Sources and contamination routes of seafood with human pathogenic Vibrio spp.: A Farm-to-Fork approach.

Vibrio spp., known human foodborne pathogens, thrive in freshwater, estuaries, and marine settings, causing vibriosis upon ingestion. The rising global vibriosis cases due to climate change necessitate a deeper understanding of Vibrio epidemiology and human transmission. This review delves into Vibrio contamination in seafood, scrutinizing its sources and pathways. We comprehensively assess the contamination of human-pathogenic Vibrio in the seafood chain, covering raw materials to processed products. A "Farm-to-Fork" approach, aligned with the One Health concept, is essential for grasping the complex nature of Vibrio contamination. Vibrio's widespread presence in natural and farmed aquatic environments establishes them as potential entry points into the seafood chain. Environmental factors, including climate, human activities, and wildlife, influence contamination sources and routes, underscoring the need to understand the origin and transmission of pathogens in raw seafood. Once within the seafood chain, the formation of protective biofilms on various surfaces in production and processing poses significant food safety risks, necessitating proper cleaning and disinfection to prevent microbial residue. In addition, inadequate seafood handling, from inappropriate processing procedures to cross-contamination via pests or seafood handlers, significantly contributes to Vibrio food contamination, thus warranting attention to reduce risks. Information presented here support the imperative for proactive measures, robust research, and interdisciplinary collaboration in order to effectively mitigate the risks posed by human pathogenic Vibrio contamination, safeguarding public health and global food security. This review serves as a crucial resource for researchers, industrials, and policymakers, equipping them with the knowledge to develop biosecurity measures associated with Vibrio-contaminated seafood.

Optimization of tools for the detection and identification of Cryptocotyle metacercariae in fish: Digestion method and viability studies.

Some trematode metacercariae, including marine digeneans belonging to the genus Cryptocotyle, induce black spots in target tissues due to the attraction of fish host melanophores. To promote precise quantification of infection, the counting of black spots has to be confirmed by reliable quantification of metacercariae after tissue digestion. This process ensures the isolation of undamaged parasites for morphological and molecular identification. The aim of this work was to optimize the pepsin digestion protocol and to assess the duration of viability of Cryptocotyle metacercariae in fish post-mortem (pm). Four digestion protocols were compared by measuring the viability rate of metacercariae. The present study shows that the orbital digestion method was the least destructive for metacercariae and allowed better quantification of Cryptocotyle infection. Moreover, morphological identification seemed reliable up to 8 days pm for Cryptocotyle infection.

Morphological and molecular identification of Cryptocotyle lingua metacercariae isolated from Atlantic cod (Gadus morhua) from Danish seas and whiting (Merlangius merlangus) from the English Channel.

Trematode larvae (metacercariae) causing black spot disease occur frequently in gills, fins, skin and the superficial muscle layers of marine fish. Species within the genus Cryptocotyle Lühe, 1899 are frequently associated with this disease. Descriptions of the metacercarial stage are relatively limited and none has hitherto been reported from fish from the English Channel. The present study reports the morphological and molecular identifications of encysted black spot-inducing parasites from whiting (Merlangius merlangus) and Atlantic cod (Gadus morhua) caught respectively from the north coast of France (English Channel) and from Danish sea waters. Metacercariae were characterised morphologically based on microscopic observations and molecularly using Sanger sequencing of fragments of the mitochondrial cox1 gene and rDNA ITS region. Morphological data were compared with available data in the literature. Phylogenetic trees including reference sequences were built to confirm morphological and molecular identifications. This survey constitutes the first description of C. lingua metacercariae in the English Channel ecosystems.

Treatment with disinfectants may induce an increase in viable but non culturable populations of Listeria monocytogenes in biofilms formed in smoked salmon processing environments.

The objectives of this study were 1) to evaluate the impact of two industrial disinfectants on the viability of Listeria monocytogenes populations in biofilm and 2) to investigate the viability state of L. monocytogenes cells present on contact surfaces in the smoked salmon processing environment. In the first step, we cultured mono species and mixed species biofilms containing L. monocytogenes on stainless steel or polyvinyl chloride (PVC) at 8 °C for 48h. The biofilms were then exposed to quaternary ammonium- and hydrogen peroxide-based disinfectants. Residual total populations of L. monocytogenes were measured by qPCR, and viable culturable (VC) cell populations were quantified using standard microbiological culture-based techniques and by a quantitative PCR (qPCR) assay coupled with a propidium monoazide treatment. Decreases in VC populations and the appearance of viable but non culturable (VBNC) populations were observed in response to treatment with the disinfectants. An 8 month sampling campaign in 4 smoked salmon processing plants was also carried out to detect L. monocytogenes in environmental samples. VBNC cells were detected mainly after the cleaning and disinfection operations. This study showed that industrial disinfectants did not inactivate all L. monocytogenes cells on inert surfaces. The presence of VBNC populations of L. monocytogenes in the smoked salmon processing environment is a public health concern.

Tracking antimicrobial resistance indicator genes in wild flatfish from the English Channel and the North Sea area: A one health concern.

Antimicrobial resistance (AMR) is a burgeoning environmental concern demanding a comprehensive One Health investigation to thwart its transmission to animals and humans, ensuring food safety. Seafood, housing bacterial AMR, poses a direct threat to consumer health, amplifying the risk of hospitalization, invasive infections, and death due to compromised antimicrobial treatments. The associated antimicrobial resistance genes (ARGs) in diverse marine species can amass and transmit through various pathways, including surface contact, respiration, and feeding within food webs. Our research, focused on the English Channel and North Sea, pivotal economic areas, specifically explores the occurrence of four proposed AMR indicator genes (tet(A), blaTEM, sul1, and intI1) in a benthic food web. Analyzing 350 flatfish samples' skin, gills, and gut, our quantitative PCR (qPCR) results disclosed an overall prevalence of 71.4% for AMR indicator genes. Notably, sul1 and intI1 genes exhibited higher detection in fish skin, reaching a prevalence of 47.5%, compared to gills and gut samples. Proximity to major European ports (Le Havre, Dunkirk, Rotterdam) correlated with increased AMR gene frequencies in fish, suggesting these ports' potential role in AMR spread in marine environments. We observed a broad dispersion of indicator genes in the English Channel and the North Sea, influenced by sea currents, maritime traffic, and flatfish movements. In conclusion, sul1 and intI1 genes emerge as robust indicators of AMR contamination in the marine environment, evident in seawater and species representing a benthic food web. Further studies are imperative to delineate marine species' role in accumulating and transmitting AMR to humans via seafood consumption. This research sheds light on the urgent need for a concerted effort in comprehending and mitigating AMR risks in marine ecosystems within the context of One Health.

Monitoring indicator genes to assess antimicrobial resistance contamination in phytoplankton and zooplankton communities from the English Channel and the North Sea.

Phytoplankton and zooplankton play a crucial role in marine ecosystems as the basis of the food webs but are also vulnerable to environmental pollutants. Among emerging pollutants, antimicrobial resistance (AMR) is a major public health problem encountered in all environmental compartments. However, the role of planktonic communities in its dissemination within the marine environment remains largely unexplored. In this study, we monitored four genes proposed as AMR indicators (tetA, blaTEM, sul1, and intI1) in phytoplankton and zooplankton samples collected in the English Channel and the North Sea. The indicator gene abundance was mapped to identify the potential sources of contamination. Correlation was assessed with environmental parameters to explore the potential factors influencing the abundance of AMR in the plankton samples. The prevalence in phytoplankton and zooplankton of sul1 and intI1, the most quantified indicator genes, ranged from 63 to 88%. A higher level of phytoplankton and zooplankton carrying these genes was observed near the French and English coasts in areas subjected to anthropogenic discharges from the lands but also far from the coasts. Correlation analysis demonstrated that water temperature, pH, dissolved oxygen and turbidity were correlated to the abundance of indicator genes associated with phytoplankton and zooplankton samples. In conclusion, the sul1 and intI1 genes would be suitable indicators for monitoring AMR contamination of the marine environment, either in phytoplankton and zooplankton communities or in seawater. This study fills a part of the gaps in knowledge about the AMR transport by marine phytoplankton and zooplankton, which may play a role in the transmission of resistance to humans through the marine food webs.

Antimicrobial susceptibility profile and molecular characterization of Vibrio parahaemolyticus strains isolated from imported shrimps.

Vibrio parahaemolyticus is a threat to human health and one of the leading bacterial causes of seafood-borne infection worldwide. This pathogen is autochtonous in the marine environment and is able to acquire antimicrobial resistance (AMR) mechanisms, which is a global concern. However, the emergence of AMR V. parahaemolyticus strains in seafood is still understudied, as interpretation criteria for this species for antimicrobial susceptibility tests are limited in the literature. In this study, we investigated the susceptibility profiles to clinically important antibiotics and the associated genetic determinants of V. parahaemolyticus isolates cultured from imported shrimps. Based on the analysis of the resistance phenotypes of 304 V. parahaemolyticus isolates, we have defined experimental epidemiological cutoff values (COWT) for 14/15 antibiotics tested. We observed that 19.1% of the bacterial isolates had acquired resistance to at least one antibiotic class. The highest number of resistance was associated with tetracycline (14.5% of the strains) and trimethoprim-sulfamethoxazole (3.6%). Moreover, seven strains were multidrug-resistant (MDR, resistant to at least three antibiotic classes). The most frequently identified genes in these strains were aph(3″)-Ib/aph(6)-Id (aminoglycoside resistance), sul2 (sulfonamide), tet(59) (tetracycline), and floR (chloramphenicol). The SXT/R391 family ICE and class 1 integron-integrase genes were detected by PCR in three and one MDR V. parahaemolyticus strains, respectively. Consequently, V. parahaemolyticus in seafood can act as a reservoir of AMR, constituting a health risk for the consumer.IMPORTANCEOur study on "Antimicrobial Resistance Profiles and Genetic Determinants of Vibrio parahaemolyticus Isolates from Imported Shrimps" addresses a critical gap in understanding the emergence of antimicrobial resistance (AMR) in this seafood-associated pathogen. Vibrio parahaemolyticus is a major cause of global seafood-borne infections, and our research reveals that 19.1% of isolates from imported shrimps display resistance to at least one antibiotic class, with multidrug resistance observed in seven strains. Importantly, we establish experimental epidemiological cutoff values for antibiotic susceptibility, providing valuable criteria specific to V. parahaemolyticus. Our findings underscore the potential risk to consumers, emphasizing the need for vigilant monitoring and intervention strategies. This study significantly contributes to the comprehension of AMR dynamics in V. parahaemolyticus, offering crucial insights for global public health. The dissemination of our research through Microbiology Spectrum ensures broad accessibility and impact within the scientific community and beyond.

Antimicrobial resistance and geographical distribution of Staphylococcus sp. isolated from whiting (Merlangius merlangus) and seawater in the English Channel and the North sea.

Staphylococcus is a significant food safety hazard. The marine environment serves as a source of food for humans and is subject to various human-induced discharges, which may contain Staphylococcus strains associated with antimicrobial resistance (AMR). The aim of this study was to assess the occurrence and geographical distribution of AMR Staphylococcus isolates in seawater and whiting (Merlangius merlangus) samples collected from the English Channel and the North Sea. We isolated and identified 238 Staphylococcus strains, including 12 coagulase-positive (CoPs) and 226 coagulase-negative (CoNs) strains. All CoPs isolates exhibited resistance to at least one of the 16 antibiotics tested. Among the CoNs strains, 52% demonstrated resistance to at least one antibiotic, and 7 isolates were classified as multi-drug resistant (MDR). In these MDR strains, we identified AMR genes that confirmed the resistance phenotype, as well as other AMR genes, such as quaternary ammonium resistance. One CoNS strain carried 9 AMR genes, including both antibiotic and biocide resistance genes. By mapping the AMR phenotypes, we demonstrated that rivers had a local influence, particularly near the English coast, on the occurrence of AMR Staphylococcus. The analysis of marine environmental parameters revealed that turbidity and phosphate concentration were implicated in the occurrence of AMR Staphylococcus. Our findings underscore the crucial role of wild whiting and seawater in the dissemination of AMR Staphylococcus within the marine environment, thereby posing a risk to human health.

Genetic population structure of Listeria monocytogenes strains isolated from salmon and trout sectors in France.

Smoked salmon and smoked trout are ready-to-eat and potentially contaminated with the pathogenic bacterium Listeria monocytogenes making them high risk for the consumer. This raises questions about the presence of hypervirulent or persistent strains in the salmon and trout industries. Knowledge of the genetic diversity of circulating strains in these sectors is essential to evaluate the risk associated with this pathogen and improve food safety. We analyzed the genetic structure of 698 strains of L. monocytogenes isolated from 2006 to 2017 in France, based on their serogroup, lineage and clonal complexes (CCs) determined by Multilocus sequence typing (MLST). Most of the CCs were identified by mapping the strains PFGE profiles and a novel high-throughput real-time PCR method for CC identification. We identified thirteen CCs and one sequence type (ST) with variable distribution in salmon and trout samples (food, environment). The three most prevalent CCs were CC121, CC26 and CC204. Strains from ST191 and CC54 were detected for the first time in these sectors, while less than 0.6% of the isolates belonged to the hyper-virulent CC1, CC6 and CC20. No CC was exclusively associated with the salmon sector. This project allowed us to assess the population diversity of CCs of L. monocytogenes in the salmon and trout industries.

Benzalkonium chloride disinfectant residues stimulate biofilm formation and increase survival of Vibrio bacterial pathogens.

Vibrio spp. are opportunistic human and animal pathogens found ubiquitously in marine environments. Globally, there is a predicted rise in the prevalence of Vibrio spp. due to increasing ocean temperatures, which carries significant implications for public health and the seafood industry. Consequently, there is an urgent need for enhanced strategies to control Vibrio spp. and prevent contamination, particularly in aquaculture and seafood processing facilities. Presently, these industries employ various disinfectants, including benzalkonium chloride (BAC), as part of their management strategies. While higher concentrations of BAC may be effective against these pathogens, inadequate rinsing post-disinfection could result in residual concentrations of BAC in the surrounding environment. This study aimed to investigate the adaptation and survival of Vibrio spp. exposed to varying concentrations of BAC residues. Results revealed that Vibrio bacteria, when exposed, exhibited a phenotypic adaptation characterized by an increase in biofilm biomass. Importantly, this effect was found to be strain-specific rather than species-specific. Exposure to BAC residues induced physiological changes in Vibrio biofilms, leading to an increase in the number of injured and alive cells within the biofilm. The exact nature of the "injured" bacteria remains unclear, but it is postulated that BAC might heighten the risk of viable but non-culturable (VBNC) bacteria development. These VBNC bacteria pose a significant threat, especially since they cannot be detected using the standard culture-based methods commonly employed for microbiological risk assessment in aquaculture and seafood industries. The undetected presence of VBNC bacteria could result in recurrent contamination events and subsequent disease outbreaks. This study provides evidence regarding the role of c-di-GMP signaling pathways in Vibrio adaptation mechanisms and suggests that c-di-GMP mediated repression is a potential avenue for further research. The findings underscore that the misuse and overuse of BAC may increase the risk of biofilm development and bacterial survival within the seafood processing chain.

Black spot diseases in seven commercial fish species from the English Channel and the North Sea: infestation levels, identification and population genetics of Cryptocotyle spp.

Fish are often speckled with "black spots" caused by metacercarial trematode infection, inducing a host response. Cryptocotyle spp. (Opisthorchiidae) are among the parasites responsible for this phenomenon. So far, the impact on human health is still unknown. In addition, few publications dealing with black spot recovery, identification, distribution and diversity among commercially important fish are available. Moreover, "black spots" have been observed by fishermen on marine fish, revealing an appreciable but unquantified presence in consumed fish. An epidemiological survey of 1,586 fish from seven commercial species (herring, sprat, whiting, pout, dab, flounder, and plaice) was conducted in the Eastern English Channel and the North Sea in January 2019 and 2020. Encysted metacercariae were found in 325 out of 1,586 fish, with a total prevalence of 20.5%. Intensity of infection varied from 1 to 1,104 parasites. The recorded encysted metacercariae were identified either by microscopic examination or with molecular tools. Partial sequences of the mtDNA cox1 gene and of the rDNA ITS region were obtained. Two species of Cryptocotyle, Cryptocotyle lingua (Creplin, 1825) and Cryptocotyle concava (Creplin, 1825) were found. Metacercariae belonging to other trematode families were also identified. Molecular phylogenetic analysis and haplotype network construction were performed to confirm the identification and to study the potential presence of different populations of Cryptocotyle spp. This survey enabled us to describe the distribution of two species of Cryptocotyle in the English Channel and North Sea ecosystems. The observed differences in infestation levels between fish species and geographical areas will contribute to better understanding of the ecology of these parasites.

Title: Maladies des points noirs chez sept espèces commerciales de poissons de la Manche et de la mer du Nord : niveaux d'infestation, identification et génétique des populations de Cryptocotyle spp. Abstract: Les poissons sont souvent parsemés de « points noirs ¼ causés par une infection par des métacercaires de trématodes induisant une réponse de l'hôte. Les Cryptocotyle spp. (Opisthorchiidae) font partie des parasites responsables de ce phénomène. Jusqu'à présent, leur impact sur la santé humaine est inconnu. De plus, il existe peu de publications traitant de la récupération, l'identification, la distribution et la diversité des « points noirs ¼ parmi les poisons d'importance commerciale. Par ailleurs, des observations de « points noirs ¼ sur les poissons marins ont été constatées par les pêcheurs révélant une présence assez importante mais non quantifiée dans les poissons consommés. Une enquête épidémiologique portant sur 1 586 poissons de sept espèces commerciales (hareng, sprat, merlan, tacaud, limande, flet et plie) a été menée en Manche orientale et en Mer du Nord, en janvier 2019 et 2020. Des métacercaires enkystées ont été trouvées chez 325 poissons parmi 1 586, avec une prévalence totale de 20,5 %. L'intensité de l'infection variait de 1 à 1 104 parasites. Les métacercaires enkystées répertoriées ont été identifiées soit par examen microscopique, soit avec des outils moléculaires. Des séquences partielles du gène cox1 de l'ADNmt et de la région ITS de l'ADNr ont été obtenues. Deux espèces de Cryptocotyle, Cryptocotyle lingua (Creplin, 1825) et Cryptocotyle concava (Creplin, 1825) ont été trouvées. Des métacercaires appartenant à d'autres familles de trématodes ont également été identifiées. Une analyse phylogénétique moléculaire et la construction d'un réseau d'haplotypes ont été effectuées pour confirmer l'identification et étudier la présence potentielle de différentes populations de Cryptocotyle spp. Cette étude a permis de décrire la distribution de deux espèces de Cryptocotyle dans les écosystèmes de la Manche et de la Mer du Nord. Les différences observées dans les niveaux d'infestation entre les espèces de poissons et les zones géographiques contribueront à une meilleure compréhension de l'écologie de ces parasites.

Deuterium isotope probing (DIP) on Listeria innocua: Optimisation of labelling and impact on viability state.

An innovative approach, Raman microspectroscopy coupled with deuterium isotope probing (Raman-DIP), can be used to evaluate the metabolism of deuterated carbon source in bacteria and also to presume different anabolic pathways. This method requires the treatment of cells with heavy water that could affect the bacterial viability state at higher concentration. In this study, we evaluated the effect of heavy water incorporation on the viability state of Listeria innocua cells. We exposed the L. innocua suspensions to different heavy water concentrations (0%, 25%, 50% and 75%) from 30 minutes to 72 h of incubation times at 37°C. The total, viable and viable culturable populations were quantified by qPCR, PMA-qPCR and plate count agar respectively. We analyzed heavy water incorporation by Raman-DIP. The exposure of L. innocua cells to different concentrations of heavy water did not alter their cell viability to 24 h incubation time. In addition, the maximum intensity for C-D band, specific for the incorporation of heavy water, was reached after 2 h of exposure in a media containing 75% v/v D2O but an early detection of the labelling was possible at t = 1 h 30 min. In conclusion, the use of D2O as a metabolic marker was validated and can be developed for the detection of L. innocua cell viability state.

Identification by High-Throughput Real-Time PCR of 30 Major Circulating Listeria monocytogenes Clonal Complexes in Europe.

Listeria monocytogenes is a ubiquitous bacterium that causes a foodborne illness, listeriosis. Most strains can be classified into major clonal complexes (CCs) that account for the majority of outbreaks and sporadic cases in Europe. In addition to the 20 CCs known to account for the majority of human and animal clinical cases, 10 CCs are frequently reported in food production, thereby posing a serious challenge for the agrifood industry. Therefore, there is a need for a rapid and reliable method to identify these 30 major CCs. The high-throughput real-time PCR assay presented here provides accurate identification of these 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations, along with the molecular serogroup of a strain. Based on the BioMark high-throughput real-time PCR system, our assay analyzes 46 strains against 40 real-time PCR arrays in a single experiment. This European study (i) designed the assay from a broad panel of 3,342 L. monocytogenes genomes, (ii) tested its sensitivity and specificity on 597 sequenced strains collected from 24 European countries, and (iii) evaluated its performance in the typing of 526 strains collected during surveillance activities. The assay was then optimized for conventional multiplex real-time PCR for easy implementation in food laboratories. It has already been used for outbreak investigations. It represents a key tool for assisting food laboratories to establish strain relatedness with human clinical strains during outbreak investigations and for helping food business operators by improving their microbiological management plans. IMPORTANCE Multilocus sequence typing (MLST) is the reference method for Listeria monocytogenes typing but is expensive and takes time to perform, from 3 to 5 days for laboratories that outsource sequencing. Thirty major MLST clonal complexes (CCs) are circulating in the food chain and are currently identifiable only by sequencing. Therefore, there is a need for a rapid and reliable method to identify these CCs. The method presented here enables the rapid identification, by real-time PCR, of 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations. The assay was then optimized on different conventional multiplex real-time PCR systems for easy implementation in food laboratories. The two assays will be used for frontline identification of L. monocytogenes isolates prior to whole-genome sequencing. Such assays are of great interest for all food industry stakeholders and public agencies for tracking L. monocytogenes food contamination.

Occurrence of Indicator Genes of Antimicrobial Resistance Contamination in the English Channel and North Sea Sectors and Interactions With Environmental Variables.

The marine environment is a potential natural reservoir of antimicrobial resistance genes (ARGs), subject to anthropogenic effluents (wastewater, industrial, and domestic), and known as a final receiving system. The aim of this study was to investigate the abundance and geographical distribution of the three blaTEM , sul1, and intI1 genes, proposed as indicators of contamination to assess the state of antimicrobial resistance in environmental settings, added to the tetA gene and the microbial population (tuf gene) in the English Channel and North Sea areas. Bacterial DNA was extracted from 36 seawater samples. The abundance of these genes was determined by quantitative PCR (qPCR) and was analyzed in association with environmental variables and geographical locations to determine potential correlations. The blaTEM and tetA genes were quantified in 0% and 2.8% of samples, respectively. The sul1 and intI1 genes were detected in 42% and 31% of samples, respectively, with an apparent co-occurrence in 19% of the samples confirmed by a correlation analysis. The absolute abundance of these genes was correlated with the microbial population, with results similar to the relative abundance. We showed that the sul1 and intI1 genes were positively correlated with dissolved oxygen and turbidity, while the microbial population was correlated with pH, temperature and salinity in addition to dissolved oxygen and turbidity. The three tetA, sul1, and intI1 genes were quantified in the same sample with high abundances, and this sample was collected in the West Netherlands coast (WN) area. For the first time, we have shown the impact of anthropogenic inputs (rivers, man-made offshore structures, and maritime activities) and environmental variables on the occurrence of three indicators of environmental contamination by antimicrobial resistance in the North Sea and English Channel seawaters.

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.

A review of molecular identification tools for the opisthorchioidea.

The superfamily Opisthorchioidea encompasses the families Cryptogonimidae, Opisthorchiidae and Heterophyidae. These parasites depend on the aquatic environment and include marine and freshwater species. Some species, such as Clonorchis sinensis and Opisthorchis viverrini, have a high impact on public health with millions of infected people worldwide and have thus been the object of many studies and tool developments. However, for many species, tools for identification and detection are scarce. Although morphological descriptions have been used and are still important, they are often not efficient on the immature stages of these parasites. Thus, during the past few decades, molecular approaches for parasite identification have become commonplace. These approaches are efficient, quick and reliable. Nonetheless, for some parasites of the superfamily Opisthorchioidea, reference genomic data are limited. This study reviews available genetic data and molecular tools for the identification and/or the detection of this superfamily. Molecular data on this superfamily are mostly based on mitochondrial and ribosomal gene sequence analyses, especially on the cytochrome c oxidase subunit 1 gene and internal transcribed spacer regions respectively.

Comparison of the performance of the biofilm sampling methods (swab, sponge, contact agar) in the recovery of Listeria monocytogenes populations considering the seafood environment conditions.

AIMS: The aim of this study was to evaluate the performance of sampling methods [contact plates, sponges, and swabs] in the recovery of biofilm Listeria monocytogenes populations considering the seafood environment conditions (nature of conditioning, of materials and bacterial species). METHODS AND RESULTS: Different materials (stainless steel, polyvinyl chloride, polyurethane) were conditioned with two fish filtrates, the ready-to-eat the most consumed in Europe (smoked salmon, cod). After, we added the suspension of Listeria monocytogenes, alone or with Pseudomonas fluorescens or Carnobacterium strains, and incubated for 48 h at 8 °C. Then, the 48 h-biofilms were sampled with different methods (contact plates, sponges, and swabs). The cultivable bacterial populations were enumerated on agar, while the L. monocytogenes total and viable populations were quantified by qPCR and propidium monoazide-qPCR (PMA-qPCR), respectively. The amount of L. monocytogenes in biofilms was affected only by the nature of the conditioning with lowest adherent bacteria with cod versus with smoked salmon conditioning. Considering the amount of total population, the swab displayed the lowest values versus the sponges and the contact plates. An explanation was that the observations of the swab by microscopy showed the bacteria trapped within it. The recovery of cultivable bacterial populations was not significantly different with the three sampling methods. On the contrary, we showed that the VBNC populations were only detached by two of three methods (contact plates, sponges) while for the dead populations, those were contact plates and swabs. CONCLUSIONS: The nature of the conditioning influenced the amount of the bacteria in biofilms. And the performance of the recovery of the bacterial populations (dead, VBNC, cultivable) was dependent on the methods used. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the seafood environmental conditions influenced the biofilm formation and the assessment of the efficiency of cleaning and disinfectant operations could be significantly affected by the used sampling methods.

European survey and evaluation of sampling methods recommended by the standard EN ISO 18593 for the detection of Listeria monocytogenes and Pseudomonas fluorescens on industrial surfaces.

The ready-to-eat products can be contaminated during processing by pathogen or spoilage bacteria, which persist in the industrial environment. Some bacterial species are able to form biofilms which protect them from environmental conditions. To check the bacterial contamination of the surfaces in the food industries, the professionals must regularly use surface sampling methods to detect the pathogen such as Listeria monocytogenes or the spoilage such as Pseudomonas fluorescens. In 2010, we designed and carried out a European survey to collect surface sampling information to detect or enumerate L. monocytogenes in food processing plants. A total of 137 questionnaires from 14 European Union Member States were returned. The outcome of this survey showed that the professionals preferred friction sampling methods with gauze pad, swab and sponges versus contact sampling methods. After this survey, we compared the effectiveness of these three friction sampling methods and the contact plates, as recommended in the standard EN ISO 18593 that was revised in 2018, on the recovery of L. monocytogenes and of P. fluorescens in mono-specie biofilms. This study showed no significant difference between the effectiveness of the four sampling methods to detach the viable and culturable bacterial population of theses mono-specie biofilms.

Construction and analysis of fractional multifactorial designs to study attachment strength and transfer of Listeria monocytogenes from pure or mixed biofilms after contact with a solid model food.

The aim of this study was to establish which of seven factors influence the adhesion strength and hence bacterial transfer between biofilms containing Listeria monocytogenes (pure and two-species biofilms) and tryptone soya agar (TSA) as a solid organic surface. The two-species biofilms were made of L. monocytogenes and one of the following species of bacteria: the nonpathogenic organisms Kocuria varians, Pseudomonas fluorescens, and Staphylococcus sciuri and CCL 63, an unidentified gram-negative bacterium isolated from the processing plant environment. We used biofilms prepared under conditions simulating open surfaces in meat-processing sites. The biofilm's adhesion strength and population were evaluated by making 12 contacts on a given whole biofilm (4.5 cm(2)), using a new slice of a sterilized TSA cylinder for each contact, and plotting the logarithm CFU . cm(-2) detached by each contact against the contact number. Three types of detachment kinetics were observed: biphasic kinetics, where the first slope may be either positive or negative, and monophasic kinetics. The bacteria that resisted a chlorinated alkaline product and a glutaraldehyde- and quaternary ammonium-based disinfectant had greater adhesion strengths than those determined for untreated biofilms. One of the four non-Listeria strains studied, Kocuria varians CCL 56, favored both the attachment and detachment of L. monocytogenes. The stainless steel had smaller bacterial populations than polymer materials, and non-Listeria bacteria adhered to it less strongly. Our results helped to evaluate measures aimed at controlling the immediate risk, linked to the presence of a large number of CFU in a foodstuff, and the delayed risk, linked to the persistence of L. monocytogenes and the occurrence of slightly contaminated foods that may become dangerous if L. monocytogenes multiplies during storage. Cleaning and disinfection reduce the immediate risk, while reducing the delayed risk should be achieved by lowering the adhesion strength, which the sanitizers used here cannot do at low concentrations.