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Univerzita Pardubice Fakulta Chemicko-Technologická Faktory

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Univerzita Pardubice Fakulta Chemicko-Technologická Faktory Univerzita Pardubice Fakulta chemicko-technologická Faktory virulence u Arcobacter spp. a možná eliminace tvorby biofilmů Barbora Vacková Diplomová práce 2017 Prohlašuji: Tuto práci jsem vypracovala samostatně. Veškeré literární prameny a informace, které jsem v práci využila, jsou uvedeny v seznamu použité literatury. Byla jsem seznámena s tím, že se na moji práci vztahují práva a povinnosti vyplývající ze zákona č. 121/2000 Sb., autorský zákon, zejména se skutečností, že Univerzita Pardubice má právo na uzavření licenční smlouvy o užití této práce jako školního díla podle § 60 odst. 1 autorského zákona, a s tím, že pokud dojde k užití této práce mnou nebo bude poskytnuta licence o užití jinému subjektu, je Univerzita Pardubice oprávněna ode mne požadovat přiměřený příspěvek na úhradu nákladů, které na vytvoření díla vynaložila, a to podle okolností až do jejich skutečné výše. Beru na vědomí, že v souladu s § 47b zákona č. 111/1998 Sb., o vysokých školách a o změně a doplnění dalších zákonů (zákon o vysokých školách), ve znění pozdějších předpisů, a směrnicí Univerzity Pardubice č. 9/2012, bude práce zveřejněna v Univerzitní knihovně a prostřednictvím Digitální knihovny Univerzity Pardubice. V Pardubicích dne 9. května 2017 Bc. Barbora Vacková Poděkování Na tomto místě bych ráda poděkovala především svému vedoucímu, panu Ing. Davidu Šilhovi, Ph.D. za odbornou pomoc, trpělivost, cenné rady a připomínky, které mi pomohly tuto diplomovou práci zpracovat. Dále děkuji své konzultantce paní Ing. Lucii Šilhové, Ph.D. Rovněž děkuji svým rodičům, příteli a sourozencům za velkou trpělivost a za morální a finanční podporu při studiu. SOUHRN Teorerická část předkládané diplomové práce je souhrnem informací o rodu Arcobacter, jeho charakteristikou, výskytem a detekcí. Dále jsou obsaženy kapitoly, které se zabývají virulenčními faktory bakterií (včetně arkobakterů) a detekcí virulentně asociovaných genů. Uvedeny jsou kapitoly věnované tvorbě bakteriálního biofilmu, detekci biofilmu a především pak možným způsobům jeho eliminace. V části experimentální je popsána metoda detekce virulentně asociovaných genů a metoda detekce tvorby biofilmu v přítomnosti přírodních látek. Tyto metody byly aplikovány na vybrané referenční a sbírkové kmeny rodu Arcobacter. Geny kódující virulenční faktory arkobakterů byly detekovány pomocí optimalizované metody multiplexní polymerázové řetězové reakce. Tvorba biofilmu těchto bakterií v přítomnosti přírodních extraktů byla detekována pomocí Christensenovy metody v mikrotitračních destičkách. Na základě získaných výsledků lze říci, že výskyt jednotlivých virulenčních faktorů v genomu testovaných kmenů je variabilní. Dále lze říci, že u všech testovaných kmenů došlo v přítomnosti přírodních extraktů k eliminaci vytvořeného biofilmu. KLÍČOVÁ SLOVA Arcobacter, faktory virulence, mPCR, biofilm, eliminace biofilmu, přírodní extrakty SUMMARY The theoretic part of this thesis is a summary of information about the genus Arcobacter, its characteristics, occurrence and detection. There are the chapters about virulence factors of bacteria (including the genus Arcobacter) and about detection of virulence-associated genes. Then there are chapters about bacterial biofilm formation, about methods for its detection and primary about methods for its elimination. The experimental part describes the method of detection virulence-associated genes and the method for detection of biofilm in the presence of natural substances. These methods were applied to selected reference and collection strains of the genus Arcobacter. Genes that encode virulence factors of Arcobacter spp. were detected using an optimized multiplex polymerase chain reaction. The biofilm of these bacteria in the presence of natural extracts was determinated using the Christensen s method in the polyethylene microtitrate plates. According to acquired results the occurrence of individual virulence factors in the genome of the tested strains is variable. In addition, biofilm was eliminated in the presence of natural extracts in all tested strains. KEYWORDS Arcobacter, virulence factors, mPCR, biofilm, biofilm elimination, natural extracts OBSAH OBSAH………………………………………………………………………………………...8 SEZNAM OBRÁZKŮ ......................................................................................................... 11 SEZNAM TABULEK .......................................................................................................... 14 SEZNAM ZKRATEK A ZNAČEK....................................................................................... 15 0 ÚVOD ............................................................................................................................... 18 1 TEORETICKÁ ČÁST ....................................................................................................... 19 1.1 Rod Arcobacter........................................................................................................... 19 1.1.1 Taxonomické zařazení .......................................................................................... 19 1.1.2 Výskyt arkobakterů............................................................................................... 19 1.1.3 Morfologie arkobakterů ........................................................................................ 20 1.1.4 Biochemické vlastnosti a citlivost k vnějším vlivům ............................................. 22 1.1.5 Epidemiologie a patogeneze ................................................................................. 22 1.1.6 Terapie infekcí způsobených Arcobacter spp. ....................................................... 23 1.1.7 Laboratorní diagnostika Arcobacter spp. ............................................................... 24 1.1.7.1 Kultivace, růst a identifikace ....................................................................... 24 1.1.7.2 Molekulárně-biologické metody ................................................................. 25 1.1.7.3 MALDI /TOF MS ....................................................................................... 28 1.1.7.4 Sekvenování genomu .................................................................................. 29 1.2 Faktory virulence ........................................................................................................ 30 1.2.1 Quorum sensing spojené s virulencí ...................................................................... 30 1.2.2 Významné virulenční faktory bakterií ................................................................... 31 1.2.2.1 Adheze mikroorganismů ............................................................................. 31 1.2.2.2 Invazivita a kolonizace ............................................................................... 32 1.2.2.3 Motilita a chemotaxe .................................................................................. 33 1.2.3 Detekce a identifikace virulenčních faktorů .......................................................... 33 1.2.4 Faktory virulence u arkobakterů............................................................................ 34 1.2.5 Detekce a identifikace virulenčních faktorů arkobakterů ....................................... 36 1.3 Biofilm ....................................................................................................................... 37 1.3.1 Historie biofilmu .................................................................................................. 38 1.3.2 Tvorba biofilmu a jeho složení.............................................................................. 38 1.3.2.1 Ochranná role extracelulárních polymerních látek ....................................... 39 1.3.2.2 Buněčná různorodost uvnitř biofilmu .......................................................... 40 1.3.3 Tvorba biofilmu u arkobakterů ............................................................................. 41 1.3.4 Metody detekce biofilmu u arkobakterů ................................................................ 43 1.3.4.1 Christensenova metoda v mikrotitračních destičkách .................................. 43 1.4 Ovlivnění tvorby biofilmu .......................................................................................... 45 1.4.1 Eliminace biofilmu ............................................................................................... 45 1.4.1.1 Antibiotika.................................................................................................. 46 1.4.1.2 Čištění ........................................................................................................ 47 1.4.2 Ovlivnění tvorby biofilmu arkobakterů ................................................................. 48 1.4.2.1 Ovlivnění růstu a tvorby biofilmu arkobakterů přírodními látkami .............. 49 2 EXPERIMENTÁLNÍ ČÁST ............................................................................................. 54 2.1 Materiál a metody ....................................................................................................... 54 2.1.1 Chemikálie ........................................................................................................... 54 2.1.2 Reagencie a primery pro PCR ............................................................................... 54 2.1.3 Pracovní roztoky ................................................................................................... 55 2.1.4 Pevná a tekutá kultivační média ...........................................................................
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