Unexplored microbial diversity from 2,500 food metagenomes and links with the human microbiome.

Complex microbiomes are part of the food we eat and influence our own microbiome, but their diversity remains largely unexplored. Here, we generated the open access curatedFoodMetagenomicData (cFMD) resource by integrating 1,950 newly sequenced and 583 public food metagenomes. We produced 10,899 metagenome-assembled genomes spanning 1,036 prokaryotic and 108 eukaryotic species-level genome bins (SGBs), including 320 previously undescribed taxa. Food SGBs displayed significant microbial diversity within and between food categories. Extension to >20,000 human metagenomes revealed that food SGBs accounted on average for 3% of the adult gut microbiome. Strain-level analysis highlighted potential instances of food-to-gut transmission and intestinal colonization (e.g., Lacticaseibacillus paracasei) as well as SGBs with divergent genomic structures in food and humans (e.g., Streptococcus gallolyticus and Limosilactobabillus mucosae). The cFMD expands our knowledge on food microbiomes, their role in shaping the human microbiome, and supports future uses of metagenomics for food quality, safety, and authentication.

Bifidobacterium fermentum sp. nov. and Bifidobacterium aquikefiricola sp. nov., isolated from water kefir.

Four strains, representing two novel Bifidobacterium species, were isolated from water kefir, a fermented beverage. 16S rRNA gene analysis suggested that the novel species share high identities (98.82-98.89%) with Bifidobacterium aquikefiri LMG 28769T. Complete genomes were assembled with a short- and long-read hybrid sequencing approach. In agreement with the 16S rRNA gene analysis, phylogenetics with 117 marker genes places the novel species closest to B. aquikefiri LMG 28769T as well. The isolates have average nucleotide identity (ANI) scores ranging from 81.46 to 84.84% and digital DNA-DNA hybridization (dDDH) scores from 23.9 to 38.5% with the closest related species, as well as ANI scores between the proposed new species of 80.50%, indicating that the isolates represent two novel species. Matrix-assisted laser desorption/ionization-time of flight chemotaxonomic analysis supported the gene-based taxonomic placement. We propose the names Bifidobacterium fermentum sp. nov. and Bifidobacterium aquikefiricola sp. nov. for these novel species within the Bifidobacterium genus. The proposed type strain B. fermentum WK012_4_13T (= LMG 33104T = DSM 116073T; GenBank accession number GCF_041080835.1) has a genome size of 2.43 Mbp, with a G+C content of 56.00 mol%. The proposed type strain for B. aquikefiricola WK041_4_12T (= LMG 33105T = DSM 116074T; GenBank accession number GCF_041080795.1) has a genome size of 2.36 Mbp and a G+C content of 53.94 mol%. B. fermentum cells are Gram-positive staining, non-motile, non-spore-forming, fructose-6-phosphate phosphoketolase (F6PPK)-positive, catalase- and oxidase-negative and bacillary club shaped. B. aquikefiricola cells are Gram-positive staining, non-motile, non-spore-forming, F6PPK-positive, catalase- and oxidase-negative and square rod shaped.

Susceptibility and transcriptomic response to plasma-activated water of Listeria monocytogenes planktonic and sessile cells.

Plasma-Activated Water (PAW) was generated from tap water using a surface dielectric barrier discharge at different discharge power (26 and 36 W) and activation time (5 and 30 min). The inactivation of a three-strain Listeria monocytogenes cocktail in planktonic and biofilm state was evaluated. PAW generated at 36 W-30 min showed the lowest pH and the highest hydrogen peroxide, nitrates, nitrites contents and effectiveness against cells on planktonic state, resulting in 4.6 log reductions after a 15-min treatment. Although the antimicrobial activity in biofilms formed on stainless steel and on polystyrene was lower, increasing the exposure time to 30 min allowed an inactivation >4.5 log cycles. The mechanisms of action of PAW were investigated using chemical solutions that mimic its physico-chemical characteristics and also RNA-seq analysis. The main transcriptomic changes affected carbon metabolism, virulence and general stress response genes, with several overexpressed genes belonging to the cobalamin-dependent gene cluster.

Novel methods of microbiome analysis in the food industry.

The study of the food microbiome has gained considerable interest in recent years, mainly due to the wide range of applications that can be derived from the analysis of metagenomes. Among these applications, it is worth mentioning the possibility of using metagenomic analyses to determine food authenticity, to assess the microbiological safety of foods thanks to the detection and tracking of pathogens, antibiotic resistance genes and other undesirable traits, as well to identify the microorganisms responsible for food processing defects. Metataxonomics and metagenomics are currently the gold standard methodologies to explore the full potential of metagenomes in the food industry. However, there are still a number of challenges that must be solved in order to implement these methods routinely in food chain monitoring, and for the regulatory agencies to take them into account in their opinions. These challenges include the difficulties of analysing foods and food-related environments with a low microbial load, the lack of validated bioinformatics pipelines adapted to food microbiomes and the difficulty of assessing the viability of the detected microorganisms. This review summarizes the methods of microbiome analysis that have been used, so far, in foods and food-related environments, with a specific focus on those involving Next-Generation Sequencing technologies.

Stress resistance associated with multi-host transmission and enhanced biofilm formation at 42 °C among hyper-aerotolerant generalist Campylobacter jejuni.

One of the emerging conundrums of Campylobacter food-borne illness is the bacterial ability to survive stressful environmental conditions. We evaluated the heterogeneity among 90 C. jejuni and 21 C. coli isolates from different sources in Egypt with respect to biofilm formation capabilities (under microaerobic and aerobic atmosphere) and resistance to a range of stressors encountered along the food chain (aerobic stress, refrigeration, freeze-thaw, heat, peracetic acid, and osmotic stress). High prevalence (63%) of hyper-aerotolerant (HAT) isolates was observed, exhibiting also a significantly high tolerance to heat, osmotic stress, refrigeration, and freeze-thaw stress, coupled with high biofilm formation ability which was clearly enhanced under aerobic conditions, suggesting a potential link between stress adaptation and biofilm formation. Most HAT multi-stress resistant and strong biofilm producing C. jejuni isolates belonged to host generalist clonal complexes (ST-21, ST-45, ST-48 and ST-206). These findings highlight the potential role of oxidative stress response systems in providing cross-protection (resistance to other multiple stress conditions) and enhancing biofilm formation in Campylobacter and suggest that selective pressures encountered in hostile environments have shaped the epidemiology of C. jejuni in Egypt by selecting the transmission of highly adapted isolates, thus promoting the colonization of multiple host species by important disease-causing lineages.

Development and validation of a regression model for Listeria monocytogenes growth in roast beefs.

Food business operators are responsible for food safety and assessment of shelf lives for their ready-to-eat products. For assisting them, a customized software based on predictive models, ListWare, is being developed. The aim of this study was to develop and validate a predictive model for the growth of Listeria monocytogenes in sliced roast beef. A challenge study was performed comprising 51 different combinations of variables. The growth curves followed the Baranyi and Roberts model with no clear lag phase and specific growth rates in the range <0.005-0.110 hr-1. A linear regression model was developed based on 528 observations and had an adjusted R-square of 0.80. The significant predictors were storage temperature, sodium lactate, interactions between sodium acetate and temperature, and MAP packaging and temperature. The model was validated in four laboratories in three countries. For conditions where the model predicted up to + log 2 cfu/g Listeria concentration, the observed concentrations were true or below the predicted concentration in 90% of the cases. For the remaining 10%, the roast beef was coated with spices and therefore different from the others. The model will be implemented in ListWare web-application for calculation of "Listeria shelf life".

Selection for Antimicrobial Resistance in Foodborne Pathogens through Exposure to UV Light and Nonthermal Atmospheric Plasma Decontamination Techniques.

This study was aimed at assessing whether the repeated exposure of 12 strains of Salmonella spp., Escherichia coli, and Listeria monocytogenes to alternative nonthermal decontamination techniques with UV light (UV-C) and nonthermal atmospheric plasma (NTAP) may cause the emergence of variants showing increased resistance to clinically relevant antibiotics (ampicillin, cefotaxime, ciprofloxacin, gentamicin, streptomycin, tetracycline, erythromycin, vancomycin, and colistin). UV-C and NTAP treatments were applied on the surface of inoculated brain heart infusion (BHI) agar plates. Survivors were recovered and after 24 h of growth in BHI broth were again subjected to the decontamination treatment; this was repeated for 10 consecutive cycles. A total of 174 strain/decontamination technique/antibiotic combinations were tested, and 12 variant strains with increased resistance to one of the antibiotics studied were identified, with the increases in the MICs in Mueller-Hinton broth ranging from 2- to 256-fold. The variant strains of Salmonella spp. isolated were further characterized through phenotypic screenings and whole-genome sequencing (WGS) analyses. Most changes in susceptibility were observed for antibiotics that act at the level of protein synthesis (aminoglycosides, tetracyclines, and glycylcyclines) or DNA replication (fluoroquinolones), as well as for polymyxins. No changes in resistance to ß-lactams were detected. WGS analyses showed the occurrence of sequence alterations in some antibiotic cellular targets (e.g., gyrA for ciprofloxacin-resistant variants, rpsL for a streptomycin-resistant variant), accompanied by variations in stress response regulators and membrane transporters likely involved in the nonselective efflux of antibiotics, which altogether resulted in a low- to medium-level increase in microbial resistance to several antibiotics.IMPORTANCE The emergence and spread of antibiotic resistance along the food chain can be influenced by the different antimicrobial strategies used from farm to fork. This study evidences that two novel, not yet widely used, nonthermal microbial decontamination techniques, UV light and nonthermal atmospheric plasma, can select variants with increased resistance to various clinically relevant antibiotics, such as ciprofloxacin, streptomycin, tetracycline, and erythromycin. Whole-genome analysis of the resistant variants obtained for Salmonella spp. allowed identification of the genetic changes responsible for the observed phenotypes and suggested that some antimicrobial classes are more susceptible to the cross-resistance phenomena observed. This information is relevant, since these novel decontamination techniques are being proposed as possible alternative green techniques for the decontamination of environments and equipment in food and clinical settings.

A Systematic Review of Bacterial Foodborne Outbreaks Related to Red Meat and Meat Products.

Our investigation focused on foodborne outbreaks related to meat and meat products, published in peer-reviewed journals in the period 1980-2015. Most of the outbreaks, investigated in this study, were caused by Escherichia coli and Salmonella, causing 33 and 21 outbreaks, respectively, mostly in Europe and the United States. In the E. coli outbreaks, the total number of reported cases was 1966, of which 1543 were laboratory confirmed. The number of cases requiring hospitalization was 476, of whom 233 cases had a hemolytic-uremic syndrome (HUS), and the reported deaths were 32. All of the E. coli outbreaks, except four, were caused by serovar O157:H7. The other four outbreaks were caused by the following serovars: O111:H8, O26:H11, O111, and O103:H25. Fresh processed meat products were the category most frequently implicated. In the Salmonella outbreaks, the total number of all reported cases was 2279, of whom 1891 were laboratory confirmed. The number of reported cases requiring hospitalization was 94, and seven were reported dead. Regarding Salmonella, eight serovars caused those outbreaks. The most common serovar causing Salmonella-related outbreaks was Salmonella Typhimurium. The food category most frequently implicated in those outbreaks was raw-cured fermented sausages. Other organisms linked to meat-associated outbreaks, but less frequently reported, were Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Clostridium botulinum, and Listeria monocytogenes. Issues of the burden of outbreaks, the challenges of comparing global outbreaks, food attribution, and how the meat industry works to meet consumer demands while maintaining food safety are discussed.

Antimicrobial resistance and virulence characterization of Staphylococcus aureus and coagulase-negative staphylococci from imported beef meat.

BACKGROUND: The objectives of this study were to characterize the diversity and magnitude of antimicrobial resistance among Staphylococcus species recovered from imported beef meat sold in the Egyptian market and the potential mechanisms underlying the antimicrobial resistance phenotypes including harboring of resistance genes (mecA, cfr, gyrA, gyrB, and grlA) and biofilm formation. RESULTS: The resistance gene mecA was detected in 50% of methicillin-resistant non-Staphylococcus aureus isolates (4/8). Interestingly, our results showed that: (i) resistance genes mecA, gyrA, gyrB, grlA, and cfr were absent in Staphylococcus hominis and Staphylococcus hemolyticus isolates, although S. hominis was phenotypically resistant to methicillin (MR-non-S. aureus) while S. hemolyticus was resistant to vancomycin only; (ii) S. aureus isolates did not carry the mecA gene (100%) and were phenotypically characterized as methicillin- susceptible S. aureus (MSS); and (iii) the resistance gene mecA was present in one isolate (1/3) of Staphylococcus lugdunensis that was phenotypically characterized as methicillin-susceptible non-S. aureus (MSNSA). CONCLUSIONS: Our findings highlight the potential risk for consumers, in the absence of actionable risk management information systems, of imported foods and advice a strict implementation of international standards by different venues such as CODEX to avoid the increase in prevalence of coagulase positive and coagulase negative Staphylococcus isolates and their antibiotic resistance genes in imported beef meat at the Egyptian market.

Effects of high hydrostatic pressure on Escherichia coli ultrastructure, membrane integrity and molecular composition as assessed by FTIR spectroscopy and microscopic imaging techniques.

High hydrostatic pressure (HHP) is a novel food processing technology that is considered as an attractive alternative to conventional heat treatments for the preservation of foods, due to its lethal effects on pathogenic and spoilage microorganisms, while causing minor effects on food quality and sensorial attributes. This study is aimed at investigating how HHP treatments at varying intensities in the range 50-900 MPa affect the viability, membrane integrity, ultrastructure and molecular composition of Escherichia coli. Results of membrane integrity tests (measurement of cellular leakage and monitoring of propidium iodide uptake through fluorescence microscopy) and ultrastructural observations by transmission electron microscopy demonstrated that HHP gave rise to cellular enlargement, membrane damage or detachment, DNA and protein denaturation and loss of intracellular contents. Fourier-transform infrared (FTIR) spectroscopy analyses evidenced minor changes in molecular composition in response to high pressures, which were mostly observed on the spectral region w4 (1200-900 cm-1), mainly informative of carbohydrates and polysaccharides of the cell wall. These findings suggest that exposure of E. coli cells to HHP causes alterations in their physical integrity while producing minor modifications in biochemical cellular composition. The current study increases the knowledge on the mechanisms of E. coli inactivation by HHP and provides valuable information for the design of more effective food preservation regimes based on the integration of mild HHP in combination with other food preservation strategies into a multi-target hurdle technology approach.

Overview of Antimicrobial Resistant ESKAPEE Pathogens in Food Sources and Their Implications from a One Health Perspective.

Antimicrobial resistance is an increasing societal burden worldwide, with ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species and Escherichia coli) pathogens overwhelming the healthcare sectors and more recently becoming predominantly a concern for their persistence in food and food industries, including agricultural settings and animal husbandry environments. The aim of this review is to explore the mechanisms by which the ESKAPEE group gained its multidrug resistance profiles, to analyse their occurrence in different foods and other related reservoirs, including water, and to address the current challenges due to their spread within the food production chain. Moreover, the repertoire of surveillance programmes available focused on monitoring their occurrence, common reservoirs and the spread of antimicrobial resistance are described in this review paper. Evidence from the literature suggests that restricting our scope in relation to multidrug resistance in ESKAPEE pathogens to healthcare and healthcare-associated facilities might actually impede unveiling the actual issues these pathogens can exhibit, for example, in food and food-related reservoirs. Furthermore, this review addresses the need for increasing public campaigns aimed at addressing this challenge, which must be considered in our fight against antimicrobial resistance shown by the ESKAPEE group in food and food-related sectors.

The resistome of the food chain: A One Health perspective.

Antimicrobial resistance (AMR) represents a significant global health problem which challenges Sustainable Development Goal 3 of the United Nations, with growing concerns about the possibility of AMR transmission through the food chain. The indiscriminate use of antimicrobials for the treatment of food production animals and for agricultural crop improvement, in addition to the direct discharge of livestock farm residues to sewage and the use of animal manure in agriculture, are among the factors that can facilitate the selection and transmission of AMR throughout the food chain. The study of food microbiomes has been boosted by the advent of next-generation sequencing techniques, which have enabled gaining in-depth understanding of the diversity of antimicrobial resistance genes present in food and associated environments (the so-called resistome). The aim of this review is to provide an accurate and comprehensive overview of the knowledge currently available on the resistome of the most frequently consumed foods worldwide, from a One Health perspective. To this end, the different metagenomic studies which have been conducted to characterize the resistome of foods are compiled and critically discussed.

In-depth characterization of food and environmental microbiomes across different meat processing plants.

BACKGROUND: Processing environments can be an important source of pathogenic and spoilage microorganisms that cross contaminate meat and meat products. The aim of this study was to characterize the microbiome of raw materials, processing environments and end products from 19 facilities producing different meat products. RESULTS: The taxonomic profiles of the microbial communities evolved along processing, from raw materials to end products, suggesting that food contact (FC) surfaces play an important role in modulating the microbiome of final products. Some species persisted with the highest relative abundance in raw materials, food processing environments and/or in the final product, including species from the genera Pseudomonas, Staphylococcus, Brochothrix, Acinetobacter and Psychrobacter. Processing environments showed a very diverse core microbiota, partially shared with the products. Pseudomonas fragi and Pseudomonas sp. Lz4W (in all sample and facility types) and Brochothrix thermosphacta, Psychrobacter sp. and Psychrobacter sp. P11F6 (in raw materials, FC surfaces and end products) were prominent members of the core microbiota for all facilities, while Latilactobacillus sakei was found as a dominant species exclusively in end products from the facilities producing fermented sausages. Processing environments showed a higher amount of antimicrobial resistance genes and virulence factors than raw materials and end products. One thousand four hundred twenty-one medium/high-quality metagenome-assembled genomes (MAGs) were reconstructed. Of these, 274 high-quality MAGs (completeness > 90%) corresponded to 210 putative new species, mostly found in processing environments. For two relevant taxa in meat curing and fermentation processes (S. equorum and L. sakei, respectively), phylogenetic variation was observed associated with the specific processing facility under study, which suggests that specific strains of these taxa may be selected in different meat processing plants, likely contributing to the peculiar sensorial traits of the end products produced in them. CONCLUSIONS: Overall, our findings provide the most detailed metagenomics-based perspective up to now of the microbes that thrive in meat, meat products and associated environments and open avenues for future research activities to better understand the microbiome functionality and potential contribution to meat quality and safety. Video Abstract.

Inter-facility characterization of bacteria in seafood processing plants: Exploring potential reservoirs of spoilage organisms and the resistome.

A study was conducted in fish processing facilities to investigate the microbial composition, microbial metabolic potential, and distribution of antibiotic resistance genes. Whole metagenomic sequencing was used to analyze microbial communities from different processing rooms, operators and fish products. Taxonomic analyses identified the genera Pseudomonas and Psychrobacter as the most prevalent bacteria. A Principal Component Analysis revealed a distinct separation between fish product and environmental samples, as well as differences between fish product samples from companies processing either Gadidae or Salmonidae fish. Some particular bacterial genera and species were associated with specific processing rooms and operators. Metabolic analysis of metagenome assembled genomes demonstrated variations in microbiota metabolic profiles of microbiota across rooms and fish products. The study also examined the presence of antibiotic-resistance genes in fish processing environments, contributing to the understanding of microbial dynamics, metabolic potential, and implications for fish spoilage.

Microbiome mapping in dairy industry reveals new species and genes for probiotic and bioprotective activities.

The resident microbiome in food industries may impact on food quality and safety. In particular, microbes residing on surfaces in dairy industries may actively participate in cheese fermentation and ripening and contribute to the typical flavor and texture. In this work, we carried out an extensive microbiome mapping in 73 cheese-making industries producing different types of cheeses (fresh, medium and long ripened) and located in 4 European countries. We sequenced and analyzed metagenomes from cheese samples, raw materials and environmental swabs collected from both food contact and non-food contact surfaces, as well as operators' hands and aprons. Dairy plants were shown to harbor a very complex microbiome, characterized by high prevalence of genes potentially involved in flavor development, probiotic activities, and resistance to gastro-intestinal transit, suggesting that these microbes may potentially be transferred to the human gut microbiome. More than 6100 high-quality Metagenome Assembled Genomes (MAGs) were reconstructed, including MAGs from several Lactic Acid Bacteria species and putative new species. Although microbial pathogens were not prevalent, we found several MAGs harboring genes related to antibiotic resistance, highlighting that dairy industry surfaces represent a potential hotspot for antimicrobial resistance (AR) spreading along the food chain. Finally, we identified facility-specific strains that can represent clear microbial signatures of different cheesemaking facilities, suggesting an interesting potential of microbiome tracking for the traceability of cheese origin.

The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits.

BACKGROUND: Artisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese. RESULTS: Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and ß-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product. CONCLUSION: Overall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese. Video Abstract.

Public health aspects of Vibrio spp. related to the consumption of seafood in the EU.

Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae are the Vibrio spp. of highest relevance for public health in the EU through seafood consumption. Infection with V. parahaemolyticus is associated with the haemolysins thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) and mainly leads to acute gastroenteritis. V. vulnificus infections can lead to sepsis and death in susceptible individuals. V. cholerae non-O1/non-O139 can cause mild gastroenteritis or lead to severe infections, including sepsis, in susceptible individuals. The pooled prevalence estimate in seafood is 19.6% (95% CI 13.7-27.4), 6.1% (95% CI 3.0-11.8) and 4.1% (95% CI 2.4-6.9) for V. parahaemolyticus, V. vulnificus and non-choleragenic V. cholerae, respectively. Approximately one out of five V. parahaemolyticus-positive samples contain pathogenic strains. A large spectrum of antimicrobial resistances, some of which are intrinsic, has been found in vibrios isolated from seafood or food-borne infections in Europe. Genes conferring resistance to medically important antimicrobials and associated with mobile genetic elements are increasingly detected in vibrios. Temperature and salinity are the most relevant drivers for Vibrio abundance in the aquatic environment. It is anticipated that the occurrence and levels of the relevant Vibrio spp. in seafood will increase in response to coastal warming and extreme weather events, especially in low-salinity/brackish waters. While some measures, like high-pressure processing, irradiation or depuration reduce the levels of Vibrio spp. in seafood, maintaining the cold chain is important to prevent their growth. Available risk assessments addressed V. parahaemolyticus in various types of seafood and V. vulnificus in raw oysters and octopus. A quantitative microbiological risk assessment relevant in an EU context would be V. parahaemolyticus in bivalve molluscs (oysters), evaluating the effect of mitigations, especially in a climate change scenario. Knowledge gaps related to Vibrio spp. in seafood and aquatic environments are identified and future research needs are prioritised.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 20: Suitability of taxonomic units notified to EFSA until March 2024.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. The TUs in the QPS list were updated based on a verification, against their respective authoritative databases, of the correctness of the names and completeness of synonyms. A new procedure has been established to ensure the TUs are kept up to date in relation to recent taxonomical insights. Of 83 microorganisms notified to EFSA between October 2023 and March 2024 (47 as feed additives, 25 as food enzymes or additives, 11 as novel foods), 75 were not evaluated because: 15 were filamentous fungi, 1 was Enterococcus faecium, 10 were Escherichia coli, 1 was a Streptomyces (all excluded from the QPS evaluation) and 48 were TUs that already have a QPS status. Two of the other eight notifications were already evaluated for a possible QPS status in the previous Panel Statement: Heyndrickxia faecalis (previously Weizmannia faecalis) and Serratia marcescens. One was notified at genus level so could not be assessed for QPS status. The other five notifications belonging to five TUs were assessed for possible QPS status. Akkermansia muciniphila and Actinomadura roseirufa were still not recommended for QPS status due to safety concerns. Rhizobium radiobacter can be recommended for QPS status with the qualification for production purposes. Microbacterium arborescens and Burkholderia stagnalis cannot be included in the QPS list due to a lack of body of knowledge for its use in the food and feed chain and for B. stagnalis also due to safety concerns. A. roseirufa and B. stagnalis have been excluded from further QPS assessment.

Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

Persistence of microbiological hazards in food and feed production and processing environments.

Listeria monocytogenes (in the meat, fish and seafood, dairy and fruit and vegetable sectors), Salmonella enterica (in the feed, meat, egg and low moisture food sectors) and Cronobacter sakazakii (in the low moisture food sector) were identified as the bacterial food safety hazards most relevant to public health that are associated with persistence in the food and feed processing environment (FFPE). There is a wide range of subtypes of these hazards involved in persistence in the FFPE. While some specific subtypes are more commonly reported as persistent, it is currently not possible to identify universal markers (i.e. genetic determinants) for this trait. Common risk factors for persistence in the FFPE are inadequate zoning and hygiene barriers; lack of hygienic design of equipment and machines; and inadequate cleaning and disinfection. A well-designed environmental sampling and testing programme is the most effective strategy to identify contamination sources and detect potentially persistent hazards. The establishment of hygienic barriers and measures within the food safety management system, during implementation of hazard analysis and critical control points, is key to prevent and/or control bacterial persistence in the FFPE. Once persistence is suspected in a plant, a 'seek-and-destroy' approach is frequently recommended, including intensified monitoring, the introduction of control measures and the continuation of the intensified monitoring. Successful actions triggered by persistence of L. monocytogenes are described, as well as interventions with direct bactericidal activity. These interventions could be efficient if properly validated, correctly applied and verified under industrial conditions. Perspectives are provided for performing a risk assessment for relevant combinations of hazard and food sector to assess the relative public health risk that can be associated with persistence, based on bottom-up and top-down approaches. Knowledge gaps related to bacterial food safety hazards associated with persistence in the FFPE and priorities for future research are provided.