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

Antimicrobial susceptibility of S. aureus strains isolated from a ham plant in France

As MRSA (methicillin resistant Staphylococcus aureus) were recently isolated from pigs in the Netherlands (1), we were incited to evaluate the resistance gene circulation inside S. aureus strains isolated from swine food products. A study, build in 2002, established the biodiversity of S. aureus isolates originated from a single raw ham industrial unit located in western France. Biotypes and pulsotypes from 106 isolates from raw material ham, end product and plant environment were determined. Isolates split up into 7 biotypes and about forty pulsotypes, which delineated a total of 23 sub-groups with 80% of homology and indicated the presence of numerous S. aureus strains in the plant

2005 French Salmonella Network data on antimicrobial resistance in the swine channels

The Salmonella Network is gathering, on a voluntary participation scheme, from approximately 150 public and private laboratories dissemmated throughout France, Salmonella strains and/or epidemiological information. Those non-human Salmonella strains are isolated either from animal health and production or food, feed and the environment sectors. Thus, in 2005, a total of 527 isolations from the swine channels were reported. The top 5 prevalent serotypes were : Typhimurium, Derby, Manhattan, lnfantis and Kedougou. Two-hundred and ninety five strains were received at the laboratory and, after double clearance, 185 strains were tested for their antimicrobial resistance against 16 antibiotics by the disk diffusion method. Twenty-one strains were associated to the animal health and production sector and 164 to the food sector

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

Multidrug-resistant Strains of Salmonella enterica Typhimurium, United States, 1997–19981

To evaluate multidrug-resistant strains of Salmonella enterica Typhimurium, including definitive type 104 (DT104) in the United States, we reviewed data from the National Antimicrobial Resistance Monitoring System (NARMS). In 1997–1998, 25% (703) of 2,767 serotyped Salmonella isolates received at NARMS were S. Typhimurium; antimicrobial susceptibility testing and phage typing were completed for 697. Fifty-eight percent (402) were resistant to >1 antimicrobial agent. Three multidrug-resistant (>5 drugs) strains accounted for 74% (296) of all resistant isolates. Ceftriaxone resistance was present in 3% (8), and nalidixic acid resistance in 1% (4), of these multidrug-resistant strains. By phage typing, 37% (259) of S. Typhimurium isolates were DT104, 30% (209) were of undefined type and 15% (103) were untypable. Fifty percent (202) of resistant (>1 drug) isolates were DT104. Multidrug-resistant S. Typhimurium isolates, particularly DT104, account for a substantial proportion of S. Typhimurium isolates; ceftriaxone resistance is exhibited by some of these strains