Monitoring system for Campylobacter spp. based on Multilocus-Sequence-Typing (MLST)

• Forschungsstelle: BLV
• Projektnummer: 1.06.04
• Projekttitel Englisch: Monitoring system for Campylobacter spp. based on Multilocus-Sequence-Typing (MLST)

Schlüsselwörter (Englisch)

Zoonosis, monitoring, internet-surveillance, database, Campylobacter

Kurzbeschreibung (Englisch)

Within the last few years Campylobacter spp. and especially C. jejuni became the major agent of food born gastroenteritis in humans in Switzerland and elsewhere. A lot of effort has been put in determining the ways of infection as well as identification and typing of Campylobacter and has elucidated possible risks and prevention measures and led to promising diagnostic methods. Despite these achievements, we do not have an up-to-date tool for the surveillance and monitoring of Campylobacter in Switzerland.
DNA sequence based methods have increasingly become the method of choice for bacterial identification and typing. Standardization of data as gene sequences and easily reproducible technology are very important for the establishment of a platform- and location-independent system, that will also allow an unambiguous identification and typing of Campylobacter isolates. We therefore intend to use and further improve well established technologies to build up a comprehensive national internet based monitoring scheme for Campylobacter species. This will include sequence determination of phylogenetic markers (16S rDNA and rpoB) for identification purposes, as well as sequencing a set of 7 defined house-keeping genes and 1 flagellar gene for typing of strains in a Multi-locus sequence typing (MLST) approach. Data will be integrated in an already available Bionumerics database containing representative Campylobacter strains from a set of well defined and characterized isolates from animal origin as well as human isolates. In order to provide the best value to the technology mentioned above, data-management tools for wide-spread diseases such as Campylobacter infections need to provide networking tools and allow decentralized data-input and analysis of data while respecting all security aspects. This could be achieved using an IDNS™ (Integrated Database Network System) based data-management tool like SmartGene GmbH, a Switzerland-based company has developed for other purposes (e.g. Influenza viruses, HIV drug resistance data, Swiss HIV Cohort study). The Institute of veterinary bacteriology is a perfect interface for this project, since it harbors the National Reference Centre for Enteropathogenic Bacteria (NENT) as well as the Centre for Zoonoses, Bacterial Animal Epizootics and Antibiotic Resistance (ZOBA). Moreover, as a research unit, it has a long-standing experience in DNA sequence based identification of bacteria as well as a well established collaboration with SmartGene.
The anticipated impact will go beyond creating a tool for a single reference laboratory and will allow integration of the Swiss monitoring system in international initiatives plus allowing Swiss diagnostic labs and food-industry to provide data and compare with a database for a potential outbreak. Such a surveillance tool is urgently needed for the whole of Switzerland in order to be prepared for putative outbreaks and take quick action to either prevent them or stop them at the very beginning. Therefore it is of major public health concern and will not only be useful for the Federal Veterinary Office but will also be vital for the Federal Office of Public Health.

Projektziele (Englisch)

The aim of the project is to safeguard a well characterized Campylobacter culture and data collection and use it to establish, in a reasonable time, a national system for Swiss Campylobacter monitoring based on gene sequence data which will not outdate with technology changes like currently used methods.

1. Transpose the AFLP technique to the ABI 3100 system
- maintain the technique and keeping the value of the existing AFLP database for Campylobacter. This data-
base and the associated culture collection of strains is of extreme value as reference and should be pre-
served.
2. Genetic identification and typing of a representative set of Campylobacter isolates from Switzerland
- use 16S rDNA and rpoB gene sequencing for an unambiguous identification of Campylobacter
- set up an extended typing scheme based on classical MLST in combination with a flagellar gene
- analyze MLST data not only by determining the classical sequence types but use phylogenetic information for
typing
- complement the current Bionumerics database with this data
3. Use the IDNS technology to build up a national Web-accessible (protected) database for identification and typing of Campylobacter
- the database shell includes the sequence data from the type strains of Campylobacter species for the identi-
fication part and the major zoonotic species for the MLST part. The database should further contain a repre-
sentative set of Swiss field isolates to cover the currently circulating types. For that purpose, an already well
established collection of strains from former projects exists.
- The IDNS system will provide all necessary analysis tools, including sequence proofreading and sequence
comparison
- Its access will be protected and data transfer and server connections will be protected using state-of-the art
technology
- hosting of data and back-ups will be organized in a professional manner using current infrastructure at
SmartGene at EPFL (Lausanne).
- IDNS will contain shared and networked data-subsets, will allow to create user-specific data-sets (for data not
to be shared) and enable to do statistic evaluations
- An embedded reference database collecting data from public sources through automated updates
- An audit-trail functionality will be implemented to track all access and changes to data
- SmartGene already applies software validation procedures using the CSV model, as recommended by the
FDA.
- SmartGene can provide with interface technology for bulk data uploads and exchanges if necessary.
- The project will result also in a data platform for the Campylobacter studies carried out previously. These data
from former studies will be complemented and brought into a format and onto a platform that suits the needs
for a national and eventually international monitoring system.

Publikationen / Ergebnisse (Englisch)

Kuhn, J. (2007), Phylogenetic Identification and Improved Multilocus Sequence Typing (MLST) of Campylobacter jejuni, Diplomarbeit der phil.-nat. Fakultät der Universität Bern

Korczak, B.M.; Kuhn, J.; Fehlmann, M.; Kuhnert, P. (2007), Online monitoring system for Campylobacter jejuni using an improved MLST scheme, 66th Annual assembly of the SSM, Interlaken, 1-2 March 2007

Korczak, B.M.; Fehlmann, M.; Kuhnert, P. (2007) Improved Multilocus Sequence Typing and Flagellin Gene-based Typing for Online Monitoring of Campylobacter jejuni and Campylobacter coli, 14th International Workshop on Campylobacter, Helicobacter and Related Organisms, Rotterdam, The Netherlands, 2–5 September 2007

Schnider, A.; Overesch, G.; Korczak, M.; Kuhnert, P. (2010) Comparison of Real-Time PCR Assays for Detection, Quantification, and Differentiation of Campylobacter jejuni and Campylobacter coli in Broiler Neck Skin Samples. Journal of Food Protection, Vol. 73, No. 6, 2010, Pages 1057-1063.

Wirz, S.E.; Overesch, G.; Kuhnert, P.; Korczak, B.M. (2010) Genotype and antibiotic resistance analysis of Campylobacter isolates from caeca and the carcasses of slaughtered broiler flocks.

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