Characterization and epidemiological investigation of Listeria monocytogenes isolates using next-generation sequencing

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 izbruhovmikrobiološko 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.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 outbreakstherefore, 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 outbreaksmicrobiologically 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

Genomic insights into the emergence and spread of methicillin-resistant Staphylococcus pseudintermedius in veterinary clinics

Staphylococcus pseudintermedius is a common cause of skin and soft tissue infections in dogs but can also cause infections in cats and humans. The frequency of methicillin-resistant S. pseudintermedius (MRSP) strains is increasing worldwide. Here, we obtained 43 MRSP isolates from dogs (n = 41), one cat (n = 1) and the small animal clinic environment (n = 1) in Slovenia from the period 2008–2018, which underwent whole-genome sequencing (WGS) and antimicrobial susceptibility testing. Five sequence types (STs) were identified, with ST71 (32/43) and ST551 (8/43) being the predominant. In Slovenia, ST551 was first detected in 2016, whereas a decrease in the frequency of ST71 was observed after 2015. All isolates were multidrug-resistant and most antimicrobial-resistant phenotypes could be linked to acquisition of the corresponding resistance genes or gene mutations. Core-genome multilocus sequence typing (cgMLST) revealed several potential MRSP transmission routes: (i) between two veterinary clinics by a single MRSP-positive dog, (ii) between the environment of a veterinary clinic and a dog, and (iii) between a canine and a feline patient through the contaminated environment of a veterinary clinic. Of the six dogs that were additionally sampled from 14 days to five months after the initial sampling, each harbored the same MRSP strain, suggesting a limited within-host diversity of MRSP in symptomatic dogs. The present results highlight the importance of MRSP-positive dogs in the spread of veterinary care-associated MRSP infections and call for the implementation of strict control measures to reduce MRSP contamination in veterinary clinic environments originating from animal-contact surfaces

Source tracking on a dairy farm reveals a high occurrence of subclinical mastitis due to hypervirulent Listeria monocytogenes clonal complexes

An extensive source investigation was conducted on a dairy farm with neurolisteriosis and subclinical mastitis cases to identify infection source and potential transmission routes of Listeria monocytogenes

Retrospective investigation of listeriosis outbreaks in small ruminants using different analytical approaches for whole genome sequencing-based typing of Listeria monocytogenes

Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both humans and animals. While listeriosis outbreaks in humans are commonly investigated in detail, routine typing of L. monocytogenes is generally not performed in animal outbreaks. Here, seven presumable listeriosis outbreaks in small ruminants were retrospectively identified based on the pulsed-field gel electrophoresis (PFGE) profiles. Outbreaks were further characterised using three different analytical approaches based on the whole-genome sequencing (WGS) data: core-genome multilocus sequence typing (cgMLST), whole-genome MLST (wgMLST) and whole-genome single nucleotide polymorphism (wgSNP) typing. A monoclonal pattern of all seven outbreaks was identified using all three approaches, indicating common-source outbreaks. The outbreak strains belonged to sequence types (STs) 1 (n = 3), ST18 (n = 1), ST21 (n = 2) and ST184 (n = 1). Two epidemiologically linked ST1 outbreaks with indistinguishable PFGE profiles showed a polyphyletic nature and differed in >78 SNPsthus, they were classified as separate outbreaks according to WGS. In ST184, the outbreak strain was also found in faeces of apparently healthy ruminants, silage and water collected from the trough, which were the most likely source(s) of infection. The outbreak-associated isolates differed in 0–7 cgMLST alleles, 0–12 wgMLST alleles and 1–13 SNPs. The minimum genetic diversity between outbreak-associated isolates and epidemiologically unrelated isolates of the same ST was low in all analysed cases, approaching the maximum diversity within the outbreak cluster. The results suggest that a fixed threshold to define the outbreak cluster should only be considered as a guide and highlight the role of epidemiological data for outbreak confirmation. The identified cgMLST clusters may be further investigated by wgMLST and/or wgSNP typing to increase confidence during investigations of outbreaks caused by highly clonal L. monocytogenes groups. This study gives an overview of the inter- and intra-outbreak genetic diversity of L. monocytogenes strains involved in animal outbreaks, hence improving their investigation

Analysis of the global population structure of Paenibacillus larvae and outbreak investigation of American foulbrood using a stable wgMLST scheme

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae. To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealedhowever, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen

Helicobacter colisuis sp. nov., isolated from caecal contents of domestic pigs (Sus scrofa domesticus)

Seven Helicobacter-like isolates were cultured from caecal contents of 100 domestic pigs (Sus scrofa domesticus) sampled as part of the EFSA-coordinated harmonized monitoring of antimicrobial resistance in Campylobacter sp. in 2015. The bacteria were isolated using the standard ISO 10272 procedure for the isolation of thermotolerant Campylobacter with extended incubation time and formed small, grey, moist and flat colonies with a metallic sheen (small Campylobacter-like colonies) on modified Charcoal-Cefoperazone-Deoxycholate Agar (mCCDA) and Skirow agar plates. Morphologically, the bacterial cells were spirilli-shaped and highly motile, 1–2 µm long and ≤0.5 µm wide, Gram-negative, oxidase-positive and catalase-positive. They could not be identified using the standard-prescribed biochemical tests and had uniform, unique and reproducible MALDI-TOF mass spectra that most closely matched those of Helicobacter pullorum. Three strains (11154-15T, 14348–15 and 16470–15) underwent whole-genome sequencing. Analysis of 16S rRNA gene sequences revealed a high similarity (≥99.8 % identity) to Helicobacter canadensis. Pairwise average nucleotide identity (ANI) values revealed that the three studied strains were closely related (ANI ≥98.9 %), but distinct from the previously described Helicobacter species (ANI ≤90.6 %). The core genome-based phylogeny confirmed that the new strains form a distinct clade most closely related to H. canadensis. The conducted polyphasic taxonomic analysis confirmed that the three strains represent a novel Helicobacter species for which the name Helicobacter colisuis sp. nov. is suggested, with strain 11154-15T (= DSM 113688T = CCUG 76053T) as the type strain

Campylobacter magnus sp. nov., isolated from caecal contents of domestic pigs (Sus scrofa domesticus)

During the 2021 European Food Safety Authority coordinated harmonized monitoring of antimicrobial resistance in Campylobacter species in Slovenia, five Campylobacter-like strains were cultured from caeca of a total of 104 domestic pigs that could not be identified using the standard-prescribed biochemical tests or MALDI-TOF MS. The isolates were obtained using the standard ISO 10272 procedure for the isolation of thermotolerant Campylobacter with prolonged cultivation time. Small Campylobacter like colonies were observed on mCCDA and CASA agar plates after 2–4days of incubationdark-field microscopy revealed relatively big spirilli-shaped bacteria exhibiting characteristic Campylobacter-like motility. The cells were 1.5–3µm long and 0.5–0.7µm wide, Gram-negative, oxidase-positive and catalase-positive. MALDI-TOF mass spectra were distinctive and consistent, but with low MALDI-TOF MS log scores and the closest matches being those of Campylobacter hyointestinalis and Campylobacter fetus. All five strains underwent whole-genome sequencing. Analysis of 16S rRNA gene sequences revealed that the isolates were most similar (98.3–98.4% identity) to Campylobacter lanienae. Pairwise average nucleotide identity (ANI) values revealed that the five studied strains shared pairwise ANI of 96.2–96.5% but were clearly distinct from the previously described Campylobacter species (ANI ≤72.8%). The core genome-based phylogeny confirmed that the new strains form a distinct and well-supported clade within the genus Campylobacter. The conducted polyphasic taxonomic analysis confirmed that the five strains represent a novel Campylobacter species for which the name Campylobacter magnus sp. nov. is suggested, with strain 46386T (=DSM 115534T =CCUG 76865T ) as the type strain

ERIC and WGS typing of Paenibacillus larvae in Slovenia

Simple Summary American foulbrood is a serious disease of honeybees caused by Paenibacillus larvae. ERIC-PCR is a widely used method for typing of P. larvae that currently divides it into five ERIC types (ERIC I–V)these differ in certain phenotypic characteristics—most importantly, virulence. In the first part of the study, we assessed the distribution of ERIC types in Slovenia in the period 2017–2019 on a set of 506 P. larvae isolates. We identified ERIC II as the predominant type (70.2%), followed by ERIC I (29.8%). In the second part of the study, we typed 59 outbreak-related ERIC I isolates using whole-genome sequencing, which revealed seven ERIC I-ST2 outbreak clusters (≤35 allele differences). The transmission of the outbreak clone within a 3-km radius was observed in all seven clusters and could be explained by the activity of honeybees. The transmission of the outbreak clone between geographically distant apiaries was observed in three clusters and could be explained by migratory beekeeping and trading of bee colonies. The present findings highlight the importance of beekeeping activities in the transmission of P. larvae over large geographic distances. Abstract Paenibacillus larvae is the causative agent of American foulbrood (AFB), a fatal disease of honeybee brood. Here, we obtained 506 P. larvae isolates originating from honey or brood samples and from different geographic regions of Slovenia in the period 2017–2019. In the first part of the study, we conducted ERIC-PCR typing to assess the frequency of ERIC types in Slovenia. Capillary electrophoresis was used for the analysis of ERIC patterns, revealing good separation efficiency and enabling easy lane-to-lane comparisons. ERIC II was the predominant type (70.2%), followed by ERIC I (29.8%)two slightly altered ERIC I banding patterns were observed but were not considered relevant for the discrimination of ERIC types. No evident spatiotemporal clustering of ERIC types was observed. To assess the clonality of the outbreak-related P. larvae ERIC I isolates, 59 isolates of this type underwent whole-genome sequencing (WGS). Whole-genome multilocus sequence typing (wgMLST) revealed seven ERIC I-ST2 outbreak clusters (≤35 allele differences) with the median intra-outbreak diversity ranging from 7 to 27 allele differences. In all seven clusters, the transmission of P. larvae outbreak clone within a 3-km radius (AFB zone) was observed, which could be explained by the activity of honeybees. In three clusters, the transmission of the outbreak clone between geographically distant apiaries was revealed, which could be explained by the activities of beekeepers such as migratory beekeeping and trading of bee colonies. The present findings reinforce the importance of beekeeping activities in the transmission of P. larvae. WGS should be used as a reference typing method for the detection of P. larvae transmission cluster

Phylogeography of Brucella suis biovar 2 with focus on Slovenian wildlife

Brucella suis commonly infects swine but occasionally also other animal species and humans. Wild boars are the most important reservoir of B. suis biovar 2, continually infecting susceptible hosts through close contact. Nevertheless, the genetic diversity of B. suis in wildlife remains understudied. Here, we typed 17 Slovenian B. suis biovar 2 isolates obtained in 2017–2019 from wild boars (n = 16) and a hare (n = 1) using whole-genome sequencing (WGS). To assess the global phylogenetic diversity of B. suis, we compared them to 126 publicly available B. suis genomes. All Slovenian isolates fell within the biovar 2 lineage, confirming the previous multiplex PCR typing results. According to MLST-21, the wild boar isolates were of sequence types (STs) ST16 (n = 8) and ST153 (n = 8)the maximum genetic distance between isolates of the same ST was 28 wgMLST alleles. The ST153 isolates were restricted to the Slovenian-Croatian border and clustered together with the Croatian ST153 isolates from swine, indicating cross-border transmission of B. suis ST153 strain. The hare isolate was of ST40 and was genetically distant (≥ 489 alleles) from the wild boar isolates. The genome-wide phylogeny clearly separated different B. suis biovars. The present study is the first report on the population structure of B. suis in wildlife in Slovenia and shows that the Slovenian B. suis population is genetically heterogeneous. At the species level, B. suis biovars are clearly separated in the WGS-based phylogenetic tree and can therefore be reliably predicted using WGS