Introduction of different methods for enrichment and detection of Faecalibacterium prausnitzii

Faecalibacterium prausnitzii je Gram-pozitivna, striktno anaerobna, komenzalna bakterija. Predstavlja od 5 do 10 odstotkov skupnega števila bakterij v vzorcih fecesa zdravih ljudi. Zmanjšanje zastopanosti F. prausnitzii povezujemo z različnimi bolezenskimi stanji (Crohnova bolezen, ulcerativni kolitis, kronična vnetna črevesna bolezen). Zaradi proizvodnje butirata, ki ima pomembno presnovno in protivnetno vlogo v črevesju, jo uvršamo med pomembnejše bakterije v človeški črevesni mikrobioti. To daje F. prausnitzii velik potencial za uporabo v probiotikih nove generacije. Magistrska naloga je razdeljena v tri sklope, in sicer optimizacijo molekularne detekcije F. prausnitzii, optimizacijo in vitro gojenja ter analizo vpliva različnih predstavnikov zdrave črevesne mikrobiote na rast F. prausnitzii. V prvem sklopu smo primerjali različne protokole verižne reakcije s polimerazo v realnem času za detekcijo F. prausnitzii. Komercialni komplet RIDA® GENE Faecalibacterium prausnitzii je na podlagi naših ugotovitev zagotavljal najvišjo specifičnost in občutljivost ter ponovljive rezultate. V drugem sklopu magistrskega dela smo uspešno optimizirali in vitro gojenje, ki podpira rast F. prausnitzii. Pokazali smo, da je vampni sok ključni faktor za uspešno gojenje bakterije F. prausnitzii. V tretjem sklopu naloge smo preverili vpliv izrabljenega gojišča izbranih vrst iz rodov Bacteroides in Bifidobacterium na rast bakterije F. prausnitzii. Nasprotno kot pričakovano smo pokazali inhibicijo rasti bakterije F. prausnitzii, ki je bila odvisna od koncentracije dodanega izrabljenega gojišča.Faecalibacterium prausnitzii is a Gram-positive, strictly anaerobic, commensal bacterium. It accounts for 5 to 10 percent of the total number of bacteria in faecal samples of healthy people. Reduction of F. prausnitzii is associated with various conditions (Crohn\u27s disease, ulcerative colitis, chronic inflammatory bowel disease). Due to the production of butyrate, which plays an important metabolic and anti-inflammatory role in the gut, we consider F. prausnitzii to be among important human beneficial bacteria in the gut. This gives F. prausnitzii great potential for use in the new generation of probiotics. Master thesis is divided into three parts, the optimisation of molecular detection of F. prausnitzii, the enrichment of this bacterium in vitro and the analysis of the influence of the different bacterial representatives of the healthy gut microbiota on the growth of F. prausnitzii. In the first part of the thesis, we compared the different protocols of real-time polymerase chain reaction for detection F. prausnitzii. Based on our findings, the commercial kit RIDA® GENE Faecalibacterium prausnitzii provided the highest specificit, sensitivity and reproducible results. In the second part of the thesis, we optimized the in vitro culture that supports the growth of F. prausnitzii. We have shown that rumen fluid is the key factor for the successful growth of F. prausnitzii. In the third part of the thesis, we examined the impact of cell-free supernatant of species from the genera Bacteroides and Bifidobacterium on the growth of F. prausnitzii. In contrast to expectations, we showed growth inhibition of F. prausnitzii, which depended on the concentration of added cell-free supernatant