Ugotavljanje značilnosti in epidemioloških povezav med izolati bakterije Listeria monocytogenes s sekvenciranjem naslednje generacije

Papić, Bojan (author), Pate, Mateja (mentor), Kušar, Darja (comentor)

Listerioza je resno obolenje ljudi in živali, ki ga povzroča bakterija Listeria monocytogenes. Sekvenciranje celotnih genomov (angl. whole-genome sequencing, WGS) postaja metoda izbora za epidemiološki nadzor listerioze, vključno s preiskavami izbruhov. Tipizacija izolatov ni sestavni del rutinskih preiskav izbruhov listerioze pri živalih, zato genotipi bakterije L. monocytogenes, ki so udeleženi pri tovrstnih izbruhih, niso dobro raziskani. V prvem sklopu naloge smo metodo WGS uporabili za podrobno genetsko opredelitev domnevno epidemiološko in mikrobiološko povezanih izolatov. Retrospektivno smo analizirali sedem izbruhov listerioze pri drobnici, ki so jih povzročili kloni ST1 (CC1), ST18 (CC18), ST21 (CC21) in ST184. Z več analitičnimi pristopi, ki temeljijo na metodi WGS, smo potrdili monoklonalno naravo izbruhov; mikrobiološko povezani izolati so se razlikovali v največ 13 polimorfizmih posameznih nukleotidov v celotnem genomu (angl. whole-genome single nucleotide polymorphism, wgSNP), sedmih alelih osrednjega genoma (angl. core-genome multilocus sequence typing, cgMLST) in 12 alelih celotnega genoma (angl. whole-genome multilocus sequence typing, wgMLST). V primeru izbruha, povzročenega s sevom ST184, smo mikrobiološko povezan sev ugotovili tudi v vodi iz napajalnika in silaži, ki sta bila najverjetnejši vir okužbe za obolele živali. Metoda WGS je imela večjo moč razlikovanja od elektroforeze v pulzirajočem električnem polju (angl. pulsed-field gel electrophoresis, PFGE), ki dveh izbruhov, povzročenih s sevoma CC1, ni uspela razlikovati. Retrospektivno smo preiskali tudi izbruh listerioze pri ljudeh, ki ga je povzročil sev ST8 (CC8). Potrdili smo, da je bil izbruh monoklonalne narave, in ovrgli domnevo, da je bil vir okužbe preiskovano živilo oz. z živilom povezano okolje. Nadalje smo metodo WGS uporabili za podrobno preučitev genetske raznolikosti in poti širjenja izolatov bakterije L. monocytogenes v kmetijskem okolju. Na kmetiji s primerom živčne oblike listerioze smo hipervirulentni klon CC4 ugotovili tudi pri kravah s subkliničnim mastitisom. Z metodo WGS smo potrdili mikrobiološko povezavo med dvema izolatoma CC5 iz kmetijskega okolja z različnim profilom PFGE. Naši rezultati kažejo, da bi metoda WGS morala nadomestiti metodo PFGE za epidemiološki nadzor bakterije L. monocytogenes. V drugem sklopu naloge smo analizirali populacijsko strukturo izolatov živalskega kliničnega izvora in iz naravnega okolja, ki je slabo poznana. Za opis populacijske strukture izolatov živalskega kliničnega izvora in iz naravnega okolja smo uporabili tipizacijo na osnovi zaporedij več lokusov (angl. multilocus sequence typing, MLST). Klon CC1 je bil značilno povezan s kliničnim izvorom in z živčno obliko listerioze. Na novo smo opisali značilno povezavo klonov CC6 in CC37 z abortivno obliko bolezni. Kloni CC9, CC14 in CC29 so bili značilno povezani z naravnim okoljem. Zbrani rezultati predstavljajo pomemben doprinos k razumevanju genetske raznolikosti bakterije L. monocytogenes v kmetijskem okolju na nacionalni in mednarodni ravni.

• Language: Slovenian
• Keywords: Molekularna epidemiologija, listerioza – mikrobiologija, Listeria monocytogenes – izolacija in čiščenje – genetika, tipizacija na osnovi zaporedij več lokusov – metode, sekvenciranje celotnega genoma – metode
• Work type: Doctoral dissertation
• Organization: VF - Veterinary Faculty
• Year: 2020
• PID: 20.500.12556/RUL-125164
• Publication date in RUL: 05.03.2021

Secondary language

• Language: English
• Title: Characterization and epidemiological investigation of Listeria monocytogenes isolates using next-generation sequencing

Abstract

Listeriosis is a serious disease of humans and animals caused by Listeria monocytogenes. Whole-genome sequencing (WGS) is becoming the method of choice for the epidemiological surveillance of listeriosis, including outbreak investigations. Bacterial typing is not an integral part of routine investigations of animal listeriosis outbreaks; therefore, L. monocytogenes genotypes involved in these outbreaks remain poorly known. In the first part of the dissertation, WGS was used for detailed genetic characterization of presumably epidemiologically and microbiologically related isolates. Seven listeriosis outbreaks in small ruminants caused by the ST1 (CC1), ST18 (CC18), ST21 (CC21) and ST184 clones were retrospectively analyzed. Several WGS-based analytical approaches confirmed the monoclonal nature of the investigated outbreaks; microbiologically related isolates differed in up to 13 polymorphisms by whole-genome single nucleotide polymorphism (wgSNP) typing, seven alleles in core-genome multilocus sequence typing (cgMLST) and 12 alleles in whole-genome multilocus sequence typing (wgMLST). In the case of an outbreak caused by the ST184 strain, a microbiologically related strain was also found in the drinking water and silage, which were the most likely source(s) of infection of the diseased animals. WGS showed a greater dicriminatory power compared with pulsed-field gel electrophoresis (PFGE), which failed to distinguish between two outbreaks caused by the CC1 strains. We retrospectively investigated the human listeriosis outbreak caused by the ST8 (CC8) strain. We confirmed the monoclonal nature of the outbreak and rejected the hypothesis that the analyzed food or food-associated isolates caused the outbreak. Furthermore, WGS was used for a comprehensive genetic characterization and elucidation of L. monocytogenes transmission pathways in the farm environment. On a farm with a neurolisteriosis case, the hypervirulent CC4 clone was also found in cows with subclinical mastitis. The microbiological link between two CC5 isolates from the farm environment with different PFGE profiles was confirmed by WGS. The results of this study suggest that WGS should replace PFGE in epidemiological surveillance of L. monocytogenes. In the second part of the dissertation, we analyzed the population structure of L. monocytogenes isolates of animal clinical origin and from the natural environment, which are poorly understood. Multilocus sequence typing (MLST) was used to describe the population structure of isolates of animal clinical origin and from the natural environment. The CC1 clone was significantly associated with a clinical origin and with neurolisteriosis. We described a novel and significant association of CC6 and CC37 with abortion. CC9, CC14 and CC29 were significantly associated with the natural environment. The results presented herein represent an important contribution to the understanding of the genetic diversity of L. monocytogenes in the farm environment at regional and international levels.

• Keywords: Molecular epidemiology, listeriosis – microbiology, Listeria monocytogenes – isolation and purification – genetics, multilocus sequence typing – methods, whole genome sequencing – methods