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Aim: We investigated different sequencing and assembling methods regarding their ability to detect resistance genes and predict the association to mobile genetic elements as plasmids in E.coli. Methods: Five E.coli isolates with distinct resistance profiles and diverse plasmid contents were characterized in detail by antimicrobial susceptibility testing, XbaI-/ S1-PFGE profiling, and filter mating studies. The biological characteristics of the isolates were compared with in silico predictions using different sequencing strategies. Raw reads were generated using the Illumina NextSeq, the long-read systems PacBio Sequel and MinION. The reads were used individually or in combinations to assess appropriate workflows for reliable in silico-based typing purposes. Results: Long-reads alone were shown to be error-prone, affecting the annotation of genes and the prediction of small plasmids. However, their use represented the best option to generate genome-scaffolds. Short-reads were reliable for detecting resistance genes, but were not suitable for linking specific resistance determinants to the chromosome or to plasmid types. Moreover, short-read sequencing missed duplicated resistance genes. The use of hybrid-assemblies led to the best consensus between in silico and in vitro results, as all duplications and small plasmids were predicted and provided closed plasmid for allmost all extrachromosomal elements. Conclusions: Hybrid assembling provided a thorough overview on extrachromosomal DNA elements, associated with antimicrobial resistances and detailed insights into the genomes genetic composition. Overall, it will be worth extending the routine sequence diagnostic to hybrid sequencing, when a reference-grade complete bacterial genome is aimed, or extrachromosomal structures needs to be fully understood. &nbsp

Aim: The prevalence and diversity of qnr-carrying isolates among (fluoro-)quinolone (FQ)-resistant, commensal E. coli from the German annual antimicrobial resistance monitoring in livestock and food in 2017 was determined. The data will be used to understand mechanisms involved in FQ-resistance development and the diversity of mobile genetic elements associated with their spread. Methods: A total of 3,409 E. coli isolates from the German National Reference Laboratory for Antimicrobial Resistance was investigated. Antimicrobial resistance testing was conducted by broth microdilution according to CLSI guidelines. MIC values for ciprofloxacin and nalidixic acid were evaluated using EUCAST epidemiological cut-off values. Isolates exhibiting resistances to FQ were subjected to qnr-PCR, XbaI-/S1-PFGE, whole genome sequencing (WGS) and bioinformatics analysis. Results: Overall, 504 isolates were classified as quinolone-resistant, while only 107 of them harbored a qnr gene. XbaI-macrorestriction demonstrated a high heterogeneity, letting us assume that no predominant E. coli lineages are associated with FQ resistance development in Germany. S1-PFGE analysis showed a variety of extrachromosomal elements of various sizes. Short-read WGS revealed qnr to be associated with bla as well as with the disinfectant qacEΔ1. The most abundant plasmid replicon type among our identified qnr reference plasmids were IncN, IncY and IncX1-IncX3. Further, we frequently detected chromosomal point mutations leading to an altered susceptibility against FQs. Conclusions: qnr genes were shown to be carried by multiple plasmid types and in frequent association with other resistance genes. This diversity of qnr plasmids in combination with their multi-resistance demonstrates the risk associated with qnr positive E. coli.

Background: Colistin represents an important antimicrobial for the veterinary and human sector. Besides its outstanding antimicrobial use in gastrointestinal infections in animals, it is currently a last-line treatment option for human infections caused by multidrug-resistant Gram-negative bacteria. Successively after the description of the first mobile colistin resistance (mcr-1) element in Escherichia coli, the identification of other genes (mcr-2 to mcr-10) and variants has forced the understanding of the colistin resistance and dissemination mechanisms in Enterobacterales. Material and Methods For comparative analysis, a collection of Spanish (n=28), German (n=14) and Portuguese (n=9) E. coli isolates of porcine origin exhibiting the mobile colistin resistance determinant mcr-4, were subjected to phenotypic and genotypic in depth characterization. The isolates were investigated for their antimicrobial susceptibility, macrorestriction profiles (XbaI-PFGE), plasmid patterns (S1-PFGE) and plasmid transmission (in vitro filter mating studies). Short-read whole-genome sequencing (WGS) data were used for in silico-based typing of the genomes. Results: Overall, the investigated isolates of the three countries differed substantially in their macrorestriction profiles. While Portuguese and Spanish isolates exhibited a closer phylogenetic identity, in relation with their geographic origin, German isolates showed high heterogeneity. Similar results were observed from S1-PFGE analysis. However all isolates showed a low size mcr-4 carrying plasmid (range10 to 25 kb). In vitro transmission to the sodium azide-resistant E. coli J53 strain was confirmed in at least 50% of the Spanish and Portuguese isolates. Interestingly, most of the transconjugants harbored two plasmids of which only one carried the mobilizable mcr-4 plasmid, while the second would probably act as a helper for the transmission. While the mcr-4 plasmids seem to be based on a highly conserved ColE10 plasmid backbone, the majority of their host are highly heterogeneous. Conclusions: Our results indicate a close relationship of the individual mcr-4 carrying plasmids of Portugal, Spain and Germany. Thus, we suppose that dissemination of the conserved plasmid-type is based on a common ancestor However, the impact of this gene is currently unknown, since no comprehensive information on mcr determinants in colistin-resistant isolates from human infections exists., L. Lestón acknowledges the Ministry of Education of Spain for her pre-doctoral grant FPU19/01127. I. García-Meniño acknowledges (FEMS-GO-2020-205). This research was funded through: PID2019- 104439RB-C21/AEI/10.13039/501100011033 from the Agencia Estatal de Investigación (AEI, Spain), cofounded by the Euro-pean Regional Development Fund of the European Union: a Way to Making Europe (FEDER). The research of J.A. Hammerl was supported by the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 773830 (FULL-FORCE)., info:eu-repo/semantics/publishedVersion

Introduction: Resistance to quinolones can be chromosomally encoded or plasmid-mediated (PMQR). One PMQR mechanism is mediated by Qnr proteins. The horizontal gene transfer of this plasmid-mediated quinolone resistance increases the threat of fallible treatment with quinolones. Objectives: To better understand the distribution of PMQR, in particular qnr genes, Escherichia (E.) coli isolates recovered in 2017 from livestock and food were phenotypically and genotypically characterized. Materials & Methods: 3,409 E. coli isolates from the German National Reference Laboratory for Antimicrobial Resistance were investigated. The isolates were received in the German national monitoring program for antimicrobial resistance. Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines. MIC values for ciprofloxacin and nalidixic acid were evaluated using EUCAST epidemiological cut-off values (MICNAL ≥16 mg/L, MICCIP ≥0.06 mg/L). E.coli resistant to quinolones were subjected to qnr-PCR, XbaI-PFGE, S1-PFGE, WGS and bioinformatic analysis. Six different qnr-PCRs were conducted to identify the respective qnr-variants. Results: Overall, 504 isolates were classified as quinolone-resistant. Of those, 107 were found to harbor a qnr gene. The most abundant qnr-variant was qnrS. PFGE profiling for the 107 qnr positive isolates demonstrated a high heterogeneity, indicating that they are not associated to a predominant E. coli clone spreading via vertical transmission. S1-PFGE plasmid profiling showed a variety of extrachromosomal elements of various sizes. 43 Isolates, selected according their XbaI- and S1-PFGE pattern were further screened for their genetic setting through short read whole genome sequencing (WGS). Sequencing confirmed the high genetic diversity of the quinolone-resistant E. coli strains. Conclusion: Quinolone-resistance could not be attributed to a specific lineage of E. coli. Further analysis is needed for a better understanding of the plasmid diversity within qnr-harboring E. coli and the prerequisites of their spread.&nbsp