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We report here the complete genome of one Salmonella Agona strain isolated in 2017 from a dried milk powder in France.
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The presence of Salmonella in dry fermented sausages is source of recalls and outbreaks. The genomic diversity of 173 Salmonella isolates from the dry fermented sausage production chains (pig carcasses, pork, and sausages) from France and Spain were investigated through their core phylogenomic relationships and accessory genome profiles. Ten different serovars and thirteen sequence type profiles were identified. The most frequent serovar from sausages was the monophasic variant of S. Typhimurium (1,4,[5],12:i:-, 72%) while S. Derby was in pig carcasses (51%). Phylogenomic clusters found in S. 1,4,[5],12:i:-, S. Derby, S. Rissen and S. Typhimurium serovars identified closely related isolates, with less than 10 alleles and 20 SNPs of difference, displaying Salmonella persistence along the pork production chain. Most of the S. 1,4,[5],12:i:- contained the Salmonella genomic island-4 (SGI-4), Tn21 and IncFIB plasmid. More than half of S. Derby strains contained the SGI-1 and Tn7. S. 1,4,[5],12:i:- genomes carried the most multidrug resistance genes (91% of the strains), whereas extended-spectrum ß-lactamase genes were found in Typhimurium and Derby serovars. Salmonella monitoring and characterization in the pork production chains, specially S. 1,4,[5],12:i:- serovar, is of special importance due to its multidrug resistance capacity and persistence in dry fermented sausages.
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Microbiologia de Alimentos , Produtos da Carne , Filogenia , Salmonella , Produtos da Carne/microbiologia , Espanha , França , Animais , Salmonella/genética , Salmonella/isolamento & purificação , Salmonella/classificação , Suínos , Fermentação , Genoma Bacteriano , Sorogrupo , Genômica/métodos , Ilhas Genômicas/genéticaRESUMO
Introduction: In north-western France, Salmonella enterica susp. enterica serovar Mbandaka (S. Mbandaka) is most frequently isolated from bovine and dairy samples. While this serovar most often results in asymptomatic carriage, for a number of years it has caused episodes of abortions, which have serious economic consequences for the sector. Interestingly, this serovar is also isolated from Gallus gallus in the same geographic zone. Despite its prevalence in bovines in north-western France, S. Mbandaka has not been broadly studied at the genomic level, and its prevalence and host adaptation are still not fully understood. Methods: In this study, we analyzed the genomic diversity of 304 strains of S. Mbandaka isolated from the bovine and poultry sectors in this area over a period of 5 years. A phylogenetic analysis was carried out and two approaches were followed to identify conserved genes and mutations related to host associations. The first approach targeted the genes compiled in the MEGARESv2, Resfinder, VFDB and SPI databases. Plasmid and phage contents were also investigated. The second approach refers to an in-house algorithm developed for this study that computes sensitivity, specificity, and accuracy of accessory genes and core variants according to predefined genomes groups. Results and discussion: All the analyzed strains belong to the multi-locus sequence type profile ST413, and the phylogenomic analysis revealed main clustering by host (bovine and poultry), emphasizing the circulation of 12 different major clones, of which seven circulate in poultry and five in the bovine sector in France and a likely food production chain adaptation of these clones. All strains present resistance determinants including heavy metals and biocides that could explain the ability of this serovar to survive and persist in the environment, within herds, and in food processing plants. To explore the wild animal contribution to the spread of this serovar in north-western France, we retrieved S. Mbandaka genomes isolated from wild birds from EnteroBase and included them in the phylogenomic analysis together with our collection. Lastly, screening of accessory genes and major variants allowed us to identify conserved specific mutations characteristic of each major cluster. These mutations could be used to design useful probes for food safety surveillance.
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The French National Reference Centre for Escherichia coli, Shigella and Salmonella (FNRC-ESS) detected two human clusters of 33 cases (median age: 10â¯years; 17 females) infected by Salmonella enterica serotype Bovismorbificans, ST142, HC5_243255 (EnteroBase HierCCcgMLST scheme) in September-November 2020 and of 11 cases (median age: 11â¯years; seven males) infected by S.â¯enterica serotype 4,12:i:-, ST34, HC5_198125 in October-December 2020. Epidemiological investigations conducted by Santé publique France linked these outbreaks to the consumption of dried pork sausages from the same manufacturer. S.â¯Bovismorbificans and S.â¯4,12:i:- were isolated by the National Reference Laboratory from different food samples, but both strains were identified in a single food sample only by qPCR. Three recalls and withdrawals of dried pork products were issued by the French general directorate of food of the French ministry for agriculture and food in November 2020, affecting eight supermarket chains. A notification on the European Rapid Alert System for Food and Feed and a European urgent enquiry on the Epidemic Intelligence Information System for Food and Waterborne Diseases and Zoonoses (EPIS-FWD) were launched. No cases were reported outside France. Outbreaks caused by multiple serotypes of Salmonella may go undetected by protocols in standard procedures in microbiology laboratories.
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Produtos da Carne , Carne de Porco , Carne Vermelha , Intoxicação Alimentar por Salmonella , Animais , Feminino , Masculino , Humanos , Suínos , Criança , Salmonella typhimurium/genética , Sorogrupo , Intoxicação Alimentar por Salmonella/epidemiologia , Intoxicação Alimentar por Salmonella/microbiologia , Carne Vermelha/microbiologia , França/epidemiologia , Surtos de DoençasRESUMO
Salmonella is the most common cause of gastroenteritis in the world. Over the past 5 years, whole-genome analysis has led to the high-resolution characterization of clinical and foodborne Salmonella responsible for typhoid fever, foodborne illness or contamination of the agro-food chain. Whole-genome analyses are simplified by the availability of high-quality, complete genomes for mapping analysis and for calculating the pairwise distance between genomes, but unfortunately some difficulties may still remain. For some serovars, the complete genome is not available, or some serovars are polyphyletic and knowing the serovar alone is not sufficient for choosing the most appropriate reference genome. For these serovars, it is essential to identify the genetically closest complete genome to be able to carry out precise genome analyses. In this study, we explored the genomic proximity of 650 genomes of the 58 Salmonella enterica subsp. enterica serovars most frequently isolated in humans and from the food chain in the United States (US) and in Europe (EU), with a special focus on France. For each serovar, to take into account their genomic diversity, we included all the multilocus sequence type (MLST) profiles represented in EnteroBase with 10 or more genomes (on 19 July 2021). A phylogenetic analysis using both core- and pan-genome approaches was carried out to identify the genomic proximity of all the Salmonella studied and 20 polyphyletic serovars that have not yet been described in the literature. This study determined the genetic proximity between all 58 serovars studied and revealed polyphyletic serovars, their genomic lineages and MLST profiles. Finally, we enhanced the open-access databases with 73 new genomes and produced a list of high-quality complete reference genomes for 48 S. enterica subsp. enterica serovars among the most isolated in the US, EU, and France.
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Salmonella enterica , Biologia Computacional , Genômica , Humanos , Tipagem de Sequências Multilocus , Filogenia , Salmonella , Salmonella enterica/genética , Sorogrupo , Estados UnidosRESUMO
BACKGROUND: Salmonella spp. is a major foodborne pathogen with a wide variety of serovars associated with human cases and food sources. Nevertheless, in Europe a panel of ten serovars is responsible for up to 80% of confirmed human cases. Clustering studies by single nucleotide polymorphism (SNP) core-genome phylogenetic analysis of outbreaks due to these major serovars are simplified by the availability of many complete genomes in the free access databases. This is not the case for outbreaks due to less common serovars, such as Welikade, for which no reference genomes are available. In this study, we propose a method to solve this problem. We propose to perform a core genome MLST (cgMLST) analysis based on hierarchical clustering using the free-access EnteroBase to select the most suitable genome to use as a reference for SNP phylogenetic analysis. In this study, we applied this protocol to a retrospective analysis of a Salmonella enterica serovar Welikade (S. Welikade) foodborne outbreak that occurred in France in 2016. Finally, we compared the cgMLST and SNP analyses. SNP phylogenetic reconstruction was carried out considering the effect of recombination events identified by the ClonalFrameML tool. The accessory genome was also explored by phage content and virulome analyses. RESULTS: Our findings revealed high clustering concordance using cgMLST and SNP analyses. Nevertheless, SNP analysis allowed for better assessment of the genetic distance among strains. The results revealed epidemic clones of S. Welikade circulating within the poultry and dairy sectors in France, responsible for sporadic and non-sporadic human cases between 2012 and 2019. CONCLUSIONS: This study increases knowledge on this poorly described serovar and enriches public genome databases with 42 genomes from human and non-human S. Welikade strains, including the isolate collected in 1956 in Sri Lanka, which gave the name to this serovar. This is the first genomic analysis of an outbreak due to S. Welikade described to date.
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Intoxicação Alimentar por Salmonella , Salmonella enterica , Surtos de Doenças , Humanos , Tipagem de Sequências Multilocus/métodos , Filogenia , Estudos Retrospectivos , Salmonella/genética , SorogrupoRESUMO
With the advent of next-generation whole-genome sequencing (WGS), the need for good-quality and well-characterised Salmonella genomes has increased over the past years. Good-quality complete genomes are often required for assembly reference mapping or phylogenetic single nucleotide polymorphism (SNP) analysis. Complete genomes or contigs from specific sources or serovars are also searched for clustering analysis or source attribution studies. Therefore, new bioinformatics tools are needed for the extraction of good-quality and well-characterised genomes from public databases. Here, we developed SalmoDEST, an open-source Python tool capable of extracting Salmonella genomes with a coverage higher than 50x and genome length over 4Mb from the GenBank database in the form of complete genomes or contigs, with verification of the serovar to which they belong and identification of the corresponding multi locus sequence type (MLST) profile. To validate the ability to SalmoDEST to screen for and retrieve genomes of good quality, we compared our results for S. Typhi complete genome with those available in the literature and extracted Salmonella genomes from bovine sources strains isolated worldwide. Finally, we provide in this study a list of 239 complete genomes for 123 serovars of Salmonella of high quality. SalmoDEST is a handy and easy-to-use open-source tool to extract complete genomes or contigs that can be routinely used in public health, food safety and research laboratories. SalmoDEST (SALMOnella Download gEnome Serotype sT) is available at https://github.com/I-Guy/SalmoDEST.
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We report here the closed genome sequence of one Salmonella enterica subsp. enterica serovar Bovismorbificans strain isolated from dried pork sausage consumed by a patient suffering from salmonellosis.
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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.
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Salmonella enterica subsp. enterica serovar Derby is one of the most frequent causes of gastroenteritis in humans. In Europe, this pathogen is one of the top five most commonly reported serovars in human cases. In France, S. Derby has been among the ten most frequently isolated serovars in humans since the year 2000. The main animal hosts of this serovar are pigs and poultry, and white meat is the main source of human contamination. We have previously shown that this serovar is polyphyletic and that three distinct genetic lineages of S. Derby cohabit in France. Two of them are associated with pork and one with poultry. In this study, we conducted a source attribution study based on single nucleotide polymorphism analysis of a large collection of 440 S. Derby human and non-human isolates collected in 2014-2015, to determine the contribution of each lineage to human contamination. In France, the two lineages associated with pork strains, and corresponding to the multilocus sequence typing (MLST) profiles ST39-ST40 and ST682 were responsible for 94% of human contaminations. Interestingly, the ST40 profile is responsible for the majority of human cases (71%). An analysis of epidemiologic data and the structure of the pork sector in France allowed us to explain the spread and the sporadic pattern of human cases that occurred in the studied period.
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The partitioning of pathogenic strains isolated in environmental or human cases to their sources is challenging. The pathogens usually colonize multiple animal hosts, including livestock, which contaminate the food-production chain and the environment (e.g. soil and water), posing an additional public-health burden and major challenges in the identification of the source. Genomic data opens up new opportunities for the development of statistical models aiming to indicate the likely source of pathogen contamination. Here, we propose a computationally fast and efficient multinomial logistic regression source-attribution classifier to predict the animal source of bacterial isolates based on 'source-enriched' loci extracted from the accessory-genome profiles of a pangenomic dataset. Depending on the accuracy of the model's self-attribution step, the modeller selects the number of candidate accessory genes that best fit the model for calculating the likelihood of (source) category membership. The Accessory genes-Based Source Attribution (AB_SA) method was applied to a dataset of strains of Salmonella enterica Typhimurium and its monophasic variant (S. enterica 1,4,[5],12:i:-). The model was trained on 69 strains with known animal-source categories (i.e. poultry, ruminant and pig). The AB_SA method helped to identify 8 genes as predictors among the 2802 accessory genes. The self-attribution accuracy was 80â%. The AB_SA model was then able to classify 25 of the 29 S. enterica Typhimurium and S. enterica 1,4,[5],12:i:- isolates collected from the environment (considered to be of unknown source) into a specific category (i.e. animal source), with more than 85â% of probability. The AB_SA method herein described provides a user-friendly and valuable tool for performing source-attribution studies in only a few steps. AB_SA is written in R and freely available at https://github.com/lguillier/AB_SA.
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Proteínas de Bactérias/genética , Biologia Computacional/métodos , Gado/classificação , Salmonella typhimurium/classificação , Animais , Bases de Dados Genéticas , Microbiologia de Alimentos , Gado/microbiologia , Modelos Logísticos , Modelos Teóricos , Salmonella typhimurium/genética , Interface Usuário-ComputadorRESUMO
The investigation of foodborne outbreaks (FBOs) from genomic data typically relies on inspecting the relatedness of samples through a phylogenomic tree computed on either SNPs, genes, kmers, or alleles (i.e., cgMLST and wgMLST). The phylogenomic reconstruction is often time-consuming, computation-intensive and depends on hidden assumptions, pipelines implementation and their parameterization. In the context of FBO investigations, robust links between isolates are required in a timely manner to trigger appropriate management actions. Here, we propose a non-parametric statistical method to assert the relatedness of samples (i.e., outbreak cases) or whether to reject them (i.e., non-outbreak cases). With typical computation running within minutes on a desktop computer, we benchmarked the ability of three non-parametric statistical tests (i.e., Wilcoxon rank-sum, Kolmogorov-Smirnov and Kruskal-Wallis) on six different genomic features (i.e., SNPs, SNPs excluding recombination events, genes, kmers, cgMLST alleles, and wgMLST alleles) to discriminate outbreak cases (i.e., positive control: C+) from non-outbreak cases (i.e., negative control: C-). We leveraged four well-characterized and retrospectively investigated FBOs of Salmonella Typhimurium and its monophasic variant S. 1,4,[5],12:i:- from France, setting positive and negative controls in all the assays. We show that the approaches relying on pairwise SNP differences distinguished all four considered outbreaks in contrast to the other tested genomic features (i.e., genes, kmers, cgMLST alleles, and wgMLST alleles). The freely available non-parametric method written in R has been designed to be independent of both the phylogenomic reconstruction and the detection methods of genomic features (i.e., SNPs, genes, kmers, or alleles), making it widely and easily usable to anybody working on genomic data from suspected samples.
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Salmonella Derby (S. Derby) is emerging in Europe as a predominant serovar in fattening turkey flocks. This serovar was recorded as being predominant in the turkey sector in 2014 in the United Kingdom (UK). Only two years later, in 2016, it was also recorded in the turkey and broiler sectors in Ireland and Spain. These S. Derby isolates were characterised as members of the multilocus sequence type (MLST) profile 71 (ST71). For the first time, we characterise by whole genome sequencing (WGS) analysis a panel of 90 S. Derby ST71 genomes to understand the routes of transmission of this emerging pathogen within the poultry/turkey food trade. Selected panel included strains isolated as early as 2010 in five leading European g countries for turkey meat production. Twenty-one of the 90 genomes were extracted from a public database-Enterobase. Five of these originated from the United States (n=3), China (n=1) and Taiwan (n=1) isolated between 1986 and 2016. A phylogenomic analysis at the core-genome level revealed the presence of three groups. The largest group contained 97.5% of the European strains and included both, turkey and human isolates that were genetically related by an average of 35 ± 15 single nucleotide polymorphism substitutions (SNPs). To illustrate the diversity, the presence of antimicrobial resistance genes and phages were characteised in 30, S. Derby ST71 genomes, including 11 belonging to this study This study revealed an emergent turkey-related S. Derby ST71 clone circulating in at least five European countries (the UK, Germany, Poland, Italy, and France) since 2010 that causes human gastroenteritis. A matter of concern is the identification of a gyrA mutation involved in resistance to quinolone, present in the Italian genomes. Interestingly, the diversity of phages seems to be related to the geographic origins. These results constitute a baseline for following the spread of this emerging pathogen and identifying appropriate monitoring and prevention measures.
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Here, we report the genome sequences of two Salmonella enterica serovar Dublin strains, 03EB8736SAL and 03EB8994SAL, isolated from raw-milk cheese and milk filtrate, respectively. Analysis of the draft genomes of the two isolates reveals the presence of the viaA, viaB, and ompB loci of the Vi capsular polysaccharide antigen (Vi antigen).
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On 18 January 2016, the French National Reference Centre for Salmonella reported to Santé publique France an excess of Salmonella enterica serotype Dublin (S. Dublin) infections. We investigated to identify the source of infection and implement control measures. Whole genome sequencing (WGS) and multilocus variable-number tandem repeat analysis (MLVA) were performed to identify microbiological clusters and links among cases, animal and food sources. Clusters were defined as isolates with less than 15 single nucleotide polymorphisms determined by WGS and/or with identical MLVA pattern. We compared different clusters of cases with other cases (case-case study) and controls recruited from a web-based cohort (case-control study) in terms of food consumption. We interviewed 63/83 (76%) cases; 2,914 controls completed a questionnaire. Both studies' findings indicated that successive S. Dublin outbreaks from different sources had occurred between November 2015 and March 2016. In the case-control study, cases of distinct WGS clusters were more likely to have consumed Morbier (adjusted odds ratio (aOR): 14; 95% confidence interval (CI): 4.8-42) or Vacherin Mont d'Or (aOR: 27; 95% CI: 6.8-105), two bovine raw-milk cheeses. Based on these results, the Ministry of Agriculture launched a reinforced control plan for processing plants of raw-milk cheeses in the production region, to prevent future outbreaks.
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Queijo/microbiologia , Surtos de Doenças/prevenção & controle , Leite/microbiologia , Intoxicação Alimentar por Salmonella/epidemiologia , Salmonella/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Bovinos , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Feminino , França/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Repetições Minissatélites , Polimorfismo de Nucleotídeo Único , Salmonella/classificação , Salmonella/genética , Intoxicação Alimentar por Salmonella/microbiologia , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
We report here the draft genome sequences of 2 Salmonella enterica subsp. enterica serovar Dublin strains from St. Nectaire and Morbier cheeses having multilocus variable-number tandem-repeat analysis (MLVA) profiles identified during the fatal outbreaks that occurred in France in 2012 and 2015 to 2016, respectively. These draft genome sequences will help uncover the virulence determinants in invasive S. Dublin strains.
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[This corrects the article DOI: 10.1128/MRA.01027-18.].
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In the European Union, Salmonella enterica subsp. enterica serovar Derby is the most abundant serotype isolated from pork. Recent studies have shown that this serotype is polyphyletic. However, one main genomic lineage, characterized by sequence type 40 (ST40), the presence of the Salmonella pathogenicity island 23, and showing resistance to streptomycin, sulphonamides, and tetracycline (STR-SSS-TET), is pork associated. Here, we describe the complete genome sequence of a strain from this lineage isolated in France.
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In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify genetic factors associated with host adaptation and markers for the monitoring of these different lineages within the corresponding animal sectors. The recognition of these four lineages is of primary importance for epidemiological surveillance throughout the food production chains and constitutes the first step toward refining monitoring and preventing dispersal of this pathogen.