة على هذا، تحوي U.intestinalis و D. dichtoma على بيكالين )C15H10O5( وكركمين )C21H20O6(. يحوي B.pulmosa على بايكالين ودايدزين )C15H10O4(؛ يحوي C. tomentosum على كيرسيتين )C15H10O7( و يحوي C.compressa على )S( نارينجين )C15H12O5(. تراوحت قيم IC50 المحددة على منحى العﻻقة Logit/Log بين 10.31 ± D. dichtoma( µg/mL3..0( إلى .C. compressa( µg/mL 3.10 ± .1.1( و من B.pulmosa( µg/mL 3.90 ± 10.11( إلى C. compressa( µg/mL 1.90 ± 01.09( مع GES-22 و OXA-1 ، على التوالي. أظهر المستخلص المثانولي للطحلب D. dichtoma تأثي ًرا مثب ًطا على GES-22 أكبر من مثبطات β-lactam الكﻻسيكية المسماة كﻻفوﻻنات، سولباكتام و تازوباكتام )IC50= 68.38±0.17µg/mL, 52.68±0.64µg/mL, 29.94±0.01( على التوالي . لم يثبط سولباكتام و كﻻفوﻻنات اﻻنزيم OXA-1، خﻻفا للتازوباكتام (IC50 =243.03± 4.53 μg/mL). أظهر المستخلص الميثانولي لـ B. pulmosa تأثي ًرا مثب ًطا على (OXA-1. (IC50 =13.22 ± 0.9 μg/mL من نتائج نفس اﻻختبارات تصنف كل المستخلصات المثانولية كمثبطات غير تنافسية لـ OXA-1 و GES-22 ، باستثناء المستخلصين لـ B.pulmosa و C. compressa اللذان يثبطان GES-22 بطريقة تنافسية وﻻ تنافسية ،على الترتيب. بينت دراسات المحاكاة إلى أن قوة التثبيط ربما ترجع إلى تكوين روابط هيدروجينية وكارهة للماء بين المركبات التي تم الكشف عنها في المستخلصات المثانولية ومركبات اخرى طبيعية من جهة واﻷحماض اﻷمينية للموقع النشط في كل من Serine وmetallo-β-Lactamases من جهة اخرى. أظهرت جميع المركبات التي تم الكشف عنها في المستخلصات المثانولية للطحالب libdock score وCDOCKER interaction energy أكبر من مثبطات β-lactam الكﻻسيكية مع ß-lactamases 11. يمكن اقتراح hesperidin وdihydromyrecitin و curcumin كمثبطات جيدة على أساس libdock score وCDOCKER interaction energy. الكلمات المفاتيح: بيتاﻻكتاماز، محاكاة، ,Ulva intestinalis, Codium intestinalis, Bryopsis pulmosa Sargassum vulgare, Dictyota dichtoma, Halopteris scoparia, Cystoseira compressa Abstract The main aim of this study was to characterize the type and the prevalence of β-lactamases coding genes in community and nosocomial Algerian Gram negative bacterial isolates, as well as to investigate the inhibitory effects of methanolic extracts of seven seaweeds Of Algerian coast [Ulva intestinalis, Codium tomentosum, Bryopsis pulmosa, Sargassum vulgare, Dictyota dichtoma, Halopteris scoparia, and Cystoseira compressa] against GES-22 and OXA-1 β-lactamases variants. The results showed that isolates harboring at least, one β-lactamase coding gene (blaTEM, blaSHV, blaCTX-M1-M2, blaGES, blaPER-2, blaNDM, bla blaOXA-M (23,24,51,58)); neither blaKPC nor blaVIM, blaIMP gene was detected. The conserved regions of class-I and II integron gene cassettes were determined alone and together in these isolates. The TPC varied among the seven seaweeds species, ranging between 0.93 ± 0.65 and 3.22 ± 0.91 mg GAEs/g DW. TPC in the MEs of green seaweed species were found to be nearly two times greater than that in brown seaweeds. However, vitamin E contents of brown seaweed MEs were higher than those of green seaweeds. Furthermore, qualitative analysis showed the existance of common compounds in the MEs including α-linolenic acid (C18:3 ω3), linoleic acid (C18:2 ω6), oleic acid (C18:1 ω9), and arachidonic acid (C20:4 ω6). Moreover, U. intestinalis and D. dichtoma contain baicalein (C15H10O5) and curcumin (C21H20O6). B. pulmosa contains baicalein and daidzein (C15H10O4); C. tomentosum contains quercetin (C15H10O7) and C. compressa contains (S) naringenin (C15H12O5). Values of IC50 of MEs determined by linear computerized regression analysis after logit/log transformation, are ranged from 13.01 ± 0.46 μg/mL (D. dichtoma) to 41.24 ± 0.23 μg/mL (C. compressa) and from 13.22 ± 0.96 μg/mL (B. pulmosa) to 62.39 ± 1.96 μg/mL (C. compressa) with GES-22 and OXA-1, respectively. ME of D.dichtoma exhibited significant inhibitory effect on GES-22 more than classical β-lactam inhibitors, clavulanate, sulbactam and tazobactam (IC50 = 68.38 ± 0.17 μg/mL, 52.68 ± 0.64 μg/mL, and 29.94 ± 0.01 μg/mL, respectively). OXA-1 was not inhibited by sulbactam and clavulanate and was moderately inhibited by tazobactam (IC50 =243.03± 4.53 μg/mL).
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