Search

Abstract Although organic light-emitting diodes (OLEDs) are considered a mature technology, further enhancements in their efficiency are of paramount importance for advancing their incorporation in high-quality displays and flexible, wearable, electronic devices. In this regard, we propose an innovative approach, focusing on strategic modifications to the hole transport layer (HTL) through the integration of core-shell nanoparticles. Silver nanoparticles (Ag-NPs) encapsulated in a tungsten polyoxometalate compound (POM) are embedded within the prototype poly(3,4-ethylenedioxythiophene)-poly(styrenesulphonate) (PEDOT:PSS) to form the modified HTL. Our work reveals the pivotal plasmonic role of Ag-NPs in enhancing OLED device performance based on commercially available conjugated polymers. Comprehensive analyses, including UV–Vis absorption spectroscopy, atomic force microscopy, photoluminescence spectroscopy, and electrical measurements, confirm the influence of the POM encapsulated Ag-NPs on improving the device efficiency. This is attributed to the synergistic influence of enhanced hole injection and conductivity and beneficial optical effects (i.e. the Localized Surface Plasmon Resonance (LSPR) and, likely, light scattering of the POM-Ag NPs in the core-shell configuration, depending on their diameter), contributing to enhanced carrier balance and exciton recombination rate. Comparison with POM gold NPs (POM-Au NPs) highlights the distinct advantages of POM-Ag NPs. Our work reveals the potential of this innovative approach to contribute to the evolution of high-performance OLEDs, ensuring a visually compelling and efficient future.

Peracetic acid (PAA), a strong oxidizing agent, has been widely used as a disinfectant in food processing settings as it does not produce harmful chlorinated by-products. In the present study, the transcriptional response of Listeria monocytogenes to a sub-lethal concentration of PAA (2.5 ppm) was assessed using RNA-sequencing (RNA-seq). Our analysis revealed 12 differentially expressed protein-coding genes, of which nine were upregulated (ohrR, ohrA, rpsN, lmo0637, lmo1973, fur, lmo2492, zurM, and lmo1007), and three were down-regulated (argG, lmo0604 and lmo2156) in PAA-treated samples compared to the control samples. A non-coding small RNA gene (rli32) was also found to be down-regulated. In detail, the organic peroxide toxicity protection (OhrA-OhrR) system, the metal homeostasis genes fur and zurM, the SbrE-regulated lmo0636-lmo0637 operon and a carbohydrate phosphotransferase system (PTS) operon component were induced under exposure of L. monocytogenes to PAA. Hence, this study identified key elements involved in the primary response of L. monocytogenes to oxidative stress caused by PAA, including the expression of the peroxide detoxification system and fine-tuning the levels of redox-active metals in the cell. The investigation of the molecular mechanism of PAA response in L. monocytogenes is of utmost importance for the food industry, as residual PAA can lead to stress tolerance in pathogens.

Abstract The objective of this opinion was to determine if any wild caught fish species, originating from specific fishing grounds and consumed in the EU/EFTA could be considered free of zoonotic parasites. In this Opinion the term ‘fishery products’ only refers to fresh finfish. As there are multiple fish species and numerous potential parasites, Anisakis sp. was used as an indicator of zoonotic parasites in marine areas. This parasite species is particularly suited as it is common in marine environments, capable of infecting multiple fish species and is the subject of the majority of published studies. On the rare occasion where Anisakis sp. data were not available, or all tests were negative, other parasites such as Contracaecum osculatum (s.l.) and/or Phocanema spp. were considered. In freshwater systems, all zoonotic parasites were investigated. Consumption, import and landing data were used to determine the most relevant fish species and, where possible, the source fishing areas were identified. The most commonly consumed wild caught fish species in the EU/EFTA include tuna, cod, Alaskan pollock, hake, herring, sardines, mackerel, trout and saithe. Although the majority of these fish are caught in the Atlantic Ocean, the Mediterranean and the Black Sea (37) as well as several areas in the Indian Ocean, imported fish may originate from any global fishing areas, with the exception of Antarctica. Based on the data, at least one zoonotic parasite has been reported in at least one fish species in each of the FAO marine fishing areas. Thus, due to relative low fish host specificity of the zoonotic parasites, the panel concluded that all wild caught fish species may be exposed to and infected with zoonotic parasites. The same applies to freshwater fishing areas, with many areas having multiple studies reporting the presence of zoonotic parasites in the wild caught fish species.

Abstract 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 81 microorganisms notified to EFSA between October 2023 and March 2024 (45 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 46 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.

Abstract The European Commission requested an estimation of the BSE risk (C‐, L‐ and H‐BSE) from gelatine and collagen derived from ovine, caprine or bovine bones, and produced in accordance with Regulation (EC) No 853/2004, or Regulation (EC) No 1069/2009 and its implementing Regulation (EU) No 142/2011. A quantitative risk assessment was developed to estimate the BSE infectivity, measured in cattle oral infectious dose 50 (CoID50), in a small size batch of gelatine including one BSE‐infected bovine or ovine animal at the clinical stage. The model was built on a scenario where all ruminant bones could be used for the production of gelatine and high‐infectivity tissues remained attached to the skull (brain) and vertebral column (spinal cord). The risk and exposure pathways defined for humans and animals, respectively, were identified. Exposure routes other than oral via food and feed were considered and discussed but not assessed quantitatively. Other aspects were also considered as integrating evidence, like the epidemiological situation of the disease, the species barrier, the susceptibility of species to BSE and the assumption of an exponential dose–response relationship to determine the probability of BSE infection in ruminants. Exposure to infectivity in humans cannot be directly translated to risk of disease because the transmission barrier has not yet been quantified, although it is considered to be substantial, i.e. much greater amounts of infectivity would be needed to successfully infect a human and greater in the oral than in the parenteral route of exposure. The probability that no new case of BSE in the cattle or small ruminant population would be generated through oral exposure to gelatine made of ruminant bones is 99%–100% (almost certain) This conclusion is based on the current state of knowledge, the epidemiological situation of the disease and the current practices, and is also valid for collagen.

Abstract 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.

In this pilot study, we compared the metagenomic profiles of different types of artisanal fermented meat products collected in Italy, Greece, Portugal, and Morocco to investigate their taxonomic profile, also in relation to the presence of foodborne pathogens and antimicrobial resistance genes. In addition, technical replicates of the same biological sample were tested to estimate the reproducibility of shotgun metagenomics. The taxonomic analysis showed a high level of variability between different fermented meat products at both the phylum and genus levels. Staphylococcus aureus was identified with the highest abundance in Italian fermented meat; Escherichia coli in fermented meat from Morocco; Salmonella enterica in fermented meat from Greece; Klebsiella pneumoniae and Yersinia enterocolitica in fermented meat from Portugal. The fungi Aspergillus, Neosartoria, Emericella, Penicillum and Debaryomyces showed a negative correlation with Lactococcus, Enterococcus, Streptococcus, Leuconostoc and Lactobacillus. The resistome analysis indicated that genes conferring resistance to aminoglycoside, macrolide, and tetracycline were widely spread in all the samples. Our results showed that the reproducibility between technical replicates tested by shotgun metagenomic was very high under the same conditions of analysis (either DNA extraction, library preparation, sequencing analysis, and bioinformatic analysis), considering both the degree of overlapping and the pairwise correlation.

This study aimed to develop an active biodegradable bio-based (polylactic acid/PLA) equilibrium modified atmosphere packaging (EMAP) containing a carvacrol-emitting sachet (created by poly-hydroxybutyrate) (PLA-PHB-CARV) to extend the shelf-life of cherry tomatoes at 15 °C and 25 °C. Cherry tomatoes in macro-perforated polypropylene (PP) films (mimicking the commercial packaging) or in PLA-based micro-perforated film without the carvacrol sachet (PLA) were also tested. Weight loss, decay, headspace gases, pH, titratable acidity (TA), total suspended solids (TSS), ripening index, color, texture, total viable counts (TVC), and sensory analysis were performed. Decay was 40% in PLA-PHB-CARV, and 97% in PP after 20 days at 25 °C. PLA-PHB-CARV showed lower weight loss (p < 0.05) and stable firmness compared to PP and PLA at both temperatures. TSS and TA were not affected by the packaging at 15 °C, while at 25 °C, the TSS accumulation was inhibited in PLA-PHB-CARV compared to in PLA and PP (p < 0.05), indicating a notable delay in the ripening process. PLA-PHB-CARV retained their red color during storage compared to PP and PLA. Carvacrol addition inhibited TVC compared to PP and PLA by ca. 2.0 log CFU/g during storage at 25 °C, while at 15 °C, the packaging did not reveal a significant effect. Overall, the results indicated that the developed active EMAP may be adequately used as an advanced and alternative packaging for tomatoes or potentially other fruits with a similar respiration rate versus their conventional packaging, showing several advantages, e.g., a reduction in petrochemical-based plastics use, shelf-life extension of the packaged food, and consequently, the perspective of limiting food waste during distribution and retail or domestic storage.

Lately, the inclusion of additional lactic acid bacteria (LAB) strains to cheeses is becoming more popular since they can affect cheese’s nutritional, technological, and sensory properties, as well as increase the product’s safety. This work studied the effect of Lactiplantibacillus pentosus L33 and Lactiplantibacillus plantarum L125 free cells and supernatants on feta cheese quality and Listeria monocytogenes fate. In addition, rapid and non-invasive techniques such as Fourier transform infrared (FTIR) and multispectral imaging (MSI) analysis were used to classify the cheese samples based on their sensory attributes. Slices of feta cheese were contaminated with 3 log CFU/g of L. monocytogenes, and then the cheese slices were sprayed with (i) free cells of the two strains of the lactic acid bacteria (LAB) in co-culture (F, ~5 log CFU/g), (ii) supernatant of the LAB co-culture (S) and control (C, UHT milk) or wrapped with Na-alginate edible films containing the pellet (cells, FF) or the supernatant (SF) of the LAB strains. Subsequently, samples were stored in air, in brine, or in vacuum at 4 and 10 °C. During storage, microbiological counts, pH, and water activity (aw) were monitored while sensory assessment was conducted. Also, in every sampling point, spectral data were acquired by means of FTIR and MSI techniques. Results showed that the initial microbial population of Feta was ca. 7.6 log CFU/g and consisted of LAB (>7 log CFU/g) and yeast molds in lower levels, while no Enterobacteriaceae were detected. During aerobic, brine, and vacuum storage for both temperatures, pathogen population was slightly postponed for S and F samples and reached lower levels compared to the C ones. The yeast mold population was slightly delayed in brine and vacuum packaging. For aerobic storage at 4 °C, an elongation in the shelf life of F samples by 4 days was observed compared to C and S samples. At 10 °C, the shelf life of both F and S samples was extended by 13 days compared to C samples. FTIR and MSI analyses provided reliable estimations of feta quality using the PLS-DA method, with total accuracy (%) ranging from 65.26 to 84.31 and 60.43 to 89.12, respectively. In conclusion, the application of bioprotective LAB strains can result in the extension of feta’s shelf life and provide a mild antimicrobial action against L. monocytogenes and spoilage microbiota. Furthermore, the findings of this study validate the effectiveness of FTIR and MSI techniques, in tandem with data analytics, for the rapid assessment of the quality of feta samples.

Abstract 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.

Abstract Two alternative methods for producing compost in a tunnel, from certain category (Cat.) 3 animal by‐products (ABP) and other non‐ABP material, were assessed. The first method proposed a minimum temperature of 55°C for 72 h and the second 60°C for 48 h, both with a maximum particle size of 200 mm. The assessment of the Panel on Biological Hazards (BIOHAZ) exclusively focused on Cat. 3 ABP materials (catering waste and processed foodstuffs of animal origin no longer intended for human consumption). The proposed composting processes were evaluated for their efficacy to achieve a reduction of at least 5 log10 of Enterococcus faecalis and Salmonella Senftenberg (775W, H2S negative) and at least 3 log10 of relevant thermoresistant viruses. The applicant provided a list of biological hazards that may enter the composting process and selected parvoviruses as the indicator of the thermoresistant viruses. The evidence provided by the applicant included: (a) literature data on thermal inactivation of biological hazards; (b) results from validation studies on the reduction of E. faecalis, Salmonella Senftenberg 775W H2S negative and canine parvovirus carried out in composting plants across Europe; (c) and experimental data from direct measurements of reduction of infectivity of murine parvovirus in compost material applying the time/temperature conditions of the two alternative methods. The evidence provided showed the capacity of the proposed alternative methods to reduce E. faecalis and Salmonella Senftenberg 775W H2S negative by at least 5 log10, and parvoviruses by at least 3 log10. The BIOHAZ Panel concluded that the two alternative methods under assessment can be considered to be equivalent to the processing method currently approved in the Commission Regulation (EU) No 142/2011.

Globally, fresh vegetables or minimally processed salads have been implicated in several foodborne disease outbreaks. This work studied the effect of Lactiplantibacillus pentosus FMCC-B281 cells (F) and its supernatant (S) on spoilage and on the fate of Listeria monocytogenes and Escherichia coli O157:H7 on fresh-cut ready-to-eat (RTE) salads during storage. Also, Fourier transform infrared (FTIR) and multispectral imaging (MSI) analysis were used as rapid and non-destructive techniques to estimate the microbiological status of the samples. Fresh romaine lettuce, rocket cabbage, and white cabbage were used in the present study and were inoculated with L. pentosus and the two pathogens. The strains were grown at 37 °C for 24 h in MRS and BHI broths, respectively, and then were centrifuged to collect the supernatant and the pellet (cells). Cells (F, ~5 log CFU/g), the supernatant (S), and a control (C, broth) were used to spray the leaves of each fresh vegetable that had been previously contaminated (sprayed) with the pathogen (3 log CFU/g). Subsequently, the salads were packed under modified atmosphere packaging (10%CO2/10%O2/80%N2) and stored at 4 and 10 °C until spoilage. During storage, microbiological counts and pH were monitored in parallel with FTIR and MSI analyses. The results showed that during storage, the population of the pathogens increased for lettuce and rocket independent of the treatment. For cabbage, pathogen populations remained stable throughout storage. Regarding the spoilage microbiota, the Pseudomonas population was lower in the F samples, while no differences in the populations of Enterobacteriaceae and yeasts/molds were observed for the C, F, and S samples stored at 4 °C. According to sensory evaluation, the shelf-life was shorter for the control samples in contrast to the S and F samples, where their shelf-life was elongated by 1–2 days. Initial pH values were ca. 6.0 for the three leafy vegetables. An increase in the pH of ca. 0.5 values was recorded until the end of storage at both temperatures for all cases of leafy vegetables. FTIR and MSI analyses did not satisfactorily lead to the estimation of the microbiological quality of salads. In conclusion, the applied bioprotective strain (L. pentosus) can elongate the shelf-life of the RTE salads without an effect on pathogen growth.

Abstract 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.

Abstract 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.

Abstract The contamination of water used in post‐harvest handling and processing operations of fresh and frozen fruit, vegetables and herbs (ffFVHs) is a global concern. The most relevant microbial hazards associated with this water are: Listeria monocytogenes, Salmonella spp., human pathogenic Escherichia coli and enteric viruses, which have been linked to multiple outbreaks associated with ffFVHs in the European Union (EU). Contamination (i.e. the accumulation of microbiological hazards) of the process water during post‐harvest handling and processing operations is affected by several factors including: the type and contamination of the FVHs being processed, duration of the operation and transfer of microorganisms from the product to the water and vice versa, etc. For food business operators (FBOp), it is important to maintain the microbiological quality of the process water to assure the safety of ffFVHs. Good manufacturing practices (GMP) and good hygienic practices (GHP) related to a water management plan and the implementation of a water management system are critical to maintain the microbiological quality of the process water. Identified hygienic practices include technical maintenance of infrastructure, training of staff and cooling of post‐harvest process water. Intervention strategies (e.g. use of water disinfection treatments and water replenishment) have been suggested to maintain the microbiological quality of process water. Chlorine‐based disinfectants and peroxyacetic acid have been reported as common water disinfection treatments. However, given current practices in the EU, evidence of their efficacy under industrial conditions is only available for chlorine‐based disinfectants. The use of water disinfection treatments must be undertaken following an appropriate water management strategy including validation, operational monitoring and verification. During operational monitoring, real‐time information on process parameters related to the process and product, as well as the water and water disinfection treatment(s) are necessary. More specific guidance for FBOp on the validation, operational monitoring and verification is needed.

Abstract 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.

Abstract 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.

Abstract 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, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (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 38 microorganisms notified to EFSA between October 2022 and March 2023 (inclusive) (28 as feed additives, 5 as food enzymes, food additives and flavourings, 5 as novel foods), 34 were not evaluated because: 8 were filamentous fungi, 4 were Enterococcus faecium and 2 were Escherichia coli (taxonomic units that are excluded from the QPS evaluation) and 20 were taxonomic units (TUs) that already have a QPS status. Three of the other four TUs notified within this period were evaluated for the first time for a possible QPS status: Anaerobutyricum soehngenii, Stutzerimonas stutzeri (former Pseudomonas stutzeri) and Nannochloropsis oculata. Microorganism strain DSM 11798 has also been notified in 2015 and as its taxonomic unit is notified as a strain not a species, it is not suitable for the QPS approach. A. soehngenii and N. oculata are not recommended for the QPS status due to a limited body of knowledge of its use in the food and feed chains. S. stutzeri is not recommended for inclusion in the QPS list based on safety concerns and limited information about the exposure of animals and humans through the food and feed chains.

Abstract The European Commission requested an analysis of the Chronic Wasting Disease (CWD) monitoring programme in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland (9 January 2017–28 February 2022). Thirteen cases were detected in reindeer, 15 in moose and 3 in red deer. They showed two phenotypes, distinguished by the presence or absence of detectable disease‐associated normal cellular prion protein (PrP) in lymphoreticular tissues. CWD was detected for the first time in Finland, Sweden and in other areas of Norway. In countries where the disease was not detected, the evidence was insufficient to rule out its presence altogether. Where cases were detected, the prevalence was below 1%. The data also suggest that the high‐risk target groups for surveillance should be revised, and ‘road kill’ removed. Data show that, in addition to differences in age and sex, there are differences in the prion protein gene (PRNP) genotypes between positive and negative wild reindeer. A stepwise framework has been proposed with expanded minimum background surveillance to be implemented in European countries with relevant cervid species. Additional surveillance may include ad hoc surveys for four different objectives, specific to countries with/without cases, focusing on parallel testing of obex and lymph nodes from adult cervids in high‐risk target groups, sustained over time, using sampling units and a data‐driven design prevalence. Criteria for assessing the probability of CWD presence have been outlined, based on the definition of the geographical area, an annual assessment of risk of introduction, sustained minimum background surveillance, training and engagement of stakeholders and a surveillance programme based on data‐driven parameters. All positive cases should be genotyped. Sample sizes for negative samples have been proposed to detect and estimate the frequency of PRNP polymorphisms. Double‐strand sequencing of the entire PRNP open reading frame should be undertaken for all selected samples, with data collated in a centralised collection system at EU level.

Abstract Nine odorless laboratory-collected hydro-distilled aqueous extracts (basil, calendula, centrifuged oregano, corn silk, laurel, oregano, rosemary, spearmint, thyme) and one industrial steam-distilled oregano hydrolate acquired as by-products of essential oils purification were screened for their in vitro antimicrobial activity against three Salmonella Typhimurium strains (4/74, FS8, FS115) at 4 and 37 °C. Susceptibility to the extracts was mainly plant- and temperature-dependent, though strain dependent effects were also observed. Industrial oregano hydrolate eliminated strains immediately after inoculation, exhibiting the highest antimicrobial potential. Hydro-distilled extracts eliminated/reduced Salmonella levels during incubation at 4 °C. At 37 °C, oregano, centrifuged oregano, thyme, calendula and basil were bactericidal while spearmint, rosemary and corn silk bacteriostatic. A strain-dependent effect was observed for laurel. The individual or combined effect of marinades and edible coatings prepared of industrial hydrolate and hydro-distilled oregano extracts with or without oregano essential oil (OEO) was tested in pork meat at 4 °C inoculated with FS8 strain. Lower in situ activity was observed compared to in vitro assays. Marinades and edible coatings prepared of industrial oregano hydrolate + OEO were the most efficient in inhibiting pathogen. Marination in oregano extract and subsequent coating with either 50% oregano extract + OEO or water + OEO enhanced the performance of oregano extract. In conclusion, by-products of oregano essential oil purification may be promising alternative antimicrobials to pork meat stored under refrigeration when applied in the context of multiple hurdle approach.

Abstract The impact of dry‐ageing of beef and wet‐ageing of beef, pork and lamb on microbiological hazards and spoilage bacteria was examined and current practices are described. As ‘standard fresh’ and wet‐aged meat use similar processes these were differentiated based on duration. In addition to a description of the different stages, data were collated on key parameters (time, temperature, pH and aw) using a literature survey and questionnaires. The microbiological hazards that may be present in all aged meats included Shiga toxin‐producing Escherichia coli (STEC), Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, enterotoxigenic Yersinia spp., Campylobacter spp. and Clostridium spp. Moulds, such as Aspergillus spp. and Penicillium spp., may produce mycotoxins when conditions are favourable but may be prevented by ensuring a meat surface temperature of −0.5 to 3.0°C, with a relative humidity (RH) of 75–85% and an airflow of 0.2–0.5 m/s for up to 35 days. The main meat spoilage bacteria include Pseudomonas spp., Lactobacillus spp. Enterococcus spp., Weissella spp., Brochothrix spp., Leuconostoc spp., Lactobacillus spp., Shewanella spp. and Clostridium spp. Under current practices, the ageing of meat may have an impact on the load of microbiological hazards and spoilage bacteria as compared to standard fresh meat preparation. Ageing under defined and controlled conditions can achieve the same or lower loads of microbiological hazards and spoilage bacteria than the variable log10 increases predicted during standard fresh meat preparation. An approach was used to establish the conditions of time and temperature that would achieve similar or lower levels of L. monocytogenes and Yersinia enterocolitica (pork only) and lactic acid bacteria (representing spoilage bacteria) as compared to standard fresh meat. Finally, additional control activities were identified that would further assure the microbial safety of dry‐aged beef, based on recommended best practice and the outputs of the equivalence assessment.

Abstract The qualified presumption of safety (QPS) provides a generic pre‐assessment of the safety of microorganisms intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. QPS assessment allows a fast track evaluation of strains belonging to QPS taxonomic units (TUs): species for bacteria, yeast, fungi, protists/microalgae and families for viruses. QPS TUs are assessed for their body of knowledge and safety. Safety concerns related to a QPS TU are reflected, when possible, as ‘qualifications’, which should be tested at strain and/or product level. Based on the possession of potentially harmful traits by some strains, filamentous fungi, bacteriophages, oomycetes, streptomycetes, Enterococcus faecium, Escherichia coli and Clostridium butyricum are excluded from the QPS assessment. Between October 2019 and September 2022, 323 notifications of TUs were received, 217 related to feed additives, 54 to food enzymes, food additives and flavourings, 14 to plant protection products and 38 to novel foods. The list of QPS‐recommended TUs is reviewed every 6 months following an extensive literature search strategy. Only sporadic infections with a few QPS status TUs in immunosuppressed individuals were identified and the assessment did not change the QPS status of these TUs. The QPS list has been updated in relation to the most recent taxonomic insights and the qualifications were revised and streamlined. The qualification ‘absence of aminoglycoside production ability’ was withdrawn for Bacillus velezensis. Six new TUs received the QPS status: Bacillus paralicheniformis with the qualification ‘absence of toxigenic activity’ and ‘absence of bacitracin production ability’; Bacillus circulans with the qualifications for ‘production purposes only’ and ‘absence of cytotoxic activity’; Haematococcus lacustris (synonym Haematococcus pluvialis) and Ogataea polymorpha, both with the qualification ‘for production purposes only’; Lactiplantibacillus argentoratensis; Geobacillus thermodenitrificans with the qualification ‘absence of toxigenic activity’.

Abstract 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, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by ‘qualifications’. In the period covered by this Statement, new information was found leading to the withdrawal of the qualification ‘absence of aminoglycoside production ability’ for Bacillus velezensis. The qualification for Bacillus paralicheniformis was changed to ‘absence of bacitracin production ability’. For the other TUs, no new information was found that would change the status of previously recommended QPS TUs. Of 52 microorganisms notified to EFSA between April and September 2022 (inclusive), 48 were not evaluated because: 7 were filamentous fungi, 3 were Enterococcus faecium, 2 were Escherichia coli, 1 was Streptomyces spp., and 35 were taxonomic units (TUs) that already have a QPS status. The other four TUs notified within this period, and one notified previously as a different species, which was recently reclassified, were evaluated for the first time for a possible QPS status: Xanthobacter spp. could not be assessed because it was not identified to the species level; Geobacillus thermodenitrificans is recommended for QPS status with the qualification ‘absence of toxigenic activity’. Streptoccus oralis is not recommended for QPS status. Ogataea polymorpha is proposed for QPS status with the qualification ‘for production purposes only’. Lactiplantibacillus argentoratensis (new species) is included in the QPS list.

Abstract An alternative method for the production of renewable fuels from rendered animal fats (pretreated using methods 1–5 or method 7 as described in Annex IV of Commission Regulation (EC) No 2011/142) and used cooking oils, derived from Category 3 animal by‐products, was assessed. The method is based on a catalytic co‐processing hydrotreatment using a middle distillate followed by a stripping step. The materials must be submitted to a pressure of at least 60 bars and a temperature of at least 270°C for at least 4.7 min. The application focuses on the demonstration of the level of reduction of spores from non‐pathogenic spore‐forming indicator bacterial species (Bacillus subtilis and Desulfotomaculum kuznetsovii), based on a non‐systematic review of published data and additional extrapolation analyses. The EFSA BIOHAZ Panel considers that the application and supporting literature contain sufficient evidence that the proposed alternative method can achieve a reduction of at least 5 log10 in the spores of B. subtilis and a 12 log10 reduction in the spores of C. botulinum. The alternative method under evaluation is considered at least equivalent to the processing methods currently approved in the Commission Regulation (EU) No 2011/142.

The presence of microbial communities on cave walls and speleothems is an issue that requires attention. Traditional cleaning methods using water, brushes, and steam can spread the infection and cause damage to the cave structures, while chemical agents can lead to the formation of toxic compounds and damage the cave walls. Essential oils (EOs) have shown promising results in disrupting the cell membrane of bacteria and affecting their membrane permeability. In this study, we identified the microorganisms forming unwanted microbial communities on the walls and speleothems of Petralona Cave using 16S and 18S rDNA amplicon sequencing approaches and evaluated the efficacy of EOs in reducing the ATP levels of these ecosystems. The samples exhibited a variety of both prokaryotic and eukaryotic microorganisms, including Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, the SAR supergroup, Opisthokonta, Excavata, Archaeplastida, and Amoebozoa. These phyla are often found in various habitats, including caves, and contribute to the ecological intricacy of cave ecosystems. In terms of the order and genus taxonomy, the identified biota showed abundances that varied significantly among the samples. Functional predictions were also conducted to estimate the differences in expressed genes among the samples. Oregano EO was found to reduce ATP levels by 87% and 46% for black and green spots, respectively. Consecutive spraying with cinnamon EO further reduced ATP levels, with reductions of 89% for black and 88% for green spots. The application of a mixture solution caused a significant reduction up to 96% in ATP levels of both areas. Our results indicate that EOs could be a promising solution for the treatment of microbial communities on cave walls and speleothems.

Abstract The transmission of antimicrobial resistance (AMR) between food‐producing animals (poultry, cattle and pigs) during short journeys ( 8 h) directed to other farms or to the slaughterhouse lairage (directly or with intermediate stops at assembly centres or control posts, mainly transported by road) was assessed. Among the identified risk factors contributing to the probability of transmission of antimicrobial‐resistant bacteria (ARB) and antimicrobial resistance genes (ARGs), the ones considered more important are the resistance status (presence of ARB/ARGs) of the animals pre‐transport, increased faecal shedding, hygiene of the areas and vehicles, exposure to other animals carrying and/or shedding ARB/ARGs (especially between animals of different AMR loads and/or ARB/ARG types), exposure to contaminated lairage areas and duration of transport. There are nevertheless no data whereby differences between journeys shorter or longer than 8 h can be assessed. Strategies that would reduce the probability of AMR transmission, for all animal categories include minimising the duration of transport, proper cleaning and disinfection, appropriate transport planning, organising the transport in relation to AMR criteria (transport logistics), improving animal health and welfare and/or biosecurity immediately prior to and during transport, ensuring the thermal comfort of the animals and animal segregation. Most of the aforementioned measures have similar validity if applied at lairage, assembly centres and control posts. Data gaps relating to the risk factors and the effectiveness of mitigation measures have been identified, with consequent research needs in both the short and longer term listed. Quantification of the impact of animal transportation compared to the contribution of other stages of the food‐production chain, and the interplay of duration with all risk factors on the transmission of ARB/ARGs during transport and journey breaks, were identified as urgent research needs.

Abstract 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, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge, safety concerns and occurrence of antimicrobial resistance. Safety concerns identified for a taxonomic unit (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 the 50 microorganisms notified to EFSA in October 2021 to March 2022 (inclusive), 41 were not evaluated: 10 filamentous fungi, 1 Enterococcus faecium, 1 Clostridium butyricum, 3 Escherichia coli and 1 Streptomyces spp. because are excluded from QPS evaluation, and 25 TUs that have already a QPS status. Nine notifications, corresponding to seven TUs were evaluated: four of these, Streptococcus salivarius, Companilactobacillus formosensis, Pseudonocardia autotrophica and Papiliotrema terrestris, being evaluated for the first time. The other three, Microbacterium foliorum, Pseudomonas fluorescens and Ensifer adhaerens were re‐assessed. None of these TUs were recommended for QPS status: Ensifer adhaerens, Microbacterium foliorum, Companilactobacillus formosensis and Papiliotrema terrestris due to a limited body of knowledge, Streptococcus salivarius due to its ability to cause bacteraemia and systemic infection that results in a variety of morbidities, Pseudonocardia autotrophica due to lack of body of knowledge and uncertainty on the safety of biologically active compounds which can be produced, and Pseudomonas fluorescens due to possible safety concerns.

Abstract Studies evaluating the safety and efficacy of lactic acid to reduce microbiological surface contamination from carcases of wild game (i.e. kangaroos and wild pigs) and small stock (i.e. goats and sheep) before chilling at the slaughterhouse were assessed. Wild pig and kangaroo hide‐on carcases may have been chilled before they arrive at the slaughterhouse and are treated after removal of the hides. Lactic acid solutions (2–5%) are applied to the carcases at temperatures of up to 55°C by spraying or misting. The treatment lasts 6–7 s per carcass side. The Panel concluded that: [1] the treatment is of no safety concern, provided that the lactic acid complies with the European Union specifications for food additives; [2] based on the available evidence, it was not possible to conclude on the efficacy of spraying or misting lactic acid on kangaroo, wild pig, goats and sheep carcases; [3] treatment of the above‐mentioned carcases with lactic acid may induce reduced susceptibility to the same substance, but this can be minimised; there is currently no evidence that prior exposure of food‐borne pathogens to lactic acid leads to the occurrence of resistance levels that compromise antimicrobial therapy; and [4] the release of lactic acid is not of concern for the environment, assuming that wastewaters released by the slaughterhouses are treated on‐site, if necessary, to counter the potentially low pH caused by lactic acid, in compliance with local rules.

Abstract High‐pressure processing (HPP) is a non‐thermal treatment in which, for microbial inactivation, foods are subjected to isostatic pressures (P) of 400–600 MPa with common holding times (t) from 1.5 to 6 min. The main factors that influence the efficacy (log10 reduction of vegetative microorganisms) of HPP when applied to foodstuffs are intrinsic (e.g. water activity and pH), extrinsic (P and t) and microorganism‐related (type, taxonomic unit, strain and physiological state). It was concluded that HPP of food will not present any additional microbial or chemical food safety concerns when compared to other routinely applied treatments (e.g. pasteurisation). Pathogen reductions in milk/colostrum caused by the current HPP conditions applied by the industry are lower than those achieved by the legal requirements for thermal pasteurisation. However, HPP minimum requirements (P/t combinations) could be identified to achieve specific log10 reductions of relevant hazards based on performance criteria (PC) proposed by international standard agencies (5–8 log10 reductions). The most stringent HPP conditions used industrially (600 MPa, 6 min) would achieve the above‐mentioned PC, except for Staphylococcus aureus. Alkaline phosphatase (ALP), the endogenous milk enzyme that is widely used to verify adequate thermal pasteurisation of cows’ milk, is relatively pressure resistant and its use would be limited to that of an overprocessing indicator. Current data are not robust enough to support the proposal of an appropriate indicator to verify the efficacy of HPP under the current HPP conditions applied by the industry. Minimum HPP requirements to reduce Listeria monocytogenes levels by specific log10 reductions could be identified when HPP is applied to ready‐to‐eat (RTE) cooked meat products, but not for other types of RTE foods. These identified minimum requirements would result in the inactivation of other relevant pathogens (Salmonella and Escherichia coli) in these RTE foods to a similar or higher extent.

Abstract The qualified presumption of safety (QPS) approach was developed to provide a generic pre‐evaluation of the safety of biological agents. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns are, where possible, confirmed at the species/strain or product level and reflected by ‘qualifications’. The QPS list was updated in relation to the revised taxonomy of the genus Bacillus, to synonyms of yeast species and for the qualifications ‘absence of resistance to antimycotics’ and ‘only for production purposes’. Lactobacillus cellobiosus has been reclassified as Limosilactobacillus fermentum. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS taxonomic units (TU)s. Of the 70 microorganisms notified to EFSA, 64 were not evaluated: 11 filamentous fungi, one oomycete, one Clostridium butyricum, one Enterococcus faecium, five Escherichia coli, one Streptomyces sp., one Bacillus nakamurai and 43 TUs that already had a QPS status. Six notifications, corresponding to six TUs were evaluated: Paenibacillus lentus was reassessed because an update was requested for the current mandate. Enterococcus lactis synonym Enterococcus xinjiangensis, Aurantiochytrium mangrovei synonym Schizochytrium mangrovei, Schizochytrium aggregatum, Chlamydomonas reinhardtii synonym Chlamydomonas smithii and Haematococcus lacustris synonym Haematococcus pluvialis were assessed for the first time. The following TUs were not recommended for QPS status: P. lentus due to a limited body of knowledge, E. lactis synonym E. xinjiangensis due to potential safety concerns, A. mangrovei synonym S. mangrovei, S. aggregatum and C. reinhardtii synonym C. smithii, due to lack of a body of knowledge on its occurrence in the food and feed chain. H. lacustris synonym H. pluvialis is recommended for QPS status with the qualification ‘for production purposes only’.

Abstract The European Commission requested EFSA to assess if different thermal processes achieve a 5 log10 reduction in Enterococcus faecalis or Salmonella Senftenberg (775W) and (if relevant) a 3 log10 reduction in thermoresistant viruses (e.g. Parvovirus) as well as if different chemical processes achieve a 3 log10 reduction of eggs of Ascaris sp., in eight groups of Category 2 and 3 derived products and animal by‐products (ABP). These included (1) ash derived from incineration, co‐incineration and combustion; (2) glycerine derived from the production of biodiesel and renewable fuels; (3) other materials derived from the production of biodiesel and renewable fuels; (4) hides and skins; (5) wool and hair; (6) feathers and down; (7) pig bristles; and (8) horns, horn products, hooves and hoof products. Data on the presence of viral hazards and on thermal and chemical inactivation of the targeted indicator microorganisms and biological hazards under relevant processing conditions were extracted via extensive literature searches. The evidence was assessed via expert knowledge elicitation. The certainty that the required log10 reductions in the most resistant indicator microorganisms or biological hazards will be achieved for each of the eight groups of materials mentioned above by the thermal and/or chemical processes was (1) 99–100% for the two processes assessed; (2) 98–100% in Category 2 ABP, at least 90–99% in Category 3 ABP; (3) 90–99% in Category 2 ABP; at least 66–90% in Category 3 ABP; (4) 10–66% and 33–66%; (5) 1–33% and 10–50%; (6) 66–90%; (7) 33–66% and 50–95%; (8) 66–95%, respectively. Data generation on the occurrence and reduction of biological hazards by thermal and/or chemical methods in these materials and on the characterisation of the usage pathways of ABP as organic fertilisers/soil improvers is recommended.

ABSTRACT The dormancy continuum hypothesis states that in response to stress, cells enter different stages of dormancy ranging from unstressed living cells to cell death, in order to ensure their long-term survival under adverse conditions. Exposure of Listeria monocytogenes cells to sublethal stressors related to food processing may induce sublethal injury and the viable-but-nonculturable (VBNC) state. In this study, exposure to acetic acid (AA), hydrochloric acid (HCl), and two disinfectants, peracetic acid (PAA) and sodium hypochlorite (SH), at 20°C and 4°C was used to evaluate the potential induction of L. monocytogenes strain Scott A into different stages of dormancy. To differentiate the noninjured subpopulation from the total population, tryptic soy agar with 0.6% yeast extract (TSAYE), supplemented or not with 5% NaCl, was used. Sublethally injured and VBNC cells were detected by comparing plate counts obtained with fluorescence microscopy and by using combinations of carboxyfluorescein and propidium iodide (viable/dead cells). Induction of sublethal injury was more intense after PAA treatment. Two subpopulations were detected, with phenotypes of untreated cells and small colony variants (SCVs). SCVs appeared as smaller colonies of various sizes and were first observed after 5 min of exposure to 5 ppm PAA at 20°C. Increasing the stress intensity from 5 to 40 ppm PAA led to earlier detection of SCVs. L. monocytogenes remained culturable after exposure to 20 and 30 ppm PAA for 3 h. At 40 ppm, after 3 h of exposure, the whole population was considered nonculturable, while cells remained metabolically active. These results corroborate the induction of the VBNC state. IMPORTANCE Sublethally injured and VBNC cells may evade detection, resulting in underestimation of a food product’s microbial load. Under favorable conditions, cells may regain their growth capacity and acquire new resistant characteristics, posing a major threat for public health. Induction of the VBNC state is crucial for foodborne pathogens, such as L. monocytogenes, the detection of which relies almost exclusively on the use of culture recovery techniques. In the present study, we confirmed that sublethal injury is an initial stage of dormancy in L. monocytogenes that is followed by the VBNC state. Our results showed that PAA induced SCVs (a phenomenon potentially triggered by external factors) and the VBNC state in L. monocytogenes, indicating that tests of lethality based only on culturability may provide false-positive results regarding the effectiveness of an inactivation treatment.

Nowadays, consumers are increasingly concerned about nutrition and health issues. “Syglino of Monemvasia” is a traditional Greek cooked and smoked, sliced pork meat product. Although this is a nutritious food, its consumption should be done in moderation due to the pickling process of its preparation. This product was thus here optimized to contain half salt (NaCl) amount and its microbial stability and shelf life was then assessed in comparison to the already available commercial product. For this, the total viable counts (TVCs) and some critical specific spoilage associations were enumerated at each product type during vacuum incubation at four different temperatures (0, 5, 10, and 15 °C). The alterations in pH, aw, color, and some other crucial sensory attributes of each product were also periodically monitored. The new low-salt product was found to remain microbiologically stable under refrigerated vacuum storage for approximately two weeks, being finally spoiled by Brochothrix thermosphacta grown above 107 CFU/g, ultimately resulting in the deterioration of taste, odor, and overall appearance of the product, and thus leading to its subsequent organoleptic rejection. Despite its limited shelf life, the 50% NaCl-reduced “Syglino” could be released in the local market provided that the cooling chain is maintained throughout its distribution (≤5 °C) and at the same time consumers are willing to accept its milder taste. Although salt replacers could have been used to improve its flavor and at the same time increase its shelf life, the product here developed without the use of such alternatives will hopefully contribute to the taste training of its consumers for less salt in their diet, keeping in parallel its clean label with no added preservatives and other food additives.

Halitzia is a traditional white-brined cheese produced by a limited number of producers in Cyprus. During a survey of the microbiome of a number of different Halitzia samples, we identified a bacterial strain that exhibited enhanced proteolytic activity compared to the other isolates. The strain was further studied, and it was assigned as Enterococcus faecalis PK23. We proceeded with sequencing of its whole genome using Illumina technology. Initial sequencing and assembly produced 116 scaffolds with a length of 3,149,036 bp. Comparison with the available E. faecalis genomes revealed that the strain PK23 exhibited high levels of identity to the genome sequence of E. faecalis isolate 26975_2#180 deposited in GenBank as a single complete contig. From the 116 scaffolds 106 could be aligned to the genome of isolate 26975_2#180 leading to a chromosomal length of 3,132,784 bp with a GC content of 37.3%. From the remaining 10 scaffolds, five showed similarity to plasmid sequences. More specifically, scaffold 54 showed high identity with most part of plasmid pEF1071 of E. faecalis strain BFE 1071, which carries the gene cluster involved in the biosynthesis of enterocins 1071A and 1071B, while scaffold 77 showed high identity with the entire sequence of the unnamed_5 cryptic plasmid of Enterococcus faecium strain PR05720-3. The other three scaffolds were only short parts of larger plasmids. The remaining five scaffolds which could not be related to any plasmid sequence most probably constitute chromosomal sequences present in strain PK23 but absent from isolate 26975_2#180. Their total length was around 2.7 kb, which does not affect the sequence of the PK23 pseudochromosome in a major way. The whole-genome sequence annotation of strain PK23 identified 3161 coding sequences and 62 RNA sequences. The results from the Rapid Annotation using Subsystem Technology (RAST) version 2.0 server indicated the presence of seven putative genes which were related to the subsystem of Protein Degradation. This dataset provides a first overview of the proteolytic and bacteriocin producing properties of E. faecalis PK23. The dataset may also be used in future experiments which could shed light on the adaptation of the strain in the dairy environment and its role in cheese production.

Abstract The specific concentrations of amprolium in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC for amprolium, it was not possible to conclude the assessment. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of amprolium in feed that showed to have an effect on growth promotion/increased yield were reported. The lack of antibacterial activity at clinically relevant concentrations for amprolium suggests that further studies relating to bacterial resistance are not a priority.

Abstract The specific concentrations of colistin in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of colistin in feed that showed to have an effect on growth promotion/increased yield were reported. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials.

Abstract The specific concentrations of apramycin, paromomycin, neomycin and spectinomycin in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC for these antimicrobials, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for apramycin and neomycin, whilst for paromomycin and spectinomycin, no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these four antimicrobials.

Abstract The specific concentrations of tetracycline, chlortetracycline, oxytetracycline and doxycycline in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for these four tetracyclines was estimated. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tetracycline, chlortetracycline, oxytetracycline, whilst for doxycycline no suitable data for the assessment were available. Uncertainties and data gaps associated with the levels reported were addressed. It was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC for these antimicrobials.

Abstract The specific concentrations of flumequine and oxolinic acid in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data are available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. No suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials.

Abstract The specific concentrations of sulfonamides in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data are available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were identified for three sulfonamides: sulfamethazine, sulfathiazole and sulfamerazine. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these antimicrobials.

Abstract The specific concentrations of tiamulin and valnemulin in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tiamulin, while for valnemulin no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these two antimicrobials.

Abstract The specific concentrations of lincomycin in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels of lincomycin in feed that showed to have an effect on growth promotion/increased yield were reported. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for lincomycin.

Abstract The specific concentrations of florfenicol and thiamphenicol in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for florfenicol was estimated. However, due to the lack of data, the calculation of the FARSC for thiamphenicol was not possible until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for florfenicol, whilst for thiamphenicol no suitable data for the assessment were available. Uncertainties and data gaps associated to the levels reported were addressed. For florfenicol, it was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC, whereas for thiamphenicol, the recommendation was to generate the data required to fill the gaps which prevented the FARSC calculation.

Abstract The specific concentrations of amoxicillin and penicillin V in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for amoxicillin, whilst for penicillin V no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these two antimicrobials.

Abstract The specific concentrations of trimethoprim in non‐target feed for food‐producing animals below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for trimethoprim was estimated. Uncertainties and data gaps associated to the levels reported were addressed. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. No suitable data for the assessment were available. It was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC for trimethoprim.

Abstract The specific concentrations of tilmicosin, tylosin and tylvalosin in non‐target feed for food‐producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. However, due to the lack of data on the parameters required to calculate the FARSC, it was not possible to conclude the assessment until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for tilmicosin and tylosin, whilst for tylvalosin no suitable data for the assessment were available. It was recommended to carry out studies to generate the data that are required to fill the gaps which prevented the calculation of the FARSC for these three antimicrobials.

Abstract The European Commission requested EFSA to assess, in collaboration with EMA, the specific concentrations of antimicrobials resulting from cross‐contamination in non‐target feed for food‐producing animals below which there would not be an effect on the emergence of, and/or selection for, resistance in microbial agents relevant for human and animal health, as well as the levels of the antimicrobials which could have a growth promotion/increase yield effect. The assessment was performed for 24 antimicrobial active substances, as specified in the mandate. This scientific opinion describes the methodology used, and the main associated data gaps and uncertainties. To estimate the antimicrobial levels in the non‐target feed that would not result in emergence of, and/or selection for, resistance, a model was developed. This ‘Feed Antimicrobial Resistance Selection Concentration’ (FARSC) model is based on the minimal selective concentration (MSC), or the predicted MSC (PMSC) if MSC for the most susceptible bacterial species is unavailable, the fraction of antimicrobial dose available for exposure to microorganisms in the large intestine or rumen (considering pharmacokinetic parameters), the daily faecal output or rumen volume and the daily feed intake. Currently, lack of data prevents the establishment of PMSC and/or FARSC for several antimicrobials and animal species. To address growth promotion, data from an extensive literature search were used. Specific assessments of the different substances grouped by antimicrobial classes are addressed in separate scientific opinions. General conclusions and recommendations were made.