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

Transfer of Microorganisms, Including Listeria monocytogenes, from Various Materials to Beef

The quantity of microorganisms that may be transferred to a food that comes into contact with a contaminated surface depends on the density of microorganisms on the surface and on the attachment strengths of the microorganisms on the materials. We made repeated contacts between pieces of meat and various surfaces (stainless steel and conveyor belt materials [polyvinyl chloride and polyurethane]), which were conditioned with meat exudate and then were contaminated with Listeria monocytogenes, Staphylococcus sciuri, Pseudomonas putida, or Comamonas sp. Attachment strengths were assessed by the slopes of the two-phase curves obtained by plotting the logarithm of the number of microorganisms transferred against the order number of the contact. These curves were also used to estimate the microbial population on the surface by using the equation of A. Veulemans, E. Jacqmain, and D. Jacqmain (Rev. Ferment. Ind. Aliment. 25:58-65, 1970). The biofilms were characterized according to their physicochemical surface properties and structures. Their exopolysaccharide-producing capacities were assessed from biofilms grown on polystyrene. The L. monocytogenes biofilms attached more strongly to polymers than did the other strains, and attachment strength proved to be weaker on stainless steel than on the two polymers. However, in most cases, it was the population of the biofilms that had the strongest influence on the total number of CFU detached. Although attachment strengths were weaker on stainless steel, this material, carrying a smaller population of bacteria, had a weaker contaminating capacity. In most cases the equation of Veulemans et al. revealed more bacteria than did swabbing the biofilms, and it provided a better assessment of the contaminating potential of the polymeric materials studied here

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

International audienceThe 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 cm2), 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 concentration

Etude des transferts microbiens par contact depuis des surfaces inertes vers un aliment (application à la situation industrielle des bandes convoyeuses utilisées dans l'industrie de la viande)

DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopic Observation

International audienceFoodborne pathogens are responsible of foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This extracellular matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can enter a viable but nonculturable (VBNC) state. VBNC cells are characterized by a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples, and thus poses a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method with a combination of propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopic Observation

International audienceFoodborne pathogens are responsible of foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This extracellular matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can enter a viable but nonculturable (VBNC) state. VBNC cells are characterized by a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples, and thus poses a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method with a combination of propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain

Tracking antimicrobial resistance indicator genes in wild flatfish from the English Channel and the North Sea area: a One Health concern

International audienceAntimicrobial 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

Phenotypic and genotypic characterization of H2S-positive and H2S-negative strains of Shewanella baltica isolated from spoiled whiting ( Merlangius merlangus )

International audienceFour 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

Optimization of tools for the detection and identification of <i>Cryptocotyle</i> 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

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