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

Introduction: The 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. Methods: The 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. Results: A 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. Discussion: The 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.

High throughput qPCR analyses suggest that Enterobacterales of French sheep and cow cheese rarely carry genes conferring resistances to critically important antibiotics for human medicine.

Bacteria present in raw milk can carry acquired or intrinsic antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). However, only a few studies have evaluated raw milk cheese as a potential reservoir of ARGs. This study thus aimed at providing new data regarding resistance markers present in raw milk cheese. Sheep (n = 360) and cow (n = 360) cheese samples produced in France were incubated in buffered peptone water supplemented with acriflavin or novobiocin; as corroborated by 16S metabarcoding, samples were enriched in Gram-negative bacteria since Escherichia coli and Hafnia alvei respectively accounted for 40 % and 20 % of the samples' microbiota. Screening of the samples for the presence of 30 ARGs and 16 MGEs by high throughput qPCR array showed that nine ARGs conferring resistances to 1st-generation beta-lactams, aminoglycosides, trimethoprim/sulfonamides and tetracyclines occurred in >75 % of both sheep and cow samples. This is neither surprising nor alarming since these resistance genes are widely spread across the One Health human, animal and environmental sectors. Conversely, genes conferring resistances to last-generations cephalosporins were rarely identified, while those conferring resistances to carbapenems or amikacin, which are restricted to human use, were never detected. Multiple MGEs were detected, the most frequent ones being IncF plasmids, confirming the potential transmission of ARGs. Our results are in line with the few studies of the resistome of milk or milk cheese showing that genes conferring resistances to 1st-generation beta-lactams, aminoglycosides and tetracyclines families are widespread, while those conferring resistances to critically important antibiotics are rare or absent.

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.

High Throughput Screening of Antimicrobial Resistance Genes in Gram-Negative Seafood Bacteria.

From a global view of antimicrobial resistance over different sectors, seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aims to provide new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) show the occurrence of sul-1, ant(3″)-Ia, aph(3')-Ia, strA, strB, dfrA1, qnrA, and blaCTX-M-9 genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates and the presence of MGEs in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. The results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria (ARB) and ARG decreases with increasing distance from potential sources of fecal contamination. This unique and original high throughput micro-array designed for the screening of ARG and MGE in Gram-negative bacteria could be easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.

New genetic biomarkers to differentiate non-pathogenic from clinically relevant Bacillus cereus strains.

OBJECTIVES: Bacillus cereus is responsible for food poisoning and rare but severe clinical infections. The pathogenicity of strains varies from harmless to lethal strains. However, there are currently no markers, either alone or in combination, to differentiate pathogenic from non-pathogenic strains. The objective of the study was to identify new genetic biomarkers to differentiate non-pathogenic from clinically relevant B. cereus strains. METHODS: A first set of 15 B. cereus strains were compared by RNAseq. A logistic regression model with lasso penalty was applied to define combination of genes whose expression was associated with strain pathogenicity. The identified markers were checked for their presence/absence in a collection of 95 B. cereus strains with varying pathogenic potential (food-borne outbreaks, clinical and non-pathogenic). Receiver operating characteristic area under the curve (AUC) analysis was used to determine the combination of biomarkers, which best differentiate between the "disease" versus "non-disease" groups. RESULTS: Seven genes were identified during the RNAseq analysis with a prediction to differentiate between pathogenic and non-pathogenic strains. The validation of the presence/absence of these genes in a larger collection of strains coupled with AUC prediction showed that a combination of four biomarkers was sufficient to accurately discern clinical strains from harmless strains, with an AUC of 0.955, sensitivity of 0.9 and specificity of 0.86. CONCLUSIONS: These new findings help in the understanding of B. cereus pathogenic potential and complexity and may provide tools for a better assessment of the risks associated with B. cereus contamination to improve patient health and food safety.

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.

The cytotoxic potential of Bacillus cereus strains of various origins.

B. cereus is a human pathogen associated with food poisoning leading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum of B. cereus ranges 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 of B. cereus in 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 during B. cereus virulence. 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 the pathogenicity of B. cereus, thereby making it possible to improve both clinical diagnosis and food safety.

Dynamics of mobile genetic elements of Listeria monocytogenes persisting in ready-to-eat seafood processing plants in France.

BACKGROUND: Listeria monocytogenes Clonal 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 of L. monocytogenes to 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 FPEs L. monocytogenes isolates, 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. RESULTS: Food 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 named Listeria genomic island 3 (LGI3), composed by plant-associated loci and chromosomally integrating cadmium-resistance determinants cadA1C. CONCLUSIONS: Genome-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 in L. monocytogenes CCs 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 resident L. monocytogenes in FPEs to prevent contamination of RTE seafood.

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.

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae, V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products.

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.

Validation of standard method EN ISO 19343 for the detection and quantification of histamine in fish and fishery products using high-performance liquid chromatography.

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).

Bacillus cereus, a serious cause of nosocomial infections: Epidemiologic and genetic survey.

Bacillus cereus is the 2nd most frequent bacterial agent responsible for food-borne outbreaks in France and the 3rd in Europe. In addition, local and systemic infections have been reported, mainly describing individual cases or single hospital setting. The real incidence of such infection is unknown and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We performed an extensive study of B. cereus strains isolated from patients and hospital environments from nine hospitals during a 5-year study, giving an overview of the consequences, sources and pathogenic patterns of B. cereus clinical infections. We demonstrated the occurrence of several hospital-cross-contaminations. Identical B. cereus strains were recovered from different patients and hospital environments for up to 2 years. We also clearly revealed the occurrence of inter hospital contaminations by the same strain. These cases represent the first documented events of nosocomial epidemy by B. cereus responsible for intra and inter hospitals contaminations. Indeed, contamination of different patients with the same strain of B. cereus was so far never shown. In addition, we propose a scheme for the characterization of B. cereus based on biochemical properties and genetic identification and highlight that main genetic signatures may carry a high pathogenic potential. Moreover, the characterization of antibiotic resistance shows an acquired resistance phenotype for rifampicin. This may provide indication to adjust the antibiotic treatment and care of patients.

MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012.

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.

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.

A Phylogenetic and Phenotypic Analysis of Salmonella enterica Serovar Weltevreden, an Emerging Agent of Diarrheal Disease in Tropical Regions.

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.

Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity.

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.