Staphylococcal Enterotoxin Gene Cluster: Prediction of Enterotoxin (SEG and SEI) Production and of the Source of Food Poisoning on the Basis of vSaß Typing.

Currently, only 5 (SEA to SEE) out of 27 known staphylococcal enterotoxins can be analyzed using commercially available kits. Six genes (seg, sei, sem, sen, seo, and seu), encoding putative and undetectable enterotoxins, are located on the enterotoxin gene cluster (egc), which is part of the Staphylococcus aureus genomic island vSaß. These enterotoxins have been described as likely being involved in staphylococcal food-poisoning outbreaks. The aim of the present study was to determine if whole-genome data can be used for the prediction of staphylococcal egc enterotoxin production, particularly enterotoxin G (SEG) and enterotoxin I (SEI). For this purpose, whole-genome sequences of 75 Staphylococcus aureus strains from different origins (food-poisoning outbreaks, human, and animal) were investigated by applying bioinformatics methods (phylogenetic analysis using the core genome and different alignments). SEG and SEI expression was tested in vitro using a sandwich enzyme-linked immunosorbent assay method. Strains could be allocated to 14 different vSaß types, each type being associated with a single clonal complex (CC). In addition, the vSaß type and CC were associated with the origin of the strain (human or cattle derived). The amount of SEG and SEI produced also correlated with the vSaß type and the CC of a strain. The present results show promising indications that the in vitro production of SEG and SEI can be predicted based on the vSaß type or CC of a strain. IMPORTANCE Besides having infectious properties in human and animals, S. aureus can produce different enterotoxins in food. The enterotoxins can cause vomiting and diarrhea, often involving many people. Most of these outbreaks remain undiscovered, as detection methods for enterotoxins are only available for a few enterotoxins but not for the more recently discovered enterotoxins G (SEG) and I (SEI). In this study, we show promising results that in vitro production of SEG and SEI can be predicted based on the whole-genome sequencing data of a strain. In addition, these data could be used to find the source (human or cattle derived) of an outbreak strain, which is the key for a better understanding of the role SEG and SEI play in foodborne outbreaks caused by S. aureus.

Influence of reduced levels or suppression of sodium nitrite on the outgrowth and toxinogenesis of psychrotrophic Clostridium botulinum Group II type B in cooked ham.

Outgrowth and toxinogenesis of Clostridium botulinum Group II (non-proteolytic) type B were studied in cooked ham prepared with different NaNO2 (ranging from 0 to 80 mg/kg) and sodium chloride (NaCl, ranging from 12 to 19 g/kg) incorporation rates. Cured ground pork batters were inoculated with a cocktail of 3 strains of C. botulinum Group II type B at 3.5 log10 CFU/g, portioned and samples of 50 g were vacuum packed then cooked and cooled based on thermal processing employed by the meat processing industry. These cooked ham model samples were stored under reasonably foreseeable conditions of use and storage i.e. for 14 days at 4 °C, followed by a cold chain break for 1 h at 20 °C then up to 33 days at 8 °C. Storage times and temperatures were used to mimic those commonly encountered along the supply chain. Enumeration of C. botulinum and detection of the botulinum neurotoxin type B (BoNT/B) were performed in triplicate at different storage times. Under these experimental conditions, incorporation rates of NaNO2 ≥ 30 mg/kg prevented the outgrowth and toxinogenesis of C. botulinum Group II type B in the cooked ham model, regardless of the NaCl concentrations tested. In contrast, total removal of nitrite allowed outgrowth and toxin production during storage of the processed meat product. Results showed that the maximum ingoing amount of nitrite (i.e. 150 mg/kg) that may be added according to the EU legislation (Regulation (EC) No 1333/2008) can be reduced in cooked ham while still ensuring control of C. botulinum Group II type B. According to the multiple factors that could affect C. botulinum behavior in processing meat products, outgrowth and toxin production of C. botulinum should be evaluated on a case by case basis, depending on the recipe, manufacturing process, food matrix and storage conditions.

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.

Effect of exposure to stress conditions on propidium monoazide (PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses.

Campylobacter quantification by qPCR is unable to distinguish viable vs. dead cells in contrast to the culture-based ISO 10272-2 reference method. Propidium monoazide (PMA) has been used to overcome this disadvantage. A Campylobacter PMA-qPCR enumeration method was evaluated for its consistency and compared to the culture-based enumeration for both artificially and natural contaminated broiler carcass rinses. The PMA effect was further evaluated on stressed cells. Five conditions, commonly encountered during the slaughter process and storage (acid, heat, cold, oxidation and freezing), were inflicted to the broiler carcass rinses artificially contaminated with Campylobacter jejuni or Campylobacter coli. A better correlation between the reference method and the qPCR enumeration was obtained when PMA was used. The two cultured-based methods used showed a significant CFU reduction for heat, cold and acid stresses although the PMA-qPCR enumeration showed that viable bacteria were underestimated. Freezing showed the highest reduction effect, while the reduction extend was also overestimated by the microbiological enumeration procedure. Exposure to a mild oxidative stress was the only stress condition applied at temperatures permitting adaptation of Campylobacter and did not lead to either reduction in CFU nor in the PMA-qPCR signal.

Androgen receptor knockout and knock-in mouse models.

Androgens play an important role in male reproductive development and function. These steroid hormones mediate their actions by binding to the androgen receptor (AR). Diseases such as androgen insensitivity syndrome, prostate cancer, Kennedy's disease, and infertility can be caused by mutations in the AR. To get a better insight into the molecular working mechanisms of the AR, several knockout and knock-in mouse models have been developed. These models are reviewed here and are compared with human diseases.