A streamlined workflow for a fast and cost-effective count of tyndallized probiotics using flow cytometry.

The use of dead probiotics and their cellular metabolites seems to exhibit immunomodulatory and anti-inflammatory properties, providing protection against pathogens. These inanimate microorganisms, often referred to as tyndallized or heat-killed bacteria, are a new class of probiotics employed in clinical practice. Safety concerns regarding the extensive use of live microbial cells have increased interest in inactivated bacteria, as they could eliminate shelf-life problems and reduce the risks of microbial translocation and infection. Culture-dependent methods are not suitable for the quality assessment of these products, and alternative methods are needed for their quantification. To date, bacterial counting chambers and microscopy have been used for tyndallized bacteria enumeration, but no alternative validated methods are now available for commercial release. The aim of the present study is to design a new method for the qualitative and quantitative determination of tyndallized bacterial cells using flow cytometric technology. Using a live/dead viability assay based on two nucleic acid stains, thiazole orange (TO) and propidium iodide (PI), we optimized a workflow to evaluate bacterial viability beyond the reproduction capacity that provides information about the structural properties and metabolic activities of probiotics on FACSVerse without using beads as a reference. The data obtained in this study represent the first analytical application that works effectively both on viable and non-viable cells. The results provided consistent evidence, and different samples were analyzed using the same staining protocol and acquisition settings. No significant discrepancies were highlighted between the declared specification of commercial strain and the analytical data obtained. For the first time, flow cytometry was used for counting tyndallized bacterial cells as a quality control assessment in probiotic production. This aspect becomes important if applied to medical devices where we cannot boast metabolic but only mechanical activities.

Verification of a Rapid Analytical Method for the Qualitative Detection of Listeria spp. and Listeria monocytogenes by a Real-Time PCR Assay according to EN UNI ISO 16140-3:2021.

Microbial contamination and foodborne infections are a significant global public health concern. For this reason, the detection, monitoring, and characterization of pathogens represent a significant challenge in quality control settings. Standard approaches, such as culture methods and biochemical tests, are known to be very time-consuming and intensive. Conversely, molecular technologies based on the genomic identification of bacteria are quick and low-cost. Listeria monocytogenes is an opportunistic pathogen and a major concern especially in food industries. It is important to understand and implement multiple quality control measures to control Listeria infection risk and prevent the contamination of products. Standardized detection and confirmation tests such as the API Listeria test, MALDI-TOF MS, and PCR analysis are available. The aim of our work is to provide a specific molecular method, designed according to the EN UNI ISO 16140-3:2021, for the specific detection, monitoring, and characterization of Listeria spp. and Listeria monocytogenes contamination. The verification of this new rapid approach by real-time PCR (qPCR) overcomes the limitations of culture-based techniques, meeting all the verification criteria required by ISO guidelines, including implementation and item confirmation. This system offers a powerful approach to the real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.

An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements.

Research surrounding health benefits from probiotics is becoming popular because of the increasing demand for safer products with protective and therapeutic effects. Proven benefits are species- or genus-specific; however, no certified assays are available for their characterization and quantification at the strain level in the food supplement industry. The objective of this study was to develop a strain-specific Real-time quantitative polymerase chain reaction (RT-qPCR)-based method to be implemented in routine tests for the identification and quantification of Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, starting from a powder mixture of food supplements. The method optimization was carried out in combination with flow cytometry to compare results between the two strategies and implement the analytical workflow with the information also regarding cell viability. These assays were validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) criteria using the plate count enumeration as the gold standard reference. Briefly, probiotic DNAs were extracted from two powder food supplements. Strain-specific primers targeting unique sequence regions of 16S RNA were identified and amplified by RT-qPCR. Primers were tested for specificity, sensitivity, and efficiency. Both RT-qPCR and flow-cytometry methods described in our work for the quantification and identification of Lactobacillus and Bifidobacterium strains were specific, sensitive, and precise, showing better performances with respect to the morphological colony identification. This work demonstrated that RT-qPCR can be implemented in the quality control workflow of commercial probiotic products giving more standardized and effective results regarding species discrimination.

Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests. METHODS: In this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples. RESULTS: The identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen's Kappa = 0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples. CONCLUSIONS: RT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs, and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.

Intra-Laboratory Validation of Alpha-Galactosidase Activity Measurement in Dietary Supplements.

INTRODUCTION: Alpha-galactosidase (α-Gal) is an enzyme responsible for the hydrolyzation of glycolipids and glycoprotein commonly found in dietary sources. More than 20% of the general population suffers from abdominal pain or discomfort caused by intestinal gas and by indigested or partially digested food residuals. Therefore, α-Gal is used in dietary supplements to reduce intestinal gases and help complex food digestion. Marketed enzyme-containing dietary supplements must be produced in accordance with the Food and Drug Administration (FDA) regulations for Current Good Manufacturing Practice (cGMPs). AIM: in this work we illustrated the process used to develop and validate a spectrophotometric enzymatic assay for α-Gal activity quantification in dietary supplements. METHODS: The validation workflow included an initial statistical-phase optimization of materials, reagents, and conditions, and subsequently a comparative study with another fluorimetric assay. A final validation of method performance in terms of specificity, linearity, accuracy, intermediate-precision repeatability, and system precision was then executed. RESULTS AND CONCLUSIONS: The proven method achieved good performance in the quantitative determination of α-Gal activity in commercial food supplements in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals (ICH) guidelines and is suitable as a rapid in-house quality control test.

A novel pharmaceutical approach for the analytical validation of probiotic bacterial count by flow cytometry.

INTRODUCTION: Flow cytometry is a powerful and sensitive technique able to characterize single cells within a heterogeneous population. Different fluorescent dyes can be combined and used together to analyze a great variety of parameters simultaneously. In particular, flow-cytometry allows to measure viability and vitality of probiotics measuring their metabolic activity, fermentation capacity, acidification potential or oxygen uptake ability (Hayouni et al., 2008). To now, plate counting is considered the gold standard in microbiological technique for probiotic enumeration. However, this approach is limited to the detection of only those viable cells which are able to proliferate and form colonies on a solid medium but is not able to recognize not cultivable bacteria and nonviable cells. AIM: The aim of the present study was to apply The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) parameters for the validation of new analytical methods in microbiology. ICH requirements, which are commonly employed for the analysis of drugs and chemical analytes, have been here applied to live cells for the comparison between a flow-cytometric assay and the traditional plate count method for the quantification of viable probiotics bacteria. METHODS AND RESULTS: Combining specific viability dyes such as thiazole orange (TO) and propidium iodide (PI), probiotic counts of Lactobacillus and Bifidobacterium species were carried out using a FACS Verse (BD Biosciences) cytometer. Analyses were conducted in parallel with the traditional plate count, on specific media. Raw data were analyzed using the FACSuite software (BD Biosciences) and then elaborated with the statistical software Neolicy (VWR International). Results indicated that flow cytometry provides very similar results in cell counting if compared to classical microbiology approaches, showing better performances (ICH parameters) than the traditional plate count method. CONCLUSIONS: This work demonstrated the analytical ICH validation of probiotic counts in food supplement products using a robust flow cytometric approach able to enumerate and to assess bacteria viability with stronger results in comparison to the traditional plate count.