The qualified presumption of safety (QPS) was developed to provide a safety pre‐assessment within EFSA for microorganisms. Strains belonging to QPS taxonomic units (TUs) still require an assessment based on a specific data package, but QPS status facilitates fast track evaluation. QPS TUs are unambiguously defined biological agents assessed for the body of knowledge, their safety and their end use. Safety concerns are, where possible, to be confirmed at strain or product level, and reflected as ‘qualifications’. Qualifications need to be evaluated at strain level by the respective EFSA units. The lowest QPS TU is the species level for bacteria, yeasts and protists/algae, and the family for viruses. The QPS concept is also applicable to genetically modified microorganisms used for production purposes if the recipient strain qualifies for the QPS status, and if the genetic modification does not indicate a concern. Based on the actual body of knowledge and/or an ambiguous taxonomic position, the following TUs were excluded from the QPS assessment: filamentous fungi, oomycetes, streptomycetes, Enterococcus faecium, Escherichia coli and bacteriophages. The list of QPS‐recommended biological agents was reviewed and updated in the current opinion and therefore now becomes the valid list. For this update, reports on the safety of previously assessed microorganisms, including bacteria, yeasts and viruses (the latter only when used for plant protection purposes) were reviewed, following an Extensive Literature Search strategy. All TUs previously recommended for 2016 QPS list had their status reconfirmed as well as their qualifications. The TUs related to the new notifications received since the 2016 QPS opinion was periodically evaluated for QPS status in the Statements of the BIOHAZ Panel, and the QPS list was also periodically updated. In total, 14 new TUs received a QPS status between 2017 and 2019: three yeasts, eight bacteria and three algae/protists.

In this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.

The provisional molecular approach, proposed by EFSA in 2013, for the pathogenicity assessment of Shiga toxin-producing Escherichia coli (STEC) has been reviewed. Analysis of the confirmed reported human STEC infections in the EU/EEA (2012-2017) demonstrated that isolates positive for any of the reported Shiga toxin (Stx) subtypes (and encoding stx gene subtypes) may be associated with severe illness (defined as bloody diarrhoea (BD), haemolytic uraemic syndrome (HUS) and/or hospitalisation). Although strains positive for stx2a gene showed the highest rates, strains with all other stx subtypes, or combinations thereof, were also associated with at least one human case with a severe clinical outcome. Serogroup cannot be used as a predictor of clinical outcome and the presence of the intimin gene (eae) is not essential for severe illness. These findings are supported by the published literature, a review of which suggested there was no single or combination of virulence markers associated exclusively with severe illness. Based on available evidence, it was concluded that all STEC strains are pathogenic in humans, capable of causing at least diarrhoea and that all STEC subtypes may be associated with severe illness. Source attribution analysis, based on 'strong evidence' outbreak data in the EU/EEA (2012-2017), suggests that 'bovine meat and products thereof', 'milk and dairy products', 'tap water including well water' and 'vegetables, fruit and products thereof' are the main sources of STEC infections in the EU/EEA, but a ranking between these categories cannot be made as the data are insufficient. Other food commodities are also potentially associated with STEC infections but rank lower. Data gaps are identified, and are primarily caused by the lack of harmonisation in sampling strategies, sampling methods, detection and characterisation methods, data collation and reporting within the EU. (VTEC). All these institutions provided data for this scientific output in the context of two consultations developed via the EU survey tool (Appendices I and J).

An increase in confirmed human salmonellosis cases in the EU after 2014 triggered investigation of contributory factors and control options in poultry production. Reconsideration of the five current target serovars for breeding hens showed that there is justification for retaining Salmonella Enteritidis, Salmonella Typhimurium (including monophasic variants) and Salmonella Infantis, while Salmonella Virchow and Salmonella Hadar could be replaced by Salmonella Kentucky and either Salmonella Heidelberg, Salmonella Thompson or a variable serovar in national prevalence targets. However, a target that incorporates all serovars is expected to be more effective as the most relevant serovars in breeding flocks vary between Member State (MS) and over time. Achievement of a 1% target for the current target serovars in laying hen flocks is estimated to be reduced by 254,400 CrI 95 [98,540; 602,700] compared to the situation in 2016. This translates to a reduction of 53.4% CrI 95 [39.1; 65.7] considering the layer-associated human salmonellosis true cases and 6.2% considering the overall human salmonellosis true cases in the 23 MSs included in attribution modelling. A review of risk factors for Salmonella in laying hens revealed that overall evidence points to a lower occurrence in non-cage compared to cage systems. A conclusion on the effect of outdoor access or impact of the shift from conventional to enriched cages could not be reached. A similar review for broiler chickens concluded that the evidence that outdoor access affects the occurrence of Salmonella is inconclusive. There is conclusive evidence that an increased stocking density, larger farms and stress result in increased occurrence, persistence and spread of Salmonella in laying hen flocks. Based on scientific evidence, an impact of Salmonella control programmes, apart from general hygiene procedures, on the prevalence of Campylobacter in broiler flocks at the holding and on broiler meat at the end of the slaughter process is not expected.

The role of food-producing environments in the emergence and spread of antimicrobial resistance (AMR) in EU plant-based food production, terrestrial animals (poultry, cattle and pigs) and aquaculture was assessed. Among the various sources and transmission routes identified, fertilisers of faecal origin, irrigation and surface water for plant-based food and water for aquaculture were considered of major importance. For terrestrial animal production, potential sources consist of feed, humans, water, air/dust, soil, wildlife, rodents, arthropods and equipment. Among those, evidence was found for introduction with feed and humans, for the other sources, the importance could not be assessed. Several ARB of highest priority for public health, such as carbapenem or extended-spectrum cephalosporin and/or fluoroquinolone-resistant Enterobacterales (including Salmonella enterica), fluoroquinolone-resistant Campylobacter spp., methicillin-resistant Staphylococcus aureus and glycopeptide-resistant Enterococcus faecium and E. faecalis were identified. Among highest priority ARGs bla CTX-M , bla VIM , bla NDM , bla OXA-48like , bla OXA-23 , mcr, armA, vanA, cfr and optrA were reported. These highest priority bacteria and genes were identified in different sources, at primary and post-harvest level, particularly faeces/manure, soil and water. For all sectors, reducing the occurrence of faecal microbial contamination of fertilisers, water, feed and the production environment and minimising persistence/recycling of ARB within animal production facilities is a priority. Proper implementation of good hygiene practices, biosecurity and food safety management systems is very important. Potential AMR-specific interventions are in the early stages of development. Many data gaps relating to sources and relevance of transmission routes, diversity of ARB and ARGs, effectiveness of mitigation measures were identified. Representative epidemiological and attribution studies on AMR and its effective control in food production environments at EU level, linked to One Health and environmental initiatives, are urgently required.

This Opinion considers the application of whole genome sequencing (WGS) and metagenomics for outbreak investigation, source attribution and risk assessment of food-borne pathogens. WGS offers the highest level of bacterial strain discrimination for food-borne outbreak investigation and sourceattribution as well as potential for more precise hazard identification, thereby facilitating more targeted risk assessment and risk management. WGS improves linking of sporadic cases associated with different food products and geographical regions to a point source outbreak and can facilitate epidemiological investigations, allowing also the use of previously sequenced genomes. Source attribution may be favoured by improved identification of transmission pathways, through the integration of spatial-temporal factors and the detection of multidirectional transmission and pathogenhost interactions. Metagenomics has potential, especially in relation to the detection and characterisation of non-culturable, difficult-to-culture or slow-growing microorganisms, for tracking of hazard-related genetic determinants and the dynamic evaluation of the composition and functionality of complex microbial communities. A SWOT analysis is provided on the use of WGS and metagenomics for Salmonella and Shigatoxin-producing Escherichia coli (STEC) serotyping and the identification of antimicrobial resistance determinants in bacteria. Close agreement between phenotypic and WGSbased genotyping data has been observed. WGS provides additional information on the nature and localisation of antimicrobial resistance determinants and on their dissemination potential by horizontal gene transfer, as well as on genes relating to virulence and biological fitness. Interoperable data will play a major role in the future use of WGS and metagenomic data. Capacity building based on harmonised, quality controlled operational systems within European laboratories and worldwide is essential for the investigation of cross-border outbreaks and for the development of international standardised risk assessments of food-borne microorganisms.

The 2011 EFSA opinion on Campylobacter was updated using more recent scientific data. The relative risk reduction in EU human campylobacteriosis attributable to broiler meat was estimated for on‐farm control options using Population Attributable Fractions (PAF) for interventions that reduce Campylobacter flock prevalence, updating the modelling approach for interventions that reduce caecal concentrations and reviewing scientific literature. According to the PAF analyses calculated for six control options, the mean relative risk reductions that could be achieved by adoption of each of these six control options individually are estimated to be substantial but the width of the confidence intervals of all control options indicates a high degree of uncertainty in the specific risk reduction potentials. The updated model resulted in lower estimates of impact than the model used in the previous opinion. A 3‐log10 reduction in broiler caecal concentrations was estimated to reduce the relative EU risk of human campylobacteriosis attributable to broiler meat by 58% compared to an estimate larger than 90% in the previous opinion. Expert Knowledge Elicitation was used to rank control options, for weighting and integrating different evidence streams and assess uncertainties. Medians of the relative risk reductions of selected control options had largely overlapping probability intervals, so the rank order was uncertain: vaccination 27% (90% probability interval (PI) 4–74%); feed and water additives 24% (90% PI 4–60%); discontinued thinning 18% (90% PI 5–65%); employing few and well‐trained staff 16% (90% PI 5–45%); avoiding drinkers that allow standing water 15% (90% PI 4–53%); addition of disinfectants to drinking water 14% (90% PI 3–36%); hygienic anterooms 12% (90% PI 3–50%); designated tools per broiler house 7% (90% PI 1–18%). It is not possible to quantify the effects of combined control activities because the evidence‐derived estimates are inter‐dependent and there is a high level of uncertainty associated with each.

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'.