Collaborative cytometric inter-laboratory ring test for probiotics quantification.

Introduction: Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. From this definition, accurate enumeration of probiotic products is a necessity. Nonetheless, this definition does not specify the methods for assessing such viability. Colony forming units is the de facto gold standard for enumerating viable in probiotic products. The notion of microbial viability has been anchored in the concept of cultivability, which refers to a cell's capacity to replicate and form colonies on agar media. However, there is a growing consensus that the term "viability" should not be exclusively tied to the ability to cultivate cells. For example, bacterial cells can exist in a Viable But Non-Culturable (VBNC) state, characterized by the maintenance of characteristics such as membrane integrity, enzymatic activity, pH gradients, and elevated levels of rRNA, despite losing the ability to form colonies. Methods: Herein we present the results of a collaborative inter-laboratory ring test for cytometric bacterial quantification. Specifically, membrane integrity fluorescence flow cytometry (FFC) method and the newer impedance flow cytometry (IFC) method have been used. Both methods interrogate single cells in solution for the presence of intact membranes. FFC exploits fluorochromes that reflect the presence or absence of an intact membrane. IFC probes membrane integrity in a label-free approach by detecting membrane-induced hindrances to the propagation of electricity. Results: A performance ring-test and comparison design on the FFC method showed that the method is robust against the exchange of equipment, procedures, materials, and operators. After initial method optimization with assessments of rehydration medium, wake-up duration, and phase shift gating on the individual strains, the IFC method showed good agreement with the FFC results. Specifically, we tested 6 distinct species of probiotic bacteria (3 Lactobacillus and 3 Bifidobacterium strains) finding good agreement between FFC and IFC results in terms of total and live cells. Discussion: Together, these results demonstrate that flow cytometry is a reliable, precise, and user-friendly culture-independent method for bacterial enumeration.

Supranational Assessment of the Quality of Probiotics: Collaborative Initiative between Independent Accredited Testing Laboratories.

Recent acquisitions about the role of the microbiota in the functioning of the human body make it possible to envisage an increasing use of beneficial microbes, and more particularly of probiotics as well as their metabolites, as nutritional supplements. National and EU authorities are engaged in assuring the safety and quality of food supplements and in defining rules to assess and communicate their efficacy on human health. The quality of probiotics, intended as strains' identification, viability, and stability over time, is a crucial factor of credibility with consumers and health professionals. Analytical technologies for the quality control of probiotics must also be adapted to new preparations, such as those including new multistrains complex combinations. Accredited laboratories face this relevant challenge on a daily basis. Through its close collaboration with the laboratory commissioned to produce the specifications for its ESLP quality label (identification and quantitative analyses) together with its scientific committee, the ESLP has been focusing on this issue for 10 years. Recently, as part of the internationalization of the ESLP quality label, a new and unique initiative in Europe for the evaluation of the quality of probiotic preparations has been carried out. The collaboration between two accredited laboratories in Belgium and in Italy represented a concrete example of supranational collaboration in the assessment of the quality of probiotic preparations. Results show that both laboratories are in line as expected in terms of performance. Common approaches to the qualitative assessment of probiotic preparations, especially for complex and composite recipes, in terms of number of strains and included substances, should be encouraged and promoted all over the EU.

Probiotics in cystic fibrosis patients: A double blind crossover placebo controlled study: Pilot study from the ESPGHAN Working Group on Pancreas/CF.

BACKGROUND: A potential positive effect of probiotics in cystic fibrosis (CF) on fecal calprotectin (FCP), pulmonary exacerbations and weight has been described in small controlled trials. METHODS: A double-blind multicenter cross-over study (2 × 4 m) was performed looking at abdominal pain, nutritional status, pulmonary function, pulmonary exacerbation, FCP and lactulose/mannitol gut permeability test. Patients kept a diary with daily scoring of abdominal pain, stool frequency and consistency as well as treatment changes. RESULTS: 31 CF patients entered the study of which 25 finished it. At start patients aged 9.3yrs (6.9-12.2), had a median BMI z-score of -0.5 (-1.5-0.08), height z-score of -0.4 (-1.1-0.05) and FEV1% of 100% (87.2-106.6). Median FCP at start was 61 µg/g (17-108) and gut permeability 0.079 (0.051-0.122). No significant changes were observed in the clinical parameters (BMI, FEV1%, abdominal pain, exacerbations). Despite being frequently abnormal (17/28 (61%) >50 mg/kg), FCP did not change significantly with probiotics. The proportion of patients with normal permeability was 8% during placebo and 32% during probiotic treatment (p = 0.031). FCP correlated to BMI z-score (p = 0.043) and gut permeability to abdominal pain (p = 0.015). The microbiome revealed a high predominance of Actinobacteria and Proteobacteriae. Probiotic supplementation did not result in a shift at the phylum nor at phylogenetic level. CONCLUSION: Normalization of gut permeability was observed in 13% of patients during probiotic treatment. However, none of the previously described effects could be confirmed.