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Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

International audience; Aim: Current European monitoring systems for antimicrobial resistance (AMR) fall short in identifying the drivers of horizontal transmission of AMR genes. Although an increasing emphasis is put on genomic-based surveillance, mobile genetic elements (MGEs) remain challenging to reconstitute from short-read sequence data due to their chimeric, modular and repetitive nature. Methods: The goal of the FULL_FORCE consortium will broadly introduce long-read sequencing in EU veterinary and public health institutes, by supplying 17 EU partners with a technological toolbox and hands-on training in MinION™ sequencing. This know-how will be applied on five study cases, and applications in metagenomics and AMR transmission models.Results: The Full Force Plasmid Assembler (FFPA v1.0) was created. This python script joins best-in-field tools to trim and QC short and long sequence reads (qcat, Trimmometric), enables species identification through Kraken and performs either Nanopore or hybrid assemblies through Unicycler. The wet and dry lab performance of each partner is being assessed in a proficiency test, which includes full-length sequencing of five multi-drug resistant Escherichia coli isolates. Moreover, the Full_Force methodology is applied in the phylogenetic analysis of IncI1, IncK plasmids in cross-sectional datasets, of epidemic ESBL plasmids from horses across Europe, in the diversity of the pESI megaplasmid in EU-wide isolated Salmonella Infantis strains, and in the plasmidome of Klebsiella pneumoniae strains isolated in a One Health setting. Conclusions: The effective implentation of long-read sequencing across public health and veterinary labs can provide a paradigm shift in EU AMR surveillance, delivering insight in dominant MGEs, which are driving resistance among commensal and pathogenic Enterobacterales in Europe.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying blaCTX-M-1 and the fos operon was identified.

The relatedness of the equine-associated Escherichia coli strain ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe (Czech Republic [n = 23], The Netherlands [n = 18], Germany [n = 9], Denmark [n = 3], and France [n = 1]) from 2008 to 2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250- SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure, and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 plasmids and one IncHI1/ST2 plasmid were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (,8 single nucleotide polymorphisms [SNP]). The majority of isolates belonged to phylogroup B1 (73/79 [92.4%]) and carried blaCTX-M-1 (58/79 [73.4%]). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62 [90.3%]) and ST2 (6/62 [9.7%]), while 84.5% (49/58) of the blaCTX-M-1 genes were associated with the presence of the IncHI1 replicon of ST9 and 6.9% (4/58) with the IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short-chain fructooligosaccharides (the fos operon) was present in 55 of 79 (69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of blaCTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (.98% similarity) regardless of country of origin and differed only in the structure and integration site of the multidrug resistance (MDR) region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically diverse strains associated with horses. A strong linkage of E. coli ST1250 with the epidemic multidrug resistance plasmid lineage IncHI1/ST9 carrying blaCTX-M-1 and the fos operon was identified.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.