A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella enterica serotype Typhimurium

<p>Abstract</p> <p>Background</p> <p>Typhimurium is the main serotype of <it>Salmonella enterica </it>subsp. <it>enterica </it>implicated in food-borne diseases worldwide. This study aimed to detect the prevalence of ten markers combined in a macro-array based on multiplex real-time PCR. We targeted characteristic determinants located on pathogenicity islands (SPI-2 to -5, virulence plasmid <it>pSLT </it>and <it>Salmonella </it>genomic island 1 (SGI1)) as well as a specific 16S-23S rRNA intergenic spacer sequence of definitive type 104 (DT104). To investigate antimicrobial resistance, the study also targeted the presence of genes involved in sulfonamide (<it>sul1</it>) and beta-lactam (<it>bla</it>_TEM) resistance. Finally, the <it>intI1 </it>determinant encoding integrase from class 1 integron was also investigated.</p> <p>Results</p> <p>A total of 538 unrelated <it>S</it>. Typhimurium strains isolated between 1999 and 2009 from various sources, including food animals, food products, human and environmental samples were studied. Based on the combined presence or absence of these markers, we distinguished 34 different genotypes, including three major genotypes encountered in 75% of the studied strains, Although SPI determinants were almost always detected, SGI1, <it>intI1</it>, <it>sul1 </it>and <it>bla</it>_TEMdeterminants were found 47%, 52%, 54% and 12% of the time respectively, varying according to isolation source. Low-marker patterns were most often detected in poultry sources whereas full-marker patterns were observed in pig, cattle and human sources.</p> <p>Conclusion</p> <p>The GeneDisc^®assay developed in this study madeit easier to explore variability within serotype Typhimurium by analyzing ten relevant gene determinants in a large collection of strains. This real-time multiplex method constitutes a valuable tool for strains characterization on epidemiological purposes.</p

Development of an SPME-GC-MS method for the specific quantification of dimethylamine and trimethylamine: use of a new ratio for the freshness monitoring of cod fillets

International audienceBACKGROUND: Fish is a highly perishable food, so it is important to be able to estimate its freshness to ensure optimum quality for consumers. The present study describes the development of an SPME‐GC‐MS technique capable of quantifying both trimethylamine (TMA) and dimethylamine (DMA), components of what has been defined as partial volatile basic nitrogen (PVB‐N). This method was used, together with other reference methods, to monitor the storage of cod fillets (Gadus morhua) conserved under melting ice.RESULTS: Careful optimisation enabled definition of the best parameters for extracting and separating targeted amines and an internal standard. The study of cod spoilage by sensory analysis and TVB‐N assay led to the conclusion that the shelf‐life of cod fillet was between 6 and 7 days. Throughout the study, TMA and DMA were specifically quantified by SPME‐GC‐MS; the first was found to be highly correlated with the values returned by steam distillation assays. Neither TMA‐N nor DMA‐N were able to successfully characterise the decrease in early freshness, unlike dimethylamine/trimethylamine ratio (DTR), whose evolution is closely related to the results of sensory analysis until the stage where fillets need to be rejected.CONCLUSION: DTR was proposed as a reliable indicator for the early decrease of freshness until fish rejection

Structure of the French farm-to-table surveillance system for Salmonella

The French surveillance system for Salmonella is based on a national system which can be traced back to 1947 for human cases and to the late 1980s for the main animal reservoirs. This system has evolved with regard to both European regulations and changes in the observed prevalence of Salmonella. European regulations establish a solid foundation on which to build an active harmonised surveillance system at the production level and for integrating data from the whole food chain. There are also passive surveillance networks in the agri-food and veterinary sectors and these allow complementary information to be obtained from other sectors or sources. The main strengths and weaknesses of these systems are described and a comparison of the different approaches is presented using a grid analysis. The results show that passive systems are very useful for detecting emerging or unusual events and for early warning of outbreaks. They also produce time series of cases or can determine the number of strains that should be used to assess the impact of interventions. Active surveillance data, due to their representativeness and reliability, are key elements in the application of risk analysis tools such as quantitative risk assessment or attribution. Thus, although data is collected and analysed by various organisations, these organisations all collaborate at a national level. Furthermore, their implication in European and international projects is effective and the main objectives of a surveillance system can be met

Variant Salmonella Genomic Island 1 Antibiotic Resistance Gene Cluster in Salmonella enterica Serovar Albany

Salmonella genomic island 1 (SGI1) contains an antibiotic resistance gene cluster and has been previously identified in multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, and Paratyphi B. We identified a variant SGI1 antibiotic-resistance gene cluster in a multidrug-resistant strain of S. enterica serovar Albany isolated from food fish from Thailand and imported to France. In this strain, the streptomycin resistance aadA2 gene cassette in one of the SGI1 integrons was replaced by a dfrA1 gene cassette, conferring resistance to trimethoprim and an open reading frame of unknown function. Thus, this serovar Albany strain represents the fourth S. enterica serovar in which SGI1 has been identified and the first SGI1 example where gene cassette replacement took place in one of its integron structures. The antibiotic resistance gene cluster of serovar Albany strain 7205.00 constitutes a new SGI1 variant; we propose a name of SGI1-F

A multi-country One Health foodborne outbreak simulation exercise: cross-sectoral cooperation, data sharing and communication

IntroductionThe awareness of scientists and policy makers regarding the requirement for an integrated One Health (OH) approach in responding to zoonoses has increased in recent years. However, there remains an overall inertia in relation to the implementation of practical cross-sector collaborations. Foodborne outbreaks of zoonotic diseases continue to affect the European population despite stringent regulations, evidencing the requirement for better ‘prevent, detect and response’ strategies. Response exercises play an essential role in the improvement of crisis management plans, providing the opportunity to test practical intervention methodologies in a controlled environment.MethodsThe One Health European Joint Programme simulation exercise (OHEJP SimEx) aimed at practicing the OH capacity and interoperability across public health, animal health and food safety sectors in a challenging outbreak scenario. The OHEJP SimEx was delivered through a sequence of scripts covering the different stages of a Salmonella outbreak investigation at a national level, involving both the human food chain and the raw pet feed industry.ResultsA total of 255 participants from 11 European countries (Belgium, Denmark, Estonia, Finland, France, Italy, Norway, Poland, Portugal, Sweden, the Netherlands) took part in national level two-day exercises during 2022. National evaluations identified common recommendations to countries aiming to improve their OH structure to establish formal communication channels between sectors, implement a common data sharing platform, harmonize laboratory procedures, and reinforce inter-laboratory networks within countries. The large proportion of participants (94%) indicated significant interest in pursuing a OH approach and desire to work more closely with other sectors.DiscussionThe OHEJP SimEx outcomes will assist policy makers in implementing a harmonized approach to cross-sector health-related topics, by highlighting the benefits of cooperation, identifying gaps in the current strategies and suggesting actions required to better address foodborne outbreaks. Furthermore, we summarize recommendations for future OH simulation exercises, which are essential to continually test, challenge and improve national OH strategies

Cross-Border Transmission of Salmonella Choleraesuis var. Kunzendorf in European Pigs and Wild Boar: Infection, Genetics, and Evolution

Salmonella enterica subspecies enterica serotype Choleraesuis is a swine adapted serovar. S. Choleraesuis variant Kunzendorf is responsible for the majority of outbreaks among pigs. S. Choleraesuis is rare in Europe, although there have been serious outbreaks in pigs including two outbreaks in Denmark in 1999–2000 and 2012–2013. Here, we elucidate the epidemiology, possible transmission routes and sources, and clonality of European S. Choleraesuis isolates including the Danish outbreak isolates. A total of 102 S. Choleraesuis isolates from different European countries and the United States, covering available isolates from the last two decades were selected for whole genome sequencing. We applied a temporally structured sequence analysis within a Bayesian framework to reconstruct a temporal and spatial phylogenetic tree. MLST type, resistance genes, plasmid replicons, and accessory genes were identified using bioinformatics tools. Fifty-eight isolates including 11 out of 12 strains from wild boars were pan-susceptible. The remaining isolates carried multiple resistance genes. Eleven different plasmid replicons in eight plasmids were determined among the isolates. Accessory genes were associated to the identified resistance genes and plasmids. The European S. Choleraesuis was estimated to have emerged in ∼1837 (95% credible interval, 1733–1983) with the mutation rate of 1.02 SNPs/genome/year. The isolates were clustered according to countries and neighbor countries. There were transmission events between strains from the United States and European countries. Wild boar and pig isolates were genetically linked suggesting cross-border transmission and transmission due to a wildlife reservoir. The phylogenetic tree shows that multiple introductions were responsible for the outbreak of 2012–2013 in Denmark, and suggests that poorly disinfected vehicles crossing the border into Denmark were potentially the source of the outbreak. Low levels of single nucleotide polymorphisms (SNPs) differences (0–4 SNPs) can be observed between clonal strains isolated from different organs of the same animal. Proper disinfection of livestock vehicles and improved quality control of livestock feed could help to prevent future spread of S. Choleraesuis or other more serious infectious diseases such as African swine fever (ASF) in the European pig production system

Présentation du projet CPER MARCO

International audienc