Isolation and characterization of bacterium Clostridium difficile from soil from different environments

Clostridium difficile je anaerobna patogena črevesna bakterija, ki se ob porušenem ravnovesju črevesne mikrobiote lahko prekomerno namnoži ter povzroči okužbo, vse od blage driske do psevdomembranoznega kolitisa. Zelo pogosta je v bolnišnicah, pa tudi v izvenbolnišničnem okolju. Osredotočili smo se na izolacijo bakterije C. difficile iz vzorcev prsti, odvzetih iz njiv, vrtov, parkov in iz vzorcev peska iz peskovnikov. Vzorčili smo na 30 različnih lokacijah v Pomurju. Posamezni vzorec smo obdelali z dvema metodama, ki sta se razlikovali v začetnih korakih – direktno metodo in metodo, ki je vključevala sonikacijo. Vse izolate smo okaraterizirali s PCR ribotipizacijo in toksinotipizacijo. C. difficile smo izolirali iz 17 pozitivnih vzorcev (56,7 %), 15 vzorcev prsti in 2 vzorca peska. Skupaj smo osamili 170 izolatov C. difficile, katere smo uvrstili v 15 različnih PCR-ribotipov, od katerih je bil le en nov – SLO 257. Netoksigenih PCR-ribotipov je bilo 7, toksigenih pa 8slednje smo uvrstili v dva različna toksinotipa, 0 in IV. Identificirani ribotipi se deloma prekrivajo z ribotipi, ki jih najdemo tudi pri ljudeh in živalih. V prsti smo našli tudi nekaj ribotipov (predvsem netoksigenih), ki prej še niso bili opisani v človeški populaciji, kar kaže na to, da v prsti lahko najdemo genotipe, ki bi lahko bili bolj prilagojeni na okolje. Iz rezultatov lahko razberemo še, da je bakterija C. difficile v prsti prisotna v večjem deležu, kot navajajo dosedanji podatki. V tem delu smo prvič opisali izolacijo C. difficile iz peskovnikov v Sloveniji.Clostridium difficile is an anaerobic pathogenic bacteriium, which can over-multiply during imbalance in gut microbiota and cause anything from mild diarrhoea to pseudomembranotic colitis. It is quite common in hospitals and also in the environment. We focused on isolation of C. difficile from soil from farming fields, gardens, parks and from sand from sandboxes. Samples were collected on 30 different locations in Pomurje region. Individual samples were treated with two methods, which differed in starting steps – direct method and method that included sonication. All isoalted were characterised by PCR ribotyping and toxinotyping. C. difficile were isolated from 17 positive samples (56,7%) – 15 samples of soil and 2 samples of sand. Of 170 C. difficile isolates obtained 15 different PCR-ribotypes were identified, of which one was new – SLO 257. Seven PCR-ribotypes were nontoxigenic and 8 were toxigenic – belongin toxinotypes 0 and IV. Ribotypes partially overlap with ribotypes found in humans and animals. In soil a few ribotypes (mostly nontoxigenic) have also been found, which haven’t been detected in human population previously, indicating that some types may be more adapted tothe environment. Our result show higher numbers of C. difficile positive soil samples than previously reported. Also, in this work isolation of C. difficile from sand in sandboxes in Slovenia, has been reported for the first time