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Klinicky Významné Rezistentní Plazmidy – Studium Biologické Zátěže a Stability Úspěšných Mobilních Genetických Elementů

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Klinicky Významné Rezistentní Plazmidy – Studium Biologické Zátěže a Stability Úspěšných Mobilních Genetických Elementů MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV EXPERIMENTÁLNÍ BIOLOGIE Klinicky významné rezistentní plazmidy – studium biologické zátěže a stability úspěšných mobilních genetických elementů Diplomová práce Tomáš Nohejl Vedoucí práce: doc. RNDr. Monika Dolejská, Ph.D. Brno 2018 Bibliografický záznam Autor: Bc. Tomáš Nohejl Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Název práce: Klinicky významné rezistentní plazmidy – studium biologické zátěže a stability úspěšných mobilních genetických elementů Studijní program: Experimentální biologie Studijní obor: Molekulární biologie a genetika Vedoucí práce: doc. RNDr. Monika Dolejská, Ph.D. Akademický rok: 2017/2018 Počet stran 78 Klíčová slova: Antibiotická rezistence; plazmid; horizontální přenos genetické informace; HGT; konjugace; PMQR; chinolony; fitness; perzistence Bibliographic entry Author: Bc. Tomáš Nohejl Faculty of Science, Masaryk University Department of Experimental Biology Title of Thesis: Clinically important resistance plasmids - the study of biological burden and stability of successful mobile genetic elements Degree programme: Experimental biology Field of Study: Molecular Biology and Genetics Supervisor: doc. RNDr. Monika Dolejská, Ph.D. Academic Year: 2017/2018 Number of Pages: 78 Keywords: Antibiotic resistance; plasmid; horizontal gene transfer; HGT; conjugation; PMQR; quinolones; fitness; persistence Abstrakt Antibiotická rezistence představuje jeden z nejzávažnějších globálních problémů současnosti. Šíření genů kódující antibiotickou rezistenci je zprostředkováno mobilními genetickými elementy, zejména pak konjugací plazmidů. Aby se předešlo případné krizi neefektivních antibiotik a rozšíření infekcí způsobených rezistentními bakteriemi, je nutná znalost vlastností a chování plazmidů. Práce je zaměřena na IncX skupinu plazmidů nesoucí geny pro plazmidově přenášenou rezistenci k chinolonům (PMQR). Cílem práce je odhalit trendy přenosu plazmidů skupiny IncX a zátěže a perzistence v hostitelských bakteriích několika druhů za různých podmínek. Frekvence přenosu byla stanovena jako poměr počtu transkonjugantů na počet recipient. Byly zjištěny signifikantní rozdíly mezi jednotlivými IncX plazmidy, jejich bakteriálními hostiteli i fázemi růstu ve prospěch podskupiny IncX1. Analýza růstových křivek prokázala, že přítomnost IncX plazmidu negativně ovlivňuje fitness hostitelské bakterie, a to především v počátečních fázích růstu. Dlouhodobý kultivační experiment poukázal na perzistenci rezistentního plazmidu i za podmínek, kdy by neměl být pro bakteriální buňku v prostředí bez antibiotik potřebný. Tato práce ukazuje na významné vlastnosti IncX plazmidů nesoucích geny antibiotické rezistence, které pravděpodobně hrají úlohu v jejich šíření a udržování v bakteriálních populacích. Předmětem dalšího výzkumu by měl být detailní popis mechanismů, které stojí za trendy pozorovanými v této diplomové práci. Abstract Antibiotic resistance represents one of the most serious global issues of present. Dissemination of genes encoding for antibiotic resistance is mediated by mobile genetic elements, in particular by conjugation of plasmids. In order to prevent possible crisis of ineffective antibiotics and spread of infections caused by resistant bacteria, knowledge of the plasmid characteristics and behaviour is necessary. Thesis is focused on IncX group of plasmids carrying genes for plasmid-mediated quinolone resistance (PMQR). The aim of this thesis is to reveal the trends of IncX plasmid transfer and burden and persistence within host bacteria of several species under distinct conditions. The frequency of transfer was determined as the ratio of the number of transconjugants to the number of recipients. Significant differences were found between the individual IncX plasmids, their bacterial hosts, and the phases of growth in favour of the IncX1 subgroup. Analysis of growth curves has shown that the presence of IncX plasmid negatively affects the fitness of host bacteria, especially in the early phases of growth. A long-term cultivation experiment pointed out the persistence of a resistant plasmid even in antibiotic-free environment where it should not be needed for a bacterial cell. This thesis shows the significant properties of IncX plasmids carrying antibiotic resistance genes that are likely to play a role in their dissemination and stability in bacterial populations. The subject of further research should be a detailed description of the mechanisms that are behind the trends observed in this diploma thesis. ww Poděkování Na tomto místě bych rád poděkoval mé vedoucí doc. RNDr. Monice Dolejské, Ph.D. za její vstřícný přístup, ochotu, čas a především trpělivost, kterou mi věnovala. Dále také mému konzultantovi a mentorovi MSc. Marcovi Minoia, Ph.D. za neocenitelné rady, podporu a neutuchající nadšení pro vědu. Poděkování rovněž patří celému týmu na VFU za vstřícné přijetí, jejich humor, pomoc a čas mně věnovaný. V neposlední řadě velké díky patří mé rodině za jejich podporu, a to především mé milující manželce Barboře, která z každé strasti dokáže vykouzlit procházku růžovým sadem. Prohlášení Prohlašuji, že jsem svoji diplomovou práci vypracoval samostatně s využitím informačních zdrojů, které jsou v práci citovány. Brno 10. května 2018 Tomáš Nohejl Table of Contents 1. Introduction ..................................................................................................................... 11 1.1 Dissemination of antibiotic resistance by horizontal gene transfer .......................... 12 1.1.1 Mechanism of horizontal gene transfer in bacteria .............................................. 12 1.2 Mobile genetic elements in the dissemination of antimicrobial resistance .............. 16 1.2.1 Transposons .......................................................................................................... 17 1.2.2 Integrons and gene cassettes ................................................................................ 18 1.2.3 Plasmids ............................................................................................................... 18 1.3 Plasmid properties and characteristics ..................................................................... 19 1.3.1 Plasmid features ................................................................................................... 19 1.3.2 Plasmid classification ........................................................................................... 22 1.3.3 IncX plasmids ....................................................................................................... 23 1.3.4 Conjugative plasmids ........................................................................................... 24 1.4 Plasmid fitness cost, stability and impact on the host bacteria ................................ 26 1.5 Principles of antibiotic resistance in bacteria ........................................................... 27 1.5.1 Antibiotics ............................................................................................................ 27 1.5.2 Antibiotic resistance ............................................................................................. 28 1.5.3 Quinolones and resistance mechanisms in bacteria ............................................. 29 2. Goals ................................................................................................................................. 31 3. Material ............................................................................................................................ 32 3.1 Microorganisms ........................................................................................................ 32 3.2 Plasmids ................................................................................................................... 33 3.3 Culture media ........................................................................................................... 34 3.4 Devices ..................................................................................................................... 35 3.5 Chemicals ................................................................................................................. 36 3.5.1 Chemicals for polymerase chain reaction (PCR) and gel electrophoresis ........... 36 3.5.2 Antimicrobials ...................................................................................................... 36 3.5.3 Commercial kits ................................................................................................... 37 3.5.4 Other chemicals .................................................................................................... 37 3.6 Other equipment ....................................................................................................... 37 4. Methods ............................................................................................................................ 38 4.1 Transformation ......................................................................................................... 38 4.2 Plasmid detection ..................................................................................................... 38 4.2.1 Polymerase chain reaction with specific primers ................................................. 38 4.2.2 Agarose gel electrophoresis ................................................................................. 40 4.3 Conjugation experiment ..........................................................................................
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