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The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

We present a case of carbapenemase-producing blaNDM-1-positive Salmonella Kottbus in an 82-year-old Danish man. The blaNDM-1 was also identified in Escherichia coli and Citrobacter freundii in the same patient on the same 43 kb IncN2 plasmid, suggesting in vivo inter-species plasmid transfer. A NCBI BLAST analysis of the plasmid (pAMA003584_NDM-1) identified 12 highly similar plasmids, all originating from east and south-east Asia. This case could be the first confirmed case of blaNDM-1-positive Salmonella not related to travel outside Europe.

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

Salmonella enterica subspecies enterica serovar Typhimurium and its monophasic variant are among the most common Salmonella serovars associated with human salmonellosis each year. Related infections are often due to consumption of contaminated meat of pig, cattle, and poultry origin. In order to evaluate novel microbial subtyping methods for source attribution, an approach based on weighted networks was applied on 141 human and 210 food and animal isolates of pigs, broilers, layers, ducks, and cattle collected in Denmark from 2013 to 2014. A whole-genome SNP calling was performed along with cgMLST and wgMLST. Based on these genomic input data, pairwise distance matrices were built and used as input for construction of a weighted network where nodes represent genomes and links to distances. Analyzing food and animal Typhimurium genomes, the coherence of source clustering ranged from 89 to 90% for animal source, from 84 to 85% for country, and from 63 to 65% for year of isolation and was equal to 82% for serotype, suggesting animal source as the first driver of clustering formation. Adding human isolate genomes to the network, a percentage between 93.6 and 97.2% clustered with the existing component and only a percentage between 2.8 and 6.4% appeared as not attributed to any animal sources. The majority of human genomes were attributed to pigs with probabilities ranging from 83.9 to 84.5%, followed by broilers, ducks, cattle, and layers in descending order. In conclusion, a weighted network approach based on pairwise SNPs, cgMLST, and wgMLST matrices showed promising results for source attribution studies.

Zoonotic Salmonella causes millions of human salmonellosis infections worldwide each year. Information about the source of the bacteria guides risk managers on control and preventive strategies. Source attribution is the effort to quantify the number of sporadic human cases of a specific illness to specific sources and animal reservoirs. Source attribution methods for Salmonella have so far been based on traditional wet-lab typing methods. With the change to whole genome sequencing there is a need to develop new methods for source attribution based on sequencing data. Four European datasets collected in Denmark (DK), Germany (DE), the United Kingdom (UK) and France (FR) are presented in this descriptor. The datasets contain sequenced samples of Salmonella Typhimurium and its monophasic variants isolated from human, food, animal and the environment. The objective of the datasets was either to attribute the human salmonellosis cases to animal reservoirs or to investigate contamination of the environment by attributing the environmental isolates to different animal reservoirs., Measurement(s)sequence_assembly • DNATechnology Type(s)sequence assembly process • DNA sequencing • Paired-End SequencingFactor Type(s)geographic location • Salmonella Typhimurium isolate sourceSample Characteristic - OrganismSalmonellaSample Characteristic - LocationKingdom of Denmark • Germany • United Kingdom • French Republic Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11816748

Prevention of the emergence and spread of foodborne diseases is an important prerequisite for the improvement of public health. Source attribution models link sporadic human cases of a specific illness to food sources and animal reservoirs. With the next generation sequencing technology, it is possible to develop novel source attribution models. We investigated the potential of machine learning to predict the animal reservoir from which a bacterial strain isolated from a human salmonellosis case originated based on whole‐genome sequencing. Machine learning methods recognize patterns in large and complex data sets and use this knowledge to build models. The model learns patterns associated with genetic variations in bacteria isolated from the different animal reservoirs. We selected different machine learning algorithms to predict sources of human salmonellosis cases and trained the model with Danish Salmonella Typhimurium isolates sampled from broilers (n = 34), cattle (n = 2), ducks (n = 11), layers (n = 4), and pigs (n = 159). Using cgMLST as input features, the model yielded an average accuracy of 0.783 (95% CI: 0.77–0.80) in the source prediction for the random forest and 0.933 (95% CI: 0.92–0.94) for the logit boost algorithm. Logit boost algorithm was most accurate (valid accuracy: 92%, CI: 0.8706–0.9579) and predicted the origin of 81% of the domestic sporadic human salmonellosis cases. The most important source was Danish produced pigs (53%) followed by imported pigs (16%), imported broilers (6%), imported ducks (2%), Danish produced layers (2%), Danish produced cattle and imported cattle (

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

Aquaculture systems are widely recognised as hotspots for horizontal gene transfer, and the need for screening for bacteria carrying antimicrobial resistance genes in aquaculture systems is becoming more important. In this study, we characterised seventeen bacterial strains (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and A. nosocomialis) resistant to colistin originating from retailed aquaculture products imported from Vietnam to the Czech Republic. The mcr-1.1 gene was found located on plasmid types IncHI2, IncI2, and IncX4, as well as on the rarely described plasmid types IncFIB-FIC and IncFIB(K), phage-like plasmid p0111, and on the chromosome of E. coli. One E. coli strain carried the mcr-3.5 gene on IncFII(pCoo) plasmid in addition to the mcr-1.1 gene located on IncHI2 plasmid. K. pneumoniae was found to carry the mcr-1.1 and mcr-8.2 genes on IncFIA(HI1) plasmid. The mcr-4.3 gene was found on similar untypeable plasmids of A. baumannii and A. nosocomialis strains, pointing to the possible interspecies transfer of plasmids carrying the mcr-4 gene. Our results highlight that some aquaculture products of Asian origin can represent an important source of variable plasmids carrying mcr genes. The results showed an involvement of phages in the incorporation of the mcr-1 gene into plasmids or the chromosome in E. coli strains from aquaculture. The detection of E. coli with the mcr-1 gene in the chromosome points to the risks associated with the stabilisation of the mcr genes in the bacterial chromosome.

We report the results of three consecutive External Quality Assessments (EQAs) for molecular subtyping of Salmonella to assess the performance of the European national public health reference laboratories (NPHRLs). The EQA included the molecular typing methods used for European enhanced surveillance of human Salmonella infections: pulsed field gel electrophoresis (PFGE), including gel analysis by the use of the software BioNumerics, and 5-locus multiple locus variable number of tandem repeat analysis (MLVA) for serovar Typhimurium. The participation in the PFGE laboratory part was higher (27/35) than in the gel analysis (19/35) and MLVA (15/35), suggestive of the need for capacity building in methods requiring specialized equipment (MLVA) or software (gel analysis). The majority (25/27) of the participating NPHRLs produced inter-laboratory comparable PFGE gel(s). Two laboratories continued to produce low-quality gels and should have additional technical assistance in the future. In particular, two gel quality evaluation parameters, measuring “image acquisition and running conditions” and “bands”, were identified to cause gel quality problems throughout the EQAs. Despite the high number of laboratories participating in the PFGE laboratory part, the participation in gel analysis was low, although increasing. In the MLVA part, the NPHRLs correctly assigned 96% (405/420) allelic profiles according to the nomenclature. In conclusion, the EQAs identified critical parameters for unsuccessful performance and helped to offer assistance to those laboratories that needed it most. The assessments supported the development of quality in molecular typing and promoted the harmonization of subtyping methods used for EU/EEA-wide surveillance of human Salmonella infections.

In April 2019, a cross-border outbreak of Yersinia entercolitica O3 was identified in Sweden and Denmark and confirmed using whole genome sequencing. Close cross-border collaboration with representatives from human and food authorities helped direct resources and investigations. Combined epidemiological and trace-back investigations pointed to imported fresh spinach as the outbreak vehicle and highlight that other vehicles of Y. enterocolitica outbreaks than pork should be considered.

In 2017, an outbreak of gastroenteritis in England attributed to Salmonella Adjame was detected and investigated. With the introduction of whole genome sequencing (WGS) for microbial typing, methods for comparing international outbreak data require evaluation. A case was defined as a person resident in England with a clinical sample from 1 June 2017 to 27 July 2017 from whom S. Adjame was isolated. Cases were interviewed and exposures analysed. Backward tracing of food provenance was undertaken. WGS was performed on isolates from cases and historical isolates and compared using Public Health England’s SnapperDB high-quality SNP pipeline and Enterobase’s Salmonella core genome multi-locus sequence typing (cgMLST) scheme. In total, 14 cases were identified. The majority were vegetarian, probably of South Asian descent, with a median age of 66.5 years with no recent international travel reported. Cases consumed a range of fresh food products including herbs and spices bought from South Asian grocers. Backward tracing did not identify a common source. WGS typing showed sub-clustering and considerable genetic variation across human samples. cgMLST allele-based analysis was comparable to SNP-derived phylogenetic analysis and clusters were defined using each method. Imported herbs or spices were suspected vehicles. The cases were linked in time and place but WGS showed marked heterogeneity, atypical of a point source Salmonella outbreak. The application of incorporating SNP or allelic differences into the case definition may not always be appropriate. With further validation, cgMLST could be used for international outbreak alerts when WGS analysis is being undertaken to facilitate comparison.

BackgroundSalmonellaInfantis (S. Infantis) is one of the most frequentSalmonellaserovars isolated from human cases of salmonellosis and the most detected serovar from animal and food sources in Europe. The serovar is commonly associated with poultry and there is increasing concern over multidrug resistant clones spreading worldwide, as the dominating clones are characterized by presence of large plasmids carrying multiple resistance genes. Increasing the knowledge of theS. Infantis population and evolution is important for understanding and preventing further spread.In this study, we analysed a collection of strains representing different decades, sources and geographic locations. We analysed the population structure and the accessory genome, in particular we identified prophages with a view to understand the role of prophages in relation to the evolution of this serovar.ResultsWe sequenced a global collection of 100 S. Infantis strains. A core-genome SNP analysis separated five strains in e-Burst Group (eBG) 297 with a long branch. The remaining strains, all in eBG31, were divided into three lineages that were estimated to have separated approximately 150 years ago. One lineage contained the vast majority of strains. In five of six clusters, no obvious correlation with source or geographical locations was seen. However, one cluster contained mostly strains from human and avian sources, indicating a clone with preference for these sources. The majority of strains within this cluster harboured a pESI-like plasmid with multiple resistance genes. Another lineage contained three genetic clusters with more rarely isolated strains of mainly animal origin, possibly less sampled or less infectious clones.Conserved prophages were identified in all strains, likely representing bacteriophages which integrated into the chromosome of a common ancestor toS. Infantis. We also saw that some prophages were specific to clusters and were probably introduced when the clusters were formed.ConclusionsThis study analysed a globalS. Infantis population and described its genetic structure. We hypothesize that the population has evolved in three separate lineages, with one more successfully emerging lineage. We furthermore detected conserved prophages present in the entire population and cluster specific prophages, which probably shaped the population structure.

This report describes one Salmonella isolate harbouring both mcr-1 and mcr-3. We also found nine other Salmonella isolates positive for the plasmid-borne colistin resistance gene, mcr-3. The strains were isolated from patients in Denmark between 2009 and 2017 and five of the patients had travelled to Asia. In addition to mcr-3, all strains were found positive for blaTEM-1 , strA, strB, sul2 and tet(A) or tet(B), and most strains were positive for blaCTX-M-55 and qnrS.

Whole-genome sequencing is rapidly replacing current molecular typing methods for surveillance purposes. Our study evaluates core-genome single-nucleotide polymorphism analysis for outbreak detection and linking of sources of Salmonella enterica serovar Typhimurium and its monophasic variants during a 7-month surveillance period in Denmark. We reanalyzed and defined 8 previously characterized outbreaks from the phylogenetic relatedness of the isolates, epidemiologic data, and food traceback investigations. All outbreaks were identified, and we were able to exclude unrelated and include additional related human cases. We were furthermore able to link possible food and veterinary sources to the outbreaks. Isolates clustered according to sequence types (STs) 19, 34, and 36. Our study shows that core-genome single-nucleotide polymorphism analysis is suitable for surveillance and outbreak investigation for Salmonella Typhimurium (ST19 and ST36), but whole genome–wide analysis may be required for the tight genetic clone of monophasic variants (ST34).

Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein-coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST-828 and ST-1150 clonal complexes. The ST-1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST-828 complex bacteria have 10-11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have

Aims: To characterize isolates of Salmonella Typhimurium DT41 obtained from infected flocks of broiler breeders by multiple-locus variable-number tandem-repeats analysis (MLVA) and compare results with a diverse strain collection from Germany and United Kingdom and isolates from Danish patients. Methods and Results: A total of 102 isolates of Salm. Typhimurium phage type DT41 were MLVA typed. MLVA typing showed 4, 12, 25, 9 and 8 different alleles at the five MLVA loci 9, 5, 6, 10 and 3, respectively. A dendrogram based on MLVA types was constructed, and one large group, nine minor groups and 29 more unrelated MLVA types were obtained. The major group included 20 of the 30 human isolates. Isolates obtained from broiler breeders demonstrated major diversity, indicating the existence of several independent introductions of DT41 at farm level. When comparison was made to isolates included from Germany and England, DT41 seems to be ubiquitous in the wild fauna which might represent a risk factor for poultry. Conclusions: Transmission from Danish broilers to humans was not demonstrated, neither was the transmission from rearing farms to broiler breeder farms. Sources of infection at broiler breeder farm level remained unidentified. Significance and Impact of the Study: Major diversity was demonstrated for DT41 MLVA types. A persisting problem with infection of broiler breeder flocks with DT41 was not reflected in broiler flocks originating from these flocks.

Salmonella enterica subsp. enterica is one of the leading causes of zoonotic food-borne disease worldwide. The consequence of these infections is a serious impact on economics of the society in the form of lost productivity and expenses for medical care. The objective of this study was to analyze the difference in genomic content between selected serovars, especially the content of pathogenicity genes and this was done with a DNA microarray. Furthermore, we investigated the phylogenetic relationship between serovars using multilocus sequence typing (MLST). We chose serovars Typhimurium and Enteritidis as they are responsible for 75% of human infections in Europe. Additionally, we included serovars Derby, Dublin, Saintpaul, 4,5,12:i:-, Java and 4,5,12:b:- which are suspected to have different degrees of virulence to humans. MLST analysis clustered strains according to serovar with the exception of Java and Derby. DNA microarray clustered strains according to serovar and serogroup except for serovar 4,5,12:b:-. Differences in content of pathogenicity related genes between serovars with various host preferences and virulence towards humans were not observed. However, our strains from the supposedly less virulent serovar Derby lacked a combination of genes important for virulence. It might be speculated that other serovars can sustain their pathogenicity lacking one or two of these genes, whereas lack of many virulence genes will result in reduced virulence. A partial lack of concordance between MLST and microarray was found and this can be explained by the underlying data. On one hand, microarray data include highly variable regions which are known to be involved in horizontal gene transfer. On the other hand, MLST data is restricted to seven sequences and disregards contribution of horizontally acquired genes when evaluating evolution. The DNA microarray and MLST analysis complement each other giving a clearer image of evolution of these serovars and, furthermore, a visualization of the horizontally acquired genes.

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks., Author Summary Pulsed-field gel electrophoresis (PFGE) is the gold standard for recognizing outbreaks of food-borne disease. Comparative genomics has been used to elucidate transmission chains and evolutionary patterns within bacterial pathogens. However, their global genetic diversity has not yet been compared across multiple food-borne outbreaks. It was uncertain whether such outbreaks are caused by closely related organisms or are due to genetic properties that were recently acquired by horizontal genetic transfer. We investigated Salmonella enterica serovar Agona, a common cause of food-borne gastroenteritis, by sequencing genomes from bacterial strains from five outbreaks, as well as sporadic disease and the environment. Each outbreak was caused by a genetically distinct variant, but closely related bacteria were also found in the environment or in animal sources. The erroneous identity of bacteria according to PFGE reflected repeated, neutral acquisitions of bacteriophages, and genetic acquisitions or losses were not uniform across outbreak strains. Our results indicate that genomic differences used by epidemiologists to reach important decisions for public health can be stochastic and unrelated to disease or fitness. Our results also define the population structure and minimal age of Agona, and suggest that many serovars of Salmonella may be of recent origin.

Three large clusters of Salmonella Typhimurium infections in Denmark in 2008 and 2009 were defined by multilocus variable number of tandem repeat analysis (MLVA). One of these proved to be the hereto largest Danish cluster of salmonellosis with 1446 cases. Two smaller clusters with a total of 197 and 89 cases, respectively, were seen concurrently. These clusters shared epidemiological characteristics such as age distribution, geography, and time. To investigate the possible genetic relationship between the cluster strains, these were further characterized by phage typing, pulsed-field gel electrophoresis, and Optical Mapping. Although the MLVA method proved robust and well-performing in detecting and defining clusters, the employment of a second typing method detected an additional fourth cluster among the isolates. The cluster strains were stable throughout the almost 2-year period, even though we detected changes in three of five MLVA loci in a small fraction of isolates. These changes were mainly due to the gain or loss of single repeats. Optical Mapping of the large cluster strain indicated no increased content of virulence genes; however, Optical Mapping did reveal a large insert, a probable prophage, in the main cluster. This probable prophage may give the cluster strain a competitive advantage. The molecular methods employed suggested that the four clusters represented four distinct strains, although they seemed to be epidemiologically linked and shared genotypic characteristics.

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

Aims: To assess whether Campylobacter coli isolated from different sources in Denmark constitute separate populations. Methods and Results: Multilocus sequence typing (MLST) was applied to 160 C. coli isolates from animal origin, food products and human cases of gastroenteritis. The isolates were collected in Denmark over a 2-year period. In total, 84 sequence types (STs) were obtained and 57 of these STs were novel to this study. Ten per cent of the isolates possessed STs that were found in both human, poultry and pig isolates. Only 10% of the isolates from pigs shared ST with isolates from humans, and these shared STs were found in poultry isolates as well. Conclusions: Great genetic diversity was seen within the Danish C. coli population. Furthermore, we found that the C. coli types isolated from Danish pigs constitute a small fraction of the C. coli causing human disease. Significance and Impact of the Study: C. coli isolates from pigs is presumably not a significant source of human campylobacteriosis in Denmark. The Danish C. coli isolates include 68% STs novel to this study, showing a great diversity compared with studies from other countries.

Background Salmonella enterica subsp. enterica is one of the leading food-borne pathogens in the USA and European countries. Outcome of human Salmonella serotype Typhimurium infections ranges from mild self-limiting diarrhoea to severe diarrhoea that requires hospitalization. Increased knowledge of the mechanisms that are responsible for causing infection and especially the severity of infection is of high interest. Results Strains were selected from patients with mild infections (n = 9) and patients with severe infections (n = 9) and clinical data allowed us to correct for known underlying diseases. Additionally, outbreak isolates (n = 3) were selected. Strains were analyzed on a DNA-DNA microarray for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype and metabolism. Strains showed highly similar profiles when comparing virulence associated genes, but differences between strains were detected in the prophage marker group. The Salmonella virulence plasmid was present in 72% of the strains, but presence or absence of the virulence plasmid did not correspond to disease symptoms. A dendrogram clustered strains into four groups. Clustering confirmed DT104 as being a clonal phagetype. Clustering of the remaining strains was mainly correlated to presence or absence of the virulence plasmid and mobile elements such as transposons. Each of the four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of infection. Conclusions We investigated clinical significance of known virulence factors of Salmonella serotype Typhimurium strains causing different disease symptoms, and conclude that the few detected differences in Salmonella serotype Typhimurium do not affect outcome of human disease.

Background Escherichia coli have been found in increased numbers in tissues from patients with Inflammatory Bowel Disease (IBD) and adherent-invasive E. coli have been found in resected ileum from patients with Crohn's disesae. This study aimed to characterize possible differences in phylogenetic group (triplex PCR), extraintestinal pathogenic E. coli (ExPEC) genes and multilocus sequence type (MLST) between E. coli strains isolated from IBD patients with past or present involvement of the left side of the colon and from controls. Results Fecal samples were collected from 18 patients and from 10 healthy controls. Disease activity was evaluated by sigmoidoscopy. Interestingly, E. coli strains of the phylogenetic group B2 were cultured from 60% of patients with IBD compared to 11% of healthy controls (p < 0.05). Furthermore, when comparing the number of E. coli B2 strains with at least one positive ExPEC gene among different groups, 86% were found positive among active IBD patients, significantly more than 13% among inactive IBD patients (p < 0.05), and 11% among healthy controls (p < 0.05). The B2 phylogenetic group was found in a specific cluster based on MLST, but no further separation between E. coli strains associated with active compared to inactive IBD was achieved. Conclusion In conclusion, E. coli of the phylogenetic group B2 were isolated more frequently from IBD patients with past or present involvement of the left side of the colon compared to healthy controls, and B2 strains with ExPEC genes were found more frequently among IBD patients with active disease compared to patients with inactive disease.