Genotipizacija bakterije Mycoplasma pneumoniae in ugotavljanje patogenosti posameznih genotipov in vitro

Kogoj, Rok (author), Keše, Darja (mentor)

V raziskavi smo želeli ugotoviti pojavnost okužb dihal povzročenih z bakterijo M. pneumoniae pri slovenskih bolnikih v obdobju desetih let, spoznati kateri genotipi so bili v tem obdobju navzoči ter proučiti, če obstajajo med posameznimi genotipi razlike v patogenosti in vitro. Pregledali smo podatke iz strokovno zdravstvene dejavnosti za dokaz okužb z M. pneumoniae in jih analizirali. Shranjene izolate M. pneumoniae smo uporabili za genotipizacijo bakterije na podlagi gena za beljakovino P1 s pirosekvenciranjem in dodatno še z metodo MLVA. Pet genotipov smo uporabili za okužbo celic A549 in merili hitrost razmnoževanja mikoplazem, citotoksični učinek na gostiteljske celice ter tvorbo provnetnih citokinov IL-1β, IL-6, IL-8, TNF-α in IFN-γ. Ugotovili smo, da smo v obdobju med 2006 in 2015 v Sloveniji imeli dve epidemiji (2010/11 in 2014/15). V prvi epidemiji je prevladoval P1 tip 2, medtem ko v drugi P1 tip 1. Skupaj smo odkrili devet genotipov MLVA od katerih so bili trije (MLVA-3,5,6,2; MLVA-3,6,6,2 in MLVA-4,5,7,2) navzoči stalno in predstavljali 95,2 % vseh izolatov. Med petimi izbranimi genotipi MLVA nismo opazili razlik v hitrosti razmnoževanja ali citotoksičnosti. Celice so se na okužbo z vsemi testiranimi genotipi, razen MLVA-3,5,6,2, odzvale enako: z visoko tvorbo IL-6 in IL-8, manjšim porastom IL-1β ter brez odziva s TNF-α ali IFN-γ. Za genotip MLVA-3,5,6,2 smo opazili zavrto tvorbo IL-6 glede na negativno kontrolo ter statistično značilno manjšo tvorbo IL-8 glede na ostale genotipe. Vse skupaj nakazuje ne morebiten vpliv genetske različice bakterije na odziv gostiteljskih celic.

• Language: Slovenian
• Keywords: Mycoplasma pneumoniae, epidemiologija, genotipizacija, patogeneza, citotoksičnost, imunski odziv
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2019
• PID: 20.500.12556/RUL-108265
• COBISS.SI-ID: 5060984
• Publication date in RUL: 23.06.2019

Secondary language

• Language: English
• Title: Mycoplasma pneumoniae genotyping and pathogenicity determination of different genotypes in vitro.

Abstract

The aims of this study were to assess the epidemiology of M. pneumoniae respiratory tract infections in Slovenian patients during a 10-year period, to recognize which genotypes were present in the study population and to investigate if different genotypes show diverse in vitro pathogenicity potentials. We analysed the results from routine diagnostics for the detection of M. pneumoniae and used stored M. pneumoniae isolates for genotyping (P1 typing and MLVA). Five genotypes were used for in vitro infection of A549 cells in order to determine bacterial growth, cytotoxicity and pro-inflammatory cytokine (IL-1β, IL-6, IL-8, TNF-α and IFN-γ) production. The results showed that in the period from 2006 to 2015 two distinct epidemics occurred (2010/11 and 2014/15) and that the dominant P1 type shifted from P1 type 2 to P1 type 1 just prior to the second epidemic. All together we detected nine MLVA genotypes from which three (MLVA-3,5,6,2; MLVA-3,6,6,2 and MLVA-4,5,7,2) were present during the whole study period and represented 95.2 % of isolates. We found no differences in bacterial growth dynamics or cytotoxicity for five selected MLVA genotypes. The host cells responded to all genotypes except MLVA-3,5,6,2 by producing IL-1β, IL-6 and IL-8 but not TNF-α or IFN-γ. For the genotype MLVA-3,5,6,2 we observed an inhibition of IL-6 production and more than half less IL-8 response. These results may indicate a possible impact of bacterial genotype on the immune response of host cells.

• Keywords: Mycoplasma pneumoniae, epidemiology, genotyping, pathogenesis, cytotoxicity, immune response