Validation of N-Light™ Salmonella Risk Test Kit for Detection of Salmonella spp. on Environmental Surfaces: AOAC Performance Tested MethodSM 072204.

BACKGROUND: The NEMIS N-Light™ Salmonella Risk method uses chemiluminescence designed for the qualitative detection of Salmonella spp. from environmental surface samples. OBJECTIVE: To validate the N-Light Salmonella Risk assay using independent and method developer validation studies according to the AOAC Performance Tested MethodsSM (PTM) program for the detection of Salmonella spp. on stainless-steel, polystyrene, and ceramic environmental surfaces. METHOD: The N-Light Salmonella Risk assay was evaluated in a matrix study in comparison to the ISO 6579-1:2017 method ("Microbiology of the Food Chain-Horizontal Method for the Detection, Enumeration, and Serotyping of Salmonella-Part 1: Detection of Salmonella spp.") using an unpaired study design. Additional PTM studies performed were inclusivity/exclusivity, robustness, product consistency, and stability. RESULTS: The N-Light Salmonella Risk assay demonstrated a specific detection of all Salmonella strains tested. In the matrix study, the N-Light Salmonella Risk assay showed no significant differences between presumptive and confirmed results or between candidate and reference method results on the three surfaces evaluated. Data for additional PTM studies met acceptance criteria requirements. CONCLUSIONS: The NEMIS Technologies N-Light Salmonella Risk assay is an effective method for the qualitative detection of Salmonella on stainless-steel, polystyrene, and ceramic environmental surfaces. HIGHLIGHTS: The NEMIS Technologies N-Light Salmonella Risk assay, which is the first chemiluminescence-based detection system that uses a novel, patented dioxetane compound, allowing for easy and rapid detection of Salmonella.

Validation of N-LightTM L. monocytogenes for Detection of Listeria monocytogenes on Environmental Surfaces: AOAC Performance Tested MethodSM 122002.

BACKGROUND: The NEMIS Technologies N-LightTM L. monocytogenes assay uses chemiluminescence designed for the qualitative detection of Listeria monocytogenes from environmental surface samples. OBJECTIVE: To validate the NEMIS Technologies N-Light L. monocytogenes assay as part of the AOAC Performance Tested MethodSM program for the detection of L. monocytogenes on stainless steel, plastic (polystyrene), and ceramic environmental surfaces. METHOD: Using the Vitl Life Science Solutions Lu-mini luminometer, the NEMIS Technologies N-Light L. monocytogenes assay was compared to the ISO 11290-1:2017: Microbiology of the Food Chain-Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 1 using a 1" × 1" stainless steel test area in an unpaired study design. RESULTS: The NEMIS Technologies N-Light L. monocytogenes assay using the Vitl Life Science Solutions Lu-mini luminometer demonstrated no statistically significant differences between presumptive and confirmed results or between candidate and reference method results. Data for additional Performance Tested MethodSM studies met acceptance criteria requirements. CONCLUSIONS: The NEMIS Technologies N-Light L. monocytogenes assay is an effective method for the qualitative detection of L. monocytogenes from stainless steel, plastic (polystyrene), and ceramic environmental surface samples. HIGHLIGHTS: The NEMIS method is the first chemiluminescence detection system based on a novel, patented, dioxetane compound.

Prediction of Genetic Groups within Brettanomyces bruxellensis through Cell Morphology Using a Deep Learning Tool.

Brettanomyces bruxellensis is described as a wine spoilage yeast with many mainly strain-dependent genetic characteristics, bestowing tolerance against environmental stresses and persistence during the winemaking process. Thus, it is essential to discriminate B. bruxellensis isolates at the strain level in order to predict their stress resistance capacities. Few predictive tools are available to reveal intraspecific diversity within B. bruxellensis species; also, they require expertise and can be expensive. In this study, a Random Amplified Polymorphic DNA (RAPD) adapted PCR method was used with three different primers to discriminate 74 different B. bruxellensis isolates. High correlation between the results of this method using the primer OPA-09 and those of a previous microsatellite analysis was obtained, allowing us to cluster the isolates among four genetic groups more quickly and cheaply than microsatellite analysis. To make analysis even faster, we further investigated the correlation suggested in a previous study between genetic groups and cell polymorphism using the analysis of optical microscopy images via deep learning. A Convolutional Neural Network (CNN) was trained to predict the genetic group of B. bruxellensis isolates with 96.6% accuracy. These methods make intraspecific discrimination among B. bruxellensis species faster, simpler and less costly. These results open up very promising new perspectives in oenology for the study of microbial ecosystems.

Recombinant human plasma phospholipid transfer protein (PLTP) to prevent bacterial growth and to treat sepsis.

Although plasma phospholipid transfer protein (PLTP) has been mainly studied in the context of atherosclerosis, it shares homology with proteins involved in innate immunity. Here, we produced active recombinant human PLTP (rhPLTP) in the milk of new lines of transgenic rabbits. We successfully used rhPLTP as an exogenous therapeutic protein to treat endotoxemia and sepsis. In mouse models with injections of purified lipopolysaccharides or with polymicrobial infection, we demonstrated that rhPLTP prevented bacterial growth and detoxified LPS. In further support of the antimicrobial effect of PLTP, PLTP-knocked out mice were found to be less able than wild-type mice to fight against sepsis. To our knowledge, the production of rhPLTP to counter infection and to reduce endotoxemia and its harmful consequences is reported here for the first time. This paves the way for a novel strategy to satisfy long-felt, but unmet needs to prevent and treat sepsis.

Co-expression of the small heat shock protein, Lo18, with ß-glucosidase in Escherichia coli improves solubilization and reveals various associations with overproduced heterologous protein, GroEL/ES.

We developed a new system to improve the overproduction of soluble proteins in E. coli based on a plasmid encoding the small heat-shock protein, Lo18, derived from the lactic acid bacterium Oenococcus oeni. The efficiency of this system was compared with that of another system based on production of the E. coli universal chaperone GroEL/ES. A compatible plasmid encoding ß-glucosidase was constructed for the overproduction and aggregation of this enzyme. Co-expression with Lo18 resulted in an increase in soluble ß-glucosidase levels similar to that obtained in the GroEL/ES co-expression system. Lo18 was found preferentially in the insoluble fraction, associated with aggregated enzyme. By contrast, GroEL/ES was more abundant in the soluble fraction.

Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium.

The tolerance of the lactic acid bacterium Oenococcus oeni to hostile wine conditions is essential for the success of malolactic fermentation (MLF). In this study, reverse transcription quantitative PCR (RT-qPCR) was used to quantify the transcript level of 13 genes that could play a role in adaptation of O. oeni in wine. To optimize survival and growth in wine, cells were adapted during growth at low pH (3.5) prior to inoculation into wine. The level of gene expression was analyzed after growth at pH 3.5 in a rich medium and during MLF in a wine-like medium. RT-qPCR analyses exhibited different expression ratios of stress genes. The data obtained showed that determination of mRNA levels could constitute a new approach to studying the stress response of O. oeni after adaptation at low pH and during growth in a wine-like medium.

A new approach for selection of Oenococcus oeni strains in order to produce malolactic starters.

The lactic acid bacterium Oenococcus oeni, mainly responsible for malolactic fermentation (MLF), is used in new winery process as starter culture for direct inoculation. The difficulty to master MLF according to the wine led us to search a new approach to select effective O. oeni strains. Biochemical and molecular tests were performed in order to characterize three strains of O. oeni selected for malolactic starter elaboration. Malolactic and ATPase activities that appeared as a great interest in MLF were measured and the expression of a small heat shock protein Lo18 was evaluated by immunoblotting and real-time PCR. These results were correlated with the performances of strains in two red wines. Physiological and molecular characteristics of the three strains showed significant differences for the global malolactic activity on intact cell at pH 3.0 and at the level of induction of the small heat shock protein Lo18. These two parameters appeared of interest to evaluate in the ability of O. oeni strains to survive into wine after direct inoculation and to perform MLF. Indeed, a tested strain that presented the highest malolactic activity on intact cells at pH 3.0 and a high level of Lo18 induction showed a high growth rate and a high specific kinetic of malate consumption. The techniques used in this work carry out more quickly and more reliable than usual for the selection of effective strains intended for direct inoculation in wines.

Determination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni.

The expression gene pattern reflects, in part, mechanisms involved in adaptation to environmental conditions. Thus, we established and validated a method that enables relative transcript quantification in different conditions in the lactic acid bacteria Oenococcus oeni, notably in a technological medium. First, we determined an internal control in our conditions by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the SYBR Green I technology. Among the seven presumed housekeeping tested genes, the ldhD gene was retained for further experiments. Then, the PCR reproducibility was verified in our conditions and the comparative critical threshold (2deltadeltaC(T)) method was applied to quantify the transcript level of genes. The quantification of transcript levels of several stress genes already studied in our laboratory by Northern blot after a heat shock and at the entry of stationary phase allowed us to validate this method. RT-qPCR appeared as a powerful tool to study O. oeni response in stress conditions and wine mimetic conditions.