Profiling Campylobacter from High selection Areas for the development of a Novel alert tool to meet the global Challenge of Enforced antimicrobial resistance

Campylobacter is an internationally underestimated foodborne pathogen and has been the most important zoonotic pathogen in the European Union since 2005, causing 137,107 reported cases of campylobacteriosis in 2022. Acute symptoms of the disease include fever, diarrhoea and abdominal cramps, but also severe secondary symptoms such as arthritis, irritable bowel syndrome and, in some cases, Guillain-Barré syndrome. Around a third of campylobacteriosis patients in Germany have been treated with antibiotics, in particular with fluoroquinolones or macrolides. Campylobacter isolates are often resistant to fluoroquinolones and tetracyclines, while resistance to macrolides, gentamicin and streptomycin is still low in Germany. Increasing antibiotic resistance jeopardises the effective treatment of infections in humans. The global antibiotic resistance situation plays a major role here. The project, therefore, focussed on a comparison of resistance determinants of German and Vietnamese Campylobacter isolates. The aim of this project was to develop novel molecular and microbial warning tools for use in routine surveillance programmes. The methods used in this project include whole genome sequencing of Campylobacter isolates from the chicken food chain, phenotypic characterisation by microdilution and real-time PCR rapid detection of resistance.

A questionnaire on antibiotic use on Vietnamese chicken farms was created and the information was collated in WP1. In WP2, the NIVR provided strains and supplied the metadata collected from WP1, such as date and place of sampling, antibiotic use, etc., using an anonymous code/ID for the respective farms of origin (results of WP1). The BfRshort forGerman Federal Institute for Risk Assessment was then responsible for the phenotypic characterisation of antimicrobial resistance using the EU-wide standardised microtitre panel EUCAMP2. In WP3, a relevant microdilution panel of antimicrobial substances that can be used for the monitoring of Campylobacter isolates and are not yet anchored in routine monitoring was identified. Antimicrobial substances that are important for human health and/or veterinary medicine were included, e.g. further carbapenem antibiotics and fosfomycin. The BfRshort forGerman Federal Institute for Risk Assessment prepared a protocol for the extended panel and both laboratories, LGL and BfRshort forGerman Federal Institute for Risk Assessment, tested reference strains and various isolates.Following harmonisation of the workflows for whole genome sequencing between the BfRshort forGerman Federal Institute for Risk Assessment and LGL, a total of 600 Campylobacter isolates from Germany and Vietnam were sequenced under WP4. The analysis of the whole genome data aimed at under WP5 showed that a large number of different resistance determinants could be identified. Vietnamese isolates carried significantly more resistance determinants than German isolates, which was consistent with the phenotypically collected data from WP2. Furthermore, it was possible to identify weaknesses in the predictive prediction of phenotypic resistance based on the whole genome data. These results were published by the BfRshort forGerman Federal Institute for Risk Assessment in a publication in the scientific journal BMC Genomics.The data collected through whole genome sequencing was subsequently used by the LGL under WP6 to design new primers and probes for a new PCR assay for the rapid routine identification of important resistance determinants in Campylobacter. Accordingly, a new multiplex real-time PCR system was established and subsequently validated under the leadership of the LGL in WP7 and WP8. In future, it will be possible to screen for resistance determinants associated with fluoroquinolone, macrolide or tetracycline resistance at relatively low cost. The results of the development and validation were published in the scientific journal Microorganisms by the LGL.As part of the public relations work (WP9), the results were presented to specialist audiences at various symposia, congresses and conferences at both national and international level. Furthermore, the knowledge of techniques in dealing with phenotypic and genotypic characterisation was imparted to the Vietnamese cooperation partners in a training course.