Countrywide multi-serotype outbreak of Salmonella Bovismorbificans ST142 and monophasic Salmonella Typhimurium ST34 associated with dried pork sausages in France, September 2020* to January 2021.

The French National Reference Centre for Escherichia coli, Shigella and Salmonella (FNRC-ESS) detected two human clusters of 33 cases (median age: 10 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: 11 years; seven males) infected by S. 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. Bovismorbificans and S. 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.

Salmonella enterica subsp. enterica Welikade: guideline for phylogenetic analysis of serovars rarely involved in foodborne outbreaks.

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.

Disentangling a complex nationwide Salmonella Dublin outbreak associated with raw-milk cheese consumption, France, 2015 to 2016.

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.

Bacillus cereus-induced food-borne outbreaks in France, 2007 to 2014: epidemiology and genetic characterisation.

The aim of this study was to identify and characterise Bacillus cereus from a unique national collection of 564 strains associated with 140 strong-evidence food-borne outbreaks (FBOs) occurring in France during 2007 to 2014. Starchy food and vegetables were the most frequent food vehicles identified; 747 of 911 human cases occurred in institutional catering contexts. Incubation period was significantly shorter for emetic strains compared with diarrhoeal strains A sub-panel of 149 strains strictly associated to 74 FBOs and selected on Coliphage M13-PCR pattern, was studied for detection of the genes encoding cereulide, diarrhoeic toxins (Nhe, Hbl, CytK1 and CytK2) and haemolysin (HlyII), as well as panC phylogenetic classification. This clustered the strains into 12 genetic signatures (GSs) highlighting the virulence potential of each strain. GS1 (nhe genes only) and GS2 (nhe, hbl and cytK2), were the most prevalent GS and may have a large impact on human health as they were present in 28% and 31% of FBOs, respectively. Our study provides a convenient molecular scheme for characterisation of B. cereus strains responsible for FBOs in order to improve the monitoring and investigation of B. cereus-induced FBOs, assess emerging clusters and diversity of strains.

Outbreak of Salmonella Enteritidis linked to the consumption of frozen beefburgers received from a food bank and originating from Poland: northern France, December 2014 to April 2015.

A prolonged outbreak of Salmonella enterica serotype Enteritidis occurred in northern France between December 2014 and April 2015. Epidemiological investigations following the initial notification on 30 December 2014 of five cases of salmonellosis (two confirmed S. Enteritidis) in young children residing in the Somme department revealed that all cases frequented the same food bank A. Further epidemiological, microbiological and food trace-back investigations indicated frozen beefburgers as the source of the outbreak and the suspected lot originating from Poland was recalled on 22 January 2015. On 2 March 2015 a second notification of S. Enteritidis cases in the Somme reinitiated investigations that confirmed a link with food bank A and with consumption of frozen beefburgers from the same Polish producer. In the face of a possible persistent source of contamination, all frozen beefburgers distributed by food bank A and from the same origin were blocked on 3 March 2015. Microbiological analyses confirmed contamination by S. Enteritidis of frozen beefburgers from a second lot remaining in cases' homes. A second recall was initiated on 6 March 2015 and all frozen beefburgers from the Polish producer remain blocked after analyses identified additional contaminated lots over several months of production.

Complete genome sequence of a Salmonella enterica strain of serovar Agona associated with an outbreak in France.

We report here the complete genome of one Salmonella Agona strain isolated in 2017 from a dried milk powder in France.

Genomic insights of Salmonella isolated from dry fermented sausage production chains in Spain and France.

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.

Tell me if you prefer bovine or poultry sectors and I'll tell you who you are: Characterization of Salmonella enterica subsp. enterica serovar Mbandaka in France.

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.

Polyphyly in widespread Salmonella enterica serovars and using genomic proximity to choose the best reference genome for bioinformatics analyses.

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.

Retrieving Good-Quality Salmonella Genomes From the GenBank Database Using a Python Tool, SalmoDEST.

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.

The Spatiotemporal Dynamics and Microevolution Events That Favored the Success of the Highly Clonal Multidrug-Resistant Monophasic Salmonella Typhimurium Circulating in Europe.

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.

Closed Genome Sequence of a Salmonella enterica Serovar Bovismorbificans Strain Isolated from Dried Pork Sausage Associated with an Outbreak in France.

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.

AB_SA: Accessory genes-Based Source Attribution - tracing the source of Salmonella enterica Typhimurium environmental strains.

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 80 %. 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 85 % 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.

Source Attribution Study of Sporadic Salmonella Derby Cases in France.

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.

Halogenase genes in nonribosomal peptide synthetase gene clusters of Microcystis (cyanobacteria): sporadic distribution and evolution.

Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S-23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains.

Identification of a polyketide synthase coding sequence specific for anatoxin-a-producing Oscillatoria cyanobacteria.

We report the identification of a sequence from the genome of Oscillatoria sp. strain PCC 6506 coding for a polyketide synthase. Using 50 axenic cyanobacteria, we found this sequence only in the genomes of Oscillatoria strains producing anatoxin-a or homoanatoxin-a, indicating its likely involvement in the biosynthesis of these toxins.

Plasticity and evolution of aeruginosin biosynthesis in cyanobacteria.

Aeruginosins are bioactive oligopeptides that are produced in high structural diversity by strains of the bloom-forming cyanobacterial genera Microcystis and Planktothrix. A hallmark of aeruginosins is the unusual Choi moiety central to the tetrapeptides, while other positions are occupied by variable moieties in individual congeners. Here we report on three aeruginosin synthetase gene clusters (aer) of Microcystis aeruginosa (strains PCC 7806, NIES-98, and NIES-843). The analysis and comparison the aer gene clusters provide the first insight into the molecular basis of biosynthetic and structural plasticity in aeruginosin pathways. Major parts of the aer gene clusters are highly similar in all strains, particularly the genes coding for the first three nonribosomal peptide synthetase (NRPS) modules except for the region coding for the second adenylation domain. However, the gene clusters differ largely in genes coding for tailoring enzymes such as halogenases and sulfotransferases, reflecting structural peculiarities in aeruginosin congeners produced by the individual strains. Significant deviations were further observed in the C-terminal NRPS modules, suggesting two distinct release mechanisms. The architecture of the gene clusters is in agreement with the particular aeruginosin variants that are produced by individual strains, the structures of two of which (aeruginosins 686 A and 686 B) were elucidated. The aer gene clusters of Microcystis and Planktothrix are proposed to originate from a common ancestor and to have evolved to their present-day diversity largely through horizontal gene transfer and recombination events.

Whole-Genome Sequences of Two Salmonella enterica Serovar Dublin Strains That Harbor the viaA, viaB, and ompB Loci of the Vi Antigen.

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).

Draft Genome Sequences of Salmonella enterica subsp. enterica Serovar Dublin Strains from St. Nectaire and Morbier Cheeses Characterized by Multilocus Variable-Number Tandem-Repeat Analysis Profiles Associated with Two Fatal Outbreaks in France.

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.

Correction for Sévellec et al., "Complete Genome Sequence of Salmonella enterica subsp. enterica Serotype Derby, Associated with the Pork Sector in France".

[This corrects the article DOI: 10.1128/MRA.01027-18.].