Improving phenotypic testing of AMRshort forAntimicrobial resistance by development of rapid and sensitive screening assays for emerging resistances and setting missing ECOFFs (EJP IMPART)

Currently, antimicrobial resistance (AMRshort forAntimicrobial resistance) poses an increasing threat to human health. In the EU Action Plan Against the rising threats from AMRshort forAntimicrobial resistance: Road Map (EU 2015) one of the topics was reinforced joint cross-border research to address AMRshort forAntimicrobial resistance, e.g. the development of diagnostic tools for the detection of bacteria and resistance markers and harmonization of monitoring. IMPART addresses four topics related to the development and harmonization of phenotypic methods for detection of AMRshort forAntimicrobial resistance: (1) selective isolation and detection of colistin-resistant Enterobacteriaceae, (2) selective isolation and detection of carbapenemase-producing Enterobacteriaceae, (3) standardization of a disk diffusion method for susceptibility testing of Clostridioides (C.) difficile, (4) setting ECOFFs for specific pathogen/antibiotic combinations. This project will result in a validated and sensitive method to detect colistin-resistant (carrying mcr) and carbapenemase-producing Enterobacteriaceae (CPE) in caecal samples from animals and in food. This is essential for the monitoring of the current prevalence and spread of bacteria carrying these emerging resistance genes in animals and the risk of transmission of these genes via food. Additionally, in order to provide a suitable and cost-effective method for antimicrobial resistance surveillance of the zoonotic pathogen C. difficile, a disk diffusion method will be optimized and standardized for a broad range of antimicrobials. The generated distributions of inhibition zone diameter will support future work on setting epidemiological cut-off values (ECOFFs). Furthermore, the setting of ECOFFs for veterinary pathogens will highly improve harmonization of monitoring of resistance in animal pathogens and support the process for defining animal species specific clinical breakpoints for veterinary antimicrobials which will ultimately lead to more effective and prudent use of antimicrobials in animals.

In WP1, different protocols and media for the selective detection of colistin-resistant enterobacteriaceae were compared. Since the discovery of mobile colistin resistance genes (mcr), resistance to colistin has become a potential problem in the treatment of severe infections in human medicine. The goal of this WP was to validate a uniform method for the detection of this resistance trait. A ring trial conducted within the different laboratories of the consortium showed 100% specificity of molecular biological confirmation of mcr genes. However, the selective detection of the resistant bacteria using different enrichment and cultivation methods could not be sufficiently standardized. In WP2 improvement of the detection of carbapenemase-producing E. coli was investigated, compared to the methods specified in the protocols of the EURL. The investigations of various influencing factors carried out as part of a dissertation at the BfRshort forGerman Federal Institute for Risk Assessment were able to show that the storage time of the sample and the selective medium have significant influence on the cultural detection of the resistant bacteria. In ring-trials between the project partners, various commercial media were compared and the results were published. In the context of the work at the BfRshort forGerman Federal Institute for Risk Assessment it could be shown that the tested self-made medium has a significantly higher sensitivity than the best tested commercial medium in the detection of bacteria with slightly elevated carbapenem tolerance.In WP3, sensitivity values of various animal pathogenic bacteria to antibiotics were determined. The results are the basis for providing more Cut Off values for the treatment of bacterial infections in farm animals in the future in the context of resistance testing. Both the investigated bacterial species and the tested antibiotics were selected according to their relevance in veterinary medicine. and the obtained results were made available for the European Committee for Antimicrobial Susceptibility Testing (EUCAST) and are used there for the establishment of Epidemiological Cut Off Values (ECOFF).
In WP4, we optimized and validated the disk diffusion as cost effective and easy-to-use alternative method compared to the current gold standard, the agar dilution, for the susceptibility testing of Clostridioides (C.) difficile. Therefore, we tested different growth media and different anaerobic conditions regarding their effect on reproducibility. In addition, we collected app. 500 test strains from our strain collection as well as from project and external partners, characterized and used them to finally propose cut-off values for resistance testing against eight different antimicrobials applying the disk diffusion method. Based on these cut-off values and an optimized method description, which where evaluated by a ring trial study, the susceptibility of C. difficile against six of the eight tested antimicrobials can be reliably predicted.
Project publications:Perrin-Guyomard, A., Granier, S.A., Slettemeås, J.S., Anjum, M., Randall, L., AbuOun, M., Pauly, N., Irrgang, A., Hammerl, J.A., Kjeldgaard, J.S., Hammerum, A., Franco, A., Skarżyńska, M., Kamińska, E., Wasyl, D., Dierikx, C., Börjesson, S., Geurts, Y., Haenni, M. and Veldman, K. (2022). Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat. Letters in Applied Microbiology. Early view. DOI: https://doi.org/10.1111/lam.13717
Dierikx C., Börjesson S., Perrin-Guyomard A., Haenni M., Norström M., Divon H. H., Ilag H. K., Granier S. A., Hammerum A., Sejer Kjeldgaard J., Pauly N., Randall L., Anjum M. F., Smialowska A., Franco A., Veldman K., Schau Slettemeås J. (2022). A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae. Journal of Microbiological Methods. 193:106418 DOI: https://doi.org/10.1016/j.mimet.2022.106418.
Pauly, N., Hammerl, J. A., Grobbel, M., Käsbohrer, A., Tenhagen, B-A., Malorny, B., Schwarz, S., Meemken, D., Irrgang, A. (2021). Identification of a blaVIM-1-Carrying IncA/C2 Multiresistance Plasmid in an Escherichia coli Isolate Recovered from the German Food Chain. Microorganisms.  9(1), 29. DOI: https://doi.org/10.3390/microorganisms9010029
Perrin-Guyomard, A., Granier, S., Haenni, M., Slettemeås, J. S., Ilag, H. K., Anjum, M., Randall, L., AbuOun, M., Pauly, N., Irrgang, A., Hammerl, J. A., Svendsen, C. A., Kjeldgaard, J. S., Hammerum, A., Franco, A., Skarżyńska, M., Kamińska, E., Wasyl, D., Dierikx, C., Börjesson, S., Geurts, Y. & Veldman, K. (2021). Multicentre evaluation of culture based methods to selectively isolate Colistin-resistant Enterobacteriaceae from food producing animals and food products. Poster presentation at OHEJP Annual Scientific Meeting 2021, Copenhagen, Denmark. 9-11  June 2021. DOI: https://doi.org/10.5281/zenodo.7390564
Pauly, N., Klaar, Y., Skladnikiewicz-Ziemer, T., Juraschek, K., Grobbel, M., Hammerl, JA., Hemmers, L., Käsbohrer, A., Schwarz, S., Meemken, D., Tenhagen, B-A., Irrgang, A. (2021). Isolation Procedure for CP E. colishort forEscherichia coli from Caeca Samples under Review towards an Increased Sensitivity. Microorganisms. 9, 1105. DOI: https://doi.org/10.3390/microorganisms9051105
Pauly, N., Hammerl, J. A., Grobbel, M., Käsbohrer, A., Tenhagen, B-A., Malorny, B., Schwarz, S., Meemken, D., Irrgang, A. (2020). Identification of a blaVIM-1-Carrying IncA/C2 Multiresistance Plasmid in an Escherichia coli Isolate Recovered from the German Food Chain. Microorganisms. 9, 29. DOI: https://doi.org/10.3390/%20microorganisms9010029
Pauly, N., Hammerl, JA., Schwarz, S., Grobbel, M., Meemken, D., Malorny, B., Tenhagen, BA., Käsbohrer, A., Irrgang, A. (2020). Co-occurrence of the blaVIM-1 and blaSHV-12 genes on an IncHI2 plasmid of an Escherichia coli isolate recovered from German livestock. Journal of Antimicrobial Chemotherapy. 76(2), 531–533. DOI: https://doi.org/10.1093/jac/dkaa436 
Diaconu, EL., Carfora, V., Alba, Pshort forphosphorus., Di Matteo, Pshort forphosphorus., Stravino, F., Buccella, C., Dell’Aira, E., Onorati, R., Sorbara, L., Battisti, A., Franco, A. (2020). Novel IncFII plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy. Journal of Antimicrobial Chemotherapy. 75(12), 3475–3479. DOI: https://doi.org/10.1093/jac/dkaa374
Irrgang, A., Tausch SH., Pauly, N., Grobbel, M., Kaesbohrer, A,. Hammerl, JA. (2020). First Detection of GES-5-Producing Escherichia coli from Livestock—An Increasing Diversity of Carbapenemases Recognized from German Pig Production. Microorganisms. 8 (10), 159. DOI: https://doi.org/10.3390/microorganisms8101593
Pauly, N., Hammerl, JA., Grobbel, M., Tenhagen, BA., Käsbohrer, A., Bisenius, S., Fuchs, J., Horlacher, S., Lingstädt, H., Mauermann, U., Mitro, S., Müller, M., Rohrmann, S., Schiffmann, AP., Stührenberg, B., Zimmermann, Pshort forphosphorus., Schwarz, S., Meemken, D., Irrgang, A. (2020). ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems. Frontiers in Microbiology. 11, 1678. DOI: https://doi.org/10.3389/fmicb.2020.01678
Irrgang, A., Pauly, N., Tenhagen, BA., Grobbel, M., Kaesbohrer, A., Hammerl, JA. (2020). Spill-Over from Public Health? First Detection of an OXA-48-Producing Escherichia coli in a German Pig Farm. Microorganisms. 8(6), 855. DOI: https://doi.org/10.3390/microorganisms8060855
Veldman, K., Slettemeås, JS., Granier, S., Perrin-Guyomard, A., Hechard, T., Dierikx, C., Maurischat, S. (2020). IMPART: an update of the study. Poster presented at One Health EJP Annual Scientific Meeting 2020. Online, 27-29th May 2020. DOI: https://doi.org/10.5281/zenodo.4066734
Veldman, K., Slettemeås, JS., Grobbel, M., Börjesson, S., Dors, A., Franco, A., Haenni, M., Turner, Oshort foroxygen., Broens, E. (2020). Susceptibility testing of veterinary pathogenic bacteria as a first step in setting new epidemiological cut-off values (ECOFFs). Poster presented at: One Health EJP Annual Scientific Meeting 2020. Online, 27-29th May 2020. DOI: https://doi.org/10.5281/zenodo.4066667
Maurischat, S., Witt, Pshort forphosphorus., Maneck, C., Grobbel, M. and the OHEJP IMPART consortium (2020). Clostridioides difficile antimicrobial susceptibility testing using disc diffusion. Poster presented at: One Health EJP Annual Scientific meeting, 2020. Online, 27-29th May 2020. DOI: https://doi.org/10.5281/zenodo.7390483 
Slettemeås, JS., Norström, M., Divon, H., Ilag, HK., Granier, S., Perrin-Guyomard, A., Haenni, M., Veldman, K., Hammerum, A., Börjesson, S., Kjeldgaard, JS., Irrgang, A., Dierikx, C., Randall, L., Smialowska, A., Franco, A. (2020). A multicenter study examining different culturing methods to detect carbapenemase-producing Enterobacteriaceae. Poster presented at One Health EJP Annual Scientific Meeting 2020. Online, 27-29th May 2020. DOI: https://doi.org/10.5281/zenodo.4066631