Integrated genomic surveillance of zoonotic pathogens (IGS-Zoo)

Typing of bacterial infectious agents using whole genome sequencing (WGS), provides a new and important decision-making tool for surveillance and outbreak detection. It contributes to a better understanding of the emergence and spread of pathogens and, in turn, to stop the spread of infections through specific control and intervention measures as quickly as possible. While some countries and institutions have already begun implementing WGS for typing of zoonotic pathogens, e.g., PulseNet of the Centers for Disease Control and Prevention (CDC) in the U.S. and PulseNet International of the European Centre for Disease Prevention and Control (ECDCshort forEuropean Centre for Disease Prevention and Control), and Public Health England in the United Kingdom (now UK Health Security Agency and Office for Health Improvement and Disparities), Germany has lacked a comparable systematic and coordinated national initiative. As a result, there has been no possibility to identify the spread of zoonotic pathogens early and systematically on the basis of typing data, and to combat them in a targeted manner. The overarching question in the project "Integrated Genomic Surveillance of zoonotic pathogens (IGS-Zoo)" is therefore how to monitor these pathogens in Germany on the basis of the core genome multilocus sequence typing (cgMLST) and how can it work. For this purpose concepts using the example of enterohemorrhagic Escherichia coli (EHECshort forenterohemorrhagic Escherichia coli), the highly virulent variant of Shiga toxin-producing E. colishort forEscherichia coli (STECshort forShiga toxin-producing Escherichia coli) shall be established. The concepts for STECshort forShiga toxin-producing Escherichia coli as a prototypical and - due to its high virulence and ability to produce virulence and the ability to cause outbreaks - extremely relevant zoonotic pathogen, shall be practically tested and evaluated. From the start, the focus was on the cooperation between the different sectors of the health system (human medicine and food monitoring (LMÜ)) and their close connection to the public health services (ÖGD).

The inventory of available and actually applied typing methods of zoonotic pathogens showed that the currently most frequently used method is the determination of Shiga toxin (stx)- and intimin (eae)-encoding genes by polymerase chain reaction (PCR). Further characterization of the pathogens by serotyping and/or WGS would be necessary for epidemiological questions, especially outbreak detection and elucidation. However, only just under half of the participating laboratories perform serotyping by determining the Oshort foroxygen- or H-antigens, and overall also just under half of the laboratories have internal or external access to WGS. The most common obstacle to further STECshort forShiga toxin-producing Escherichia coli/EHECshort forenterohemorrhagic Escherichia coli typing was cited as the fact that even when the stx gene is clearly detected by PCR, isolation of the pathogen from sample material is often unsuccessful. Training or incentives for isolate collection would therefore be useful. WGS is used in all participating federal laboratories. In the state laboratories, the method can already be used internally at just under a quarter of the facilities, but additional laboratories may use external options. However, most of the participants that cannot currently use WGS plan to establish the method in their laboratories, with funding constraints and lack of qualified personnel seen as problematic for implementation. In parallel to the survey of the status quo, standards for the exchange of isolate-specific sequence and metadata were developed within the project - taking into account data protection aspects - in order to define a common basis for the further course of the project and to create the technical prerequisites for genomic surveillance during the targeted prospective proof-of-principle study. To this end, interfaces were created between the software selected for genomic surveillance (Ridom SeqSphere+) and a cgMLST allele nomenclature server as well as a project-internal database to ensure secure data exchange. In addition, an alert function was added to the analysis software to ensure that the institutions participating in the consortium will be informed promptly about clustering or outbreak situations in the future and can initiate further steps to clarify possible epidemiological relationships between isolates. In order to achieve a uniformly high typing quality within the project, 10 selected STECshort forShiga toxin-producing Escherichia coli/EHECshort forenterohemorrhagic Escherichia coli isolates (outbreak isolates and isolates of sporadic cases) each were characterized by the participants in two round proficiency tests by means of genome sequencing and analyzed under surveillance aspects. Despite different analysis pipelines, typing and cluster analysis of both proficiency tests were performed by all members with consistent results. In parallel, a harmonized approach was established in two screening assays for the rapid development of rapid tests in outbreak investigations. Based on single nucleotide polymorphisms (SNP), first real time PCR-based detection methods were developed and subsequently outbreak isolates were identified either by high-resolution melting point analysis (HRM) and/or the use of TaqMan probes (target vs. non-target strains). The latter was successfully achieved by all participating project partners within the given deadlines despite a partly very different approach. Finally, the elaborated concept was subjected to a long-term test in a one-year proof-of-principle study, in which more than 300 STECshort forShiga toxin-producing Escherichia coli isolates were analyzed by the project members and compared via the consortium server. Several clusters of different serotypes were identified, confirming the functionality of the system. The results and experience gained have subsequently been incorporated into the development of work instructions and training materials for users and future stakeholders. Overall, it can be stated that the project was successful despite difficult conditions. The intended concept could be implemented as planned, so that cross-sector surveillance for STECshort forShiga toxin-producing Escherichia coli was made possible. The project has led to a significant shortening of the timeline in WGS cluster analysis and surveillance and an improvement in communication channels within the ÖGD and the agencies associated for foodborne outbreak analysis. Finally, the jointly built database will continue to be used by the project partners in the future and will be expanded by additional institutions.