Impact of gut microbiota modulation on colonization with Clostridium difficile

Bakterijo Clostridium difficile uvrščamo med najpogostejše povzročitelje okužb gastrointestinalnega trakta, pri čemer je ključen pogoj za uspešno kolonizacijo bakterije predhodno porušena črevesna mikrobiota. V okviru doktorske disertacije smo proučevali vpliv modulacije črevesne mikrobiote na kolonizacijo z bakterijo C. difficile s ciljem da identificiramo mikroorganizme ali konzorcije mikroorganizmov, ki sodelujejo v kolonizacijski odpornosti proti tej bakteriji. V populacijski študiji smo analizirali skupino hospitaliziranih bolnikov s kroničnimi vnetnimi črevesnimi boleznimi (KVČB, n = 40), saj je pri njih mikrobiota modulirana zaradi kroničnega obolenja in so posledično izpostavljena skupina za razvoj okužbe z bakterijo C. difficile. V primerjavi z ostalimi hospitaliziranimi bolniki (prva kontrolna skupina, n = 81) ter skupino zdravih prostovoljcev (druga kontrolna skupina, n = 160) smo pokazali, da je črevesna mikrobiota bolnikov s KVČB povezana z bolj izrazitimi znaki porušenja. Stopnja kolonizacije z bakterijo C. difficile se med skupinama hospitaliziranih bolnikov ni razlikovala, je pa bila značilno višja kot v zdravi populaciji. Glivna združba se ni razlikovala med koloniziranimi ter ne-koloniziranimi z bakterijo C. difficile, smo pa v povezavi s koloniziranostjo pokazali razlike v bakterijski združbi bolnikov brez KVČB. Poročamo 11 diferencialno zastopanih bakterijskih skupin, med njimi sta najbolj izrazita s kolonizacijo povezan porast predstavnikov iz rodu Enterococcus ter upad rodu Faecalibacterium. Z in vitro modulacijo črevesne mikrobiote z antibiotiki in ekstrakti polifenolov (PE) smo pokazali, da PE granatnega jabolka (mezokarp) ter PE borovnic v nasprotju s pričakovanji ne povečata kolonizacijske rezistence proti bakteriji C. difficile. Ugotovili smo, da specifični vzorci v bakterijski združbi neodvisno korelirajo z rastjo bakterije C. difficile ter aktivnostjo toksinov TcdA in TcdB, ki jih bakterija proizvaja. Uspešno smo osamili tri bakterijske izolate ter pokazali, da bakterija Clostridium sporogenes v ko-kulturi značilno zmanjša aktivnost toksinov bakterije C. difficile, medtem ko na rast bakterije ne vpliva.Infection with Clostridium difficile (CDI) is one of the most prevalent hospital acquired gastrointestinal infection. A key condition for a successful colonization with C. difficile is a disrupted gut microbiota. Here we present our research work on the effect of modulated microbiota on the colonization with C. difficile, with the main objective to identify microorganism or consortium of microorganisms that participate in the resistance against colonization with this bacterium. In the population study we analysed a group of hospitalized patients with inflammatory bowel diseases (IBD, n=40). Due to chronic inflammations they typically show signs of highly disrupted microbiota and are therefore at an elevated risk of developing CDI. Compared to other hospitalized patients (first control group, n=81) and group of healthy volunteers (second control group, n=160) they show more prominent patterns of disrupted microbiota. The rate of colonization with C. difficile did not differ between both groups of hospitalized patients, but it was significantly higher compared to the group of healthy volunteers. We did not find any patterns in fungal gut community associated to C. difficile colonization status, while on the other hand bacterial community differentiated colonized against non-colonized in the group of hospitalized patients without IBD. We report 11 differentially represented OTUs, most prominent being colonization associated increase in Enterococcus and decrease in Faecalibacterium. With an in vitro modulation of gut microbiota with polyphenolic extracts (PE) we showed, that against our expectations, neither pomegranate PE nor blueberry PE increased colonization resistance against C. difficile. We described different patterns that independently correlate with growth and activity of C. difficile toxins TcdA and TcdB. We successfully isolated three bacterial strains and showed that Clostridium sporogenes is able to reduce the activity of toxins without observable effect on the growth of C. difficile