Proof of concept: real-time viability and metabolic profiling of probiotics with isothermal microcalorimetry.

Isothermal microcalorimetry (IMC) is a potent analytical method for the real-time assessment of microbial metabolic activity, which serves as an indicator of microbial viability. This approach is highly relevant to the fields of probiotics and Live Biotherapeutic Products (LBPs), offering insights into microbial viability and growth kinetics. One important characteristic of IMC is its ability to measure microbial metabolic activity separately from cellular enumeration. This is particularly useful in situations where continuous tracking of bacterial activity is challenging. The focus on metabolic activity significantly benefits both probiotic research and industrial microbiology applications. IMC's versatility in handling different media matrices allows for the implementation of viability assessments under conditions that mirror those found in various industrial environments or biological models. In our study, we provide a proof of concept for the application of IMC in determining viability and growth dynamics and their correlation with bacterial count in probiotic organisms. Our findings reinforce the potential of IMC as a key method for process enhancement and accurate strain characterization within the probiotic sector. This supports the broader objective of refining the systematic approach and methods used during the development process, thereby providing detailed insights into probiotics and LBPs.

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.

Assessment of shelf-life and metabolic viability of a multi-strain synbiotic using standard and innovative enumeration technologies.

The number of live bacterial cells is the most used parameter to assess the quality of finished probiotic products. Plate counting (PC) is the standard method in industry to enumerate cells. Application of PC implies critical aspects related to the selection of optimal nutrient media and growth conditions and underestimation of viable but not cultivable (VBNC) cells. Flow-cytometry (FC) is a culture-independent methodology having the potential to selectively enumerate live, damaged, and dead cells representing a powerful tool for in-depth monitoring of probiotic products. We monitored the shelf life of a clinical batch of a synbiotic composition PDS-08 targeting the pediatric population by means of PC and FC according to International Conference on Harmonization (ICH) pharma guidelines testing the Arrhenius model as predictive tool; PC enumeration revealed higher destruction rate than FC suggesting a faster reduction in cultivability than membrane integrity and thus a possible shift of the bacteria into a VBNC status. PDS-08 maintained acidification capability over time, when re-suspended in nutrient medium, even in samples tested sub-optimally for CFU detection (below 1 billion cells/dose). Due to similar kinetics described by the study of metabolic activity and membrane integrity, FC might be suggested as a valid tool for the study of functional stability of a probiotic product.

New insights in enumeration methodologies of probiotic cells in finished products.

The number of bacterial cells is currently recognized as the most important parameter for the efficacy and quality of finished probiotic or live biotherapeutic products (LBP). Cell enumeration is generally performed by culture-dependent methodologies like plate count (PC). These techniques are able to reveal the number of viable cells able to replicate and generate a colony. However, they are limited by their dependence on the combination of culture conditions (e.g. nutrients, temperature) selected for cell recovery. Additionally, they do not provide information on the heterogeneity of a bacterial culture, namely they do not detect the cells in a viable but not cultivable (VBNC) status. Flow-cytometry (FC) is a culture-independent methodology having the potential to enumerate selectively live and damaged or dead cells. FC relies on the use of specific probes for different cell targets (e.g. membrane, enzymes) to unveil information on the cell structure and physiological statuses within a bacterial population. In this context, we monitored three batches of freeze-dried Lactobacillus rhamnosus GG (ATCC 53103) during a 3 year of storage at different conditions of temperature and relative humidity, according to ICH guidelines, by means of PC and FC. The Arrhenius model was applied to assess the suitability of the model to predict the mortality of probiotic cells in finished products. The higher destruction rate (k) obtained by PC data compared to FC data suggests a faster reduction of cultivability compared to membrane integrity, probably representing a dynamic shift of the bacterial population into a VBNC/dormant status during storage time. Interestingly, this mechanistic approach works both for PC and FC methodologies increasing the chances to monitor biological phenomenon within a mathematical modelling. The combined use of PC and FC shed lights on the true bacterial potency within a closed system like a finished product and the complexity of its heterogeneity.

Micronized Cells of the Probiotic Strain Bifidobacterium lactis BS01 Activate Monocyte Polarization: A New Approach.

GOALS: The aim of this research was to evaluate whether micronized cells (MCs) from selected biotherapeutic bacteria have the ability to effectively modulate the polarization of monocyte/macrophage subpopulations to advantageously provide a first line of defense against infections. BACKGROUND: Inflammation is a reaction of the host to viral and bacterial infections with the physiological purpose of restoring tissue homeostasis. However, uncontrolled or unresolved inflammation can lead to tissue damage, giving rise to a plethora of chronic inflammatory diseases. The monocytes/macrophages play a key role in the initiation and resolution of inflammation through different activation programs. STUDY: MCs were obtained from Bifidobacterium lactis BS01 strain using a Bioimmunizer extraction protocol. Monocytes were stimulated with the probiotic strain and/or MCs (10 mg/mL) for 24 hours and 5 days. Monocyte/macrophage differentiation was evaluated by cytometry analysis of surface markers and the activity of the 2 subpopulations on oxidative stress was assessed in an in vitro oxidative stress model with a spectrophotometric test. RESULTS: The MCs have been shown to modulate considerably the 2 subpopulations of human monocytes/macrophages, both the "patrolling subpopulation" and the "inflammatory subpopulation," thus highlighting a strong immunostimulatory effect. In addition, MCs are able to mitigate significantly the oxidative stress induced by homocysteine in an in vitro model. CONCLUSIONS: Our findings suggest that MCs derived from the biotherapeutic strain BS01 could represent a possible therapy aimed to effectively prevent and/or cure viral, bacterial, fungal, or protozoal diseases, as well as prevent and/or treat inflammatory processes triggered by external pathogenic agents.

Gut Microbiota, Probiotics, and Sport: From Clinical Evidence to Agonistic Performance.

Human beings harbor clusters of bacteria in different parts of the body, such as the surface or the deep layers of the skin, the mouth, the lungs, the intestine, the vagina, and all the surfaces exposed to the outer world. The majority of microbes resides in the gut, have a weighty influence on human physiology and nutrition and are vital for human life. There is growing evidence showing that the gut microbiota plays important roles in the maturation of the immune system and the protection against some infectious agents. In addition, there are several well-known effects of exercise on gut physiology. Exercise volume and intensity have been shown to exert an influence on gastrointestinal health status. An estimated 20% to 60% of athletes suffer from stress caused by excessive exercise and inadequate recovery. Supplementing the diet with prebiotics and/or probiotics able to improve the metabolic, immune, and barrier function can be a therapy for athletes. A recent study showed the effects of coadministration of 2 probiotic strains (Bifidobacterium breve BR03 and Streptococcus thermophilus FP4) on measures of skeletal muscle performance, damage, tension, and inflammation following a bout of strenuous exercise. Probiotic supplementation likely enhanced isometric average peak torque production from 24 to 72 hours into the recovery period following exercise. The active formulation also moderately increased resting arm angle at 24 and 48 hours following exercise. In conclusion, selected beneficial bacteria could positively affect athletes undergoing periods of intense training and may assist in the performance recovery.

Flow Cytometry: Evolution of Microbiological Methods for Probiotics Enumeration.

GOALS: The purpose of this trial was to verify that the analytical method ISO 19344:2015 (E)-IDF 232:2015 (E) is valid and reliable for quantifying the concentration of the probiotic Lactobacillus rhamnosus GG (ATCC 53103) in a finished product formulation. BACKGROUND: Flow cytometry assay is emerging as an alternative rapid method for microbial detection, enumeration, and population profiling. The use of flow cytometry not only permits the determination of viable cell counts but also allows for enumeration of damaged and dead cell subpopulations. Results are expressed as TFU (Total Fluorescent Units) and AFU (Active Fluorescent Units). In December 2015, the International Standard ISO 19344-IDF 232 "Milk and milk products-Starter cultures, probiotics and fermented products-Quantification of lactic acid bacteria by flow cytometry" was published. This particular ISO can be applied universally and regardless of the species of interest. STUDY: Analytical method validation was conducted on 3 different industrial batches of L. rhamnosus GG according to USP39<1225>/ICH Q2R1 in term of: accuracy, precision (repeatability), intermediate precision (ruggedness), specificity, limit of quantification, linearity, range, robustness. RESULTS: The data obtained on the 3 batches of finished product have significantly demonstrated the validity and robustness of the cytofluorimetric analysis. CONCLUSIONS: On the basis of the results obtained, the ISO 19344:2015 (E)-IDF 232:2015 (E) "Quantification of lactic acid bacteria by flow cytometry" can be used for the enumeration of L. rhamnosus GG in a finished product formulation.

Searching for the Perfect Homeostasis: Five Strains of Bifidobacterium longum From Centenarians Have a Similar Behavior in the Production of Cytokines.

GOALS: To investigate the modulation of human cytokines by Bifidobacterium longum strains isolated from Centenarians. In particular, we measured the production of interleukin (IL)-12p70, interferon-γ, IL-17A, and IL-4 from human peripheral blood mononuclear cells after stimulation with live bacteria. BACKGROUND: Probiotics may inhibit pathogens and modulate the immune system, bringing a beneficial effect on human health. Among the probiotic strains, bifidobacteria play a key role in the maturation of the host's immune system. At present, only a few comparative data are available on the effects of bifidobacteria associations on cytokine production. STUDY: Peripheral blood mononuclear cells were isolated, cultured, and stimulated (ratio 1:1) with B. longum DLBL07, B. longum DLBL08, B. longum DLBL09, B. longum DLBL10, or B. longum DLBL11, either alone or in association. Cytokine production was determined by an enzyme-linked immunosorbent assay. RESULTS: Both the B. longum DLBL mixture and the individual B. longum DLBL strains induced similar levels of IL-4, interferon-γ, and IL-17A. Under all conditions tested, no IL-12p70 release was detected. CONCLUSIONS: The fact that B. longum strains were obtained from Centenarians suggests a perfect homeostasis between this specific species and the host. Moreover all the B. longum strains from Centenarians used in our study share some biological similarities.

The Possible Innovative Use of Bifidobacterium longum W11 in Association With Rifaximin: A New Horizon for Combined Approach?

GOALS: The aim of the study was to unequivocally demonstrate the nontransmissibility of the genes mediating the resistance of the strain Bifidobacterium longum W11 (LMG P-21586) to rifaximin. BACKGROUND: Most antibiotic treatments can induce unfavorable side effects such as antibiotic-associated diarrhea, which is largely attributable to the disruption of the intestinal microbiota. The parallel intake of probiotic bacteria might reduce these events, even if with generally very poor results. In this regard, the use of antibiotic-resistant beneficial bacteria could represent a worthy strategy. STUDY: Rifaximin was tested in parallel with rifampicin, rifapentine, and rifabutin, all rifamycin derivates, using 5 different concentrations. Susceptibility tests were performed by the disc diffusion method of Kirby-Bauer, and inhibition zones were measured after incubation at 37°C. B. longum BL03 was used as comparison. The B. longum W11 genome was sequenced on Illumina MiSeq with a 250 PE reads module. After mapping the reads with the reference bacterial genome, the alignment data were processed using FreeBayes software. RESULTS: B. longum BL03 was inhibited by all antibiotics even at the lowest concentration. In contrast, the W11 strain was inhibited by rifampicin, rifabutin, and rifaximin only at the highest concentration (512 µg/mL). The genomic analysis showed a mutation into the chromosomal DNA. No transposable elements were found, and the genetic locus was not flanked by close mobile genetic elements. CONCLUSIONS: B. longum W11 could be used in combined therapy with rifaximin, thus opening new focused frontiers in the probiotic era while preserving the necessary safety of use for consumers.

In Vitro Activity of Lactobacillus fermentum LF5 Against Different Candida Species and Gardnerella vaginalis: A New Perspective to Approach Mixed Vaginal Infections?

GOALS: This study was undertaken to demonstrate the ability of Lactobacillus fermentum LF5 (DSM 32277) to inhibit in vitro different Candida species and Gardnerella vaginalis to weigh its potential effectiveness even in mixed vaginal infections. BACKGROUND: A wide female population is suffering from various vulvovaginal infections. These diseases are often associated with a decrease in the concentration of Lactobacilli in the vagina. Mixed vaginal infections represent >20% of women with vulvovaginal infection. STUDY: LF5 strain was cocultured in De Man, Rogosa and Sharpe with Candida according to a 1:100 ratio in favor of the yeast. Each culture was sampled after 24 hours of incubation for the selective enumeration of the yeasts performed on yeast extract glucose chloramphenicol agar medium.The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants of L. fermentum LF5 ranging from 5% to 20% was quantified by means of optical density at 600 nm (OD600). RESULTS: L. fermentum LF5 demonstrated the ability to inhibit significantly the growth of the 5 species of Candida by at least 4 logarithms.Furthermore, L. fermentum LF5 showed a significant activity after both 24 and 48 hours (46% and 82% with 20% of neutralized supernatant, respectively). A significant dose-dependent growth inhibition was recorded in particular after 48 hours of incubation, even achieving a 80% inhibition of G. vaginalis growth. CONCLUSIONS: The biotherapeutic LF5 could be the only documented strain effective in mixed forms. For this purpose, a human clinical trial is in progress.

The In Vitro Effectiveness of Lactobacillus fermentum Against Different Candida Species Compared With Broadly Used Azoles.

GOALS: To investigate the possible use of Lactobacillus strains in the prophylaxis and/or adjuvant therapy of acute vulvovaginal candidiasis and other vaginal infections sustained by Candida yeasts. BACKGROUND: The incidence of Candida infections has substantially increased in recent years. Treatment of vaginal infections with lactobacilli has a long tradition, starting with Döderlein's description of the vaginal microbiota. MATERIALS AND METHODS: We assessed the activity of serially diluted fluconazole and miconazole (from 3 ng/mL to 1 mg/mL) against Candida strains. Serial dilutions of the azoles were prepared in Sabouraud Dextrose Broth in the presence of Candida strains. Broths were incubated under aerobic condition at 30°C, and the optical density was measured at 560 nm. Minimum inhibitory concentration was defined as the lowest concentration of the antibiotic that completely inhibited visible growth. RESULTS: An evident resistance to the azoles used was recorded for all species of Candida, with the exception of Candida parapsilosis. For this species, a minimum inhibitory concentration ≤1 mg/mL was obtained, thus confirming the slight sensitivity to fluconazole and miconazole.All Lactobacillus strains tested, namely LF5, LF09, LF10, and LF11, have the ability to significantly inhibit the growth of the five species of Candida of at least 4 logarithms. Furthermore, the best result obtained with miconazole on C. parapsilosis is still 2 logarithms lower. CONCLUSIONS: The use of beneficial bacteria, especially lactobacilli, could be regarded as a good alternative for the prevention and treatment of Candida infections.

In vitro sensitivity of probiotics to human pancreatic juice.

BACKGROUND: The resistance of gut flora with probiotic activity to pancreatic juice is usually tested with artificial pancreatic fluid. Previous studies evaluated the sensitivity of diverse probiotics to human gastric and biliary secretion; none tested the resistance of probiotics to human pancreatic juice. As most bacteria sensitive to artificial pancreatic fluid in vitro have a high rate of isolation from feces, the resistance to human pancreatic juice could be higher. AIM: The aim of this study was to compare the sensitivity of different strains of probiotics to artificial and human pancreatic juice. MATERIALS AND METHODS: The viability of 8 strains of Lactobacillus and 4 strains of Bifidobacterium was tested with standard artificial and human pancreatic juice withdrawn from 16 patients during endoscopic retrograde cholangiopancreatography procedure. The mortality rate (%) of various bacteria was measured after 5, 30, and 60 minutes contact time. The results were normalized for mortality rate induced by hypotonic condition and time exposure to 37 degrees C. RESULTS: When incubated with artificial and human pancreatic juice, the mortality rate of all strains of Lactobacillus at 5, 30, and 60 minutes was 10.1 versus 7.6, 20.5 versus 19.7, and 28.6 versus 29.8, respectively; whereas the mortality rate of all strains of Bifidobacterium was 8.0 versus 9.2, 33.3 versus 28.9, and 42.2 versus 44.4, respectively. CONCLUSIONS: All the tested strains were sensitive to artificial and human pancreatic juice depending on time contact. Bifidobacterium strains seem to be more sensitive than Lactobacillus strains in particular at higher time contact. There is no significant difference between sensitivity to simulated and human pancreatic juice. For this reason, probiotics activity may be tested with artificial pancreatic fluid using a standardized, easier, and less costly procedure.