Opredeljevanje patogenosti izolatov Escherichia coli, osamljenih iz blata bolnikov z drisko, z uporabo komercialnega kompleta »Diarrhoeagenic E. coli PCR kit«

Brišar, Nuša (author), Pirš, Mateja (mentor)

Bakterija Escherichia coli se nahaja kot komenzal v črevesju ljudi in jo lahko izoliramo iz blata večine ljudi, obstajajo pa tudi patogeni sevi E. coli. Nekateri med njimi povzročajo črevesna obolenja (angl. intestinal pathogenic E. coli, IPEC) oz. diareagene E. coli (angl. diarrheagenic E. coli, DEC). DEC delimo na šest glavnih patotipov: enterohemoragična oz. verotoksigena E. coli (EHEC oz. VTEC), enterotoksigena E. coli (ETEC), enteroinvazivna E. coli (EIEC), enteropatogena E. coli (EPEC), enteroagregativna E. coli (EAEC) in difuzno adherentna E. coli (DAEC). Namen magistrske naloge je bil ugotoviti, kako pogosto se pojavljajo klinično najpomembnejši patotipi DEC (knDEC) med izolati E. coli, osamljeni iz blata bolnikov s težje potekajočo drisko (ECB), pridobljeno v izvenbolnišničnem okolju, ki so potrebovali hospitalizacijo, in kontrolne skupine oseb brez driske (ECK). Preučevali smo 90 izolatov ECB in 40 izolatov ECK. Skupina bolnikov je bila nadalje razdeljena v skupino bolnikov, ki so drisko pridobili izven Evrope, ZDA ali Avstralije (ECBP), in bolnikov, ki so drisko pridobili v domačem okolju (ECBD). Z molekularno metodo PCR z uporabo komercialnega kompleta »Diarrhoeagenic E. coli PCR kit« proizvajalca SSI Diagnostica smo pri vseh 135 preučevanih izolatih iskali knDEC na podlagi značilnih molekularnih označevalcev (eltA, estA, vtx1, vtx2, eae in ipaH), s katerimi smo določili štiri glavne knDEC-patotipe – EIEC, EPEC, EHEC/VTEC, ETEC. Z metodo serotipizacije in naborom antiserumov proizvajalca SSI Diagnostica in Denka Seiken smo vsem izolatom knDEC določali serološko skupino O. Med izolati knDEC ECB smo zasledili EPEC – A/EEC (14/95 izolatov; 15 %) in ETEC (7/95; 7 %), med izolati knDEC ECK pa EPEC – A/EEC (7/40 izolatov; 18 %) in VTEC (2/40; 5 %). Izolatov EIEC nismo zaznali v nobeni skupini. Med izolati ECB in ECK ni bilo statistično pomembnih razlik v pojavnosti knDEC (p = 0,960), zato lahko našo hipotezo, da se knDEC pogosteje pojavljajo pri bolnikih s težje potekajočo drisko kot v kontrolni skupini, zavržemo. Med izolati knDEC ECBD smo zasledili EPEC – A/EEC (8/49 izolatov; 16 %) in ETEC (1/49; 2 %), med izolati knDEC ECBP pa EPEC – A/EEC (6/46 izolatov; 13 %) in ETEC (6/46, 13 %), razlike v pojavnosti med skupinama niso bile statistično pomembne (p = 0,365). Razlike v pojavljanju knDEC med ECBD in ECK niso bile statistično značilne (p = 0,521). Pri primerjavi pojavljanja izolatov knDEC med ECBP in ECK sicer ni bilo statistično značilnih razlik v pojavljanju knDEC (p = 0,699), opazili pa smo statistično značilno pogostejše pojavljanje patotipa ETEC med ECBP v primerjavi z ECK (p = 0,042). Primerjava naše študije z drugimi v Evropi ter z epidemiološkim spremljanjem okužb z E. coli v Sloveniji, ki jih izvaja NIJZ, je pokazala primerljive rezultate pojavljanja okužb s štirimi knDEC-patotipi – EIEC, EPEC, EHEC/VTEC, ETEC.

• Language: Slovenian
• Keywords: Escherichia coli, črevesna obolenja, EPEC, VTEC, EAEC, DAEC, ETEC, EIEC, antigeni O, vzorci blata, bolniki z drisko, izvenbolnišnično okolje, pojavljanje okužb, gojišče UriSelect, molekularne metode, PCR, serotipizacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [N. Brišar]
• Year: 2018
• PID: 20.500.12556/RUL-105846
• UDC: 579.61:616-092:577.2.083
• COBISS.SI-ID: 5010040
• Publication date in RUL: 20.12.2018

Secondary language

• Language: English
• Title: Evaluation of pathogenicity of Escherichia coli isolates from stool samples from patients with diarrhea using the commercial "Diarrhoeagenic E. coli PCR Kit"

Abstract

The bacterium Escherichia coli coexists as a commensal in human intestines and can be isolated from the stool of most people. However, there are also pathogenic strains of E. coli. Some of these are capable of causing intestinal diseases, namely intestinal pathogenic E. coli (IPEC) or diarrheagenic E. coli (DEC). DEC are divided into six main pathotypes: enterohemorrhagic or verotoxigenic E. coli (EHEC or VTEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC). The purpose of the master's thesis was to determine how often the clinically most important DEC pathotypes (knDEC) occur among E. coli isolates, isolated from stool samples from patients with severe diarrhea (ECB), obtained in an outpatient environment requiring hospitalization, and control group of people without diarrhea (ECK). We examined 90 ECB isolates and 40 ECK isolates. The patients with diarrhea were further divided into patients who had acquired diarrhea outside Europe, the USA or Australia (ECBP), and patients who acquired diarrhea in domestic environment (ECBD). Using the molecular PCR method and commercialy available "Diarrhoeagenic E. coli PCR kit", manufactured by SSI Diagnostica, we examined knDEC in all 135 studied isolates by identifiying specific molecular markers (eltA, estA, vtx1, vtx2, eae and ipaH), which identified four major knDEC pathotypes – EIEC, EPEC, EHEC/VTEC, ETEC. Using the serotyping method and the antiserum sets manufactured by SSI Diagnostica and Denka Seiken, O serogroup was determined for all knDEC isolates. Among the knDEC ECB isolates we observed EPEC – A/EEC (14/95 isolates; 15 %) and ETEC (7/95; 7 %). Among the knDEC ECK we observed EPEC – A/EEC (7/40 isolates; 18 %) and VTEC (2/40; 5 %). We detected no EIEC isolates in any of the groups. Among ECB and ECK, there were no statistically significant differences in the incidence of knDEC (p = 0.960), therefore our hypothesis that knDEC is more common in patients with severe diarrhea can be discarded. Among the knDEC ECBD we observed EPEC – A/EEC (8/49 isolates; 16 %) and ETEC (1/49; 2 %) Among the knDEC ECBP we observed EPEC – A/EEC (6/46 isolates; 13 %) and ETEC (6/46, 13 %). The differences in incidence among the groups were not statistically significant (p = 0.365). The differences in the incidence of knDEC among ECBD and ECK were not statistically significant (p = 0.521). In comparing the occurrence of knDEC isolates among the ECBP and ECK, there were no statistically significant differences in the incidence of knDEC (p = 0.699), but the statistically significantly more frequent occurrence of the ETEC pathotype among the ECBP was observed compared to ECK (p = 0.042). Comparing our study to others in Europe and to the epidemiological monitoring of E. coli infections in Slovenia, performed by the Slovenian Public Health Institute, it showed comparable results of infections with the four main knDEC pathotypes – EIEC, EPEC, EHEC/VTEC, ETEC.

• Keywords: Escherichia coli, intestinal diseases, EPEC, VTEC, EAEC, DAEC, ETEC, EIEC, O antigens, stool samples, patients with diarrhea, outpatient environment, infection occurrence, UriSelect medium, molecular methods, PCR, serotipisation