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© MSU l K. Stepnitz EDAR 2017 4th INTERNATIONAL SYMPOSIUM ON THE ENVIRONMENTAL DIMENSION OF ANTIBIOTIC RESISTANCE 13–17 AUG 2017 LANSING, MICHIGAN l UNITED STATES Abstracts www.antibiotic-resistance.de Session 1 – Natural resistome, selection and evolution O1 Selection and co-selection for antibiotic resistance: hunting high and low in a complex community Aimee Murray1, Lihong Zhang1, Angus Buckling1, Tong Zhang2, Jason Snape3, William Gaze1 1University of Exeter, Penryn, United Kingdom 2University of Hong Kong, Hong Kong, Hong Kong 3AstraZeneca, Alderly, United Kingdom The Minimum Selective Concentration (MSC) is the lowest antibiotic concentration that can select for antimicrobial resistance (AMR). MSCs have been determined previously in single species assays for both chromosomal and plasmid-mediated resistance mechanisms, and in open experimental systems in biofilms. However, there have been no studies that have directly compared MSCs determined in single species and complex community assays in the same experimental system. Development of reliable, environmentally relevant assays is a necessity in order to generate more data for rigorous environmental risk assessment (ERA). This study determined the MSC of cefotaxime for the clinically important class of resistance genes the CTX-Ms, found worldwide in clinical pathogens and a variety of natural environments. Single species or complex community (untreated waste water) inocula were used. qPCR tracked prevalence of CTX- M genes in closed, experimental evolution systems which were passaged daily for 8 days. This was used to determine MSCs both statistically and using selection coefficients. Metagenomic analyses of evolved populations at selected concentrations at the end of the community experiment were performed to assess the level of co-selection for other resistance determinants. Results show the MSC of cefotaxime in the community assay was low, at 30 – 125µg/L (based on selection coefficients and statistical analyses respectively). This suggests selection for CTX-M genes may be occurring in aquatic systems, based on previously measured environmental concentrations. However, the MSC in the single species assay was much higher (8mg/L), demonstrating single species assays may not always yield meaningful results for ERA. This elevated MSC is likely to be in part due to indirect selection, which allows growth of susceptible strains well above their MIC due to antibiotic degradation by the resistant population. Metagenomics revealed that several AMR gene classes were also enriched by cefotaxime exposure, including aminoglycoside, sulphonamide and trimethoprim resistance genes, amongst others. Session 1 – Natural resistome, selection and evolution O2 Minimal selective concentration of ciprofloxacin for Escherichia coli grown in complex aquatic bacterial biofilms Nadine Kraupner1,2, Stefan Ebmeyer1,2, Johan Bengtsson-Palme1,2, Jerker Fick3, Carl-Fredrik Flach1,2, D. G. Joakim Larsson1,2 1University of Gothenburg, Department of Infectious Diseases, Gothenburg, Sweden 2Centre for Antibiotic Resistance Research (CARe), Gothenburg, Sweden 3Umeå University, Department of Chemistry, Umeå, Sweden Introduction To assess and manage risks with antibiotics in the environment, it is important to know their Minimal Selective Concentrations (MSC). In the environment, bacteria exist in microbial communities, and estimates of MSCs in simplified assays may not be directly translated to such complex conditions. Objectives The aim is to assess the MSC of ciprofloxacin for E. coli grown in complex aquatic bacterial biofilms by phenotypic and genotypic assays. Methods Biofilms were established on glass slides in a flow through aquaria system and continuously exposed to different concentrations of ciprofloxacin. Diluted treated sewage effluent served as inoculum. Biofilms were harvested after 9 days and administered onto selective CHROMagar™ ECC media. Relative counts of E. coli on plates with and without ciprofloxacin were used as a measure of phenotypic resistance. Allele specific PCR was performed to identify genetic changes in the quinolone resistance-determining regions (QRDR) in gyrA and parC of E. coli isolates. Additionally, community DNA was isolated for metagenomic analysis and amplicon sequencing of the E. coli QRDR. Results There was a low, stable proportion of resistant E. coli isolates in communities exposed to 0, 0.1 and 1 µg/L, while a clear increase was seen at 10 µg/L (Figure 1). Irrespective of antibiotic exposure, the most common genetic change in the QRDR of resistant isolates was a triple mutation (gyrA S83L, gyrA D87N and parC S80I) conferring a MIC of >32 µg/L for ciprofloxacin. Sequencing of gyrA and parC amplicons from the biofilms will provide a more complete picture of the abundance and distribution of resistance mutations across all studied

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