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1.
IZOLACIJA IN KARAKTERIZACIJA BAKTERIJ IZ RIZOPLANA HLADNIKIJE
Tamara Mithans, 2009, undergraduate thesis

Abstract: Na površini korenin, imenovani rizoplan, lahko najdemo zelo različne skupine mikroorganizmov. Ti lahko na rast rastlin vplivajo pozitivno, lahko pa s svojo prisotnostjo in delovanjem rastlini škodujejo. Cilj diplomskega dela je bil izolacija in identififikacija bakterij, ki naseljujejo rizoplan hladnikije — slovenske endemične vrste. Iz treh rastlin smo osamili 17 bakterijskih sevov. Izolirali smo bakterijsko DNA in s pomočjo verižne reakcije s polimerazo (PCR) pomnožili 16S rDNA. Glede na primerjavo pomnožene 16S rDNa z referenčnimi bazami podatkov (GenBank, Ribosomal database project) smo izolirane bakterije identificirali. Izolirane bakterije so pripadale rodovom Bacillus, Enterobacter, Pseudomonas, Erwinia in Pantoea.
Keywords: Hladnikija, Hladnikia pastinacifolia, 16S rDNA, PCR, verižna reakcija s polimerazo, rizosfera, rizoplan, rizobakterije
Published: 25.11.2009; Views: 2583; Downloads: 254
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2.
Vpliv netoksinogenega seva bakterije Clostridium difficile s hemolitično aktivnostjo na morfologijo celic mišjih fibroblastov
Natalija Pungerl, 2011, undergraduate thesis

Abstract: Bakterija Clostridium difficile je normalno prisotna v naravi in prebavilih sesalcev. Je po Gramu pozitivna, anaerobna, sporogena bakterija. Spore so zelo odporne proti izsuševanju, kemikalijam ter ekstremnim temperaturam. Najpogosteje se okužijo starejši hospitalizirani ljudje, ki se jim poruši ravnovesje v črevesni flori, običajno zaradi daljšega zdravljenja z antibiotiki. Sevi, ki povzročajo bolezen, proizvajajo toksine, med katerimi sta najpomembnejša toksin A in toksin B. V diplomski nalogi smo se osredotočili na vpliv netoksinogenih sevov bakterije Clostridium difficile s hemolitično aktivnostjo na citoskelet mišjih fibroblastov. V raziskavi smo uporabili celice McCoy, ki smo jih tretirali s supernatantom, barvali aktinske filamente in jedra ter opazovali učinek na aktinskem citoskeletu s konfokalnim mikroskopom. Ugotovili smo, da je prišlo pri celicah tretiranih s supernatantom s hemolitično aktivnostjo do določenih sprememb, predvsem na aktinskem citoskeletu, medtem ko na jedrih ni bilo večjih razlik. Spremembe so bile vidne na aktinskih filamentih, ki so bili prekinjeni in združeni v skupke.
Keywords: Clostridium difficile, aktinski filamenti, celice McCoy, konfokalni mikroskop, supernatant, toksini
Published: 29.02.2012; Views: 2146; Downloads: 181
.pdf Full text (985,00 KB)

3.
MIKROSKOPSKE IN MAKROSKOPSKE MORFOLOŠKE ZNAČILNOSTI BAKTERIJE CLOSTRIDIUM DIFFICILE
Mateja Medved, 2012, undergraduate thesis

Abstract: Bakterija Clostridium difficile je najpogostejši povzročitelj bolnišničnih črevesnih infekcij. V zadnjem času v epidemiologiji okužb prihaja do sprememb, saj pogostnost in resnost bolezni narašča tudi zunaj bolnišničnega okolja. Bakterija je splošno razširjena zaradi zmožnosti tvorbe spor, kontaminacija okolja s sporami pa je ključni dejavnik za okužbo z bakterijo C. difficile (CDI; angl. Clostridium difficile infection). V diplomskem delu so nas zanimale morfološke značilnosti kolonij (oblika, velikost, barva, površina in oblika roba) ter morfološke značilnosti celic izbranih sevov bakterije Clostridium difficile. Morfologijo smo primerjali na treh trdnih gojiščih (COH, CLO, CDALT) z uporabo stereolupe in svetlobne mikroskopije. Ugotovili smo, da sta si rast in tvorba vidnih kolonij na različnih trdnih gojiščih istega seva zelo podobni, medtem ko se nekateri sevi med seboj razlikujejo. Ugotovili smo tudi, da je pri nekaterih sevih delež prostih spor večji kot delež spor, ki so še znotraj vegetativnih celic, in obratno. Namen dela je bil ugotoviti, ali je morfologija kolonij odvisna od posameznega PCR-ribotipa in ali se kolonije razlikujejo na različnih trdih gojiščih.
Keywords: Clostridium difficile, svetlobna mikroskopija, stereolupa, mikroskopska in makroskopska morfologija.
Published: 04.01.2013; Views: 2453; Downloads: 151
.pdf Full text (1,08 MB)

4.
NOVI MEHANIZMI TOKSIČNOSTI PRI BAKTERIJI Clostridium difficile
Mateja Zemljič, 2010, dissertation

Abstract: Clostridium difficile proizvaja tri toksine. Toksin A (TcdA, enterotoksin in citotoksin) in toksin B (TcdB, močen citotoksin) sta glavna dejavnika virulence, ki povzročata simptome driske in kolitisa. Nekateri sevi pa tvorijo še binarni toksin CDT. Prav tako bi določeni sevi lahko proizvajali tudi toksine, ki jih do sedaj pri tej bakteriji še ne poznamo. Toksin A in toksin B sta enoverižna proteina, sestavljena iz štirih funkcionalnih domen: encimske domene na N-terminalnem delu, cisteinske proteazne domene, centralne hidrofobne domene in vezavne domene na C-terminalnem delu proteina. Rekombinantni protein N-terminalne encimske domene toksina B in del rekombinantne C-terminalne vezavne domene toksina A imata lastnosti enake holotoksinu (celična smrt, imunski odgovor). Podobno delovanje za C-terminalno vezavno domeno toksina B do sedaj še ni bilo opisano. V raziskavo smo vključili seve bakterije C. difficile s poznanimi in atipičnimi toksičnimi učinki na celice. Uporabili smo TcdB referenčnega seva VPI 10463 in variantnega seva 8864 in njuni rekombinantni vezavni domeni rec-TcdB3VPI in rec-TcdB38864. Za in vitro študije interakcij med črevesnimi epitelnimi celicami in bakterijskimi toksini smo uporabili celični liniji epitelnih celic črevesja HT-29 in T84. Za proučevanje virulence sevov bakterije C. difficile pa smo izbrali živalski nevretenčarski model Caenorhabditis elegans. Rekombinantni vezavni domeni toksina B tako kot holotoksina poškodujeta epitelij, povzročita razpad tesnih stikov ter nekrozo oziroma apoptozo. Z vezavo na črevesne epitelijske celice vzpodbudita celice imunskega sistema in sprožita izločanje interlevkina 8. Holotoksin B sproži še proces avtofagije v črevesnih epitelijskih celicah. Nekateri sevi bakterije C. difficile so hemolitični. Poznavanje mehanizmov njihovega delovanja je tako bistvenega pomena za razumevanje bolezenskih znakov, ki jih povzročajo. Živalski nevretenčarski model C. elegans se je izkazal kot neprimeren živalski model za preučevanje virulence, vendar je njegova uporaba šele na začetku. Prvič smo opisali toksičnost C-terminalne domene TcdB, ki je neodvisna od modifikacije majhnih GTPaz in zelo verjetno predstavlja pomemben del pri okužbi z bakterijo C. difficile. Prav tako je poznavanje mehanizma delovanja toksina B pomembno za razumevanje njegove vloge pri celični smrti. Ker C. difficile pripada rodu z veliko in zelo razširjeno skupino toksinov, je velika verjetnost, da sevi C. difficile proizvajajo tudi toksine, ki celice lizirajo (hemolizini). S poznavanjem mehanizma delovanja toksinov lahko razložimo nekatere osnovne, pa tudi na prvi pogled nejasne fiziološke procese.
Keywords: Clostridium difficile, vezavna domena, toksin B, citotoksičnost, patogeneza, Caenorhabditis elegans
Published: 18.02.2013; Views: 1977; Downloads: 201
.pdf Full text (4,13 MB)

5.
IZOLACIJA BAKTERIJ IZ RODU Clostridium IZ VZORCEV PRSTI
Tanja Rikanovič, 2013, undergraduate thesis

Abstract: Klostridiji so paličaste, obligatno anaerobne, po Gramu pozitivne bakterije, ki tvorijo izjemno odporne spore. Zaradi slednjega so klostridiji ubikvitarni, saj jih lahko najdemo v okoljih kot so prst, okoljske vode, prisotni pa so tudi v človeškem in živalskem prebavilu. Ena izmed pomembnih vrst tega rodu je bakterija Clostridium difficile, ki je trenutno v svetu eden izmed pomembnejših povzročiteljev bolnišničnih okužb. V zadnjem času pa narašča tudi število izvenbolnišničnih okužb. Bolezen povzročajo le toksigeni sevi, ki izdelujejo toksin A in/ali toksin B, ki sta glavna dejavnika virulence C. difficile. V diplomskem delu smo iz 6 različnih vrst/tipov prsti osamili več klostridijskih vrst, tudi C. difficile. Izolate smo identificirali s komercialnim sistemom MALDI Biotyper (Bruker Daltonics), ki je komercialna fenotipska metoda za identifikacijo in temelji na analizi proteinov s pomočjo laserske svetlobe. Izolate C. difficile smo še karakterizirali, in sicer z dvema dobro razširjenima genotipizacijskima metodama: toksinotipizacijo in PCR ribotipizacijo. Skupno smo osamili 34 klostridijskih izolatov, ki so pripadali 7 različnim vrstam. Med njimi so prevladovale vrste Clostridium glycolicum (n=11), Clostridium bifermentans (n=5) in Clostridium mayombei (n=4). C. difficile (n=10) smo zaznali le v vzorcu sedimenta reke Drave, s selektivno gojitveno metodo. Skupno deset izolatov iz tega vzorca je bilo netoksigenih in PCR ribotipa 010. Največje število in raznolikost klostridijev smo določili v vzorcu komposta. Z raziskavo v diplomskem delu smo potrdili, da lahko v različnih okoljih pričakujemo različne predstavnike rodu Clostridium, raznolikost vrst pa je odvisna od tipa okolja nabranih vzorcev. Bakterije C. difficile sicer nismo našli v različnih vzorcih prsti, pač pa v rečnem sedimentu, kjer do sedaj v Sloveniji še ni bil opisan.
Keywords: klostridiji, prst, Clostridium difficile, identifikacija, toksinotipizacija, PCR ribotipizacija, raznolikost vrst
Published: 13.11.2013; Views: 1560; Downloads: 206
.pdf Full text (1,29 MB)

6.
Fourteen-genome comparison identifies DNA markers for severe-disease-associated strains of Clostridium difficile
Vincenzo Forgetta, Maja Rupnik, 2011, original scientific article

Abstract: Clostridium difficile is a common cause of infectious diarrhea in hospitalizedpatients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, theexistence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising 3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxinA-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.
Published: 05.06.2012; Views: 1023; Downloads: 81
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7.
Isolation and characterization of Clostridium difficile from shellfish and marine environments
Vincenzo Pasquale, Maja Rupnik, 2011, original scientific article

Abstract: This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater,sediment, and zooplankton were collected at five sampling stations inthe Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marinesediment. Majority of the C. difficile isolates were toxin A/B positive.Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.
Published: 05.06.2012; Views: 882; Downloads: 50
URL Link to full text

8.
Gut microbiota patterns associated with colonization of different clostridium difficile ribotypes
Jure Škraban, Sašo Džeroski, Bernard Ženko, Domen Mongus, Simon Gangl, Maja Rupnik, 2013, original scientific article

Abstract: Abstract Introduction Materials and Methods Results Discussion Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract C. difficile infection is associated with disturbed gut microbiota and changes in relative frequencies and abundance of individual bacterial taxons have been described. In this study we have analysed bacterial, fungal and archaeal microbiota by denaturing high pressure liquid chromatography (DHPLC) and with machine learning methods in 208 faecal samplesfrom healthy volunteers and in routine samples with requested C. difficile testing. The latter were further divided according to stool consistency, C. difficile presence or absence and C. difficile ribotype (027 or non-027). Lower microbiota diversity was a common trait of all routine samples and not necessarily connected only to C. difficile colonisation. Differences between the healthy donors and C. difficile positive routine samples were detected in bacterial, fungal and archaeal components. Bifidobacterium longum was the single most important species associated with C. difficile negative samples. However, by machine learning approaches we haveidentified patterns of microbiota composition predictive for C. difficile colonization. Those patterns also differed between samples with C. difficile ribotype 027 and other C. difficile ribotypes. The results indicate that not only the presence of a single species/group is important but that certain combinations of gut microbes are associated with C. difficile carriage and that some ribotypes (027) might be associated with more disturbed microbiota than the others.
Keywords: microbiota, clostridium difficile, chromatography
Published: 19.06.2017; Views: 392; Downloads: 184
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9.
Clostridium difficile ribotypes in humans and animals in Brazil
Rodrigo Otávio Silveira Silva, Maja Rupnik, Amanda Nádia Diniz, Eduardo Garcia Vilela, Francisco Carlos Faria Lobato, 2015, original scientific article

Abstract: Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen.
Keywords: Clostridium difficile, pseudomembranous colitis, zoonosis
Published: 07.08.2017; Views: 472; Downloads: 181
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10.
Highly divergent Clostridium difficile strains isolated from the environment
Sandra Janežič, Mojca Potočnik, Valerija Tkalec, Maja Rupnik, 2016, original scientific article

Abstract: Clostridium difficile is one of the most important human and animal pathogens. However, the bacterium is ubiquitous and can be isolated from various sources. Here we report the prevalence and characterization of C. difficile in less studied environmental samples, puddle water (n = 104) and soil (n = 79). C. difficile was detected in 14.4% of puddle water and in 36.7% of soil samples. Environmental strains displayed antimicrobial resistance patterns comparable to already published data of human and animal isolates. A total of 480 isolates were grouped into 34 different PCR ribotypes. More than half of these (52.9%; 18 of 34) were already described in humans or animals. However, 14 PCR ribotypes were new in our PCR ribotype library and all but one were non-toxigenic. The multilocus sequence analysis of these new PCR ribotypes revealed that non-toxigenic environmental isolates are phylogenetically distinct and belong to three highly divergent clades, two of which have not been described before. Our data suggest that environment is a potential reservoir of genetically diverse population of C. difficile.
Keywords: clostridium difficile, environment, isolation
Published: 19.06.2017; Views: 372; Downloads: 201
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