Występowanie oraz charakterystyka molekularna oporności na azole wśród klinicznych i środowiskowych izolatów Aspergillus fumigatus
Abstract
Celem badań prowadzonych w ramach niniejszej pracy doktorskiej było sprawdzenie występowania oporności na azole grzybów A. fumigatus pochodzących z Polski oraz jej molekularna charakterystyka. W toku badań utworzono kolekcję 200 izolatów A. fumigatus pochodzących z różnych miejsc Polski. W skład kolekcji wchodziły izolaty kliniczne, środowiskowe i weterynaryjne. Przynależność gatunkową badanych grzybów potwierdzono metodami diagnostyki klasycznej oraz molekularnej. Następnie oznaczono lekowrażliwość badanych izolatów na posakonazol itrakonazol i worikonazol. W toku przeprowadzonych badań wykazano obecność izolatów opornych na azole. W ramach pracy nad niniejszą rozprawą przeanalizowano występowanie różnych mechanizmów oporności na azole u badanych izolatów: mutacje punktowe w genie CYP51A, występowanie tandemowych powtórzeń w rejonie promotora genu CYP51A (TR34), ekspresję genów CYP51A, CYP51B, MDR1, MDR2, MDR3, ATRF. W toku przeprowadzonych badań zaobserwowano występowanie różnych mutacji w genie CYP51A w tym jednej, którą udało się powiązać z opornością na azole (mutacja L98H skorelowana z TR34), a także zaobserwowano różnice w poziomach ekspresji badanych genów pomiędzy izolatami opornymi i wrażliwymi na azole. W trakcie badań własnych sprawdzono także wpływ obecności azoli w medium hodowlanym na grzyby z gatunku A. fumigatus. Sprawdzono wpływ obecności worikonazolu, posakonazolu i itrakonazolu w medium hodowlanym na poziomy ekspresji genów i zmiany wartości MIC. W toku przeprowadzonych badań zaobserwowano nabycie oporności na azole w wyniku długotrwałej ekspozycji, zmiany poziomów ekspresji badanych genów oraz zmiany w morfologii grzybów w obecności posakonazolu i itrakonazolu. W toku przeprowadzonych badań oznaczono także wrażliwość izolatów na izawukonazol - nowy związek azolowy dopuszczony do stosowania u ludzi. Zaobserwowano występowanie oporności na izawukonazol zarówno izolatów opornych jak i wrażliwych na pozostałe azole.
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- Category:
- Thesis, nostrification
- Type:
- praca doktorska pracowników zatrudnionych w PG oraz studentów studium doktoranckiego
- Language:
- Polish
- Publication year:
- 2018
- Bibliography: test
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- W Polsce występują zarówno kliniczne, środowiskowe i weterynaryjne izolaty A. fumigatus oporne na azole. Wśród przebadanych 200 izolatów oporność wynosiła 8,5% (w tym wśród klinicznych 3,6%, wśród środowiskowych 2,2% wśród weterynaryjnych 78,6%).
- Wśród izolatów A. fumigatus występujących w Polsce istnieją różne mechanizmy oporności na azole. Należą do nich mutacja TR 34 L98H w genie CYP51A oraz nadekspresja genów kodujących pompy błonowe i genów odpowiedzialnych za poprawną budowę ściany i błony komórkowej grzybów. open in new tab
- Obecność w medium hodowlanym posakonazolu lub itrakonazolu może powodować zmiany w kolorze grzybni grzybów z gatunku A. fumigatus. open in new tab
- Obecność w medium hodowlanym badanych azoli indukuje u grzybów z gatunku A. fumigatus zwiększoną produkcję zarodników. open in new tab
- Długotrwała obecność itrakonazolu, posakonazolu albo worikonazolu w środowisku może indukować oporność na azole wśród szczepów A. fumigatus. open in new tab
- Obecność w medium hodowlanym itrakonazolu i worikonazolu powoduje wzrost ekspresji genów MDR1, MDR2, MDR3, ATRF, CYP51A, CYP51B grzybów A. fumigatus bez względu na mutacje występujące w genie CYP51A. open in new tab
- Obecność w medium hodowlanym posakonazolu powoduje wzrost poziomu ekspresji genów MDR1, MDR2, MDR3, ATRF, CYP51A, CYP51B izolatów A. fumigatus bez mutacji TR 34 L98H, zaś w przypadku izolatów z tą mutacją powoduje spadek poziomu ekspresji genów CYP51B, MDR2, MDR3, ATRF a także nie powoduje istotnych zmian w poziomach ekspresji genów CYP51A i MDR1. open in new tab
- Nadekspresja genów kodujących transportery błonowe oraz genów odpowiedzialnych za poprawną budowę ściany i błony komórkowej grzybów z gatunku A. fumigatus może przyczynić się do nabycia oporności na azole. open in new tab
- Izolaty A. fumigatus oporne wyłącznie na itrakonazol charakteryzują się nadekspresją genu CYP51A.
- Izolaty oporne na worikonazol, itrakonazol i posakonazol nie są wrażliwe na działanie izawukonazolu, a inne izolaty A. fumigatus mogą być oporne na izawukonazol pomimo braku oporności na pozostałe triazole. BIBLIOGRAFIA 1. open in new tab
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- Kurzyk E.: "Budowa biofilmu chorobotwórczych drożdży i jego wpływ na oporność." Dokonania Młodych Naukowców nr 5 s. 266-267, 2014
- Prezentacje ustne na konferencjach: Kurzyk E., "Oporność izolatów Aspergillus fumigatus na izawukonazol" Ogólnopolska Konferencja Biotechnologów i Mikrobiologów BioMillenium 2017, Gdańsk 6-8 wrzesień 2017.
- Kurzyk E.: "Quorum sensing -rola w patogenezie drożdży Candida. Wpływ Młodych Naukowców na osiągnięcia Polskiej Nauk, VI Edycja, Gdańsk 25.04.2014
- Plakaty na konferencjach: Nawrot U., Wieliczko A., Kurzyk E., Brillowska-Dąbrowska A.: "Low frequency of itraconazole resistance fund among avian Aspergillus fumigatus isolates" 8 th Trends in Medical Mycology, Mycoses. Serbia, 06-09.10.2017.
- Kurzyk E., Mroczyńska M., Nawrot U., Włodarczyk K., Brillowska-Dąbrowska A.: " Oporność izolatów Aspergillus fumigatus na azole" XXVII Zjazd Polskiego Towarzystwa Mikrobiologów. Bydgoszcz 25-27.09.2016
- Kurzyk E., Nawrot U., Włodarczyk K., Brillowska-Dąbrowska A.: "Występowanie tandemowych powtórzeń (TR 34 ) w rejonie promotora genu CYP51A oraz mutacji w tym genie jako czynników predysponujących do oporności na azole wśród izolatów Aspergillus fumigatus" II Ogólnopolska Konferencja "Drobnoustroje w świecie człowieka -Drobnoustroje oportunistyczne" Bydgoszcz 21-22.05.2016. -praca wyróżniona I miejscem w sesji plakatowej Mroczyńska M., Kurzyk E., Brillowska-Dąbrowska A., Gucwa K., Szweda P.: Genotypowanie klinicznych izolatów Candida albicans metodą MP PCR" II Ogólnopolska Konferencja "Drobnoustroje w świecie człowieka -Drobnoustroje oportunistyczne" Bydgoszcz 21-22.05.2016.
- Kurzyk E., Mroczyńska M., Nawrot U., Włodarczyk K., Brillowska-Dąbrowska A.: "Czy istnieje konieczność oznaczania oporności na azole dla szczepów Aspergillus fumigatus izolowanych w Polsce?" BioMedSession, Gdańsk 12.12.2015
- Kurzyk E., Nawrot U., Mroczyńska M., Włodarczyk K., Ussowicz M., Zdziarski P., Arendrup MC., Brillowska-Dąbrowska A.: Detection of clinical Aspergillus fumigatus isolates resistant to triazoles. 7th Trends in Medical Mycology. Vol. 58 Supplement S4. P006. str. 53-54. Lizbona 9-12 październik 2015
- Nawrot U., Kurzyk E., Włodarczyk K., Ussowicz M., Arendrup MC., Brillowska-Dąbrowska A.: The first Polish report of azole resistant clinical Aspergillus fumigatus isolate. 6th International Weigl Conference on Microbiology. Acta Biochemica Polonica. Vol. 62 Supplement 2/2015. V.P.38. str. 104. Gdańsk, 8-10 lipiec 2015
- Kurzyk E., Kadzewicz K., Romanowska E., Dzierżanowska-Fangrat K., Brillowska-Dąbrowska A., Szweda P.: "Genotypowanie klinicznych izolatów Candida krusi. " I Ogólnopolska Konferencja "Drobnoustroje oportunistyczne" Bydgoszcz 18-20 wrzesień 2014
- Sources of funding:
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- Praca współfinansowana ze środków NCN w ramach projektu OPUS nr 2013/11/B/NZ7/04935:
- Verified by:
- Gdańsk University of Technology
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