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Abstract The European Commission requested scientific and technical assistance in the preparation of a EU‐wide baseline survey of antimicrobial resistance (AMR) in bacteria from aquaculture animals. It is recommended that the survey would aim at estimating the occurrence of AMR in Aeromonas spp. isolated from Atlantic Salmon (Salmo salar), European seabass (Dicentrarchus labrax) and trout (Salmo trutta, Salvelinus fontinalis, Oncorhynchus mykiss) intended to consumption, at harvesting (at farm/slaughter), at the EU level and in addition, at estimating the occurrence and diversity of AMR of Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Vibrio parahaemolyticus and Vibrio alginolyticus in blue mussel (Mytilus edulis) and Mediterranean mussel (Mytilus galloprovincialis) from production areas and at dispatch centres at the EU level. These technical specifications define the target populations, the sample size for the survey, sample collection requirements, the analytical methods (for isolation, identification, phenotypic susceptibility testing and further genotypic analysis of some of the bacteria targeted) and the data reporting requirements. The data to be reported by the EU Member States to support this baseline survey are presented in three data models. The results of the survey should be reported using the EFSA reporting system.

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.

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 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.

IntroductionExtended-spectrum β-lactamase- (ESBL) and AmpC- β-lactamase-producing Enterobacterales are widely distributed and emerging in both human and animal reservoirs worldwide. A growing concern has emerged in Europe following the appearance of carbapenemase-producing Escherichia coli (E. coli) in the primary production of food animals. In 2013, the European Commission (EC) issued the Implementing Decision on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. The European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) was tasked with providing two laboratory protocols for samples derived from meat and caecal content, respectively, for the isolation of ESBL- and AmpC-producing E. coli (part 1) and carbapenemase-producing (CP) E. coli (part 2). In this study, we describe the current protocols, including the preparatory work for the development.MethodsUp to nine laboratory procedures were tested using minced meat as the matrix from beef, pork, and chicken as well as six procedures for the caecal content of cattle, pigs, and chicken. Variables included sample volume, pre-enrichment volume, pre-enrichment broth with and without antimicrobial supplementation, and incubation time/temperature. The procedures were evaluated against up to nine E. coli strains harboring different AMR genes and belonging to the three β-lactamase groups.Results and discussionThe laboratory procedures tested revealed that the most sensitive and specific methodologies were based on a Buffered Peptone Water pre-enrichment of 225 ml to 25 g or 9 ml to 1 g for minced meat and caecal content, respectively, incubated at 37°C overnight, followed by inoculation onto MacConkey agar supplemented with 1 mg/L cefotaxime for detecting ESBL- and AmpC-producing E. coli and Chrom ID SMART (Chrom ID CARBA and OXA) for CP E. coli, incubated overnight at 37 and 44°C, respectively. We provided two isolation protocols for the EU-specific monitoring of ESBL- and AmpC- producing E. coli (part 1) and CP E. coli (part 2) from fresh meat (protocol 1) and caecal (protocol 2) samples, which have been successfully implemented by all EU Member States for the monitoring period 2014–2027 (EU 2020/1729).

Societal Impact Statement Mycorrhizal associations are acknowledged as key components of global ecosystem functioning. This is especially relevant in the context of global change, since they contribute to the amelioration of adverse soil conditions and play crucial roles in agriculture. Generally speaking, the lay public is uninformed on the importance of mycorrhizal fungi and symbiosis to our planet. Therefore, mycorrhizal scientific outreach activities are of paramount importance in order to bridge the aforementioned gap. We think that informing people about the benefits of mycorrhizal fungi and symbiosis in the face of global change, will raise general awareness of relevant research and aid conservation efforts. Summary Science outreach has become a particularly important duty in shortening the knowledge gap between scientists and the public, in order to strengthen societal decision‐making power in the global change crisis. Mycorrhizal fungi and mycorrhizal symbioses are key components of terrestrial ecosystems that contribute significantly to endure and reduce certain negative global change effects. Their importance has been gaining recognition in academic circles, but not among the general public. The aim of this article is to encourage as many mycorrhizal fungi researchers around the world as possible to build, through science outreach, a bridge between their scientific work and public interest. To this end, we conducted a review and discussed the relationship between global change and the mycorrhizal symbiosis. We highlight potential audiences, tools, resources, activities, outreach models, pros and cons, as well as the quantification potential for the outreach activities success. We extend an invitation to all mycorrhizologists around the world to contribute with mycorrhizal outreach material. Contributions will become available on the South American Mycorrhizal Research Network website for individuals or organizations interested in starting or innovating in mycorrhizal science outreach activities. Finally, the hashtag #mycorrhizalscienceoutreach is proposed to be used whenever a mycorrhizal fungi‐related science outreach activity is shared in social media.

Abstract The European Commission requested scientific and technical assistance in the preparation of a EU‐wide baseline survey protocol for a European Union (EU) coordinated monitoring programme on the prevalence of methicillin‐resistant Staphylococcus Aureus (MRSA) in pigs. The objective of the survey is to estimate the MRSA prevalence in batches of fattening pigs at slaughter at both European and national levels, with a 95% level of confidence and a level of precision of 10% considering an expected prevalence of 50%. The survey protocol defines the target population, the sample size for the survey, sample collection requirements, the analytical methods (for isolation, identification, phenotypic susceptibility testing and further genotypic testing of MRSA isolates), the data reporting requirements and the plan of analysis. The samples are to be analysed according to the laboratory protocols available on the European Union Reference Laboratory (EURL‐AR) website. Generalised linear models will be used to estimate proportion (with 95% confidence intervals) of batches of slaughter pigs tested positive to MRSA. The necessary data to be reported by the EU Member States to support this baseline survey are presented in three data models. The results of the survey should be reported using the EFSA data collection framework.

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.

Adipocytes exhibit different morphological and functional characteristics, depending on their anatomical location, developmental origin, and stimulus. While white adipocytes tend to accumulate energy as triglycerides, brown and beige adipocytes tend to direct carbon sources to fuel thermogenesis. White and beige adipocytes originate from common progenitor cells, which are distinct from brown adipocyte precursors. Having a method to study white vs. beige vs. brown adipocyte differentiation may help to unveil the mechanisms driving distinct adipogenic programs. Preadipocytes can be cultured and differentiated in vitro using a combination of compounds to stimulate adipogenesis. Here, we describe and compare protocols designed to stimulate adipocyte differentiation and induce brown/beige-like or white-like characteristics in differentiating adipocytes. The protocols consist in exposing murine preadipocytes to pharmacological stimuli aimed at triggering adipogenesis and inducing (or not) a thermogenic gene expression program. After 8 days of differentiation with a pro-browning cocktail, immortalized preadipocytes isolated from interscapular brown fat (9B cells) or inguinal white fat (9W cells) from the same mouse expressed higher levels of brown/beige adipocyte markers (e.g., Ucp1) and pan-adipocyte differentiation markers (e.g., Pparg, Cebpa and aP2) when compared to the same cells differentiated with a cocktail that lacked brown/beige adipogenic inducers (i.e., rosiglitazone, T3, and indomethacin). Consistent with a higher thermogenic potential of brown vs. beige adipocytes, differentiated 9B cells expressed higher Ucp1 levels than differentiated 9W cells. This simple protocol may help researchers to understand mechanisms of adipogenesis and how adipocytes become thermogenic.

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.

Abstract 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 blaCTX‐M, blaVIM, blaNDM, blaOXA‐48-like, blaOXA‐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.

Abstract 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 source‐attribution 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 pathogen–host 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 WGS‐based 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.

Abstract Proposals to update the harmonised monitoring and reporting of antimicrobial resistance (AMR) from a public health perspective in Salmonella, Campylobacter coli, Campylobacter jejuni, Escherichia coli, Enterococcus faecalis, Enterococcus faecium and methicillin‐resistant Staphylococcus aureus (MRSA) from food‐producing animals and derived meat in the EU are presented in this report, accounting for recent trends in AMR, data collection needs and new scientific developments. Phenotypic monitoring of AMR in bacterial isolates, using microdilution methods for testing susceptibility and interpreting resistance using epidemiological cut‐off values is reinforced, including further characterisation of those isolates of E. coli and Salmonella showing resistance to extended‐spectrum cephalosporins and carbapenems, as well as the specific monitoring of ESBL/AmpC/carbapenemase‐producing E. coli. Combinations of bacterial species, food‐producing animals and meat, as well as antimicrobial panels have been reviewed and adapted, where deemed necessary. Considering differing sample sizes, numerical simulations have been performed to evaluate the related statistical power available for assessing occurrence and temporal trends in resistance, with a predetermined accuracy, to support the choice of harmonised sample size. Randomised sampling procedures, based on a generic proportionate stratified sampling process, have been reviewed and reinforced. Proposals to improve the harmonisation of monitoring of prevalence, genetic diversity and AMR in MRSA are presented. It is suggested to complement routine monitoring with specific cross‐sectional surveys on MRSA in pigs and on AMR in bacteria from seafood and the environment. Whole genome sequencing (WGS) of isolates obtained from the specific monitoring of ESBL/AmpC/carbapenemase‐producing E. coli is strongly advocated to be implemented, on a voluntary basis, over the validity period of the next legislation, with possible mandatory implementation by the end of the period; the gene sequences encoding for ESBL/AmpC/carbapenemases being reported to EFSA. Harmonised protocols for WGS analysis/interpretation and external quality assurance programmes are planned to be provided by the EU‐Reference Laboratory on AMR.

During antimicrobial drug resistance testing for Vibrio spp. from coastal waters of Germany, we identified 4 nontoxigenic, carbapenem-resistant V. cholerae isolates. We used whole-genome sequencing to identify the carbapenemase gene blaVCC-1. In addition, a molecular survey showed that more blaVCC-1–harboring isolates are present in coastal waters of Germany.

Carbapenems are last-resort antibiotics used in human medicine. The increased detection of carbapenem-resistant Enterobacteriaceae (CRE) is therefore worrying. In 2011 we reported the first livestock-associated VIM-1-producing Salmonella (S.) enterica serovar Infantis (R3) isolate from dust, sampled in a German chicken fattening farm. Due to this observation we retrospectively investigated more than 536 stored bacterial cultures, isolated from 45 chicken fattening farms during the years 2011 and 2012. After a non-selective overnight incubation, the bacteria were transferred to selective media. Escherichia (E.) coli and Salmonella growing on these media were further investigated, including antibiotic susceptibility testing, carbapenemase gene screening and whole genome sequencing (WGS). In total, four CRE were found in three out of 45 investigated farms: Besides R3, one additional Salmonella (G-336-1a) as well as two E. coli isolates (G-336-2, G-268-2). All but G-268-2 harbored the blaVIM-1 gene. Salmonella isolates R3 and G-336-1 were closely related although derived from two different farms. All three blaVIM-1-encoding isolates possessed identical plasmids and the blaVIM-1- containing transposon showed mobility at least in vitro. In isolate G-268-2, the AmpC beta-lactamase gene blaCMY-2 but no known carbapenemase gene was identified. However, a transfer of the phenotypic resistance was possible. Furthermore, G-268-2 contained the mcr-1 gene, combining phenotypical carbapenem- as well as colistin resistance in one isolate. Carbapenem-resistant Enterobacteriaceae have been found in three out of 45 investigated chicken flocks. This finding is alarming and emphasizes the importance of intervention strategies to contain the environmental spread of resistant bacteria in animals and humans.

Extended-spectrum beta-lactamases (ESBL) mediating resistance to 3rd generation cephalosporins are a major public health issue. As food may be a vehicle in the spread of ESLB-producing bacteria, a study on the occurrence of cephalosporin-resistantu Escherichia coli in food was initiated. A total of 404 ESBL-producing isolates were obtained from animal-derived food samples (e.g., poultry products, pork, beef and raw milk) between 2011 and 2013. As CTX-M-15 is the most abundant enzyme in ESBL-producing E. coli causing human infections, this study focusses on E. coli isolates from food samples harboring the blaCTX-M-15 gene. The blaCTX-M-15 gene was detected in 5.2% (n = 21) of all isolates. Molecular analyses revealed a phylogenetic group A ST167 clone that was repeatedly isolated from raw milk and beef samples over a period of 6 months. The analyses indicate that spread of CTX-M-15-producing E. coli in German food samples were associated with a multireplicon IncF (FIA FIB FII) plasmid and additional antimicrobial resistance genes such as aac(6)-Ib-cr, blaOXA−1, catB3, different tet-variants as well as a class 1 integron with an aadA5/dfrA17 gene cassette. In addition, four phylogenetic group A ST410 isolates were detected. Three of them carried a chromosomal copy of the blaCTX-M-15 gene and a single isolate with the gene on a 90 kb IncF plasmid. The blaCTX-M-15 gene was always associated with the ISEcp1 element. In conclusion, CTX-M-15-producing E. coli were detected in German food samples. Among isolates of different matrices, two prominent clonal lineages, namely A-ST167 and A-ST410, were identified. These lineages may be important for the foodborne dissemination of CTX-M-15-producing E. coli in Germany. Interestingly, these clonal lineages were reported to be widely distributed and especially prevalent in isolates from humans and livestock. Transmission of CTX-M-15-harboring isolates from food-producing animals to food appears probable, as isolates obtained from livestock and food samples within the same time period exhibit comparable characteristics as compared to isolates detected from human. However, the routes and direction of transmission need further investigation.

El tratamiento de residuales líquidos es prioridad para el sistema de salud en Cuba. Los altos precios de los coagulantes químicos y la escasez de materias primas para producirlos nacionalmente propician el empleo de las semillas de la especie vegetal Moringa oleífera como alternativa para la sustitución de coagulantes químicos. En este trabajo se utilizó el residuo después de la extracción de aceite para el tratamiento de agua residual de una planta municipal, determinando parámetros indicadores de calidad de los residuales líquidos. La demanda química de oxígeno obtenida fue de 80 mg/L con una dosis de coagulante de 69,70 mg/L, lográndose una reducción superior al 90 %. Se analizó el contenido de sólidos, dureza, alcalinidad y fósforo. Con la utilización del producto evaluado se obtuvieron ventajas ambientales, económicas y sociales. Palabras clave: coagulantes, semillas de Moringa oleífera, tratamiento de residuales líquidos.

Abstract EFSA and EMA have jointly reviewed measures taken in the EU to reduce the need for and use of antimicrobials in food‐producing animals, and the resultant impacts on antimicrobial resistance (AMR). Reduction strategies have been implemented successfully in some Member States. Such strategies include national reduction targets, benchmarking of antimicrobial use, controls on prescribing and restrictions on use of specific critically important antimicrobials, together with improvements to animal husbandry and disease prevention and control measures. Due to the multiplicity of factors contributing to AMR, the impact of any single measure is difficult to quantify, although there is evidence of an association between reduction in antimicrobial use and reduced AMR. To minimise antimicrobial use, a multifaceted integrated approach should be implemented, adapted to local circumstances. Recommended options (non‐prioritised) include: development of national strategies; harmonised systems for monitoring antimicrobial use and AMR development; establishing national targets for antimicrobial use reduction; use of on‐farm health plans; increasing the responsibility of veterinarians for antimicrobial prescribing; training, education and raising public awareness; increasing the availability of rapid and reliable diagnostics; improving husbandry and management procedures for disease prevention and control; rethinking livestock production systems to reduce inherent disease risk. A limited number of studies provide robust evidence of alternatives to antimicrobials that positively influence health parameters. Possible alternatives include probiotics and prebiotics, competitive exclusion, bacteriophages, immunomodulators, organic acids and teat sealants. Development of a legislative framework that permits the use of specific products as alternatives should be considered. Further research to evaluate the potential of alternative farming systems on reducing AMR is also recommended. Animals suffering from bacterial infections should only be treated with antimicrobials based on veterinary diagnosis and prescription. Options should be reviewed to phase out most preventive use of antimicrobials and to reduce and refine metaphylaxis by applying recognised alternative measures.

Abstract EFSA was requested to: 1) assess the risk for the development of antimicrobial resistance (AMR) due to feeding on farm of calves with colostrum potentially containing residues of antibiotics; 2) assess the risk for the development of AMR due to feeding on farm of calves with milk of cows treated during lactation with an antibiotic and milked during the withdrawal period, and 3) propose possible options to mitigate the risk for the development of AMR derived from such practices. Treatment of dairy cows during the dry period and during lactation is common in the EU Member States. Penicillins, alone or in combination with aminoglycosides, and cephalosporins are most commonly used. Residue levels of antimicrobials decrease with the length of the dry period. When the interval from the start of the drying‐off treatment until calving is as long as or longer than the minimum specified in the Summary of Product Characteristics of the antimicrobial, faecal shedding of antimicrobial‐resistant bacteria will not increase when calves are fed colostrum from treated cows. Milk from cows receiving antimicrobial treatment during lactation contains substantial residues during the treatment and withdrawal period. Consumption of such milk will lead to increased faecal shedding of antimicrobial‐resistant bacteria by calves. A range of possible options exist for restricting the feeding of such milk to calves, which could be targeting the highest priority critically important antimicrobials. β‐Lactamases can reduce the concentration of β‐lactams which are the most frequently used antimicrobials in milking cows. Options to mitigate the presence of resistant bacteria in raw milk or colostrum are mainly based on thermal inactivation.

In developing countries, Salmonella enterica serovar Enteritidis causes substantial illness and death, and drug resistance is increasing. Isolates from the United Kingdom containing virulence-resistance plasmids were characterized. They mainly caused invasive infections in adults linked to Africa. The common features in isolates from these continents indicate the role of human travel in their spread.

Las enfermedades transmitidas por alimentos constituyen un problema cada vez más importante a nivel internacional. Según la Organización Mundial de la Salud, entre un 70 y un 80 % de los casos de diarrea que se producen se deben a la ingestión de agua y alimentos contaminados, constituyendo actualmente un desafío, puesto que se desconoce su real incidencia. Motivados por la necesidad de incrementar la vigilancia de los brotes de estas enfermedades y de realizar un adecuado estudios de los mismos, se realizó una investigación epidemiológica observacional descriptiva de un brote de enfermedades transmitidas por alimentos, ocurrido en una instalación hotelera en Varadero, en agosto de 2009. La muestra estuvo constituida por 58 turistas: 29 sanos e igual número de enfermos, a los que se le aplicó la encuesta validada para estudiar estos eventos; de ella se obtuvo la información. Se calculó la Mediana para determinar período de incubación, haciéndolo corresponder con el posible agente etiológico. Se halló la tasa de ataque específica, la cual indicó el alimento sospechoso. Se realizó estudios de laboratorio a muestras de alimentos, especímenes y a manipuladores de alimentos. Se demostró que la no aplicación de las buenas prácticas de manufactura de los alimentos, unido al deterioro del cuadro higiénico de la instalación, fue la causa directa del brote; el agente causal la Samonella D, aislada en el revoltillo y en los especímenes.The diseases transmitted by food are a more and more important problem at the international level. According to the World Health Organization, between the 70 and the 80 % of the diarrheic cases are due to the ingestion of contaminated water and food, being a challenge nowadays, because its real incidence is unknown. Motivated for the necessity of increasing the surveillance of these diseases outbreaks and adequately studying them, we carried out the descriptive, observational, epidemiologic survey of an outbreak of diseases transmitted by foods that took place in a hotel in Varadero, in August 2009. The sample was formed by 59 tourists: 28 healthy and the same number of sick. They applied a survey validated to study these events; the information was taken from it. The media was calculated to determine the incubation period, making it correspond with the possible etiologic agent. We calculated the specific attack rate, indicating the suspecting food. We made laboratory studies of food samples, specimens and food manipulators. It was showed that not applying good practices of food elaboration, together with the worsening of the health situation of the installation, was the direct cause of the outbreak; the causal agent was the Salmonella D, isolated in the scrambled eggs and the specimens.

Non-typhoidal serovars of Salmonella enterica (NTS) are a leading cause of food-borne disease in animals and humans worldwide. Like other zoonotic bacteria, NTS have the potential to act as reservoirs and vehicles for the transmission of antimicrobial drug resistance in different settings. Of particular concern is the resistance to critical “last resort” antimicrobials, such as carbapenems. In contrast to other Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli, and Enterobacter, which are major nosocomial pathogens affecting debilitated and immunocompromised patients), carbapenem resistance is still very rare in NTS. Nevertheless, it has already been detected in isolates recovered from humans, companion animals, livestock, wild animals, and food. Five carbapenemases with major clinical importance—namely KPC (Klebsiella pneumoniae carbapenemase) (class A), IMP (imipenemase), NDM (New Delhi metallo-β-lactamase), VIM (Verona integron-encoded metallo-β-lactamase) (class B), and OXA-48 (oxacillinase, class D)—have been reported in NTS. Carbapenem resistance due to the production of extended spectrum- or AmpC β-lactamases combined with porin loss has also been detected in NTS. Horizontal gene transfer of carbapenemase-encoding genes (which are frequently located on self-transferable plasmids), together with co- and cross-selective adaptations, could have been involved in the development of carbapenem resistance by NTS. Once acquired by a zoonotic bacterium, resistance can be transmitted from humans to animals and from animals to humans through the food chain. Continuous surveillance of resistance to these “last resort” antibiotics is required to establish possible links between reservoirs and to limit the bidirectional transfer of the encoding genes between S. enterica and other commensal or pathogenic bacteria.

No presente artigo, os autores discutem brevemente sobre a leptospirose, realçando a forma pulmonar da doença. Revê-se a patologia, achados clínicos, diagnóstico por métodos de imagem e broncoscopia e tratamento da leptospirose pulmonar. É também lembrado o diagnóstico clínico e radiológico precoces, para que se possa iniciar terapêutica adequada. Os autores concluem que a forma pulmonar da leptospirose deve ser sempre considerada como causa e diagnóstico diferencial da hemorragia alveolar difusa e síndroma de dificuldade respiratória do adulto.In this article, the authors discuss briefly the leptospirosis, emphasizing mainly the pulmonary form of disease. The authors review pathology, clinical findings, imaging and broncoscopy diagnosis, treatment of pulmonary leptospirosis. It is also remembered about early clinics and radiology diagnosis to start therapeutics. The authors conclude that pulmonary form of disease must always be remembered and considered as cause and differential diagnosis of Diffuse Alveolar Hemorrhage and Adult Respiratory Distress Syndrome.

Resumo: No presente artigo, os autores discutem brevemente sobre a leptospirose, realçando a forma pulmonar da doença. Revê-se a patologia, achados clínicos, diagnóstico por métodos de imagem e broncoscopia e tratamento da leptospirose pulmonar. à também lembrado o diagnóstico clínico e radiológico precoces, para que se possa iniciar terapêutica adequada. Os autores concluem que a forma pulmonar da leptospirose deve ser sempre considerada como causa e diagnóstico diferencial da hemorragia alveolar difusa e síndroma de dificuldade respiratória do adulto.Rev Port Pneumol 2007; XIII (6): 827-839 Abstracts: In this article, the authors discuss briefly the leptospirosis, emphasizing mainly the pulmonary form of disease. The authors review pathology, clinical findings, imaging and broncoscopy diagnosis, treatment of pulmonary leptospirosis. It is also remembered about early clinics and radiology diagnosis to start therapeutics. The authors conclude that pulmonary form of disease must always be remembered and considered as cause and differential diagnosis of Diffuse Alveolar Hemorrhage and Adult Respiratory Distress Syndrome.Rev Port Pneumol 2007; XIII (6): 827-839 Palavras-chave: Leptospirose, manifestações respiratórias, hemorragia alveolar difusa e síndroma da dificuldade respiratória do adulto, Key-words: Leptospirosis, respiratory manifestations, diffuse alveolar hemorrhage and adult respiratory distress syndrome

RESUMO: Relato de caso de paracoccidiodomicose forma crónica, tipo adulta com lesões genitais e forma residual, apresentada como disfagia e disfonia no mesmo paciente. Diagnóstico confirmado pelo exame direto das lesões cutâneas. Após tratamento específico, houve regressão das lesões e sintomas desapareceram.REV PORT PNEUMOL 2003; IX (2): 173-178 ABSTRACT: Case report of paracoccidiodomycosis Chronic Form, adult type, with genital lesions and residual form presented as dysphagia and dysphonia at the same patient. Diagnosis accomplished by direct examination of clinical specimens, in this case cutaneous lesions. After specific treatment there was regression of lesions and symptons had desapeared.REV PORT PNEUMOL 2003; IX (2): 173-178 Palavras-chave: Paracoccidiodomicose Crónica, forma crónica, tipo adulta, forma residual, diagnóstico exame directo, Key-words: Paracoccidiodomycosis Chronic Form, adult type, residual form, diagnosis direct examination

Beta-lactamase resistant bacteria and especially ESBL producing Enterobacteriaceae are an increasing problem worldwide. For this reason a major interest in efficient and reliable methods for rapid screening of high sample numbers is recognizable. Therefore, a multiplex real-time PCR was developed to detect the predominant class A beta-lactamase genes blaCTX-M, blaSHV, blaTEM and CIT-type AmpCs in a one-step reaction. A set of 114 Enterobacteriaceae containing previously identified resistance gene subtypes and in addition 20 undefined animal and environmental isolates were used for the validation of this assay. To confirm the accessibility in variable settings, the real-time runs were performed analogous in two different laboratories using different real-time cyclers. The obtained results showed complete accordance between the real-time data and the predetermined genotypes. Even if sequence analyses are further necessary for a comprehensive characterization, this method was proofed to be reliable for rapid screening of high sample numbers and therefore could be an important tool for e. g. epidemiological purposes or support infection control measures.

Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3' conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and blaTEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla(CTX-M-group-1) dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla(CTX-M-group-1) and bla(OXA-1-like) genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6')-Ib, catB3, bla(OXA-1-like) and bla(CTX-M-group-1). forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

We found antibodies to leptospires in 25 (18%) of 141 wild boars from Berlin (95% confidence interval 12–25). Seropositivity was associated with chronic interstitial nephritis (odds ratio 10.5; p = 0.01), and leptospires were detected in kidney tissues. Wild boars represent a potential source for human leptospirosis in urban environments.