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Raw reads of the 34 genomes of S. Bovismorbificans and 12 genomes of S. Typhimurium from human and food origin in this study were submitted to the European Nucleotide Archive under project numbers PRJEB41645 and PRJEB49565.; International audience; 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 HierCC‑cgMLST 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.

In contaminated fish, bacterial decarboxylases produce histamine from histidine, thereby causing scombroid fish poisoning. European Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs requires using a fully validated, standardized reference HPLC method for detecting and quantifying histamine. After optimizing this reference method for the quantification of histamine in fish muscle, we organized an inter-laboratory study in 2013 across nine laboratories from seven European countries using defined criteria of method performance. The optimized, validated method was standardized (Standard EN ISO19343) as part of Mandate M381 from the European Commission to the European Committee for Standardization (CEN), signed in December 2010. The standard method was validated for three types of foodstuffs (fish with enzymatic maturation, fish without enzymatic maturation and fish sauce).

https://doi.org/10.1016/j.fm.2021.103759 B. cereusis a human pathogen associated withfood poisoningleading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum ofB. cereusranges from strains used as probiotics in humans to lethal highly toxic strains. In this study, we gathered a collection of 100 strains representative of the pathological diversity ofB. cereusin humans, and characterized these strains for their cytotoxic potential towards human cells. We analyzed the correlation between cytotoxicity to epithelial and macrophage cells and the combination of 10 genes suspected to play a role duringB. cereusvirulence. We highlight genetic differences among isolates and studied correlations between genetic signature, cytotoxicity and strain pathological status. We hope that our findings will improve our understanding of thepathogenicityofB. cereus,thereby making it possible to improve both clinical diagnosis and food safety.

BackgroundListeria monocytogenesClonal Complexes (CCs) have been epidemiologically associated with foods, especially ready-to-eat (RTE) products for which the most likely source of contamination depends on the occurrence of persisting clones in food-processing environments (FPEs). As the ability ofL. monocytogenesto adapt to environmental stressors met in the food chain challenges the efforts to its eradication from FPEs, the threat of persistent strains to the food industry and public health authorities continues to rise. In this study, 94 food and FPEsL. monocytogenesisolates, representing persistent subtypes contaminating three French seafood facilities over 2–6 years, were whole-genome sequenced to characterize their genetic diversity and determine the biomarkers associated with long-term survival in FPEs.ResultsFood and FPEs isolates belonged to five CCs, comprising long-term intra- and inter-plant persisting clones. Mobile genetic elements (MGEs) such as plasmids, prophages and transposons were highly conserved within CCs, some of which harboured genes for resistance to chemical compounds and biocides used in the processing plants. Some of these genes were found in a 90.8 kbp plasmid, predicted to be” mobilizable”, identical in isolates from CC204 and CC155, and highly similar to an 81.6 kbp plasmid from isolates belonging to CC7. These similarities suggest horizontal transfer between isolates, accompanied by deletion and homologous recombination in isolates from CC7. Prophage profiles characterized persistent clonal strains and several prophage-loci were plant-associated. Notably, a persistent clone from CC101 harboured a novel 31.5 kbp genomic island that we namedListeriagenomic island 3 (LGI3), composed by plant-associated loci and chromosomally integrating cadmium-resistance determinantscadA1C.ConclusionsGenome-wide analysis indicated that inter- and intra-plant persisting clones harbour conserved MGEs, likely acquired in FPEs and maintained by selective pressures. The presence of closely related plasmids inL. monocytogenesCCs supports the hypothesis of horizontal gene transfer conferring enhanced survival to FPE-associated stressors, especially in hard-to-clean harbourage sites. Investigating the MGEs evolutionary and transmission dynamics provides additional resolution to trace-back potentially persistent clones. The biomarkers herein discovered provide new tools for better designing effective strategies for the removal or reduction of residentL. monocytogenesin FPEs to prevent contamination of RTE seafood.

International audience; This study presents a miniaturized 3-methyl-2-benzothiazolinone hydrazone hydrochloride-assay (MBTHassay) 1 , optimized for high-throughput measurement of shrimp phenol oxidase activities and the screening of potential inhibitors of post-mortem melanosis. To validate this approach, assay parameters, including limit of detection and dynamic range, were compared to values obtained for DOPAchrome-assay, used here as a reference method. A reliable high-throughput automatized analysis was also performed, under standardized parameters, in order to compare and characterize 40 potential inhibitors of phenol oxidase activities. Among the selected molecules, twenty showed inhibitory action. Enzyme-inhibitor dissociation constant (K i), median inhibitor concentration (IC 50) and inhibition modes could be determined for nine of them. 4-Hexylresorcinol, Nphenylthiourea L-cystein and glutathione displayed K i values lower than sulfite salts (Na 2 S 2 O 5 and Na 2 SO 3) used as positive controls.

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.

International audience; Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872-1:2007 and TS 21872-2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.

BACKGROUND 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. © 2015 Society of Chemical Industry

BACKGROUND: Monitoring of early stages of freshness decay is a major issue for the fishery industry to guarantee the best quality for this highly perishable food matrix. Numerous techniques have been developed, but most of them have the disadvantage of being reliable only either in the last stages of fish freshness or for the analysis of whole fish. This study describes the development of a qPCR method targeting the torA gene harboured by fish spoilage microorganisms. torA encodes an enzyme that leads to the production of trimethylamine responsible for the characteristic spoiled‐fish odour. RESULTS: A degenerate primer pair was designed. It amplified torA gene of both Vibrio and Photobacterium with good efficiencies on 7‐log DNA dilutions. The primer pair was used during a shelf‐life monitoring study achieved on modified atmosphere packed, chilled, whiting (Merlangius merlangus) fillets. The qPCR approach allows the detection of an increase of torA copies throughout the storage of fillets in correlation with the evolution of both total volatile basic nitrogen (−0.86) and trimethylamine concentrations (−0.81), known as spoilage markers. CONCLUSION: This study described a very promising, sensitive, reliable, time‐effective, technique in the field of freshness characterisation of processed fish. © 2015 Society of Chemical Industry

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations.

UNLABELLED Four strains were isolated from a spoiled whiting (Merlangius merlangus). All of them were able to grow aerobically from 4 to 30°C and also able to develop anaerobically in the presence of trimethylamine N-Oxide (TMAO) at 25°C. Biochemical characterization did not allow identification of the strains species but showed that one of the four strains was unable to produce H2 S. Two strains synthetized an ornithine decarboxylase being potential putrescine producers. Results of carbon source use highlighted that the four strains were able to use citrate and d-sucrose and one strain was not able to use l-arabinose. Genotypic characterization of the strains thanks to 16S rRNA and gyrB partial gene sequencing led to their identification as members of Shewanella baltica species. These observations suggest that H2 S production may not be the most appropriate screening parameter for Shewanella species and further to monitor the development of spoilage flora. SIGNIFICANCE AND IMPACT OF THE STUDY Shewanella is a complex genus composed of numerous and heterogeneous species. One of them Shewanella baltica has previously been described as one of the most important H2 S-producing bacterial species in iced stored fish and may act as spoilage organism through the reduction of trimethylamine N-Oxide (TMAO). Four strains of S. baltica were isolated from spoiled whiting (Merlangius merlangus), and description of three H2 S-positive strains and one H2 S-negative strain of S. baltica is highlighted in this short paper. Consequently, H2 S production might not be the most appropriate screening parameter to assess the development of spoilage organisms.

International audience

A major community outbreak of salmonellosis occurred in France in October 2010. Classical epidemiological investigations led to the identification of beef burgers as the cause of the outbreak and the presence of the emerging monophasic Salmonella Typhimurium 4,5,12:i:−. The objective of this study was to understand the events that led to this large outbreak, that is to say, what are the contributing factors associated with consumer exposure to Salmonella. To this end, intensive microbiological investigations on several beef burgers were conducted and a risk assessment model was built. The microbiological results confirm the presence of Salmonella in all analysed frozen burgers at high levels of contamination above 1000 MPN/g. These results in frozen burgers combined with a model of thermal destruction were used to estimate the dose ingested by the exposed persons. Most people that consumed cooked beef burgers were exposed from 1.6 to 3.1 log10 (MPN). The number of sick people predicted with a dose–response relationship for Salmonella is consistent with the observed number of salmonellosis cases. The very high initial contamination level in frozen beef burgers is the primary cause of this large outbreak rather than bad cooking practices. Intensive investigations, modelling of the initial contamination and quantitative exposure and risk assessments are complementary to epidemiological investigation. They can be valuable elements for the assessment of missing information or the identification of the primary causes of outbreaks.

This study aimed at updating knowledge on the prevalence and antimicrobial resistance characteristics of Salmonella isolated from poultry in the province of N'Djamena, Chad. The results collected during this study provide the first baseline data on the prevalence of contamination by Salmonella in laying hens and broiler chicken farms in N'Djamena. All samples were collected from sixteen poultry farms over two periods of six months each: from August 2010 to January 2011 and from September 2011 to February 2012. Diagnostic methods used during this study allowed to isolate eighty four Salmonella strains, belonging to twenty seven different serotypes. The most frequent serotypes were Salmonella Colindale (19%) followed by S. Minnesota (18%) S. Havana and S. Riggil (each 6%), S. Kottbus and S. Amager (4.7%), S. Idikan, Mississipi, and Muenchen (3.6%). Other serotypes were poorly represented. The majority of these serotypes were susceptible to all antibiotics tested (CLSI Standards), except some S. Colindale isolates that exhibited a decreased susceptibility to fluoroquinolones, S. Limete resistant to three antibiotics and S. Minnesota isolates resistant to five different antimicrobial classes. The different serotypes and antibiotic resistance profiles that were observed highlight the substantial diversity of Salmonella in Chad, the contribution of avian isolates to human salmonellosis and Salmonella's capacity to colonize all types of environment worldwide.

Salmonella enterica serovar Weltevreden (S. Weltevreden) is an emerging cause of diarrheal and invasive disease in humans residing in tropical regions. Despite the regional and international emergence of this Salmonella serovar, relatively little is known about its genetic diversity, genomics or virulence potential in model systems. Here we used whole genome sequencing and bioinformatics analyses to define the phylogenetic structure of a diverse global selection of S. Weltevreden. Phylogenetic analysis of more than 100 isolates demonstrated that the population of S. Weltevreden can be segregated into two main phylogenetic clusters, one associated predominantly with continental Southeast Asia and the other more internationally dispersed. Subcluster analysis suggested the local evolution of S. Weltevreden within specific geographical regions. Four of the isolates were sequenced using long read sequencing to produce high quality reference genomes. Phenotypic analysis in Hep-2 cells and in a murine infection model indicated that S. Weltevreden were significantly attenuated in these models compared to the classical S. Typhimurium reference strain SL1344. Our work outlines novel insights into this important emerging pathogen and provides a baseline understanding for future research studies., Author Summary Organisms belonging to the species Salmonella enterica are a major cause of infection globally. Such infections can be zoonotic in origin or transmitted between humans. One of the most notable features of the genus Salmonella is that the dominant serovars that cause human infections change over time, with new threats periodically emerging. These trends often go unnoticed and are underreported in low-income locations. There is good evidence that Salmonella Weltevreden is emerging (particularly in low-income countries in the tropics) as a significant cause of diarrhea and sometimes invasive bacterial disease in humans. However, little is known about the phylogenetic structure or virulence potential of this unstudied serovar. Here, we provide a detailed phylogenetic analysis of S. Weltevreden through whole genome sequencing and bioinformatics tools. We identify that specific phylogenetic clusters are associated with geographical regions, providing novel data regarding the global distribution of different clades and high quality reference genomes to facilitate future work. We additionally show that S. Weltevreden has a distinct virulence-associated phenotype in conventional laboratory Salmonella pathogenicity assays that will guide future investigations on this serovar.

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.

Corrigendum: Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. [Nat Genet. 2017]Erratum: Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. [Nat Genet. 2017]Comment in Biodiversity and hypervirulence of Listeria monocytogenes. [Nat Genet. 2016] Bacterial GWAS: not just gilding the lily. [Nat Rev Microbiol. 2016]; International audience; Microbial pathogenesis studies are typically performed with reference strains, thereby overlooking within-species heterogeneity in microbial virulence. Here we integrated human epidemiological and clinical data with bacterial population genomics to harness the biodiversity of the model foodborne pathogen Listeria monocytogenes and decipher the basis of its neural and placental tropisms. Taking advantage of the clonal structure of this bacterial species, we identify clones epidemiologically associated either with food or with human central nervous system (CNS) or maternal-neonatal (MN) listeriosis. The latter clones are also most prevalent in patients without immunosuppressive comorbidities. Strikingly, CNS- and MN-associated clones are hypervirulent in a humanized mouse model of listeriosis. By integrating epidemiological data and comparative genomics, we have uncovered multiple new putative virulence factors and demonstrate experimentally the contribution of the first gene cluster mediating L. monocytogenes neural and placental tropisms. This study illustrates the exceptional power in harnessing microbial biodiversity to identify clinically relevant microbial virulence attributes

Variant strains of Salmonella enterica serovar Typhimurium, lacking one or both flagellar phases have been widely reported. The monophasic S.1,4,[5],12:i:- variant has emerged worldwide in the past few years and has become one of the most frequently encountered in many countries. In contrast, monophasic S.1,4,[5],12:-:1,2 and nonmotile S.1,4,[5],12:-:- strains are rarely described. This study investigated seven molecular markers to identify and delineate monophasic S.1,4,[5],12:i:- (n = 90), S.1,4,[5],12:-:1,2 (n = 25), nonmotile S.1,4,[5],12:-:- (n = 17) strains, and some serovar Typhimurium strains (n = 124) collected through the French Salmonella network between 2001 and 2010. Three markers were commonly detected in serovar Typhimurium and in all variant strains: STM2757, mdh and fliA-B. Monophasic S.1,4,[5],12:i:- were genotypically confirmed by the absence of the fljB, fljA, and hin genes. Nevertheless, 13 (14.5%) of them were positive for these last three genes, revealing monophasic strains named “inconsistent” as previously described. All nonmotile 1,4,[5],12:-:- strains had the fliC, fljA, fljB, and hin genes and the fliC gene was detected in 88% of monophasic S.1,4,[5],12:-:1,2 strains. The combination of the seven markers detection enables to recognize eight different genotypes within the S.1,4,[5],12:i:- collection, among which the Spanish and the U.S. clones previously described could be distinguished and assigned to a genotype. Based on this molecular approach, 71% of the French S.1,4,[5],12:i:- collection belonged to the Spanish clone, whereas only 2% were assigned to the U.S. clone. This study highlights the usefulness of these molecular markers and genotypes for identifying lineages, especially among the epidemiologically important monophasic S.1,4,[5],12:i:- variant.

Attributing foodborne illnesses to food sources is essential to conceive, prioritize, and assess the impact of public health policy measures. The Bayesian microbial subtyping attribution model by Hald et al. is one of the most advanced approaches to attribute sporadic cases; it namely allows taking into account the level of exposure to the sources and the differences between bacterial types and between sources. This step forward requires introducing type and source-dependent parameters, and generates overparameterization, which was addressed in Hald's paper by setting some parameters to constant values. We question the impact of the choices made for the parameterization (parameters set and values used) on model robustness and propose an alternative parameterization for the Hald model. We illustrate this analysis with the 2005 French data set of non-typhi Salmonella. Mullner's modified Hald model and a simple deterministic model were used to compare the results and assess the accuracy of the estimates. Setting the parameters for bacterial types specific to a unique source instead of the most frequent one and using data-based values instead of arbitrary values enhanced the convergence and adequacy of the estimates and led to attribution estimates consistent with the other models' results. The type and source parameters estimates were also coherent with Mullner's model estimates. The model appeared to be highly sensitive to parameterization. The proposed solution based on specific types and data-based values improved the robustness of estimates and enabled the use of this highly valuable tool successfully with the French data set. tel: +33 1 47 10 79 08; fax: +33 1 47 14 49 03; laurence.watier@inserm.fr.

Summary Aims: Methicillin-resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. Methods and results: Twelve specific pathogen-free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 104 colony-forming units per animal, and three naive pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naive contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. Conclusion: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. Significance and Impact of the Study: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 104 colony-forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.

Monophasic variants of Salmonella enterica serotype Typhimurium, lacking either the second phase H-antigen with the antigenic formula 1,4,[5],12:i:- or lacking the first phase H-antigen with the antigenic formula 1,4,[5],12:-:1,2, and a non-motile variant lacking both flagellar phases were phenotypically and genotypically characterised. The genetic diversity of a collection of 58 isolates of Salmonella enterica 1,4,[5],12:i:- (n = 23), 1,4,[5],12:-:1,2 (n = 17), 1,4,[5],12:-:- (n = 18) mainly from swine, bovine and poultry sources using a novel multiplex real-time PCR to simultaneously target different classes of genes (i.e. 10 loci) was investigated in regards to antimicrobial resistance and pulsed field gel electrophoresis (PFGE) typing. The majority of the S. 1,4,[5],12:i:- strains were genotypically confirmed to be variant of serotype Typhimurium, except two mdh negative isolates. Five fljB positive S. 1,4,[5],12:i:- variants were detected and could be considered as “inconsistent” variant. Moreover, presence of the fliC and fljB amplicons in the phenotypically monophasic S. 1,4,[5],12:-:1,2 and non-motile isolates suggest that their expressions should be blocked by mutation or deletion in the promoter regions linked to the flagellar phase expression. Based on the combination of detection of the following markers: Salmonella Pathogenic Islands (SPI-2 to SPI-5), virulence plasmid (spvC), antimicrobial resistance markers (blaTEM and sul1), Salmonella Genomic island (SGI-1 left junction), the integrase from class 1 integron (intI1) and DT104 16S-23S spacer region, thirteen different genotypes were identified among the 58 isolates. All investigated isolates showed virulence potential as determined by the presence of four SPI markers, which was exhibited in the genotype A9 mainly encountered for S. 1,4,[5],12:-:1,2 isolates. In contrast, high-frequency of markers were encountered for S. 1,4,[5],12:i:- assigned to genotypes B7 gathering almost all 10 markers except SGI-1. Frequencies of SGI-1 markers typically detected in serotype Typhimurium strains differed strongly among the three variants ranging from 0% for fljB negative S. 1,4,[5],12:i:- to 58.8% for S. 1,4,[5],12:-:-. The three variants were clearly phenotypically and genotypically distinct and varied in their resistance-types as well as in their PFGE types and genotypes newly described in this study. This first study on the French collection from non human sources could suggest presence of the Spanish clone in France as well as, to a lesser extent, some “inconsistent” variants exhibiting the resistance-type AM S SSS TE previously described in different European countries.

Listeriosis is a severe infection that mainly affects pregnant women, neonates, and immuno-compromised adults. The commercially available semi-automated repetitive-sequence-based polymerase chain reaction assay system, DiversiLab, has been successfully used for subtyping several species of bacteria. In this article we compare the DiversiLab System with macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), which is currently the gold standard for molecular subtyping of Listeria monocytogenes. We used a panel of 116 human and food L. monocytogenes isolates for the comparative evaluation. Among these isolates, there were 4 pairs of duplicates, 13 strains were epidemiologically related, and the remaining food isolates were epidemiologically unrelated. The isolates of different serotypes represented distinct DiversiLab types (DTs) and ApaI/AscI-PFGE types except for one DT-containing isolates of two serotypes, 4b and 1/2b. The four duplicates displayed the same DT and ApaI/AscI PFGE type demonstrating the good reproducibility of the two methods. The epidemiologically related strains were clustered in the same DT and PFGE type. The Simpson's index of diversity was 0.954; 0.988; 0.994; and 0.998 for DiversiLab, AscI-PFGE, ApaI-PFGE, and AscI/ApaI-PFGE, respectively. Thus, PFGE was more discriminating than DiversiLab. However, for 1/2a serotype strains, six AscI-PFGE, three ApaI-PFGE, and one ApaI/AscI PFGE type were divided into different DTs. DiversiLab enabled a good discrimination between serotype 1/2a strains. DiversiLab is less labor intensive than PFGE and provides results in24 hours compared with 30 hours to 3 days for PFGE from the time a pure culture of the bacteria has been obtained. On the basis of these results, DiversiLab may be useful for tracking the source of contamination in food-processing facilities and their environments. Also, DiversiLab may be more appropriate for long-term epidemiological studies where less discrimination is needed.

Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L. monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L. monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L. monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L. monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L. monocytogenes serotypes.

We describe the characterization of a novel CTX-M β-lactamase from Salmonella enterica . Four S. enterica isolates (three of serotype Westhampton and one of serotype Senftenberg) resistant to extended-spectrum cephalosporins (cefotaxime and ceftazidime) were recovered in 2004 from living cockles in three supermarkets located in distant geographic areas in France, which got their supplies from the same fishery. The isolates were found to produce a novel extended-spectrum β-lactamase (ESBL) belonging to the CTX-M-1 phylogenetic group and named CTX-M-53. The CTX-M-53 β-lactamase harbored the substitution Asp240Gly, like the CTX-M-15 enzyme, which is specifically implicated in a higher catalytic efficiency against ceftazidime. The bla CTX-M-53 gene was located on a mobilizable 11-kb plasmid, pWES-1. The complete sequence of pWES-1 revealed the presence of a novel insertion sequence, IS Sen2 , and an IS 26 element upstream and downstream of the bla CTX-M-53 gene, respectively; however, transposition assays of the bla CTX-M-53 gene were unsuccessful. IS 26 elements may have contributed to the acquisition of the bla CTX-M-53 gene. Interestingly, the mobilization module of the pWES-1 plasmid was similar to that of quinolone resistance plasmids (carrying the qnrS2 gene) from aquatic sources. Although belonging to two serotypes differentiated on the basis of the O-antigen structure (E1 or E4 groups), the isolates were found to be genetically indistinguishable by pulsed-field gel electrophoresis. Multilocus sequence typing showed that the isolates of serotype Westhampton had a sequence type, ST14, common among isolates of serotype Senftenberg. This is the first characterization of the CTX-M-53 ESBL, which represents an additional ceftazidime-hydrolyzing CTX-M enzyme.

The present study provides the first data about the prevalence of Salmonella contamination of broilers and slaughterhouses in the region of Constantine, Algeria. The serotypes and anti-microbial resistance phenotypes of the isolates were determined, and risk factors contributing to the contamination were evaluated. A total of 2490 samples, 1800 originating from 30 broiler farms and 690 from 15 slaughterhouses, were taken during two periods: March 2005-June 2006 and September 2006-March 2007. Salmonella contamination concerned 37% of the broiler farms and 53% of the slaughterhouses. Among the 55 isolates recovered, 10 different serotypes were identified. The most frequently recovered serotypes in both slaughterhouses and breeder farms were S. Hadar (36%, n = 20), S. Virchow (16%, n = 9), S. Infantis (10.9%, n = 6), S. Albany (11%, n = 6) and S. Carnac (7%, n = 4). Isolates belonging to S. Heidelberg (2%, n = 1) and S. Rissen (2%, n = 1) were found only in farms, while those belonging to S. Typhimurium (9%, n = 5), S. Enteritidis (4%, n = 2) and S. Montevideo (2%, n = 1) were recovered only from slaughterhouses. Thirty-nine isolates (80%) were resistant to at least one anti-microbial and 51% were multi-resistant, i.e. resistant to two or more anti-microbial molecules. About 58% (n = 32) were resistant to streptomycin, 36% (n = 20) to tetracyclines, 27% (n = 15) to nalidixic acid, 13% (n = 7) to ofloxacin and one isolate to enrofloxacin. Finally, seven distinct anti-microbial resistance profiles were identified. In parallel, four risk factors were found to be significantly associated with Salmonella contamination. Together with the huge spread of Salmonella in the broiler production chain in Constantine, Algeria, these risk factors highlight the hazards of the broiler channels, particularly linked to poor technical and hygiene practices.

Seventy-eight isolates of Salmonella spp. isolated from beef sampled from the official city slaughterhouse and from retailers in Dakar, Senegal were analyzed using serotyping, antimicrobial testing and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (PFGE). These analyses were done to identify clonal relationships and potential transmission routes in beef channel. XbaI macrorestriction allowed defining 17 genotypes among the six main analyzed serotypes: Salmonella bredeney (3 genotypes), S. muenster (6), S. waycross (1), S. corvallis (3), S. kentucky (1) and S. brandenburg (3). The cross analysis of PFGE profiles and origin of the beef samples reveals a wide range of contamination sources in the beef channel in Dakar. Comparison of PFGE and antimicrobial resistance types shows that the Salmonella contamination sources are equally shared by the slaughterhouse (56% of the isolates) and by the distribution channel (44% of the isolates) by handlings and houseflies.

Nontyphoid Salmonella is one of the main causes of bacterial gastroenteritis worldwide and is responsible for 65% of reported outbreaks of foodborne diseases in France. Serotyping is widely used for isolate preliminary identification, but it poorly discriminates strains. Rapid, efficient molecular subtyping tools have therefore been developed for the investigation of outbreaks. We evaluated the performance of the pulsed-field gel electrophoresis (PFGE) method for discrimination of 31 Salmonella serotypes frequently isolated in France. We set up a genomic database of Salmonella strains isolated from food, animals, the environment, and humans to improve the management of contamination and reactions to foodborne disease outbreaks. We studied 1128 isolates by PFGE, according to the standardized PulseNet protocol. We identified 452 PFGE patterns, 67.5% of which corresponded to a single isolate. The ability of this method to distinguish between isolates was estimated by calculating the Simpson index and the 95% confidence interval. Values obtained ranged between 0.33 (0.11-0.54) to 0.99 (0.96-1.00), depending on serotype. Epidemiological information about isolates was used for analyses of intra- and interserotype diversity results and for determining whether PFGE patterns were linked to the source of the isolate. Clustering analysis of the PFGE patterns obtained confirmed that serotype and PFGE genotype were closely linked. Some PFGE patterns were identified as major patterns, each of these patterns being found in at least 10 isolates. The database generated has already proved its effectiveness in epidemiological investigations in livestock production and foodborne outbreaks.

Staphylococcal food poisoning outbreaks (SFPOs) are frequently reported in France. However, most of them remain unconfirmed, highlighting a need for a better characterization of isolated strains. Here we analyzed the genetic diversity of 112 Staphylococcus aureus strains isolated from 76 distinct SFPOs that occurred in France over the last 30 years. We used a recently developed multiple-locus variable-number tandem-repeat analysis (MLVA) protocol and compared this method with pulsed field gel electrophoresis (PFGE), spa-typing and carriage of genes (se genes) coding for 11 staphylococcal enterotoxins (i.e., SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEP, SER). The strains known to have an epidemiological association with one another had identical MLVA types, PFGE profiles, spa-types or se gene carriage. MLVA, PFGE and spa-typing divided 103 epidemiologically unrelated strains into 84, 80, and 50 types respectively demonstrating the high genetic diversity of S. aureus strains involved in SFPOs. Each MLVA type shared by more than one strain corresponded to a single spa-type except for one MLVA type represented by four strains that showed two different-but closely related-spa-types. The 87 enterotoxigenic strains were distributed across 68 distinct MLVA types that correlated all with se gene carriage except for four MLVA types. The most frequent se gene detected was sea, followed by seg and sei and the most frequently associated se genes were sea-seh and sea-sed-sej-ser. The discriminatory ability of MLVA was similar to that of PFGE and higher than that of spa-typing. This MLVA protocol was found to be compatible with high throughput analysis, and was also faster and less labor-intensive than PFGE. MLVA holds promise as a suitable method for investigating SFPOs and tracking the source of contamination in food processing facilities in real time.

An epidemiological investigation was carried out on one hundredSalmonellaisolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), Insertion Sequence200-PCR (IS200-PCR), and Pulsed Field Gel Electrophoresis (PFGE) resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidalSalmonella. Altogether, our results seem to indicate that nontyphoidal avianSalmonellamay play an important role in human salmonellosis in the Constantine region.

A study was made of Salmonella contamination in beef sampled from a slaughterhouse and from retailers in Dakar, Senegal. The serotypes as well as antibiotic-resistance patterns of the Salmonella isolates were determined. A total of 435 meat samples (236 from the slaughterhouse, 199 from retailers) were tested. Among them, 275 (63%) were positive for Salmonella, 43% (101/236) from the slaughterhouse and 87% (174/199) from the retailers. Furthermore, 97% of the investigated retailers had at least one beef sample contaminated by Salmonella. The 286 Salmonella isolates were divided into 51 serotypes. The most prevalent serotypes were Salmonella bredeney (25%), S. muenster (8%), S. waycross (7%), S. corvallis (4%) and S. kentucky (4%). About 62% of the isolates were resistant to nitrofurans. Resistance rates were lower to streptomycin (22%), sulfamethoxazole (15%), spectinomycin (1%), chloramphenicol (1%), and tetracycline (0,4%) while low-level resistance to quinolones was detected. About 16% of the Salmonella strains were multiresistant to two or more antibiotic families. Finally, ten resistance profiles have been identified. This study shows the huge spread of Salmonella in the beef production chain in Dakar, Senegal. Finally, this study provides the very first data about Salmonella prevalence in sub-saharian Africa.

As a part of our effort in quantitative risk analysis of food-borne diseases, we carried out an epidemiologic study to estimate the prevalence of multidrug resistant (MDR) Salmonella in dairy herds situated in western France. The study population consisted of 489 farms in the region and manure or slurry was sampled from these operations and tested for the Salmonella spp. All strains isolated during the study were serotyped and tested for their antimicrobial susceptibility. Salmonella spp. was isolated from 8.1% (95% confidence interval (CI 95%): 4.5–13.3%) of the sampled herds. The herd prevalence of MDR Salmonella among the sampled herds was 1.9% (CI 95%: 0.5–5.4%). Spatial statistics were used to check for sampling representativeness and to determine if infected herds were clustered spatially.

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.

Nat. Genet. 48, 308–313 (2016); published online 1 February 2016; corrected after print 6 March 2017 In the version of this article initially published, the titles of the x axes in Figure 5b and 5c should have been “Brain/blood CFU ratio” instead of “Blood/brain CFU ratio,” and the title of the z axis in Figure 2c should have been “% of isolates” instead of “Number of isolates.

Nat. Genet. 48, 308–313 (2016); published online 1 February 2016; corrected after print 6 March 2017 In the version of this article initially published, in Figure 2b, in the panel called “CNS infections” the bar of CC3 should have been represented in red and the one of CC121 should have been represented in blue.

In order to assess antimicrobial resistance in Listeria monocytogenes , 202 food and environmental isolates from 1996 to 2006 were tested. Only four strains displayed acquired resistance. Resistance to erythromycin, tetracycline-minocycline, and trimethoprim was evidenced, and the genes erm (B), tet (M), and dfrD , already found in L. monocytogenes , were detected.

Epidemiologic relationships were investigated in 187 ampicillin-resistant Salmonella typhimurium strains (86 human, 101 animal) from >2000 strains isolated in 1994. Of 23 resistance patterns, the most frequent (ampicillin [Am], chloramphenicol [Cm], tetracycline [Tc], streptomycin and spectinomycin [Sm], and sulfonamides [Su]) was found in 69.5% of human and 64.8% of animal isolates. Four beta-lactamase genes were identified, blaTEM (24%), blaPSE-1 (78%), and blaSHV and oxa-2 (each

Milk and dairy products harbour a natural microbial flora and/or other micro-organisms, which vary within the wide range of products available on the French market. The origin of contamination by pathogenic bacteria varies with the type of product and the mode of production and processing. Contamination of milk and dairy products by pathogenic micro-organisms can be of endogenous origin, following excretion from the udder of an infected animal. Contamination may also be of exogenous origin, through direct contact with infected herds or through the environment (e.g. water, personnel). Treatment and processing of milk can inhibit or encourage the multiplication of micro-organisms. The authors describe the relevant aspects of bacterial physiology and ecology, the occurrence of bacteria in dairy products, and the public health significance for each of the principal micro-organisms found in such products. Bacteria most frequently involved are mycobacteria, Brucella sp., Listeria monocytogenes, Staphylococcus aureus and enterobacteria (including toxigenic Escherichia coli and Salmonella). At present, systems of testing and surveillance are required for the control of pathogenic bacteria in milk and dairy products, as specified by regulations currently being developed for all countries in the European Union. Preventive measures should take into account the well-established facts concerning the potential microbiological impact of pathogenic bacteria on milk and dairy products. There should be increased recourse to risk analysis methods to assess the threat to the consumer with regard to the presence of pathogenic bacteria in food.

The European Union Reference Laboratory ( EURL ) for Listeria monocytogenes ( Lm ) collaborates with a network of 35 National Reference Laboratories (NRLs) throughout Europe. Most of these NRLs are in charge of detecting and typing Lm strains from food, environment and animals, which are isolated nationally. The past few years EURL activities have enabled NRLs to reinforce typing capabilities according to standardised protocols. Consequently the need to exchange typing data within the NRL network has emerged. That is why the EURL has recently set up a EURL Lm Database (EURL Lm DB). Each NRL contributes data, which is then shared within the network. Data include strain-typing-results (PFGE and serotyping) and epidemiological information on the strains. This article describes (1) the EURL typing activities that led to the creation of the EURL Lm DB, (2) the different steps involved in developing the EURL Lm DB, and (3) the usefulness of this database for public health. The combined use of this database, with databases on human strains, is being integrated into the European surveillance system of Lm strains circulating throughout Europe. It should improve the detection of this pathogen and provide support for outbreak investigations.