1. Ribotype classification of Clostridioides difficile isolates Is not predictive of the amino acid sequence diversity of the toxin virulence factors TcdA and TcdBZhenghui Li, Kwok Lee, Urvi Rajyaguru, C. Hal Jones, Sandra Janežič, Maja Rupnik, Annaliesa S. Anderson, Paul Liberator, 2020, izvirni znanstveni članek Opis: Clostridioides (Clostridium) difficile is the most commonly recognized cause of infectious diarrhea in healthcare settings. Currently there is no vaccine to prevent initial or recurrent C. difficile infection (CDI). Two large clostridial toxins, TcdA and TcdB, are the primary virulence factors for CDI. Immunological approaches to prevent CDI include antibody-mediated neutralization of the cytotoxicity of these toxins. An understanding of the sequence diversity of the two toxins expressed by disease causing isolates is critical for the interpretation of the immune response to the toxins. In this study, we determined the whole genome sequence (WGS) of 478 C. difficile isolates collected in 12 countries between 2004 and 2018 to probe toxin variant diversity. A total of 44 unique TcdA variants and 37 unique TcdB variants were identified. The amino acid sequence conservation among the TcdA variants (>98%) is considerably greater than among the TcdB variants (as low as 86.1%), suggesting that different selection pressures may have contributed to the evolution of the two toxins. Phylogenomic analysis of the WGS data demonstrate that isolates grouped together based on ribotype or MLST code for multiple different toxin variants. These findings illustrate the importance of determining not only the ribotype but also the toxin sequence when evaluating strain coverage using vaccine strategies that target these virulence factors. We recommend that toxin variant type and sequence type (ST), be used together with ribotype data to provide a more comprehensive strain classification scheme for C. difficile surveillance during vaccine development objectives.
Ključne besede: Clostridioides difficile, Clostridium difficile, TCDB, TcdA, vaccines, whole genome sequencing
Objavljeno v DKUM: 28.01.2025; Ogledov: 0; Prenosov: 5
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2. Clostridioides difficile ribotype distribution in a large teaching hospital in SerbiaMiloš Korać, Maja Rupnik, Nataša Nikolić, Milica Jovanović, Tanja Tošić, Jovan Malinić, Nikola Mitrović, Marko Marković, Ankica Vujović, Sanja Peruničić, Ksenija Bojović, Vladimir V. Đorđević, Aleksandra Barać, Ivana Milošević, 2020, izvirni znanstveni članek Opis: Background: The global epidemic of nosocomial diarrhea caused by Clostridioides (Clostridium) difficile started in 2000, with high mortality rates and emergence of a new hypervirulent strain NAP1/BI/027. The aim of this study was to assess the presence of ribotype 027 and other C. difficile ribotypes in a Serbian University Hospital, compare the temporal variability of ribotypes 3 years apart, as well as to compare clinical, demographic and laboratory characteristics and disease outcome among patients infected with 027 and non-027 ribotype. This was a prospective observational cohort study addressing 4-month intervals during 2014/2015 and 2017/2018.
Results: Ribotyping was performed in 64 non-duplicate C. difficile strains. Ribotype 027 was the most prevalent, and was detected in 53 (82.8%) patients (43/45 and 10/19 patients in 2014-2015 and 2017/2018, respectively). Other detected ribotypes were 001/072 in 4 (6.3%), 002 in 4 (6.3%), 014/020 in 2 (3.1%) and 176 in 1 (1.5%) patient. The percentage of the patients infected with ribotype 027 significantly decreased during the 3-year period, from 95.6 to 52.6% (p < 0.001). Ribotype 027 infection was associated with fluoroquinolone treatment more frequently than infection with other ribotypes [33 (62.3%) vs. 2 (18.2%), p = 0.010)]. A severe C. difficile infection was diagnosed more often in patients with the detected ribotype 027 compared to those infected with non-027 ribotypes (p = 0.006). No significant difference in the mortality and recurrence rates was found between the patients infected with ribotype 027 and those infected with other ribotypes [10/53 (18.8%) vs. 2/11 (18.2%), p = 0.708, and 10/35 (28.6%) vs. 0/2 (0%), p = 1.000, respectively].
Conclusion: Clostridium difficile ribotype 027 was the most prevalent ribotype among patients in a large Serbian hospital, but there is a clear decreasing trend.
Ključne besede: Clostridium difficile, ribotype 027, Serbia
Objavljeno v DKUM: 27.01.2025; Ogledov: 0; Prenosov: 5
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3. High contamination rates of shoes of veterinarians, veterinary support staff and veterinary students with Clostridioides difficile sporesJoanna Wojtacka, Beata Wysok, Aleksander Kocuvan, Maja Rupnik, 2022, izvirni znanstveni članek Opis: Clostridioides difficile is often found in animals and their environment. However, not much has been reported on veterinary clinics environment in terms of the spore load, prevalence and PCR ribotype diversity. The aim of this study was to assess the prevalence of C. difficile on shoe soles of veterinarians, veterinary support staff and veterinary students at the Veterinary Faculty campus. Altogether, 50 shoe sole swabs were collected, and the positivity rates ranged from 86.7% in swabs from veterinarians to 100% in swabs from support staff and students. Non-toxigenic and toxigenic strains representing toxinotypes 0, IV and XIX were isolated and distributed into 17 different PCR ribotypes, most common being 010, 014/020, SLO002 and 009. PCR ribotype 010 was the most prevalent and isolated from shoe soles sampled in 6/7 areas. Students' shoes had highest ribotype diversity (15/17 PCR ribotypes) but showed a low overlap with ribotype isolated from vets and support staff shoes. Veterinary students are likely the main vectors of C. difficile spores transmissions among veterinary teaching clinics and the hospital.
Ključne besede: Clostridioides (Clostridium) difficile, environment, footwear, One Health, transmission, veterinary clinic
Objavljeno v DKUM: 22.10.2024; Ogledov: 0; Prenosov: 1
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4. Epidemiološki in mikrobiološki pristopi za spremljanje bolnišničnih okužb v UKC MariborBožena Kotnik Kevorkijan, 2019, doktorsko delo/naloga Opis: Bolnišnične okužbe (BO) so okužbe, povezane z zdravstveno oskrbo (OPZ), ki nastanejo v bolnišnici in predstavljajo najpogostejši zaplet zdravljenja v bolnišnici. Spremljanje njihove pogostosti se lahko uporablja kot kazalnik kakovosti za bolnišnice. K spremljanju spreminjanja pogostosti BO lahko pristopimo tudi s ponavljajočimi se presečnimi raziskavami BO. Evropski center za preprečevanje in obvladovanje bolezni (angl. European Centre for Disease Prevention and Control, ECDC) vsakih pet let koordinira izvedbo evropske presečne raziskave BO.
Leta 2011 smo v Sloveniji opravili drugo Slovensko nacionalno presečno raziskavo BO (SNPRBO II), prevalenčno presečno raziskavo na dan (angl. point prevalence survey - PPS) v okviru evropske prevalenčne presečne raziskave BO. V raziskavi je sodeloval tudi Univerzitetni klinični center (UKC) Maribor. Visoko usposobljena zdravnica za obvladovanje in preprečevanje BO (ZOBO) je v UKC Maribor opravila vzporedno validacijo metode zbiranja podatkov, ki so jih zbrali anketarji v skladu s standardizirano metodologijo zbiranja podatkov, ki jo priporoča ECDC. Preverila je pravilnost zabeleženih podatkov za vse bolnike, pri katerih so anketarji prepoznali BO, in za vsakega petega bolnika, pri katerem anketarji niso prepoznali BO.
Ob spremljanju širjenja mikroorganizmov znotraj bolnišnice in prepoznavanju izbruhov je pomembna tipizacija mikroorganizmov, ki so povzročili BO. V UKC Maribor smo se odločili za tipizacijo proti meticilinu odpornih zlatih stafilokokov (angl. Methicillin resistant Staphylococcus aureus, MRSA), saj je spremljanje MRSA obvezni kazalnik kakovosti v slovenskih bolnišnicah. Tipizirali smo tudi bakterijo Clostridium difficile (CD), saj marsikje v zdravstveno razvitih državah CD predstavlja kazalnik kakovosti in povzroča klinično pomembne okužbe. Za MRSA smo uporabili spa tipizacijo, za CD pa ribotipizacijo.
Cilji doktorske naloge so vključevali: razvoj modificirane, delovno intenzivnejše metode za validacijo uporabljene »običajne ECDC metode« za prepoznavanje BO v presečni raziskavi (»modificirana ECDC metoda«); ocenili smo prevalence BO ob uporabi »običajne ECDC metode« in »modificirane ECDC metode«, razvite v okviru doktorske naloge; občutljivost in specifičnost »običajne ECDC metode« za prepoznavanje BO v primerjavi z »modificirano ECDC metodo« ter potrebo po nadomestitvi »običajne ECDC metodo« z našo »modificirano ECDC metodo«. Opisali smo značilnosti bolnikov, vključenih v presečno raziskavo, izpostavljenost invazivnim postopkom, BO, izolirane mikroorganizme in odpornost nekaterih mikroorganizmov na izbrane antibiotike ali skupine antibiotikov, ter uporabo antibiotikov. Spremljali smo dva izbrana povzročitelja BO, MRSA in CD, v časovnem obdobju enega leta in ocenili pomen molekularnih tipizacij za prepoznavanje in nadzor BO.
V PPS smo vključili 991 bolnikov, to je vse bolnike, ki so bili hospitalizirani v UKC Maribor na dan raziskave. O vsakem smo zabeležili številne podatke, tudi o dejavnikih tveganja za BO, zdravljenju s protimikrobnimi sredstvi in BO. Z obema metodama smo prepoznali 52 bolnikov, ki so na dan raziskave imeli vsaj eno BO oziroma so bili na dan raziskave še vedno zdravljeni zaradi BO. Ustrezna ocena prevalence BO je bila 5,2 % (s 95 % intervalom zaupanja 3,9 % - 6,8 %). Največ BO so imeli bolniki v enotah za intenzivno zdravljenje (EIZ) 25,0 %, na kirurških oddelkih je imelo BO 6,2 % bolnikov, na ginekološkem in porodnem oddelku 4,4 %, na internih oddelkih 3,1 % bolnikov.
Z univariatnimi in multivariatnimi analizami smo opredelili neodvisne dejavnike tveganja za BO: prisotnost vsadka v zadnjem letu, prisotnost urinskega katetra, operacija v zadnjih 30 dneh in intubacija.
Skupno smo prepoznali 66 BO. Najpogostejše so bile okužbe spodnjih dihal (25 %), okužbe kirurške rane in okužbe sečil. 9 % BO je bilo prisotnih že ob sprejemu bolnika v UKC Maribor, 91 % BO pa je bilo pridobljenih v času aktualne hospitalizacije, največ v obdobju 4.–7. dne po sprejemu. V
Ključne besede: bolnišnične okužbe, presečna raziskava, prevalenca, proti meticilinu odporen Staphylococcus aureus - MRSA, Clostridium difficile, spa tipizacija, ribotipizacija
Objavljeno v DKUM: 14.01.2020; Ogledov: 1926; Prenosov: 457
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5. Interakcije različnih sevov bakterije Clostridium difficile s črevesno mikrobiotoSabina Horvat, 2019, doktorska disertacija Opis: 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.
Ključne besede: črevesna mikrobiota, Clostridium difficile, 16S-metagenom, sekvenciranje naslednje generacije, in vitro modeli
Objavljeno v DKUM: 14.01.2020; Ogledov: 1870; Prenosov: 225
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6. Izolacija in karakterizacija bakterije Clostridum difficile s površine krompirjaTamara Simonič, 2018, magistrsko delo/naloga Opis: Clostridium difficile je po Gramu pozitivna, anaerobna, sporogena bakterija, ki povzroča črevesne okužbe, najpogosteje po zdravljenju z antibiotiki, kemoterapiji in po kirurških posegih, ki privedejo do porušenja normalne črevesne mikrobiote. Bakterijo lahko najdemo v različnih naravnih okoljih, črevesju živali in tudi hrani. Namen magistrskega dela je bil izolirati in karakterizirati bakterijo C. difficile s površine krompirjev, kupljenih v različnih lokalnih trgovinah ter iz čim več različnih držav. Iz 8 različnih trgovin smo pridobili 23 vzorcev krompirja, ki so izvirali iz 10 različnih držav. Prisotnost C. difficile smo dokazali pri 14 vzorcih (60,9 %). Iz vseh vzorcev smo osamili 162 izolatov C. difficile, katerim smo najprej s PCR-pomnoževanjem potrdili za C. difficile specifični gen cdd3, ter jih opredelili s PCR-ribotipizacijo in toksinotipizacijo. Vseh 162 izolatov smo uvrstili v 9 različnih PCR-ribotipov: 012, 014/020, 023, 027, 053, 126, 150, SLO 129 in SLO187. Najpogostejši PCR-ribotip je bil 014/020, sledila sta ribotipa 053 in 126. Toksigenih je bilo 137 izolatov, kasneje uvrščenih v 5 različnih toksinotipov – toksinotip 0, I, III, IV in V, 26 izolatov pa je bilo netoksigenih (s PCR nismo mogli dokazati prisotnosti toksinskih genov). Naši rezultati kažejo, da je krompir kontaminiran s sporami bakterije C. difficile v visokem deležu in da lahko na krompirju najdemo enake PCR-ribotipe, kot se pojavljajo pri ljudeh, živalih in v okolju.
Ključne besede: Clostridium difficile, krompir, PCR-ribotipizacija, toksinotipizacija
Objavljeno v DKUM: 27.09.2018; Ogledov: 2133; Prenosov: 137
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8. Evaluating the effect of Clostridium difficile conditioned medium on fecal microbiota community structureSabina Horvat, Aleksander Mahnič, Martin Breskvar, Sašo Džeroski, Maja Rupnik, 2017, izvirni znanstveni članek Opis: Clostridium difficile infection (CDI) is typically associated with disturbed gut microbiota and changes related to decreased colonization resistance against C. difficile are well described. However, nothing is known about possible effects of C. difficile on gut microbiota restoration during or after CDI. In this study, we have mimicked such a situation by using C. difficile conditioned medium of six different C. difficile strains belonging to PCR ribotypes 027 and 014/020 for cultivation of fecal microbiota. A marked decrease of microbial diversity was observed in conditioned medium of both tested ribotypes. The majority of differences occurred within the phylum Firmicutes, with a general decrease of gut commensals with putative protective functions (i.e. Lactobacillus, Clostridium_XIVa) and an increase in opportunistic pathogens (i.e. Enterococcus). Bacterial populations in conditioned medium differed between the two C. difficile ribotypes, 027 and 014/020 and are likely associated with nutrient availability. Fecal microbiota cultivated in medium conditioned by E. coli, Salmonella Enteritidis or Staphylococcus epidermidis grouped together and was clearly different from microbiota cultivated in C. difficile conditioned medium suggesting that C. difficile effects are specific. Our results show that the changes observed in microbiota of CDI patients are partially directly influenced by C. difficile.
Ključne besede: Clostridium difficile, infection, gut microbiota
Objavljeno v DKUM: 12.12.2017; Ogledov: 1601; Prenosov: 437
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9. Clostridium difficile ribotypes in humans and animals in BrazilRodrigo Otávio Silveira Silva, Maja Rupnik, Amanda Nádia Diniz, Eduardo Garcia Vilela, Francisco Carlos Faria Lobato, 2015, izvirni znanstveni članek Opis: 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.
Ključne besede: Clostridium difficile, pseudomembranous colitis, zoonosis
Objavljeno v DKUM: 07.08.2017; Ogledov: 1511; Prenosov: 397
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10. Identification of risk factors influencing Clostridium difficile prevalence in middle-size dairy farmsPetra Bandelj, Rok Blagus, France Briški, Olga Frlic, Aleksandra Vergles-Rataj, Maja Rupnik, Matjaž Ocepek, Modest Vengušt, 2016, izvirni znanstveni članek Opis: Farm animals have been suggested to play an important role in the epidemiology of Clostridium difficile infection (CDI) in the community. The purpose of this study was to evaluate risk factors associated with C. difficile dissemination in family dairy farms, which are the most common farming model in the European Union. Environmental samples and fecal samples from cows and calves were collected repeatedly over a 1 year period on 20 mid-size family dairy farms. Clostridium difficile was detected in cattle feces on all farms using qPCR. The average prevalence between farms was 10% (0-44.4%) and 35.7% (3.7-66.7%) in cows and calves, respectively. Bacterial culture yielded 103 C. difficile isolates from cattle and 61 from the environment. Most C. difficile isolates were PCR-ribotype 033. A univariate mixed effect model analysis of risk factors associated dietary changes with increasing C. difficile prevalence in cows (P = 0.0004); and dietary changes (P = 0.004), breeding Simmental cattle (P = 0.001), mastitis (P = 0.003) and antibiotic treatment (P = 0.003) in calves. Multivariate analysis of risk factors found that dietary changes in cows (P = 0.0001) and calves (P = 0.002) increase C. difficile prevalence; mastitis was identified as a risk factor in calves (P = 0.001). This study shows that C. difficile is common on dairy farms and that shedding is more influenced by farm management than environmental factors. Based on molecular typing of C. difficile isolates, it could also be concluded that family dairy farms are currently not contributing to increased CDI incidence.
Ključne besede: farm animals, clostridium difficile infection (CDI), dairy farms
Objavljeno v DKUM: 29.06.2017; Ogledov: 1334; Prenosov: 416
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