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.

Interlaboratory proficiency tests for the detection of staphylococcal enterotoxin type A in food.

Staphylococcal enterotoxins (SEs) are among the leading causes of food intoxications, affecting consumer health even in nanogram (ng) amounts. In the European Union, certain food safety criteria are specified, including the absence of SEs in cheeses, milk powder and whey powder. Until 2019, the analytical reference method used was the European Screening Method, which was replaced by EN ISO 19020. For the official laboratories involved in food control, the German Reference Laboratory for coagulase-positive staphylococci including Staphylococcus aureus organized three interlaboratory proficiency tests (ILPTs) to detect SE type A in food during the years 2013-2018. The selected food products (cream cheese and vanilla pudding) were successfully tested beforehand with regard to easy handling, homogeneity and stability of the added toxin. In 2013, ILPT participants overall were not competent in detecting SE type A in food. The following factors were identified to improve the performance: (i) concentration of sample extract using dialysis; (ii) selection of a sensitive detection kit; and (iii) proper sample handling. By taking these factors into account and instructing and training the laboratories, their competence greatly improved. In 2018, all performance criteria (specificity, sensitivity and accuracy) were >90%, even at very low concentrations of SE type A of approximately 0·01 ng g-1 food.

Enterotoxin genes and antimicrobial resistance in Staphylococcus aureus isolated from food products in Algeria.

AIMS: The aim of this study was to characterize Staphylococcusaureus isolates of food origin (dairy and meat products, pastries and sandwiches) determining the carriage in enterotoxin genes and the antimicrobial resistance pheno/genotypes. METHODS AND RESULTS: A total of 300 food samples were collected and analysed for the detection of S. aureus. The presence of enterotoxin genes was investigated by multiplex PCRs. Resistance of isolates to 11 antimicrobials was determined using disc diffusion method and molecular characterization of methicillin-resistant S. aureus was carried out by spa typing and multilocus sequence typing. Overall, 51 out of 300 samples (17%) were contaminated with S. aureus, and 104 isolates were recovered. In all, 65 of these isolates (62·5%) harboured one or more genes encoding for staphylococcal enterotoxins, being seg and sei the most observed genes. The highest resistance profile was ascribed to penicillin G (95·19%). Five isolates were methicillin-resistant (MRSA) harbouring the mecA gene. All MRSA isolates belonged to the sequence type ST5 and to two different spa types (t450 and t688); the MRSA-t450 isolate carried the scn gene (specific marker of the immune evasion cluster system), but the four MRSA-t688 isolates were scn negative. The MRSA isolates carried enterotoxin genes but were negative for the genes of the Panton Valentine leukocidine (lukF/S-PV). CONCLUSION: The presence of enterotoxigenic S. aureus isolates, including MRSA, in food samples can represent a risk for public health. SIGNIFICANCE AND IMPACT OF THIS STUDY: This work describes the molecular characteristics of MRSA strains isolated from foods in Algeria and it can contribute to an extended database concerning the S. aureus isolated from food origin.

Probabilistic multicompartmental model for interpreting DGT kinetics in sediments.

Extensive research has been performed on the use of the DIFS (DGT-Induced Fluxes in Soils and Sediments) model to interpret diffusive gradients in thin-film, or DGT, measurements in soils and sediments. The current report identifies some areas where the DIFS model has been shown to yield poor results and proposes a model to address weaknesses. In particular, two major flaws in the current approaches are considered: (i) many studies of accumulation kinetics in DGT exhibit multiple kinetic stages and (ii) several combinations of the two fitted DIFS parameters can yield identical results, leaving the question of how to select the 'best' combination. Previously, problem (i) has been addressed by separating the experimental data sets into distinct time segments. To overcome these problems, a model considering two types of particulate binding sites is proposed, instead of the DIFS model which assumed one single particulate pool. A probabilistic approach is proposed to fit experimental data and to determine the range of possible physical parameters using Probability Distribution Functions (PDFs), as opposed to single values without any indication of their uncertainty. The new probabilistic model, called DGT-PROFS, was tested on three different formulated sediments which mainly differ in the presence or absence of iron oxides. It was shown that a good fit can be obtained for the complete set of data (instead of DIFS-2D) and that a range of uncertainty values for each modeling parameter can be obtained. The interpretation of parameter PDFs allows one to distinguish between a variety of geochemical behaviors, providing useful information on metal dynamics in sediments.

Development and validation of the Standard method EN ISO 19020 - microbiology of the food chain - Horizontal method for the immunoenzymatic detection of staphylococcal enterotoxins in foodstuffs.

In the frame of the CEN Mandate M/381 from the European Commission to CEN (European Committee for Standardization), a method for the detection of staphylococcal enterotoxins in foodstuffs has been developed, validated and standardized. An extraction procedure based on dialysis concentration followed by an immuno-enzymatic detection has been defined. In addition, performance criteria (minimum values of sensitivity, specificity and level of detection) to be achieved by the commercially available immuno-enzymatic kits that could be used to detect staphylococcal enterotoxins in food matrices, were developed. A 2-stage validation study was conducted: The first stage aimed at selecting the commercial kits to be included in the second stage, which consisted in an interlaboratory study, using eight matrices covering five food categories (ready-to-eat food, meat products, milk products, dessert and fish). Results showed that two detection kits included in the study met the pre-defined performance criteria. The implementation of dialysis concentration step increased significantly the sensitivity of the method. The method developed allowed to achieve the Benchmark Dose lower limit (BMD10) estimated at 6.1 ng. In 2019, finally, the European Commission recognized this standard as the European Union reference method for the detection of staphylococcal enterotoxins in food.

Development of a reference material for Staphylococcus aureus enterotoxin A in cheese: feasibility study, processing, homogeneity and stability assessment.

Staphylococcal food poisoning is caused by enterotoxins excreted into foods by strains of staphylococci. Commission Regulation 1441/2007 specifies thresholds for the presence of these toxins in foods. In this article we report on the progress towards reference materials (RMs) for Staphylococcal enterotoxin A (SEA) in cheese. RMs are crucial to enforce legislation and to implement and safeguard reliable measurements. First, a feasibility study revealed a suitable processing procedure for cheese powders: the blank material was prepared by cutting, grinding, freeze-drying and milling. For the spiked material, a cheese-water slurry was spiked with SEA solution, freeze-dried and diluted with blank material to the desired SEA concentration. Thereafter, batches of three materials (blank; two SEA concentrations) were processed. The materials were shown to be sufficiently homogeneous, and storage at ambient temperature for 4weeks did not indicate degradation. These results provide the basis for the development of a RM for SEA in cheese.

Mobility of Cd and Cu in formulated sediments coated with iron hydroxides and/or humic acids: a DGT and DGT-PROFS modeling approach.

The diffusive gradients technique in thin films (DGT) was used to investigate the kinetic resupply of Cd and Cu to pore water from the solid phase. For the sake of simplification, experiments were performed using formulated sediments that differed in the presence or absence of humic acids (HA) and/or of iron hydroxides (i.e., goethite and ferrihydrite). The effects of the time after the contamination of the solid phase (aging effect) on formulated sediments that were coated with goethite and HA and spiked with Cd were also evaluated. Kinetic DGT results were interpreted using the newly developed, multi-compartmental model DGT-PROFS. Due to Cu humate formation, the addition of HA slightly increased the Cu concentration in the pore water independent of the effect of the iron hydroxide coating on the formulated sediments and slightly decreased that of Cd. The impact of 8-190d of aging resulted in a significant decrease in the Cd concentration of the pore water over an increasing incubation time. Modeling our results with DGT-PROFS led to the following conclusions concerning the impact of HA and iron hydroxides on Cd and Cu availability. First, in the presence of HA and absence of iron hydroxides, Cd is associated mainly with weak sites, while Cu is bound to strong sites. Similarly, in the presence of both iron hydroxides and HA, Cu appeared to be more heavily associated with the strong sites than did Cd. When the incubation time increased from 8 to 190d, a proportion of Cd initially adsorbed onto weak sites transferred to the strong sites, suggesting that the adsorption of Cd on sediments is controlled partially by slow kinetic processes.