Challenging the "gold standard" of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

[Salmonella spp. prevalence and contamination risk factors in broiler and broiler meat of Gallus gallus in Germany and the European Union]. / Prävalenz und Kontaminationsrisikofaktoren für Salmonella spp. in Hähnchen und Hähnchenfleisch in Deutschland und der Europäischen Union.

In order to reduce the prevalence of the Salmonella enterica serovars Typhimurium and Enteritidis as a main causative agent of human salmonellosis originating from poultry flocks and products, the EU regulations 2160/2003 and 2073/2005 and the German Hühner-Salmonellen-Verordnung were established ten years ago. A literature review shows that this aim could be reached to a large extend in many areas of the food production chain, e.g. in breeding and husbandry facilities in most EU member states including Germany. Nevertheless some exceptions exist, and there are other S. enterica serovars which have a human pathogenic potential comparable to S. Typhimurium and S. Enteritidis. Furthermore recent publications show, that especially processes in transport and slaughter of poultry can prevent successful husbandry sanitation measures. Especially in these areas a reasonable potential for hygiene improvements still exists. Based on the prevalence data obtained between 1996 and 2011 this review summarizes recent knowledge concerning possible risks of Salmonella cross contamination and suggests potential starting points for their mitigation.