Induction into viable but non culturable state and outgrowth heterogeneity of Listeria monocytogenes is affected by stress history and type of growth.

Exposure to sublethal stresses related to food-processing may induce a heterogenous mixture of cells that co-exist, comprising healthy, sublethally injured, dormant and dead cells. Heterogeneity in survival capacity and dormancy of single cells may impede the detection of foodborne pathogens. In this study, we exposed Listeria monocytogenes Scott A strain, to peracetic acid (PAA; 20-40 ppm) and to acidic conditions (hydrochloric (HCl) and acetic (AA) acid, adjusted to pH 2.7-3.0, to evaluate the resuscitation capacity and outgrowth kinetics of metabolically active cells in two different media. Injury and the viable-but-non-culturable (VBNC) status of cells were assessed by flow cytometry using CFDA (metabolically active) and PI (dead) staining. Stressed CFDA+PI- cells were sorted on Tryptic Soy (TS) Agar or in TS broth, both supplemented with 0.6 % Yeast Extract (TSAYE or TSBYE), to evaluate culturability. Resuscitation capacity of CFDA+PI-sorted cells (10 events/well) was monitored by visual inspection on TSAYE and by optical density measurement in TSBYE for 5 days. Sorting of L. monocytogenes viable cells (CFDA+PI-) in Ringer's solution on TSAYE and TSBYE showed 100 % recovery in both media (control condition), while the mean lag time in TSBYE was 9.6 h. Treatment with 20 ppm PAA for 90 and 180 min resulted in 74.79 % and 85.82 % of non-culturable cells in TSBYE and increased the average lag time to 41.7 h and 43.8 h, respectively, compared to the control (9.6 h). The longest average lag time (79.5 h) was detected after treatment with 30 ppm PAA for 90 min, while at the same condition sorting of CFDA+PI- cells resulted in 95.05 % and 93.94 % non-culturable cells on TSAYE and TSBYE, respectively. The highest percentage of wells with non-culturable cells (96.17 %) was detected on TSAYE after treatment with 40 ppm PAA for 30 min. Fractions of VBNC cells were detected in TSBYE after treatment with HCl pH 3.0 for 60 and 240 min, and in TSAYE and TSBYE after exposure to AA pH 2.7. Treatment with AA pH 2.7 for 150-300 min increased the range of recorded lag time values compared to 60 min, from 8.6 h up to 13.3 h, as well as the mean lag times in TSBYE. Modelling of the outgrowth kinetics comparing the two types of stress (oxidative vs acid) and the two systems of growth (colonial vs planktonic) revealed that low starting concentrations hindered the detection of viable L. monocytogenes cells, either due to VBNC induction or cell heterogeneity.

Shotgun metagenomic investigation of foodborne pathogens and antimicrobial resistance genes in artisanal fermented meat products from the Mediterranean area.

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 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.

Evaluation of alternative methods of tunnel composting (submitted by the European Composting Network) II.

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.

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

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

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

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

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.

Microbiological hazards associated with the use of water in the post-harvest handling and processing operations of fresh and frozen fruits, vegetables and herbs (ffFVHs). Part 1 (outbreak data analysis, literature review and stakeholder questionnaire).

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.

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.

16S and 18S rDNA Amplicon Sequencing Analysis of Aesthetically Problematic Microbial Mats on the Walls of the Petralona Cave: The Use of Essential Oils as a Cleaning Method.

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.

Impact of preculture temperature on peracetic acid-induced inactivation and sublethal injury of L. monocytogenes and subsequent growth potential of single cells.

The disinfectant peracetic acid (PAA) that is used in the food industry can cause sublethal injury in L. monocytogenes. The effect of preculture temperature on the inactivation and sublethal injury of L. monocytogenes cells due to PAA was evaluated by plating on non-selective and selective agar medium supplemented with 5 % (w/v) NaCl. L. monocytogenes cells were precultured at 30 °C, 20 °C or 4 °C, and the former was used as reference temperature. Preculture of cells at 20 °C or 4 °C and subsequent exposure to PAA at the respective growth temperatures caused higher injury compared to cells grown at 30 °C and exposed to PAA 20 °C and PAA 4 °C, respectively. Survival was also affected by the preculture temperature; 20 °C-grown cultures resulted in lower survival at PAA 20 °C. Nevertheless, preculture at 4 °C resulted in a similar number of surviving cells when exposed to PAA 4 °C compared to cells precultured at 30 °C and exposed to PAA at 4 °C. Flow cytometry was subsequently used to quantify outgrowth capacity of stressed and sublethal damaged populations following sorting of single cells in nutrient rich medium (Tryptone soy broth supplemented with yeast extract [TSBY]). PAA treatment affected the outgrowth of L. monocytogenes at single-cell level resulting in increased outgrowth-times reflecting higher single cell heterogeneity. To conclude, the response of L. monocytogenes when exposed to PAA depended on the preculture conditions, and the highly heterogeneous outgrowth potential of PAA-injured cells may affect their detection accuracy and pose a food safety risk.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 18: Suitability of taxonomic units notified to EFSA until March 2023.

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.

Use of risk assessment and predictive microbiology in regulatory science related to the scientific opinions of the EFSA BIOHAZ Panel.

EFSA's Panel on Biological Hazards (BIOHAZ Panel) deals with questions on biological hazards relating to food safety and food-borne diseases. This covers food-borne zoonoses, transmissible spongiform encephalopathies, antimicrobial resistance, food microbiology, food hygiene, animal-by products, and associated waste management issues. The scientific assessments are diverse and frequently the development of new methodological approaches is required to deal with a mandate. Among the many risk factors, product characteristics (pH, water activity etc.), time and temperature of processing and storage along the food supply chain are highly relevant for assessing the biological risks. Therefore, predictive microbiology becomes an essential element of the assessments. Uncertainty analysis is incorporated in all BIOHAZ scientific assessments, to meet the general requirement for transparency. Assessments should clearly and unambiguously state what sources of uncertainty have been identified and their impact on the conclusions of the assessment. Four recent BIOHAZ Scientific Opinions are presented to illustrate the use of predictive modelling and quantitative microbial risk assessment principles in regulatory science. The Scientific Opinion on the guidance on date marking and related food information, gives a general overview on the use of predictive microbiology for shelf-life assessment. The Scientific Opinion on the efficacy and safety of high-pressure processing of food provides an example of inactivation modelling and compliance with performance criteria. The Scientific Opinion on the use of the so-called 'superchilling' technique for the transport of fresh fishery products illustrates the combination of heat transfer and microbial growth modelling. Finally, the Scientific Opinion on the delayed post-mortem inspection in ungulates, shows how variability and uncertainty, were quantitatively embedded in assessing the probability of Salmonella detection on carcasses, via stochastic modelling and expert knowledge elicitation.

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.

Studying the metabolic factors that may impact the growth of co-cultured Listeria monocytogenes strains at low temperature.

The simultaneous presence of more than one strains of Listeria monocytogenes in the same food product may affect the growth capacity of each strain. The present study evaluated the metabolites composition that may potentially influence the growth of individual L. monocytogenes strains in a dual strain composite. Based on previous studies, L. monocytogenes strains, C5 (4b) and 6179 (1/2a) were selected due to the remarkable interaction, which was observed during their co-culture. The selected strains were inoculated (2.0 - 3.0 log CFU/mL) in Tryptic Soy Broth with 0.6% Yeast Extract (TSB-YE) in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic conditions (AC). Their resistance to different antibiotics enabled the selective enumeration of each strain in the co-culture. After reaching stationary phase, single and dual cultures were centrifuged and filtered. The cell-free spent medium (CFSM) was either characterized by Fourier transform infrared (FTIR-ATR) spectrometry or re-inoculated, after the addition of concentrated TSB-YE (for nutrient replenishment), with single and two-strain cultures for the evaluation of growth under the influence of metabolites produced from the same singly and co-cultured strains in the different combinations of strains and CFSM origin (7 °C/AC) (n = 2x3). By the end of storage, singly-cultured C5 and 6179 had reached 9.1 log CFU/mL, while in dual culture, 6179 was affected by the presence of C5 attaining only 6.4 ± 0.8 log CFU/mL. FTIR-ATR spectra of CFSM produced by singly-cultured 6179 and the co-culture were almost identical. Characteristic peaks in FTIR-ATR spectrum of CFSM of singly-cultured C5 at 1741, 1645 and 1223 cm-1 represent functional groups which were not present in the CFSM of the co-culture. These molecules may be located intracellularly or mounted on bacterial cell surface and removed from the supernatant during cell filtration of the co-culture. Both singly- and co-cultured 6179 managed to grow similarly regardless of CFSM origin. Contrarily, both singly- and co-cultured C5 managed to outgrow 6179 in CFSM which contained high concentration of C5 metabolites, while in CFSM produced by singly-cultured 6179, C5 did not grow, suggesting that the produced metabolites of strain 6179 appears to be harmful to strain C5. However, during co-culture, C5 may produce molecules that counteract the inhibitory effect of 6179. The findings shed more light on the mechanism behind the inter-strain interactions of L. monocytogenes indicating that both contact of cells and extracellular metabolites may influence the behavior of the different co-existing strains.

Monitoring of chronic wasting disease (CWD) (IV).

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.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 17: suitability of taxonomic units notified to EFSA until September 2022.

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.

Microbiological safety of aged meat.

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.

Influence of temperature on regulation of key virulence and stress response genes in Listeria monocytogenes biofilms.

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis.

Deciphering the induction of Listeria monocytogenes into sublethal injury using fluorescence microscopy and RT-qPCR.

The adaptive response of bacterial cells to changing environmental conditions depends on the behavior of single cells within the population. Exposure of Listeria monocytogenes to sublethal acidic conditions in foods or in the gastrointestinal track of the host may induce injuries relevant to difficult physiological states within the dormancy continuum. In this study, exposure to acidic conditions (acetic-AA and hydrochloric acid-HCl adjusted to pH 3.0, 2.7, 2.5 at 20 °C for 5 h) was used to evaluate injury of L. monocytogenes, Scott A strain. To differentiate the resistant sub-population from the total, Tryptic Soy Agar with 0.6 % Yeast Extract (TSAYE) supplemented or not with 5 % NaCl were comparatively used. Sublethally injured cells were detected by comparing plate counts with fluorescence microscopy, using combinations of CFDA (viability) and Propidium-Iodide (death). Effect of acid stress on the relative transcription of clpP, mazE, mazF, relA, gadC, gadD, gadB, sigB, inlA and prfA upon transition of total population into different physiological stages was evaluated through RT-qPCR. AA treated cells showed measurable logarithmic reduction at pH 2.7 and 2.5, while there was a significant percentage of CFDA-/PI+ cells. Evaluation of the potentially culturable population on TSAYE, from the percentage of CFDA/PI-stained cells, revealed that unstained cells represented a non-culturable sub-population. Exposure to Ringer's solution pH 2.7, adjusted with AA, resulted in higher percentages of non-esterase active with membrane integrity cells (CFDA-/PI-) compared to the percentages of the enumerated culturable cells on TSAYE after 4 and 5 h. Under the same conditions, after 1 h of exposure macroscopic observation revealed size colony variations (SCVs) of the total population (CFU on TSAYE). L. monocytogenes retained its culturability after hydrochloric acid exposure, while cells remained metabolically active (CFDA+). However, a stochastic change in cell's shape, was detected after exposure to pH 3.0 and 2.5, adjusted with HCl, for 2 h at 20 °C. A pattern of gene up-regulation was observed during treatment with AA pH 2.7 and HCl pH 3.0 at the 3rd h of exposure. Deciphering L. monocytogenes sublethal injury sheds light into the physiological and molecular characteristics of this state and provides the food science community with quantitative data to improve risk assessment.