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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.

The effectiveness of antibiotics has been reduced since their discovery. Surveillance and monitoring systems are key elements to collect and assess the trends of antibiotic resistance (ABR). Livestock data on ABR are traditionally collected from bacterial populations of clinical and non-clinical isolates. Resistance data on non-clinical isolates are based on the Decision 2013/652/EU in Europe. For clinical isolates, different standards, laboratory methods and methodologies are applied to collect ABR data. Further, a clinical interpretation is preferred in clinical isolates, while an epidemiological approach is frequently used in non-clinical isolates. Lack of harmonization between data types (clinical vs. non-clinical isolates) prevents the data comparison. The Normalized Resistance Interpretation (NRI) method was applied to circumvent the lack of ABR data harmonization between and within countries. Analyses were performed to identify associations between resistance to four antibiotics (ampicillin, gentamicin, nalidixic acid and tetracycline) and the data type variable per animal category (broiler, turkey and calf) within countries. Within countries, higher resistance proportions were found in clinical isolates vs commensal isolates to: gentamicin in broilers from France and in calves from Germany and France, nalidixic acid in calves from France and Germany and tetracycline for calves from France and Germany. In contrast, a higher probability of resistance in non-clinical isolates was encountered for tetracycline in broilers and turkeys from Germany and France and to gentamicin in turkeys from Germany. It seems that the higher presence of resistance in one data type (i.e. clinical or non-clinical isolates) is strongly associated with the relationship between the animal species and the antibiotic. The NRI identifies the wild-type distribution providing approximate epidemiological cut-offs that allow comparing quantitative results from different ABR systems with a different level of harmonization. This method might be regularly used in veterinary medicine and in One Health studies to compare not harmonized ABR systems until international harmonization of ABR is achieved.

PhD02-LIN-RES

PhD02-LIN-RES

Routine antimicrobial susceptibility testing (AST) is a prerequisite for understanding and treating Clostridioides (formerly Clostridium, C.) difficile infections (CDI) which cause thousands of fatal cases in the EU each year. The disc diffusion (DD) methodology proved to bea suitable and convenient alternative to the current gold standard, the agar dilution (AD) for moxifloxacin, metronidazole and vancomycin [1]. Yet, DD has major drawbacks regarding reproducibility and the unknown influence of anaerobic conditions on the testresult. The aim of this study within the One Health European Joint Programme project IMPART (IMproving Phenotypic AntimicrobialResistance Testing) project is to optimize and propose a protocol for a robust DD method for C. difficile for a broader range ofantimicrobials and to find out, under which conditions and cut-off values a resistance determination is reliable.

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

drugs. 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 some antibiotics. To better understand the qnr PMQR pathway as well as the distribution of qnr genes, Escherichia (E.) coli isolates recovered in 2016 and 2017 from livestock and food were phenotypically and genotypically characterized. Materials/methods: 6,817 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 in zoonotic bacteria, recovered from livestock and food. Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines. MIC values were evaluated using EUCAST epidemiological cut-off values. 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: Of 6,817 E. coli tested, more than 800 isolates were classified as quinolone-resistant (MICNAL ≥16 mg/L and/or MICCIP ≥0.06 mg/L). The most abundant qnr-variant was qnrS. With the exception of qnrD, other qnr-variants were found evenly distributed. In general, E. coli isolates with qnr-genes were found more frequently in the feces of animals than in the meat of the same animal species. PFGE with XbaI-digestion was performed to examine genetic relatedness of isolates. PFGE profiling demonstrated a rather high heterogeneity. The highly diverse PFGE pattern indicates that the screened isolates 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. Isolates, selected according their PFGE pattern were further screened for their genetic setting through short read whole genome sequencing (WGS). Sequencing of those isolates confirmed the high genetic diversity of the quinolone-resistant E. coli strains. Conclusions: Quinolone-resistance could not be attributed to a specific lineage of E. coli. Further analysis is needed for better understanding of the plasmid diversity within qnr-harboring E. coli and the prerequisites of their spread.

Background and objectives: Colistin is considered as highest priority critically important antibiotic, only used to treat human infections caused by multidrug-resistant Gram-negative bacteria. So far, seven different mobile colistin resistance-genes are known. Here, characteristics of mcr-4-positive colistin-resistant E.coli isolates from the national monitoring for antimicrobial resistance in zoonotic agents from the food chain were summarized. Materials and methods: Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines and EUCAST epidemiological cut-off values. Resistant E.coli were subjected to mcr-PCR, S1-PFGE, MiSeq-sequencing (WGS) and bioinformatics. Transferability of mcr-4 carrying plasmids was investigated by filter mating experiments. Results: Up to now, 13 mcr-4-positive out of 756 colistin-resistant E.coli isolates, recovered between 2010 and 2017, were identified. Sanger sequencing revealed that two variants, mcr-4.2 and mcr-4.3, are prevalent among these isolates. WGS showed that the isolates differ in their MLST-, sero- and fim-type. However, all of them carry a highly conserved ColE-plasmid prototype that partially differs in size and genetic composition. Conclusion: Our findings indicate that mcr-4 occurs sporadically among colistin-resistant E.coli from food and livestock. However, further information on the biology and genetics are needed to assess the impact of this resistance determinant on public health. &nbsp

drugs. 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 some antibiotics. To better understand the qnr PMQR pathway as well as the distribution of qnr genes, Escherichia (E.) coli isolates recovered in 2016 and 2017 from livestock and food were phenotypically and genotypically characterized. Materials/methods: 6,817 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 in zoonotic bacteria, recovered from livestock and food. Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines. MIC values were evaluated using EUCAST epidemiological cut-off values. 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: Of 6,817 E. coli tested, more than 800 isolates were classified as quinolone-resistant (MICNAL ≥16 mg/L and/or MICCIP ≥0.06 mg/L). The most abundant qnr-variant was qnrS. With the exception of qnrD, other qnr-variants were found evenly distributed. In general, E. coli isolates with qnr-genes were found more frequently in the feces of animals than in the meat of the same animal species. PFGE with XbaI-digestion was performed to examine genetic relatedness of isolates. PFGE profiling demonstrated a rather high heterogeneity. The highly diverse PFGE pattern indicates that the screened isolates 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. Isolates, selected according their PFGE pattern were further screened for their genetic setting through short read whole genome sequencing (WGS). Sequencing of those isolates confirmed the high genetic diversity of the quinolone-resistant E. coli strains. Conclusions: Quinolone-resistance could not be attributed to a specific lineage of E. coli. Further analysis is needed for better understanding of the plasmid diversity within qnr-harboring E. coli and the prerequisites of their spread. &nbsp

OHEJP Project: IMPART. WP3 - Development of missing epidemiological cut-off values (ECOFFs)., This project was co funded by the Dutch Ministry of Agriculture, Nature and Food Quality.

Questions: Colistin is considered as an important antibiotic of the last-resort, which will be only used for the treatment of severe human infections with multidrug-resistant Gram-negative bacteria. Since 2015, several mobile colistin resistance genes were described coding for enzymes of the phosphoethanolamine-transferase family. To date, eight different mcr-genes have been characterized, mediating resistance to colistin in different bacterial genera (especially in Enterobacteriaceae). Material & Methods: By molecular screening on mcr-1 to -5 using the multiplex PCR of Rebelo et al. (2018), an E.coli isolate recovered in 2013 from chicken feces was identified to carry a mcr-5 resistance gene. Antimicrobial resistance testing according to the CLSI-guideline was performed. MIC-data were interpreted using the ECOFFS of EUCAST. The genome of the mcr-5-plasmid was deduced by whole-genome sequencing using different platforms (MiSeq, Illumina and MinIon, Nanopore). Bioinformatic analyses were performed to determine the genome structure and composition of the plasmid and isolate. Results: Within this study, a novel mcr-5 plasmid-prototype was identified in the E.coli isolate from the German national monitoring of zoonoses in food and livestock in 2013/2014. The genome of the plasmid pEC1897-13 was 38kb in size. Bioinformatics revealed that the plasmid belongs to the IncFII group, but represents a novel pMLST-allele that is closely related to the allele FII-82. Interestingly, pEC1897-13 obviously comprises all necessary components of a functional IncF conjugative-transfer system. However, up to now no self-transmission of the plasmid was observed by filter mating studies. Conclusion: The impact of the plasmid pEC1897-13 for the transmission of colistin resistance is unknown. In contrast to most of the described mcr-5 carrying plasmids, pEC1897-13 carries a complex IncF-like transfer system that might be functional under specific circumstances although currently it is not transferred under tested experimental conditions.

Background and objectives: Colistin is considered as highest priority critically important antibiotic, only used to treat human infections caused by multidrug-resistant Gram-negative bacteria. So far, seven different mobile colistin resistance-genes are known. Here, characteristics of mcr-4-positive colistin-resistant E. coli isolates from the national monitoring for antimicrobial resistance in zoonotic agents from the food chain were summarized. Materials and methods: Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines and EUCAST epidemiological cut-off values. Resistant E. coli were subjected to mcr-PCR, S1-PFGE, MiSeq-sequencing (WGS) and bioinformatics. Transferability of mcr-4 carrying plasmids was investigated by filter mating experiments. Results: Up to now, 13 mcr-4-positive out of 756 colistin-resistant E. coli isolates, recovered between 2010 and 2017, were identified. Sanger sequencing revealed that two variants, mcr-4.2 and mcr-4.3, are prevalent among these isolates. WGS showed that the isolates differ in their MLST-, sero- and fim-type. However, all of them carry a highly conserved ColE-plasmid prototype that partially differs in size and genetic composition. Conclusion: Our findings indicate that mcr-4 occurs sporadically among colistin-resistant E. coli from food and livestock. However, further information on the biology and genetics are needed to assess the impact of this resistance determinant on public health.

Questions: Quinolones are important antibiotics that 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 some antibiotics. To better understand the qnr PMQR pathway as well as the distribution of qnr genes, Escherichia (E.) coli isolates recovered in 2016 and 2017 from livestock and food were phenotypically and genotypically characterized. Materials & Methods: 6,817 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 in zoonotic bacteria, recovered from livestock and food. Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines. MIC values were evaluated using EUCAST epidemiological cut-off values. 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: Of 6,817 E. coli tested, more than 800 isolates were classified as quinolone-resistant (MICNAL ≥16 mg/L and/or MICCIP ≥0.06 mg/L). The most abundant qnr-variant was qnrS. With the exception of qnrD, other qnr-variants were found evenly distributed within the investigated matrices. In general, E. coli isolates with qnr-genes were found more frequently in the feces of animals than in the meat of the same animal species. PFGE with XbaI-digestion was performed to examine genetic relatedness of isolates. PFGE profiling demonstrated a rather high heterogeneity. The highly diverse PFGE pattern indicates that the screened isolates 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. Isolates, selected according their XbaI- and S1-PFGE pattern were further screened for their genetic setting through short read whole genome sequencing (WGS). Sequencing of those isolates 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 better understanding of the plasmid diversity within qnr-harboring E. coli and the prerequisites of their spread.

Background: Quinolones are important antibiotics belonging to a family of synthetic broad-spectrum drugs. 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 some antibiotics. To better understand the qnr PMQR pathway as well as the distribution of qnr genes, Escherichia (E.) coli isolates recovered in 2016 and 2017 from livestock and food were phenotypically and genotypically characterized. Materials/methods: 6,817 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 in zoonotic bacteria, recovered from livestock and food. Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines. MIC values were evaluated using EUCAST epidemiological cut-off values. 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: Of 6,817 E. coli tested, more than 800 isolates were classified as quinolone-resistant (MICNAL ≥16 mg/L and/or MICCIP ≥0.06 mg/L). The most abundant qnr-variant was qnrS. With the exception of qnrD, other qnr-variants were found evenly distributed. In general, E. coli isolates with qnr-genes were found more frequently in the feces of animals than in the meat of the same animal species. PFGE with XbaI-digestion was performed to examine genetic relatedness of isolates. PFGE profiling demonstrated a rather high heterogeneity. The highly diverse PFGE pattern indicates that the screened isolates 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. Isolates, selected according their PFGE pattern were further screened for their genetic setting through short read whole genome sequencing (WGS). Sequencing of those isolates confirmed the high genetic diversity of the quinolone-resistant E. coli strains. Conclusions: Quinolone-resistance could not be attributed to a specific lineage of E. coli. Further analysis is needed for better understanding of the plasmid diversity within qnr-harboring E. coli and the prerequisites of their spread.

Background: Quinolones are important antibiotics belonging to a family of synthetic broad-spectrum drugs. 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 some antibiotics. To better understand the qnr PMQR pathway as well as the distribution of qnr genes, Escherichia (E.) coli isolates recovered in 2016 and 2017 from livestock and food were phenotypically and genotypically characterized. Materials/methods: 6,817 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 in zoonotic bacteria, recovered from livestock and food. Antimicrobial resistance was determined by broth microdilution according to CLSI guidelines. MIC values were evaluated using EUCAST epidemiological cut-off values. 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: Of 6,817 E. coli tested, more than 800 isolates were classified as quinolone-resistant (MICNAL ≥16 mg/L and/or MICCIP ≥0.06 mg/L). The most abundant qnr-variant was qnrS. With the exception of qnrD, other qnr-variants were found evenly distributed. In general, E. coli isolates with qnr-genes were found more frequently in the feces of animals than in the meat of the same animal species. PFGE with XbaI-digestion was performed to examine genetic relatedness of isolates. PFGE profiling demonstrated a rather high heterogeneity. The highly diverse PFGE pattern indicates that the screened isolates 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. Isolates, selected according their PFGE pattern were further screened for their genetic setting through short read whole genome sequencing (WGS). Sequencing of those isolates confirmed the high genetic diversity of the quinolone-resistant E. coli strains. Conclusions: Quinolone-resistance could not be attributed to a specific lineage of E. coli. Further analysis is needed for better understanding of the plasmid diversity within qnr-harboring E. coli and the prerequisites of their spread.&nbsp

Questions: Colistin is considered as an important antibiotic of the last-resort, which will be only used for the treatment of severe human infections with multidrug-resistant Gram-negative bacteria. Since 2015, several mobile colistin resistance genes were described coding for enzymes of the phosphoethanolamine-transferase family. To date, eight different mcr-genes have been characterized, mediating resistance to colistin in different bacterial genera (especially in Enterobacteriaceae). Material & Methods: By molecular screening on mcr-1 to -5 using the multiplex PCR of Rebelo et al. (2018), an E. coli isolate recovered in 2013 from chicken feces was identified to carry a mcr-5 resistance gene. Antimicrobial resistance testing according to the CLSI-guideline was performed. MIC-data were interpreted using the ECOFFS of EUCAST. The genome of the mcr-5-plasmid was deduced by whole-genome sequencing using different platforms (MiSeq, Illumina and MinIon, Nanopore). Bioinformatic analyses were performed to determine the genome structure and composition of the plasmid and isolate. Results: Within this study, a novel mcr-5 plasmid-prototype was identified in the E. coli isolate from the German national monitoring of zoonoses in food and livestock in 2013/2014. The genome of the plasmid pEC1897-13 was 38 kb in size. Bioinformatics revealed that the plasmid belongs to the IncFII group, but represents a novel pMLST-allele that is closely related to the allele FII-82. Interestingly, pEC1897-13 obviously comprises all necessary components of a functional IncF conjugative-transfer system. However, up to now no self-transmission of the plasmid was observed by filter mating studies. Conclusion: The impact of the plasmid pEC1897-13 for the transmission of colistin resistance is unknown. In contrast to most of the described mcr-5 carrying plasmids, pEC1897-13 carries a complex IncF-like transfer system that might be functional under specific circumstances although currently it is not transferred under tested experimental conditions.