A pan-European ring trial to validate an International Standard for detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in seafoods.

Globally, vibrios represent an important and well-established group of bacterial foodborne pathogens. The European Commission (EC) mandated the Comite de European Normalisation (CEN) to undertake work to provide validation data for 15 methods in microbiology to support EC legislation. As part of this mandated work programme, merging of ISO/TS 21872-1:2007, which specifies a horizontal method for the detection of V. parahaemolyticus and V. cholerae, and ISO/TS 21872-2:2007, a similar horizontal method for the detection of potentially pathogenic vibrios other than V. cholerae and V. parahaemolyticus was proposed. Both parts of ISO/TS 21872 utilized classical culture-based isolation techniques coupled with biochemical confirmation steps. The work also considered simplification of the biochemical confirmation steps. In addition, because of advances in molecular based methods for identification of human pathogenic Vibrio spp. classical and real-time PCR options were also included within the scope of the validation. These considerations formed the basis of a multi-laboratory validation study with the aim of improving the precision of this ISO technical specification and providing a single ISO standard method to enable detection of these important foodborne Vibrio spp.. To achieve this aim, an international validation study involving 13 laboratories from 9 countries in Europe was conducted in 2013. The results of this validation have enabled integration of the two existing technical specifications targeting the detection of the major foodborne Vibrio spp., simplification of the suite of recommended biochemical identification tests and the introduction of molecular procedures that provide both species level identification and discrimination of putatively pathogenic strains of V. parahaemolyticus by the determination of the presence of theromostable direct and direct related haemolysins. The method performance characteristics generated in this have been included in revised international standard, ISO 21872:2017, published in July 2017.

Implementation of the Bacillus cereus microbiological plate used for the screening of tetracyclines in raw milk samples with STAR protocol - the problem with false-negative results solved.

In antibiotic residue analyses the first step of screening is just as important as the following steps. Screening methods need to be quick and inexpensive, but above all sensitive enough to detect the antibiotic residue at or below the maximum residue limit (MRL). In the case of a positive result, the next step is conducted and further methods are used for confirmation. MRLs stated in European Union Regulation 37/2010 for tetracyclines in raw milk are: 100 µg kg(-1) for tetracycline, 100 µg kg(-1) for oxytetracycline, 100 µg kg(-1) for chlortetracycline and no limit for doxycycline because it is prohibited for use in animals from which milk is produced for human consumption. The current five-plate microbiological screening method for the detection of antibiotic residues in raw milk was found to be simple and inexpensive, but not specific, sensitive and reliable enough to detect tetracycline at MRL in routine raw milk screening procedures. Spiking samples with tetracycline at the MRL level and applying them on Bacillus cereus ATCC 11778 microbiological plates often gave false-negative results, indicating that tetracyclines may have to be inactivated or masked. Tetracyclines seem to bind to a certain component in milk. Consequently, when applying samples to the B. cereus microbiological plate the antibiotic cannot inhibit the growth of B. cereus which disables the formation of inhibition zones on the test plate. After adding the appropriate amount of citric acid into the milk samples, we solved the problem of false-negative results. During the validation 79 samples of milk were spiked with tetracyclines at different concentrations: 100 µg kg(-1) for tetracycline, 100 µg kg(-1) for oxytetracycline, 80 µg kg(-1) for chlortetracycline and 30 µg kg(-1) for doxycycline. Concentrations used in the validation matched the requirements for MRLs (they were either at or below the MRLs) stated in European Union Regulation 37/2010. The sensitivity of the validation was 100%.