Interakcije različnih sevov bakterije Clostridium difficile s črevesno mikrobioto

Horvat, Sabina

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Title:	Interakcije različnih sevov bakterije Clostridium difficile s črevesno mikrobioto
Authors:	ID Horvat, Sabina (Author) ID Rupnik, Maja (Mentor) More about this mentor...
Files:	DOK_Horvat_Sabina_2019.pdf (3,96 MB) MD5: 05C3BE64FE79497378F13DA1EA2A6DE1 PID: 20.500.12556/dkum/0f5e76d6-0098-4b84-bf17-6cb27b05150b
Language:	Slovenian
Work type:	Doctoral dissertation
Typology:	2.08 - Doctoral Dissertation
Organization:	MF - Faculty of Medicine
Abstract:	Bakterija Clostridium difficile (Clostridioides difficile) povzroča črevesne okužbe, ki se najpogosteje kažejo kot lažja driska ali kolitis, redkeje pa kot pseudomembranozni kolitis. Okužba s C. difficile se najpogosteje razvije pri starejših hospitaliziranih bolnikih zaradi zdravljenja z antibiotiki širokega spektra, saj je predpogoj za uspešno kolonizacijo s C. difficile porušena črevesna mikrobiota. V zadnjem času je opazen porast okužb tudi pri mlajših generacijah, ki niso bile v stiku z bolnišničnim okoljem in niso prejemale antibiotične terapije. Mehanizmi kolonizacijske rezistence (tj. odpornosti), s katerimi funkcionalna črevesna mikrobiota preprečuje kolonizacijo in posledično okužbo s C. difficile, so relativno dobro raziskani. Ni pa znano, ali in kako C. difficile vpliva na črevesno mikrobioto med okužbo. V okviru doktorske naloge smo želeli ugotoviti, ali obstaja vpliv bakterije C. difficile na fekalno mikrobioto, ki je dober približek črevesni mikrobioti. Različne tipe fekalne mikrobiote (nezrelo, zdravo in porušeno) smo gojili in vitro ob prisotnosti vegetativnih celic C. difficile in/ali izrabljenega gojišča C. difficile. Testirali smo različne netoksigene in toksigene seve C. difficile različnih PCR-ribotipov, in sicer 010, 014/020, 027, 078 in 176, ki so v Evropi najbolj razširjeni. Za primerjavo smo fekalno mikrobioto gojili v izrabljenih gojiščih drugih bakterijskih vrst (Escherichia coli, Salmonella enterica serovar Enteritidis in Staphylococcus epidermidis). Spremembam v sestavi in pestrosti fekalne mikrobiote v različnih razmerah smo sledili s sekvenciranjem 16S-metagenoma na platformi MiSeq Illumina. V kokulturah fekalne mikrobiote s C. difficile smo spremljali rast in frekvenco sporulacije (tj. tvorbo spor) C. difficile. V določenem številu vzorcev smo z NMR-spektroskopijo pregledali tudi profile zunajceličnih metabolitov. Z raziskavo smo dokazali neposredni in specifični učinek bakterije C. difficile na pestrost in sestavo fekalne mikrobiote, ki je najverjetneje posledica spremenjene dostopnosti hranil. Fekalna mikrobiota, gojena v izrabljenih gojiščih E. coli, S. enterica serovar Enteritidis in S. epidermidis, je bila statistično značilno drugačna od fekalne mikrobiote, gojene v izrabljenem gojišču C. difficile. Naše ugotovitve so pokazale, da je učinek odvisen od tipa fekalne mikrobiote in ribotipa C. difficile. Nezrela in porušena mikrobiota sta bili in vitro bolj dovzetni za spremembe v pestrosti in sestavi zaradi C. difficile in sta omogočili boljše pogoje za sporulacijo C. difficile. Različni ribotipi C. difficile so podobno učinkovali na mikrobno pestrost, deloma pa se je razlikoval njihov vpliv na mikrobno sestavo, in sicer na nižjih taksonomskih nivojih. Spremembe, ki smo jih opazili, so podobne, kot so bile opisane pri pacientih, okuženih s C. difficile, in vključujejo manjšo mikrobno pestrost, povečan delež oportunističnih patogenov (enterokokov in enterobakterij) in zmanjšan delež bakterijskih skupin, povezanih s kolonizacijsko rezistenco (Lachnospiraceae in Ruminococcaceae). Z analizo metaboloma smo identificirali metabolite, ki bi lahko bili pomembni dejavnik pri okužbi s C. difficile. Predvsem povišane koncentracije tiramina, etanola in fenolnih kislin bi lahko pomembno doprinesle k disbiozi, ki je značilna za okužbo s C. difficile. Naši rezultati so pokazali, da lahko C. difficile aktivno vzdržuje črevesno mikrobioto v stanju neravnovesja. Spremembe v črevesni mikrobioti torej lahko razumemo ne le kot vzrok za okužbo s C. difficile, ampak tudi kot posledico prisotnosti C. difficile v črevesnem okolju.
Keywords:	črevesna mikrobiota, Clostridium difficile, 16S-metagenom, sekvenciranje naslednje generacije, in vitro modeli
Place of publishing:	Maribor
Year of publishing:	2019
PID:	20.500.12556/DKUM-73659
COBISS.SI-ID:	303326720
NUK URN:	URN:SI:UM:DK:HO3PE2VL
Publication date in DKUM:	14.01.2020
Views:	2063
Downloads:	238
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Categories:	MF
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Licences

License:	CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International

Link:	http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:	The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:	30.05.2019

Secondary language

Language:	English
Title:	Interactions between various strains of Clostridium difficile and gut microbiota
Abstract:	Clostridium difficile (Clostridioides difficile) is a causative agent of intestinal infections, which are most commonly manifested as diarrhea or colitis, and rarely as pseudomembranous colitis. C. difficile infection is common in elderly hospitalized patients, receiving a wide spectrum antibiotic treatment, since the precondition for intestinal colonization is disrupted gut microbiota. In recent years, however, there has been a noticeable increase in infections in younger generations, who have not been in contact with hospital environment, nor have they received antibiotic treatment. Mechanisms of colonization resistance, by which functional gut microbiota prevents colonization and, consequently, C. difficile infection, are relatively well described. However, it is not known whether C. difficile could influence gut microbiota during infection. The aim of the doctoral thesis was to determine whether C. difficile can influence fecal microbiota, which is a good approximation of the gut microbiota. Various types of fecal microbiota (i.e. immature, healthy and dysbiotic) were cultured in vitro with C. difficile vegetative cells and/or in C. difficile conditioned medium. Various nontoxigenic and toxigenic C. difficile strains of ribotypes 010, 014/020, 027, 078 and 176, which are commonly found in Europe, were used for interactions. For comparison, microbiota was cultured in medium conditioned by other bacteria (i.e. Escherichia coli, Salmonella enterica serovar Enteritidis and Staphylococcus epidermidis). Changes in the fecal microbiota composition and diversity under different conditions were determined by 16S metagenome sequencing on the MiSeq Illumina platform. C. difficile growth and sporulation frequency was monitored in co-cultures of fecal microbiota and C. difficile. In a certain number of samples, the profiles of extracellular metabolites were examined by NMR spectroscopy. The study demonstrated direct and specific effect of C. difficile on the diversity and composition of fecal microbiota that is likely associated with nutrient availability. Fecal microbiota cultivated in medium conditioned by E. coli, S. enterica serovar Enteritidis or S. epidermidis was clearly different from microbiota cultivated in C. difficile conditioned medium. Our results showed that the effect depended on the type of fecal microbiota and C. difficile ribotype. Immature and dysbiotic microbiota were in vitro more susceptible to changes in the diversity and composition due to C. difficile and provided better conditions for the sporulation of C. difficile. Different C. difficile ribotypes had a similar effect on microbial diversity, and partly their effect on microbial composition at lower taxonomic levels differed. The changes we observed are similar to those described in C. difficile patients and include lower microbial diversity, an increased proportion of opportunistic pathogens (enterococci and enterobacteria) and a reduced proportion of bacterial groups associated with colonization resistance (Lachnospiraceae and Ruminococcaceae). With the analysis of the metabolome we identified metabolites that could be important factor in C. difficile infection. Particularly increased concentrations of tyramine, ethanol and phenolic acids could significantly contribute to dysbiosis observed in C. difficile infection. Our results suggest that C. difficile can actively maintain gut microbiota in the state of an imbalance. Changes in gut microbiota can therefore be understood not only as a cause for C. difficile infection, but also as a consequence of C. difficile presence in the gut.
Keywords:	gut microbiota, Clostridium difficile, 16S metagenome, next generation sequencing, in vitro models

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