Abstract Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens, able to cause severe disease in humans. In the DiSCoVeR project ( https://onehealthejp.eu/jrp-discover/ ) a STEC inventory from human and non-human sources from 11 European countries was set up and ≥ 3500 strains were sequenced to perform comparative genomics analysis. We used this dataset to assess STEC population structure and to investigate potential associations between genomic features, host reservoirs and symptoms. Most STEC isolates analysed by Whole Genome Sequencing (WGS) in this study were collected between years 2010-2020. An ad hoc pipeline was deployed for a harmonised characterization of the STEC in the database, allowing the determination of serotyping, stx gene subtyping, 7-loci MLST, virulotyping and cgMLST. The results were analysed with Principal Component Analysis (PCoA) in relation with isolation source to assess clustering of STEC subpopulations. When human STEC data were analysed, the PCoA revealed three distinct human STEC subpopulations (STEC_1, STEC_2 and STEC_3), which were further analysed for associations between genomic features, symptoms and variance. The non-human STEC showed a more dispersed distribution, except for one subpopulation with genes linked to specific host species, and some virulence profiles overlapping with the STEC_1 population. In conclusion, our analysis identified distinct STEC subpopulations from human cases, each characterized by specific genetic features and associated with varying proportions of severe disease outcomes. These findings provide novel insights supporting the risk assessment of STEC. Impact statement [ This lay summary of your article should be no more than 200 words, and should a) provide a perspective of how this article adds to the literature in the field; b) identify breadth of interest/utility; and c) state the significance of output (incremental or step), in terms of relevance .] This study is based on the establishment of a One Health STEC genomes database, including sequences from isolates of different sources. Most of the isolates had been isolated in the ten-years’ time span 2010-2020, in 11 different countries, for surveillance and monitoring activities or specific surveys and research purposes. The final dataset included the whole genome sequencing of 3,418 STEC isolates, mainly from human cases of infections. The metadata included the host symptoms, where available, for human STEC strains and the animal source the strains had been isolated from. We set up a pipeline for the harmonized analysis of STEC WGS, called Discover, made available though ARIES webserver or GitHub. The analysis allowed a deep characterization of STEC strains circulating in Europe. We used this resource to assess STEC population structure and to investigate potential associations between genomic features, host reservoirs, and various symptoms associated with STEC infection by PCoA. This analysis highlighted the presence of subpopulation of human STEC associated with specific features. We provide new information useful for risk characterization, as well as a large dataset genome database and associated metadata compiled from STEC strains, representing a valuable resource for the scientific community, enabling further investigations into STEC diversity, evolution, source attribution and public health relevance. Data summary The authors confirm all supporting data, including sequence data accession numbers, code and protocols have been provided within the article or through supplementary data files. One supplementary method and five supplementary tables are available with the online version of this article

Multidrug-resistant Salmonella enterica subsp. enterica serovar Infantis clone, harbouring the pESI megaplasmid, first described in Israel in 2014, is consistently reported in poultry and humans worldwide. This study aimed to investigate the genomic epidemiology of S. Infantis collected by nine European Public Health Institutions from samples of different origins (human, food and animal sources) and understand the evolutionary dynamics of pESI-like in Europe. The resolved pESI-like sequences have also been compared with complete publicly available pESI-like sequences from other countries, in a One Health context. The circulation of a S. Infantis clone in Europe carrying the mosaic megaplasmid pESI-like has been associated with resistance to sulphonamides (sul), tetracycline (tet), streptomycin and spectinomycin (aadA1). In recent years, bla CTX-M-1-positive pESI-like plasmids have been increasingly detected in the extended-spectrum beta-lactamase-producing S. Infantis clone. Using a combined short- and long-read sequencing approach, two main types of pESI variants have been identified, differing in the accessory gene content, including the bla CTX-M variant, indicating a certain stability of pESI variants detected in different geographical regions and sources over time (2011-2021). Moreover, the differences were related to the acquisition of resistance, virulence or fitness-enhancing genes that would potentially benefit the Salmonella host.

IntroductionInfections caused by Campylobacter spp. represent a severe threat to public health worldwide. National action plans have included source attribution studies as a way to quantify the contribution of specific sources and understand the dynamic of transmission of foodborne pathogens like Salmonella and Campylobacter. Such information is crucial for implementing targeted intervention. The aim of this study was to predict the sources of human campylobacteriosis cases across multiple countries using available whole-genome sequencing (WGS) data and explore the impact of data availability and sample size distribution in a multi-country source attribution model.MethodsWe constructed a machine-learning model using k-mer frequency patterns as input data to predict human campylobacteriosis cases per source. We then constructed a multi-country model based on data from all countries. Results using different sampling strategies were compared to assess the impact of unbalanced datasets on the prediction of the cases.ResultsThe results showed that the variety of sources sampled and the quantity of samples from each source impacted the performance of the model. Most cases were attributed to broilers or cattle for the individual and multi-country models. The proportion of cases that could be attributed with 70% probability to a source decreased when using the down-sampled data set (535 vs. 273 of 2627 cases). The baseline model showed a higher sensitivity compared to the down-sampled model, where samples per source were more evenly distributed. The proportion of cases attributed to non-domestic source was higher but varied depending on the sampling strategy. Both models showed that most cases could be attributed to domestic sources in each country (baseline: 248/273 cases, 91%; down-sampled: 361/535 cases, 67%;).DiscussionThe sample sizes per source and the variety of sources included in the model influence the accuracy of the model and consequently the uncertainty of the predicted estimates. The attribution estimates for sources with a high number of samples available tend to be overestimated, whereas the estimates for source with only a few samples tend to be underestimated. Reccomendations for future sampling strategies include to aim for a more balanced sample distribution to improve the overall accuracy and utility of source attribution efforts.

Non-typhoidal Salmonella is the second most frequently reported zoonotic pathogen in the European Union and European Economic Area. Most human infections are caused by serovars Enteritidis and Typhimurium. Genomic characterisation of Salmonella isolates from humans and animals has become a routine public health surveillance tool in many countries. In this study, the relative contributions of several potential sources of human infection of the five frequently reported Salmonella serovars were estimated using machine-learning methods based on a large, cross-sectional collection of genomes from human cases, and animal and environmental sources, across ten European countries. To define the population structure, core-genome Multilocus Sequence Typing was performed. A supervised machine-learning approach was applied for source attribution in the form of a Random Forest classifier. The source and country attribution models achieved moderate accuracy (F1=0.6-0.9), which is lower than in previous studies using machine-learning on Whole Genome Sequencing data. However, attributions of human clinical isolates to different sources were generally in line with previous findings for these five serovars. While the lack of clonality in some sources hindered their prediction, it is also likely that certain sources (e.g., pets) do not serve as major contributors to human infection. Therefore, in most cases attributing these sources to the livestock species they are typically associated with, is likely appropriate. Country attributions showed that substantial human cases are attributable to countries other than their own, indicating geographical interrelatedness of sources. This highlights the value of internationally harmonised Salmonella-control policies in the food production chain.

Objectives: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. Methods: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. Results: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncI gamma and IncFII plasmids. Conclusions: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the leading causes of foodborne infections associated with broilers and laying hens. Portugal has had the lowest notification rates of salmonellosis in recent years, due to the vaccinations of layer and breeder flocks and strict compliance with biosecurity measures. However, data about the genetic diversity of S. Enteritidis in Portugal are scarce. In this study, 102 S. Enteritidis isolates selected from human (n = 63) and non-human sources (n = 39) were characterized by serotyping, antimicrobial susceptibility, and whole genome sequencing. The S. Enteritidis population was mainly resistant to fluoroquinolones, and a sole isolate showed resistance to extended-spectrum cephalosporins. ST11 was the most frequent sequence type, and three novel STs from human isolates (ST9236, ST4457, and ST9995) were assigned. Several Salmonella pathogenic islands (SPI) and Putative SPI were present in the genomes, namely SPI-1, 2, 3, 4, 5, 9, 10, 12, 13, and 14, C63PI, CS54_island, and 170 virulence genes were identified. The phylogenetic analysis revealed that strains from Portugal are genetically heterogeneous regarding sample type, collection date, and genetic content. This study increases the available data, essential to a better characterization of strains in a global context.

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage.

Antimicrobial resistance encoded by mobile colistin resistance (mcr) genes is a global and emergent threat. In this study, we report the occurrence of two different populations of colistin-resistant Escherichia coli harboring mcr-1 and mcr-4 variants in the intestinal microbiome of a healthy pig. Following antimicrobial susceptibility determination, the presence of mcr genes in two E. coli strains, isolated according to different selective microbiological procedures, was screened by PCR. Whole-genome sequencing confirmed that both strains were multidrug-resistant; INIAV_002EC was an AmpC producer carrying blaCMY-2, blaTEM-1B, qnrS1, mcr-1.1 genes, and INIAV_001EC carrying blaTEM-1A, tetB, and mcr-4.1 genes, along with mutations in quinolone resistance-determining regions. In addition, both strains harbored sul3, dfrA, and aadA1 determinants. Further genome analysis revealed different plasmid replicons associated with the mcr genes, IncX4 associated with mcr-1.1, and ColE10 with mcr-4.1. In addition, other replicons, including IncFIA, IncI1-Iγ, IncX1, IncY, in INIAV_002EC, and IncX1, IncI1, and p0111, in INIAV_001EC, were identified. Furthermore, both strains belonged to ST215 serotype O68:H12 and ST156 serotype O25:H28, respectively. This finding highlights the pig gut flora as a potential reservoir of mobile colistin resistance genes and reports the presence of the mcr-4.1 gene found for the first time in Portugal.

Antimicrobial resistance (AMR) is one of the top public health threats nowadays. Among the most important AMR pathogens, Escherichia coli resistant to extended spectrum cephalosporins (ESC-EC) is a perfect example of the One Health problem due to its global distribution in animal, human, and environmental sources and its resistant phenotype, derived from the carriage of plasmid-borne extended-spectrum and AmpC β-lactamases, which limits the choice of effective antimicrobial therapies. The epidemiology of ESC-EC infection is complex as a result of the multiple possible sources involved in its transmission, and its study would require databases ideally comprising information from animal (livestock, companion, wildlife), human, and environmental sources. Here, we present the steps taken to assemble a database with phenotypic and genetic information on 10,763 ESC-EC isolates retrieved from multiple sources provided by 13 partners located in eight European countries, in the frame of the DiSCoVeR Joint Research project funded by the One Health European Joint Programme (OH-EJP), along with its strengths and limitations. This database represents a first step to help in the assessment of different geographical and temporal trends and transmission dynamics in animals and humans. The work performed highlights aspects that should be considered in future international efforts, such as the one presented here.

Enterococci are part of the commensal gut microbiota of mammals, with Enterococcus faecalis and Enterococcus faecium being the most clinically relevant species. This study assesses the prevalence and diversity of enterococcal species in cattle (n = 201) and pig (n = 249) cecal samples collected in 2017. Antimicrobial susceptibility profiles of E. faecium (n = 48) and E. faecalis (n = 84) were assessed by agar and microdilution methods. Resistance genes were screened through PCR and nine strains were analyzed by Whole Genome Sequencing. A wide range of enterococci species was found colonizing the intestines of pigs and cattle. Overall, the prevalence of resistance to critically important antibiotics was low (except for erythromycin), and no glycopeptide-resistant isolates were identified. Two daptomycin-resistant E. faecalis ST58 and ST93 were found. Linezolid-resistant strains of E. faecalis (n = 3) and E. faecium (n = 1) were detected. Moreover, oxazolidinone resistance determinants optrA (n = 8) and poxtA (n = 2) were found in E. faecalis (ST16, ST58, ST207, ST474, ST1178) and E. faecium (ST22, ST2138). Multiple variants of optrA were found in different genetic contexts, either in the chromosome or plasmids. We highlight the importance of animals as reservoirs of resistance genes to critically important antibiotics.

The intensive and extensive broiler production systems imply different veterinary interventions, including the use of antimicrobials. This study aimed to compare the antimicrobial susceptibility profiles of Escherichia coli isolated from both systems, characterize resistance mechanisms to β-lactams and polymyxins, and identify genetic elements such as integrons. E. coli isolates recovered from broiler cecal samples were assayed for antimicrobial susceptibility through the broth microdilution technique. The molecular characterization of acquired resistance mechanisms to β-lactams and colistin and the detection of integrons was performed by a multiplex PCR. For most antibiotics tested, the prevalence of reduced susceptibility is higher in commensal and extended-spectrum β-lactamases (ESBL)/AmpC producers from broilers raised in the intensive system, compared with those raised under extensive conditions. SHV-12 was the most common ESBL enzyme found in both production systems. Other ESBL variants such as CTX-M-1, CTX-M-55, CTX-M-14, CTX-M-32, CTX-M-9, TEM-52, and plasmid-encoded AmpC enzyme CMY-2 were also present. MCR-1 was identified in a colistin-resistant isolate from broilers raised under the intensive system. This study highlights the differences in E. coli antibiotic susceptibility from both production types and emphasizes that a great deal of work remains to decrease consumption and antimicrobial resistance levels.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 is mainly found in Europe and North America, colonizing the nasal cavity of pigs. This study characterized the MRSA isolates recovered from pig nasal swabs (n = 171) by evaluating the antimicrobial susceptibility profile by broth microdilution and characterizing the genetic lineages by spa-typing. Three linezolid-resistant isolates were subjected to Whole-Genome Sequencing (WGS). All strains harbored the mecA gene and were resistant to tetracycline and susceptible to vancomycin. A high frequency of multidrug resistance (97.6%) was evidenced, with 55 different multidrug resistance profiles identified. The MRSA strains were found to belong to 17 spa-types, three being novel. The linezolid-resistant strains appeared to belong to the ST398 type, spa-type t011, and SCCmec_type_Vc and to harbor the cfr, fexA, blaZ, mecA, tetM, and tetK genes. The cfr gene was predicted to be carried in the plasmid, flanked by ISSau9 and the transposon TnpR. MRSA from Portuguese fattening pigs present a high diversity of genetic lineages. The presence of cfr-positive LA-MRSA may represent a risk of transmission to humans, mainly to those in contact with livestock.

In this study, a rare strain of Escherichia coli co-harboring blaCTX-M-32 and blaCTX-M-2 genes was characterized by whole genome sequencing. Escherichia coli strain was recovered from a bovine cecal sample collected at slaughter. The strain was resistant to third and fourth generation cephalosporins, fluoroquinolones, aminoglycosides, tetracycline and sulfamethoxazole, which was in concordance with the genotype: blaCTX-M-32 and blaCTX-M-2, aac(3)-Via and tet(A) and sul1 genes, respectively. The E. coli strain was characterized as pathogenic, and belongs to the ST226 (CC226), serotype O141ab/ac:H5, phylogenetic group A and fumC27/fimH41 types. No virulence genes were found in the genome. According to the plasmid analysis, IncFIB, IncHI2 and p0111 were present. The analysis of the contigs revealed that the blaCTX-M-32 was located in the chromosome, upstream IS5-like/ISKpn26 and IS1380-like/ISEc9 sequences, whereas blaCTX-M-2 was in the p0111 plasmid, within an integron class 1, and flanked by Orf3/qacEΔ1 and IS91/ISCR1. To our knowledge, this is the first report of a multidrug resistant Escherichia coli strain co-harboring two CTX-M enzymes in a healthy bovine from Portugal. This finding allows a better understanding on the dissemination of these resistance mechanism among bacteria of animal origin.

The emergence and dissemination of resistance to third- and fourth-generation cephalosporins among Enterobacteriaceae from different sources impose a global public health threat. Here, we characterized by whole-genome sequencing four Escherichia coli strains harboring the blaCTX–M–65 gene identified among 49 isolates from beef and pork collected at retail. The genomic content was determined using the Center for Genomic Epidemiology web tools. Additionally, the prediction and reconstruction of plasmids were conducted, the genetic platform of the blaCTX–M–65 genes was investigated, and phylogenetic analysis was carried out using 17 other genomes with the same sequence type and harboring the blaCTX–M–65 gene. All strains harbored blaCTX–M–65, blaOXA–1, and blaTEM–1B, and one also carried the blaSHV–12 gene. Other resistance genes, namely, qnrS2, aac(6′)-Ib-c, dfrA14, sul2, tetA, and mphA, were present in all the genomes; the mcr-1.1 gene was identified in the colistin-resistant strains. They belong to sequence type 2179, phylogenetic group B1, and serotype O9:H9 and carried plasmids IncI, IncFIC(FII), and IncFIB. All strains share an identical genetic environment with IS903 and ISEcp1 flanking the blaCTX–M–65 gene. It seems likely that the blaCTX–M–65 gene is located in the chromosome in all isolates based on deep in silico analysis. Our findings showed that the strains are clonally related and belong to two sub-lineages. This study reports the emergence of CTX-M-65-producing E. coli in Portugal in food products of animal origin. The chromosomal location of the blaCTX–M–65 gene may ensure a stable spread of resistance in the absence of selective pressure.

The present study aimed to characterize the extended-spectrum β-lactamases and plasmid-mediated AmpC β-lactamases (ESBL/PMAβ) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAβ [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6')-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

The main aim of this study was the characterization of antibiotic resistance mechanisms in 82 Staphylococcus aureus strains isolated from humans and animals. Antibiotic susceptibility testing was performed on all S. aureus isolates accordingly, and antibiotic-resistant genes were investigated by genotypic methods. The genetic diversity of S. aureus was studied through spa, multilocus sequence typing (MLST), and agr typing methods. The majority of S. aureus from human sources were resistant to cefoxitin (and harbor the mecA gene) and fluoroquinolones, whereas only four strains of S. aureus from animal sources revealed resistance to ciprofloxacin. In the set of S. aureus isolated from humans, the most frequent spa, MLST, and agr group were t032, ST22, and I, respectively. In strains from animal origin the most common spa, MLST, and agr group found were t2383, ST398, and III/not typable, respectively. S. aureus from humans and animals were identified either in clonal complexes CC5, CC30, and CC398, suggesting that they have the same putative founder in their evolution. Considering the three CCs encompassing strains from human and animal reservoirs with different spa-types, we can hypothesize that this might reflect an adaptation to different phylogenetic lineages in those reservoirs (host species) probably associated to genetic diversification of pre-existing strains.

OBJECTIVES:WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS:The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS:Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS:WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

We studied a commensal colistin-resistant Escherichia coli isolated from a swine cecum sample collected at a slaughter, in Portugal. Antimicrobial susceptibility phenotype of E. coli LV23529 showed resistance to colistin at a minimum inhibitory concentration of 4 mg/L. Whole genome of E. coli LV23529 was sequenced using a MiSeq system and the assembled contigs were analyzed for the presence of antibiotic resistance and plasmid replicon types using bioinformatics tools. We report a novel mcr-1 gene variant (mcr-1.9), carried by an IncX4 plasmid, where one-point mutation at nucleotide T1238C leads to Val413Ala substitution. The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1. Bioinformatic analysis also revealed genes conferring resistance to β-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus bla CTX-M-8, a bla ESBL gene rarely detected in Europe in isolates of animal origin; these two genes were located on different plasmids with 33,303 and 89,458 bp, respectively. MCR-1.9-harboring plasmid showed high identity to other X4-type mcr-1-harboring plasmids characterized worldwide, which strongly suggests that the presence of PMCR-encoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally.

We screened 1840 Enterobacteriaceae isolates from food-producing animals, meat, meat products and animal feed, for the detection of plasmid-mediated colistin resistance, during 2010-2015. The mcr-1 gene was detected in 8.0% (97/1206) Escherichia coli and in 0.47% (3/634) Salmonella enterica isolates, with a high number of mcr-1 positive E. coli isolates (45.7%) being extended-spectrum β-lactamase or plasmid-mediated AmpC β-lactamase co-producers. No mcr-2 gene was detected. Our findings highlight the spread of mcr-1 genes within a wide-ranging sample of food-producing animals and meat, in Portugal.

A total of 502 Campylobacter jejuni isolates from poultry in 12 different European countries (10 of them the largest poultry production countries in Europe) were whole genome sequenced to examine the genomic diversity of fluoroquinolone resistant (FQ-R) and susceptible (FQ-S) C. jejuni across the poultry producing European countries and to determine whether the emergence of fluoroquinolone resistance among C. jejuni is related to the transmission through countries or to the selection through fluoroquinolone use in the individual countries. A high genomic diversity was observed. The isolates clustered in four main clusters. All trees revealed that the isolates were clustered according to the presence/absence of the gyrA mutations causing fluoroquinolone resistance and ST-types. The cgMLST trees of only FQ-R and FQ-S isolates showed that isolates from the same country of origin were distributed into multiple clusters similarly to the trees combining FQ-R and FQ-S isolates. The different phylogenetic methods, ranging from single nucleotide polymorphisms analysis to gene-bygene approaches such as rMLST, cgMLST, wgMLST and core genome tree, provided concordant results, but it is not known which is the most accurate method for identifying the country of origin of the isolates. Allele frequency analysis of isolates under this study and a selection of previously published C. jejuni genomes in ENA showed association of geographical origin of poultry C. jejuni populations between Romania-Poland, Italy-Germany-England, Portugal-The Netherlands and USALuxemburg. Allele frequency and phylogenetic analysis indicated that the isolates from Finland were genetically different from C. jejuni populations from other European countries included in this study. GenCamp – Genomics of quinolone-resistant and susceptible C. jejuni of poultry origin in EU countries www.efsa.europa.eu/publications 2 EFSA Supporting publication 2018:EN-1398 Trade pattern and antimicrobial use in livestock were not significantly associated with allele frequency or populations of C. jejuni, but data available to investigate these associations were limited. © Technical University of Denmark National Food Institute, 2018