Validation of EN ISO 6579-1 - Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1 detection of Salmonella spp.

The European and International Standard method for the detection of Salmonella spp. in samples from the primary production stage, EN ISO 6579:2002/Amd.1:2007, was validated by an interlaboratory study in the frame of Mandate M/381, ordered by the European Commission and accepted by the European Standardisation Organisation (CEN). In addition to this study, results from two interlaboratory studies organised earlier by the European Union Reference Laboratory (EURL) for Salmonella were used for determination of the performance characteristics. Parallel to the performance evaluation for the Mandate, the revision of EN ISO 6579:2002 started. Part of this revision was the incorporation of the standardised method for detection of Salmonella in samples from the primary production stage (EN ISO 6579:2002/Amd.1:2007) and its performance characteristics in the new part 1 of EN ISO 6579. The 2002 version of EN ISO 6579 already contained performance characteristics for the detection of Salmonella in food samples, but LOD50 values (contamination level at which 50% of the samples are found positive) were not yet included. To be in line with the performance characteristics determined for detection of Salmonella spp. in samples from the primary production stage, LOD50 values for detection of Salmonella in food samples were calculated from the raw data of the validation studies performed in 2000. In this paper, the performance characteristics of EN ISO 6579-1:2017 are determined based not only on the results of the interlaboratory study carried out in 2013 under the Mandate, but also on several other interlaboratory studies. These performance characteristics consist of specificity, sensitivity and LOD50.

Phenotypic Prediction: Linking in vitro Virulence to the Genomics of 59 Salmonella enterica Strains.

The increased availability of whole-genome-sequencing techniques generates a wealth of DNA data on numerous organisms, including foodborne pathogens such as Salmonella. However, how these data can be used to improve microbial risk assessment and understanding of Salmonella epidemiology remains a challenge. The aim of this study was to assess variability in in vitro virulence and genetic characteristics between and within different serovars. The phenotypic behavior of 59 strains of 32 different Salmonella enterica serovars from animal, human and food origin was assessed in an in vitro gastro-intestinal tract (GIT) system and they were analyzed for the presence of 233 putative virulence genes as markers for phenotypic prediction. The probability of in vitro infection, P(inf), defined as the fraction of infectious cells passing from inoculation to host cell invasion at the last stage of the GIT system, was interpreted as the in vitro virulence. Results showed that the (average) P(inf) of Salmonella serovars ranged from 5.3E-05 (S. Kedougou) to 5.2E-01 (S. Typhimurium). In general, a higher P(inf) on serovar level corresponded to higher reported human incidence from epidemiological reporting data. Of the 233 virulence genes investigated, only 101 showed variability in presence/absence among the strains. In vitro P(inf) was found to be positively associated with the presence of specific plasmid related virulence genes (mig-5, pef, rck, and spv). However, not all serovars with a relatively high P(inf), > 1E-02, could be linked with these specific genes. Moreover, some outbreak related strains (S. Heidelberg and S. Thompson) did not reveal this association with P(inf). No clear association with in vitro virulence P(inf) was identified when grouping serovars with the same virulence gene profile (virulence plasmid, Typhoid toxin, peg operon and stk operon). This study shows that the in vitro P(inf) variation among individual strains from the same serovar is larger than that found between serovars. Therefore, ranking P(inf) of S. enterica on serovar level alone, or in combination with a serovar specific virulence gene profile, cannot be recommended. The attribution of single biological phenomena to individual strains or serovars is not sufficient to improve the hazard characterization for S. enterica. Future microbial risk assessments, including virulence gene profiles, require a systematic approach linked to epidemiological studies rather than revealing differences in characteristics on serovar level alone.

Quantification of Salmonella Survival and Infection in an In vitro Model of the Human Intestinal Tract as Proxy for Foodborne Pathogens.

Different techniques are available for assessing differences in virulence of bacterial foodborne pathogens. The use of animal models or human volunteers is not expedient for various reasons; the use of epidemiological data is often hampered by lack of crucial data. In this paper, we describe a static, sequential gastrointestinal tract (GIT) model system in which foodborne pathogens are exposed to simulated gastric and intestinal contents of the human digestive tract, including the interaction of pathogens with the intestinal epithelium. The system can be employed with any foodborne bacterial pathogens. Five strains of Salmonella Heidelberg and one strain of Salmonella Typhimurium were used to assess the robustness of the system. Four S. Heidelberg strains originated from an outbreak, the fifth S. Heidelberg strain and the S. Typhimurium strain originated from routine meat inspections. Data from plate counts, collected for determining the numbers of surviving bacteria in each stage, were used to quantify both the experimental uncertainty and biological variability of pathogen survival throughout the system. For this, a hierarchical Bayesian framework using Markov chain Monte Carlo (MCMC) was employed. The model system is able to distinguish serovars/strains for in vitro infectivity when accounting for within strain biological variability and experimental uncertainty.

Aspergillus vadensis, a new species of the group of black Aspergilli.

A strain from the group of black Aspergilli was analysed in detail to determine the species to which it belongs. A detailed analysis of morphology, RFLP patterns and metabolite profiles was carried out. In addition, a phylogenetic tree was constructed for the black Aspergilli using the ITS and the beta-tubulin sequences of the individual strains. The new species differs by its poor growth on glycerol and galacturonate and its unique extrolite profile consisting of aurasperone B, nigragillin, asperazine and kotanins. RFLP analysis using three genes as probes also resulted in a unique pattern. These data indicate that the strain was closely related but not identical to Aspergillus foetidus, Aspergillus niger and Aspergillus tubingensis. It was therefore designated as a novel species and named Aspergillus vadensis.

Mucoraceous moulds involved in the commercial fermentation of Sufu Pehtze.

Sufu is a fermented cheese-like soybean product in China and Vietnam, obtained by fungal solid-state fermentation of soybean curd (tofu), which results in moulded tofu or 'pehtze'. The final product sufu is obtained by maturing pehtze in a brine containing alcohol and salt during a period of several months. The present report deals with the identity and phylogenetic relationships of mould starter cultures used for the preparation ofpehtze. Starter cultures used in commercial pehtze fermentation were obtained from factories located in several provinces of China and Vietnam, isolated from their pehtze and some were obtained from culture collections. They were identified as Actinomucor repens, Actinomucor taiwanensis, Mucor circinelloides, Mocur hiemalis, Mocur racemosus, and Rhizopus microsporus var. microsporus. Phylogenetic relations based on sequencing of genomic DNA of these starters and of relevant control strains from collections indicate that the genera Mucor, Actinomucor and Rhizopus form distinct and homogenous clusters, with Mucor and Actinomucor showing a slightly closer relationship with each other than with Rhizopus.