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.

Pharmacy-Based Infectious Disease Management Programs Incorporating CLIA-Waived Point-of-Care Tests.

There are roughly 48,000 deaths caused by influenza annually and an estimated 200,000 people who have undiagnosed human immunodeficiency virus (HIV). These are examples of acute and chronic illnesses that can be identified by employing a CLIA-waived test. Pharmacies across the country have been incorporating CLIA-waived point-of-care tests (POCT) into disease screening and management programs offered in the pharmacy. The rationale behind these programs is discussed. Additionally, a summary of clinical data for some of these programs in the infectious disease arena is provided. Finally, we discuss the future potential for CLIA-waived POCT-based programs in community pharmacies.

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.

Characteristics and genetic determinants of bacteriocin activities produced by Carnobacterium piscicola CP5 isolated from cheese.

Carnobacterium piscicola CP5, isolated from a French mold-ripened soft cheese, produced a bacteriocin activity named carnocin CP5, which inhibited Carnobacterium, Enterococcus and Listeria spp. strains, and among the Lactobacillus spp. only Lactobacillus delbrueckii spp. [24]. The activity was purified by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography followed by reverse-phase high-performance liquid chromatography (RP-HPLC). This latter step separated two peaks with anti-listerial activity (CP51 and CP52). Carnocin CP51 was partially sequenced, and the N-terminal part revealed the presence of the "pediocin-like consensus" sequence-Tyr-Gly-Asn-Gly-Val-. Then, a degenerated 24-mer oligonucleotide probe was constructed from the N-terminal sequence and used to detect the structural gene. It was localized on a plasmid of about 40 kb. Cloning of restriction fragments of this one, followed by DNA sequencing, revealed the presence of the second anti-Listeria bacteriocin gene (CP52). By comparing sequences in data banks and confirming results with PCR reactions, carnocin CP51 shared homologies with carnobacteriocin BM1, and carnocin CP52 was similar to carnobacteriocin B2, both produced by C. piscicola LV17 [2]. However, carnobacteriocin A from C. piscicola LV17 gene was lacking in C. piscicola CP5, and the two microorganisms have been isolated from different ecological environments: C. piscicola CP5 and C. piscicola LV17 were isolated from soft cheese and vacuum-packed meat respectively. This fact could allow different application perspectives for C. piscicola CP5.