Monitoring antibiotic resistance in wastewater – developing an innovative method for quality assurance

Antimicrobial resistance (AMR) is one of the greatest health threats of our time. In Europe, around 35,000 people die every year from bacteria against which many antibiotics are no longer effective. From July 2027, the revised EU Urban Waste Water Treatment Directive (EU-KARL) will therefore require larger treatment plants to monitor such resistance in wastewater using PCR tests. However, a wide variety of methods are currently being used for this purpose, and it is not yet clear which of these are best suited to wastewater samples and sufficiently accurate. In particular, there is a lack of suitable materials for verifying the quality of the measurements. The AMRready4KARL project (April 2026 to March 2027) aims to close this gap. The project uses so-called virus-like particles (VLPs) as a safe test material. They contain the genetic information required to detect antibiotic resistance, but are themselves harmless and non-infectious. Thanks to their stable envelope, they are well suited for testing in wastewater. This allows PCR testing methods to be reliably assessed and better compared with one another.
 
AMRready4KARL employs so-called virus-like particles (VLPs) as an innovative solution. This involves transferring established VLP technology from molecular virology to environmental microbiology. These are small, spherical structures that can be produced synthetically; they are composed of viral envelope proteins but do not contain any functional genetic material from a virus. However, they can be loaded with various DNA fragments, such as those containing the genetic information of resistance genes required for PCR detection. The fact that this DNA is protected by the virus-like capsid in wastewater, yet is non-infectious, makes it an ideal vehicle for quality control in wastewater-based epidemiology in accordance with EU-KARL. These properties enable risk-free handling and make them ideally suited for the validation of PCR-based detection methods for AMR in wastewater. 

The principle behind the method: Given the known exact quantity of VLPs spiked into the wastewater, it is possible to verify whether the existing method for detecting AMR-carrying bacteria in wastewater is able to detect this exact quantity in the results. This would, for the first time, also enable nationwide laboratory comparisons for quality assurance in PCR-based monitoring of antibiotic resistance in wastewater, as has already been carried out for SARS-CoV-2 as part of AMELAG 2024. 

The planned work packages for AMRready4KARL initially comprise a method validation and an assessment of the current status of the PCR methods currently in use within the AMELAG network (wastewater monitoring for the RKI’s epidemiological situation assessment), the development of a wastewater-optimised and quality-assured PCR assay, VLP production and characterisation, and the conduct of a pilot interlaboratory comparison using the technology. The planned outcomes include the provision of two end products specifically tailored to regional and national user groups: 1) AMR-VLP reference material for internal laboratory quality control and calibration of testing procedures. 2) Preparation of AMR-VLP-spiked interlaboratory test samples to conduct laboratory comparisons and support the harmonisation of analytical methods.

The project is rounded off by the development of a guide on the application of the technology in routine AMR surveillance in Germany and the creation of a short explanatory video to communicate the scientific findings and the application of VLP technology for quality control in an easily understandable way. The aim is ultimately to further develop the technology to a commercially viable scale, so that an EU-wide roll-out can take place when the EU-KARL comes into force.
The research consortium comprises the Institute of Medical Virology and the Institute of Medical Microbiology and Hospital Hygiene at Goethe University Frankfurt am Main, as well as the Research Institute for Water Management and Climate Future at RWTH Aachen University (FiW). The project is supported by the Emschergenossenschaft and Lippeverband (EGLV), the State Office for Nature, Environment and Climate of North Rhine-Westphalia (LANUK), and the Frankfurt am Main Public Health Department as associated partners.