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.

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.

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.

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.

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.

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.

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.

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.

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.

Microbiome mapping in beef processing reveals safety-relevant variations in microbial diversity and genomic features.

The microbiome of surfaces along the beef processing chain represents a critical nexus where microbial ecosystems play a pivotal role in meat quality and safety of end products. This study offers a comprehensive analysis of the microbiome along beef processing using whole metagenomics with a particular focus on antimicrobial resistance and virulence-associated genes distribution. Our findings highlighted that microbial communities change dynamically in the different steps along beef processing chain, influenced by the specific conditions of each micro-environment. Brochothrix thermosphacta, Carnobacterium maltaromaticum, Pseudomonas fragi, Psychrobacter cryohalolentis and Psychrobacter immobilis were identified as the key species that characterize beef processing environments. Carcass samples and slaughterhouse surfaces exhibited a high abundance of antibiotic resistance genes (ARGs), mainly belonging to aminoglycosides, ß-lactams, amphenicols, sulfonamides and tetracyclines antibiotic classes, also localized on mobile elements, suggesting the possibility to be transmitted to human pathogens. We also evaluated how the initial microbial contamination of raw beef changes in response to storage conditions, showing different species prevailing according to the type of packaging employed. We identified several genes leading to the production of spoilage-associated compounds, and highlighted the different genomic potential selected by the storage conditions. Our results suggested that surfaces in beef processing environments represent a hotspot for beef contamination and evidenced that mapping the resident microbiome in these environments may help in reducing meat microbial contamination, increasing shelf-life, and finally contributing to food waste restraint.

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.

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.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 19: Suitability of taxonomic units notified to EFSA until September 2023.

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. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was Enterococcus faecium, 5 were Escherichia coli, 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: Ensifer adhaerens and Heyndrickxia faecalis did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and Burkholderia ubonensis also due to its ability to generate biologically active compounds with antimicrobial activity; Klebsiella pneumoniae, Serratia marcescens and Pseudomonas putida due to safety concerns. K. pneumoniae is excluded from future QPS evaluations. Chlamydomonas reinhardtii is recommended for QPS status with the qualification 'for production purposes only'; Clostridium tyrobutyricum is recommended for QPS status with the qualification 'absence of genetic determinants for toxigenic activity'; Candida oleophila has been added as a synonym of Yarrowia lipolytica. The Panel clarifies the extension of the QPS status for genetically modified strains.

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.

Exploitation of microbial activities at low pH to enhance planetary health.

Awareness is growing that human health cannot be considered in isolation but is inextricably woven with the health of the environment in which we live. It is, however, under-recognized that the sustainability of human activities strongly relies on preserving the equilibrium of the microbial communities living in/on/around us. Microbial metabolic activities are instrumental for production, functionalization, processing, and preservation of food. For circular economy, microbial metabolism would be exploited to produce building blocks for the chemical industry, to achieve effective crop protection, agri-food waste revalorization, or biofuel production, as well as in bioremediation and bioaugmentation of contaminated areas. Low pH is undoubtedly a key physical-chemical parameter that needs to be considered for exploiting the powerful microbial metabolic arsenal. Deviation from optimal pH conditions has profound effects on shaping the microbial communities responsible for carrying out essential processes. Furthermore, novel strategies to combat contaminations and infections by pathogens rely on microbial-derived acidic molecules that suppress/inhibit their growth. Herein, we present the state-of-the-art of the knowledge on the impact of acidic pH in many applied areas and how this knowledge can guide us to use the immense arsenal of microbial metabolic activities for their more impactful exploitation in a Planetary Health perspective.

Statement on how to interpret the QPS qualification on 'acquired antimicrobial resistance genes'.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms intended for use in the food or feed chains. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications' which should be assessed at strain and/or product level by EFSA's Scientific Panels. The generic qualification 'the strains should not harbour any acquired antimicrobial resistance (AMR) genes to clinically relevant antimicrobials' applies to all QPS bacterial TUs. The different EFSA risk assessment areas use the same approach to assess the qualification related to AMR genes. In this statement, the terms 'intrinsic' and 'acquired' AMR genes were defined for the purpose of EFSA's risk assessments, and they apply to bacteria used in the food and feed chains. A bioinformatic approach is proposed for demonstrating the 'intrinsic'/'acquired' nature of an AMR gene. All AMR genes that confer resistance towards 'critically important', 'highly important' and 'important' antimicrobials, as defined by the World Health Organisation (WHO), found as hits, need to be considered as hazards (for humans, animals and environment) and need further assessment. Genes identified as responsible for 'intrinsic' resistance could be considered as being of no concern in the frame of the EFSA risk assessment. 'Acquired' AMR genes resulting in a resistant phenotype should be considered as a concern. If the presence of the 'acquired' AMR gene is not leading to phenotypic resistance, further case-by-case assessment is necessary.

Sequencing-based analysis of the microbiomes of Spanish food processing facilities reveals environment-specific variation in the dominant taxa and antibiotic resistance genes.

In the last years, advances in high throughput sequencing technologies have opened the possibility to broaden environmental monitoring activities in facilities processing food, offering expanded opportunities for characterizing in an untargeted manner the microbiome and resistome of foods and food processing environments (FPE) with huge potential benefits in food safety management systems. Here the microbiome and resistome of FPE from slaughterhouses (n = 3), dairy (n = 12) and meat (n = 10) processing plants were assessed through whole metagenome sequencing of 2 composite samples for each facility, comprising 10 FPE swabs taken from food contact surfaces and 10 FPE samples from non-food contact surfaces, respectively. FPE from slaughterhouses had more diverse microbiomes and resistomes, while FPE from dairy processing plants showed the highest ß-dispersion, consistent with a more heterogeneous microbiome and resistome composition. The predominant bacterial genera depended on the industry type, with Pseudomonas and Psychrobacter being highly dominant in surfaces from slaughterhouses and meat industries, while different lactic acid bacteria predominated in dairy industries. The most abundant antimicrobial resistance genes (ARG) found were associated with resistance to aminoglycosides, tetracyclines and quaternary ammonium compounds (QAC). ARGs relating to resistance to aminoglycosides and tetracyclines were significantly more prevalent in slaughterhouses than in food processing plants, while QAC resistance genes were particularly abundant in some food contact surfaces from dairy and meat processing plants, suggesting that daily sanitation under suboptimal conditions may be selecting for persistent microbiota tolerant to these biocides in some facilities. The taxonomic mapping of ARG pointed to specific bacterial genera, such as Escherichia, Bacillus, or Staphylococcus, as carriers of the most relevant resistance determinants. About 63% of all ARG reads were assigned to contigs classified as plasmid-associated, indicating that the resistome of FPE may be strongly shaped through the spread of mobile genetic elements. Overall, the relevance of FPE as reservoirs of ARG was confirmed and it was demonstrated that next generation sequencing technologies allowing a deep characterisation of sources and routes of spread of microorganisms and antimicrobial resistance determinants in food industry settings hold promise to be integrated in monitoring and food safety management programmes.

Selection of lactic acid bacteria as biopreservation agents and optimization of their mode of application for the control of Listeria monocytogenes in ready-to-eat cooked meat products.

In order to meet consumers´ demands for more natural foods and to find new methods to control foodborne pathogens in them, research is currently being focused on alternative preservation approaches, such as biopreservation with lactic acid bacteria (LAB). Here, a collection of lactic acid bacteria (LAB) isolates was characterized to identify potential biopreservative agents. Six isolates (one Lactococcus lactis, one Lacticaseibacillus paracasei and four Lactiplantibacillus plantarum) were selected based on their antimicrobial activity in in vitro assays. Whole genome sequencing showed that none of the six LAB isolates carried known virulence factors or acquired antimicrobial resistance genes, and that the L. lactis isolate was potentially a nisin Z producer. Growth of L. monocytogenes was successfully limited by L. lactis ULE383, L. paracasei ULE721 and L. plantarum ULE1599 throughout the shelf-life of cooked ham, meatloaf and roasted pork shoulder. These LAB isolates were also applied individually or as a cocktail at different inoculum concentrations (4, 6 and 8 log10 CFU/g) in challenge test studies involving cooked ham, showing a stronger anti-Listerial activity when a cocktail was used at 8 log10 CFU/g. Thus, a reduction of up to ~5.0 log10 CFU/g in L. monocytogenes growth potential was attained in cooked ham packaged under vacuum, modified atmosphere packaging or vacuum followed by high pressure processing (HPP). Only minor changes in color and texture were induced, although there was a significant acidification of the product when the LAB cultures were applied. Remarkably, this acidification was delayed when HPP was applied to the LAB inoculated batches. Metataxonomic analyses showed that the LAB cocktail was able to grow in the cooked ham and outcompete the indigenous microbiota, including spoilage microorganisms such as Brochothrix. Moreover, none of the batches were considered unacceptable in a sensory evaluation. Overall, this study shows the favourable antilisterial activity of the cocktail of LAB employed, with the combination of HPP and LAB achieving a complete inhibition of the pathogen with no detrimental effects in physico-chemical or sensorial evaluations, highlighting the usefulness of biopreservation approaches involving LAB for enhancing the safety of cooked meat products.

Assessment on the efficacy of methods 2 to 5 and method 7 set out in Commission Regulation (EU) No 142/2011 to inactivate relevant pathogens when producing processed animal protein of porcine origin intended to feed poultry and aquaculture animals.

An assessment was conducted on the level of inactivation of relevant pathogens that could be present in processed animal protein of porcine origin intended to feed poultry and aquaculture animals when methods 2 to 5 and method 7, as detailed in Regulation (EU) No 142/2011, are applied. Five approved scenarios were selected for method 7. Salmonella Senftenberg, Enterococcus faecalis, spores of Clostridium perfringens and parvoviruses were shortlisted as target indicators. Inactivation parameters for these indicators were extracted from extensive literature search and a recent EFSA scientific opinion. An adapted Bigelow model was fitted to retrieved data to estimate the probability that methods 2 to 5, in coincidental and consecutive modes, and the five scenarios of method 7 are able to achieve a 5 log10 and a 3 log10 reduction of bacterial indicators and parvoviruses, respectively. Spores of C. perfringens were the indicator with the lowest probability of achieving the target reduction by methods 2 to 5, in coincidental and consecutive mode, and by the five considered scenarios of method 7. An expert knowledge elicitation was conducted to estimate the certainty of achieving a 5 log10 reduction of spores of C. perfringens considering the results of the model and additional evidence. A 5 log10 reduction of C. perfringens spores was judged: 99-100% certain for methods 2 and 3 in coincidental mode; 98-100% certain for method 7 scenario 3; 80-99% certain for method 5 in coincidental mode; 66-100% certain for method 4 in coincidental mode and for method 7 scenarios 4 and 5; 25-75% certain for method 7 scenario 2; and 0-5% certain for method 7 scenario 1. Higher certainty is expected for methods 2 to 5 in consecutive mode compared to coincidental mode.