Human Campylobacteriosis Cases Traceable to Chicken Meat—Evidence for Disseminated Outbreaks in Finland

Campylobacter jejuni (C. jejuni) is the most common cause of human bacterial gastroenteritis in the world. Food-borne campylobacteriosis is thought to be commonly caused by the handling and consumption of undercooked chicken meat, but the epidemiology of this disease is complex and remains poorly characterized, especially in the Nordic countries. Here, we used state-of-the-art methods in genetic epidemiology combined with patient background and temporal association data to trace domestically acquired human C. jejuni infections (n = 50) to chicken meat, in a midsize Nordic town in Finland during a seasonal peak. Although 59.2% of the human isolates shared a sequence type (ST) with a chicken batch slaughtered prior to the onset of disease, further analysis at the whole-genome level (core genome and whole-genome multilocus sequence typing, cgMLST and wgMLST, respectively) traced a mere nine cases (18.4%) to fresh chicken meat. Human isolates also shared genotypes with isolates collected from chicken batches slaughtered after the onset of the human disease, highlighting the role of alternative transmission pathways from chickens to humans besides the food chain, or a shared third source. The high resolution offered by wgMLST, combined with simple metadata, offers a more accurate way to trace sporadic cases to possible sources and reveal disseminated outbreak clustering in time, confirming the importance of complementing epidemiological investigations with molecular epidemiological data

Population genetics and molecular epidemiology of Campylobacter jejuni

During the last 40 years, Campylobacter has emerged as the number one cause of human gasteroenteritis in the developed world, of which C. jejuni accounts for the majority of cases. This Campylobacter species has an extremely broad host-range, and has been isolated from arctic penguins to cattle. Broiler chickens has traditionally been considered as the reservoir of importance for public health, but evidence suggests that other source, both known and unknown, are relevant. To decrease the number of human infections, targeted control efforts to reduce C. jejuni exposure to humans are needed. Such control efforts relies on knowledge on the nature of C. jejuni in both established and candidate sources such as broilers and wild birds, respectively, and reliable methods to trace and attribute human infections. Therefore, this thesis evaluated the usefulness of three metabolic markers in source attribution, resolved a Campylobacter outbreak using whole-genome sequence and characterized the dynamics and epidemiology of C. jejuni in Finnish chickens and barnacle geese. First, the possible host association of three metabolic markers, gamma-glutamyl transpeptidase, and the genes of secretory L-asparaginase and fucose permease, was investigated by examining their distribution among isolates collected from a variety of reservoirs and human patients, and by assessing their association with C. jejuni lineages as expressed by multilocus sequence typing. According to our results, the presence and absence of these three traits were linked to the lineages, and no evidence for host association independently of population structure was found. Therefore, these metabolic markers were deemed unsuitable as the sole subtyping scheme in source attribution. Secondly, in an attempt to identify possible new sources for human campylobacteriosis, the character, dynamics, and epidemiology of C. jejuni in barnacle geese and broiler chickens were investigated by multilocus sequence typing and whole-genome sequencing. In line with other studies, the C. jejuni population in barnacle geese was specific to that host, as certain genotypes (ST-702 and ST-1034 CC) were overrepresented in the collection. Furthermore, we proved that the C. jejuni in this wild bird species generally differed from the C. jejuni population found in agricultural animals and C. jejuni infecting humans. Therefore, barnacle geese are most probably not a major source for human campylobacteriosis. Tracing zoonotic pathogens from humans to the source of infection using whole-genome sequencing is a hot topic in the research community. To provide knowledge on how to utilize whole-genome data to profit future real-time investigations of Campylobacter outbreaks, next generation sequencing was used to reinvestigate a waterborne C. jejuni outbreak. Our results revealed that the pulsed-field gel electrophoresis performed in the original outbreak investigation overestimated the clonal relationship between some of the apparently related strains. Through the reanalysis, it became clear that the outbreak was caused by at least two different strains, or an unrelated, sporadic human case was mistakenly classified as part of the outbreak. Whole-genome sequence was therefore needed to infer the correct epidemiology of the studied human infections. To increase the knowledge on C. jejuni in a well-established reservoir, we characterized nearly 90% of all C. jejuni-positive chicken flocks by multilocus sequence typing and linked this information to the isolates metadata, such as farm and collection time-points. We found that the C. jejuni population on Finnish chicken farms was dominated by one genotype (ST-45 CC). Furthermore, our statistical analysis showed that slaughterhouse and year of collection affected the C. jejuni population in chickens mildly, while season influenced the presence of only one genotype (ST-45). Furthermore, the chicken farms were sporadically and infrequently colonized by C. jejuni, as is typical when no persistent colonization source is present on the farms. This thesis utilized an array of subtyping methods on both long- and short-time scales. The results highlight that more traditional methods, such as pulsed-field gel electrophoresis, still have their use in the current genomic era, and stress the usefulness of multilocus sequence typing as a preliminary screening tool to determine the population structure in the C. jejuni collection being investigated. However, we are convinced that whole-genome sequencing will be the subtyping scheme of choice in the near future, as it provides the full resistome, toxiome and virulome concurrently with the genotyping depth of need in a single operation.  Campylobacter har varit den främsta orsaken till human gastroenterit i den västliga värden under dom senaste 40 åren, och C. jejuni står for brorparten av dessa fallen. Denna Campylobacter-arten har ett extremt brett spektrum av värdorganismer, och finns i allt ifrån arktiska pingviner till nötkreatur. Broilerkyckling har traditionellt ansetts vara det reservoar av störst betydning för folkhälsan, men mycket tyder på at andra, alternativa källor, båda kända och okända, är relevanta. För att minska antalet infektioner hos människor, måste kontrollinsatser inriktade på att minska den mänskliga exponeringen för C. jejuni implementeras. Sådana kontrollinsatser bygger på kunskap om C. jejuni i olika källor såsom slaktkycklingar och vilda fåglar, samt tillförlitliga metoder för att spåra och attribuera människors Campylobacter infektioner. Detta doktorand-arbete utvärderar därför nyttan av tre metaboliska markörer till att attribuera campylobacterinfektioner och helgenomsekvensering i utbrotts uppklarning av Campylobacter utbrott. Vidare utforskar arbetet dynamiken och epidemiologin av C.jejuni i finländska kycklingar och vitkindade gäss. En möjlig association mellan värdorganism och förekomsten av tre metaboliska markörer i C. jejuni undersöktes ved att studera deras förekomst i bakteriestammar från olika reservoarer och patienter, samt att bedöma markörens association med multilocus sekvens typer. Enligt våra resultat är närvaron och frånvaro av dessa tre egenskaper kopplade till bakterielinjer, och inga bevis för en association med värdorganismen oberoende av populationsstrukturen hittades. Dessa metaboliska markörer anses därför vara olämpliga som enda använda subtyp-metod i Campylobacter attributionstudier. I ett försök att identifiera nya källor för människors campylobacterinfektion, ble karaktär, dynamik och epidemiologi av C. jejuni i vitkindade gäss och slaktkycklingar undersökt med multilocus sekvens typning och helgenomsekvensering. C. jejuni populationen i vitkindade gäss var värd-specifik och vissa genotyper (ST-702 och ST-1034 CC) var överrepresenterade i samlingen. Vi bevisade vidare att C.jejuni i denna vilda fågelarten skilde sig från C. jejuni i jordbruksdjur och patienter, och vitkindade gäss är derför sannolikt inte en viktig källa för mänsklig campylobacterinfektion. Spårning av zoonotiska patogen från människor till smittkällan med helagenomsekvensering är ett hett ämne i forskarvärlden. För att bidra med kunskap om hur man använder helagenomsekvensering till utbrotts uppklarning, omvärderades ett vattenburen C. jejuni utbrott från 2000 ved komparativ genomik och nästa generations sekvensering. Genom helgenomsekvensering stod det klart att utbrottet orsakades av en minst två olika C. jejuni bakterier, eller att et obesläktad, sporadiskt fall var klassificerad som en del av utbrottet. Helagenomsekvensen behövdes för att sluta sig till den rätta epidemiologin. För att öka kunskapen om C. jejuni i en väletablerad reservoar, typade vi nästan 90 % av alla C. jejuni-positiva kycklingflockar och kopplade detta till information om isolatens ursprung och insamlings tidspunkt. Vi fann att C.jejuni i finska kycklingar domineras av en genotyp (ST-45 CC). Dessutom visade vår statistiska analys att slakteri och insamlings-år svakt bestämde vilka typ av C. jejuni som hittas i kycklingar, medan säsong bara påverkar närvaron av endast en genotyp (ST-45). Vidarea var kyckling gårdarna sällan och tillfäldig koloniserad med C.jejuni, vilket är typiskt när ingen beständig källa föreligger på gårdarna. Denna avhandling utnyttjade en hel rad subtypmetoder, båda över lång och kort tid. Traditionella metoder, såsom pulsad-fält-gelelektrofores, är fortfarande aktuella i denna genomiska era, och multilocus sekvens typning är nyttig som et preliminärt screening verktyg för C. jejuni. Vi är dock övertygade om att helagenomsekvensering kommer att vara subtypning för val inom en snar framtid, eftersom denna metod ger hela resistom, toxiom och virulom och subtyping i en enda operation

Predominant Campylobacter jejuni Sequence Types Persist in Finnish Chicken Production

Peer reviewe

HUNT-One Health Enabling human-animal health studies

publishedVersio

Investigating Major Recurring Campylobacter jejuni Lineages in Luxembourg Using Four Core or Whole Genome Sequencing Typing Schemes

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, which has motivated the monitoring of genetic profiles circulating in Luxembourg since 13 years. From our integrated surveillance using a genotyping strategy based on an extended MLST scheme including gyrA and porA markers, an unexpected endemic pattern was discovered in the temporal distribution of genotypes. We aimed to test the hypothesis of stable lineages occurrence by implementing whole genome sequencing (WGS) associated with comprehensive and internationally validated schemes. This pilot study assessed four WGS-based typing schemes to classify a panel of 108 strains previously identified as recurrent or sporadic profiles using this in-house typing system. The strain collection included four common lineages in human infection (N = 67) initially identified from recurrent combination of ST-gyrA-porA alleles also detected in non-human samples: veterinary (N = 19), food (N = 20), and environmental (N = 2) sources. An additional set of 19 strains belonging to sporadic profiles completed the tested panel. All the strains were processed by WGS by using Illumina technologies and by applying stringent criteria for filtering sequencing data; we ensure robustness in our genomic comparison. Four typing schemes were applied to classify the strains: (i) the cgMLST SeqSphere+ scheme of 637 loci, (ii) the cgMLST Oxford scheme of 1,343 loci, (iii) the cgMLST INNUENDO scheme of 678 loci, and (iv) the wgMLST INNUENDO scheme of 2,795 loci. A high concordance between the typing schemes was determined by comparing the calculated adjusted Wallace coefficients. After quality control and analyses with these four typing schemes, 60 strains were confirmed as members of the four recurrent lineages regardless of the method used (N = 32, 12, 7, and 9, respectively). Our results indicate that, regardless of the typing scheme used, epidemic or endemic signals were detected as reflected by lineage B (ST2254-gyrA9-porA1) in 2014 or lineage A (ST19-gyrA8-porA7), respectively. These findings support the clonal expansion of stable genomes in Campylobacter population exhibiting a multi-host profile and accounting for the majority of clinical strains isolated over a decade. Such recurring genotypes suggest persistence in reservoirs, sources or environment, emphasising the need to investigate their survival strategy in greater depth

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak

Peer reviewe

INNUENDO: A cross-sectoral platform for the integration of genomics in the surveillance of food-borne pathogens

Abstract In response to the EFSA call New approaches in identifying and characterizing microbial and chemical hazards, the project INNUENDO (https://sites.google.com/site/theinnuendoproject/) aimed to design an analytical platform and standard procedures for the use of whole-genome sequencing in surveillance and outbreak investigation of food-borne pathogens. The project firstly attempted to identify existing flaws and needs, and then to provide applicable cross-sectorial solutions. The project focused in developing a platform for small countries with limited economical and personnel resources. To achieve these goals, we applied a user-centered design strategy involving the end-users, such as microbiologists in public health and veterinary authorities, in every step of the design, development and implementation phases. As a result, we delivered the INNUENDO Platform V1.0 (https://innuendo.readthedocs.io/en/latest/), a stand-alone, portable, open-source, end-to-end system for the management, analysis, and sharing of bacterial genomic data. The platform uses Nextflow workflow manager to assemble analytical software modules in species-specific protocols that can be run using a user-friendly interface. The reproducibility of the process is ensured by using Docker containers and throught the annotation of the whole process using an ontology. Several modules, available at https://github.com/TheInnuendoProject, have been developed including: genome assembly and species confirmation; fast genome clustering; in silico typing; standardized species-specific phylogenetic frameworks for Campylobacter jejuni, Yersinia enterocolitica, Salmonella enterica and Escherichia coli based on an innovative gene-by-gene methodology; quality control measures from raw reads to allele calling; reporting system; a built-in communication protocols and a strain classification system enabling smooth communication during outbreak investigation. As proof-of-concepts, the proposed solutions have been thoroughly tested in simulated outbreak conditions by several public health and veterinary agencies across Europe. The results have been widely disseminated through several channels (web-sites, scientific publications, organization of workshops). The INNUENDO Platform V1.0 is effectively one of the models for the usage of open-source software in genomic epidemiology.Peer reviewe

Replication Region Analysis Reveals Non-lambdoid Shiga Toxin Converting Bacteriophages

Shiga toxin is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the gene encoding it is carried within the genome of Shiga toxin-converting phages (Stx phages). Numerous Stx phages have been sequenced to gain a better understanding of their contribution to the virulence potential of EHEC. The Stx phages are classified into the lambdoid phage family based on similarities in lifestyle, gene arrangement, and nucleotide sequence to the lambda phages. This study explores the replication regions of non-lambdoid Stx phages that completely lack the O and P genes encoding the proteins involved in initiating replication in the lambdoid phage genome. Instead, they carry sequences encoding replication proteins that have not been described earlier, here referred to as eru genes (after EHEC phage replication unit genes). This study identified three different types of Eru-phages, where the Eru1-type is carried by the highly pathogenic EHEC strains that caused the Norwegian O103:H25 outbreak in 2006 and the O104:H4 strain that caused the large outbreak in Europe in 2011. We show that Eru1-phages exhibit a less stable lysogenic state than the classical lambdoid Stx phages. As production of phage particles is accompanied by production of Stx toxin, the Eru1-phage could be associated with a high-virulence phenotype of the host EHEC strain. This finding emphasizes the importance of classifying Stx phages according to their replication regions in addition to their Stx-type and could be used to develop a novel strategy to identify highly virulent EHEC strains for improved risk assessment and management.publishedVersio

Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species

The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant “Staggered-type” (S-Ena) and the shorter “Ladder-like” type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation

Replication Region Analysis Reveals Non-lambdoid Shiga Toxin Converting Bacteriophages

Shiga toxin is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the gene encoding it is carried within the genome of Shiga toxin-converting phages (Stx phages). Numerous Stx phages have been sequenced to gain a better understanding of their contribution to the virulence potential of EHEC. The Stx phages are classified into the lambdoid phage family based on similarities in lifestyle, gene arrangement, and nucleotide sequence to the lambda phages. This study explores the replication regions of non-lambdoid Stx phages that completely lack the O and P genes encoding the proteins involved in initiating replication in the lambdoid phage genome. Instead, they carry sequences encoding replication proteins that have not been described earlier, here referred to as eru genes (after EHEC phage replication unit genes). This study identified three different types of Eru-phages, where the Eru1-type is carried by the highly pathogenic EHEC strains that caused the Norwegian O103:H25 outbreak in 2006 and the O104:H4 strain that caused the large outbreak in Europe in 2011. We show that Eru1-phages exhibit a less stable lysogenic state than the classical lambdoid Stx phages. As production of phage particles is accompanied by production of Stx toxin, the Eru1-phage could be associated with a high-virulence phenotype of the host EHEC strain. This finding emphasizes the importance of classifying Stx phages according to their replication regions in addition to their Stx-type and could be used to develop a novel strategy to identify highly virulent EHEC strains for improved risk assessment and management