Live Biotherapeutic Products, A Road Map for Safety Assessment.

Recent developments in the understanding of the relationship between the microbiota and its host have provided evidence regarding the therapeutic potential of selected microorganisms to prevent or treat disease. According to Directive 2001/83/EC, in the European Union (EU), any product intended to prevent or treat disease is defined as a medicinal product and requires a marketing authorization by competent authorities prior to commercialization. Even if the pharmaceutical regulatory framework is harmonized at the EU level, obtaining marketing authorisations for medicinal products remains very challenging for Live Biotherapeutic Products (LBPs). Compared to other medicinal products currently on the market, safety assessment of LBPs represents a real challenge because of their specific characteristics and mode of action. Indeed, LBPs are not intended to reach the systemic circulation targeting distant organs, tissues, or receptors, but rather exert their effect through direct interactions with the complex native microbiota and/or the modulation of complex host-microbiota relation, indirectly leading to distant biological effects within the host. Hence, developers must rely on a thorough risk analysis, and pharmaceutical guidelines for other biological products should be taken into account in order to design relevant non-clinical and clinical development programmes. Here we aim at providing a roadmap for a risk analysis that takes into account the specificities of LBPs. We describe the different risks associated with these products and their interactions with the patient. Then, from that risk assessment, we propose solutions to design non-clinical programmes and First in Human (FIH) early clinical trials appropriate to assess LBP safety.

A prospective longitudinal study on the microbiota composition in amyotrophic lateral sclerosis.

BACKGROUND: A connection between amyotrophic lateral sclerosis (ALS) and altered gut microbiota composition has previously been reported in animal models. This work is the first prospective longitudinal study addressing the microbiota composition in ALS patients and the impact of a probiotic supplementation on the gut microbiota and disease progression. METHODS: Fifty patients and 50 matched controls were enrolled. The microbial profile of stool samples from patients and controls was analyzed via PCR-Denaturing Gradient Gel Electrophoresis, and the main microbial groups quantified via qPCR. The whole microbiota was then analyzed via next generation sequencing after amplification of the V3-V4 region of 16S rDNA. Patients were then randomized to receive probiotic treatment or placebo and followed up for 6 months with ALSFRS-R, BMI, and FVC%. RESULTS: The results demonstrate that the gut microbiota of ALS patients is characterized by some differences with respect to controls, regardless of the disability degree. Moreover, the gut microbiota composition changes during the course of the disease as demonstrated by the significant decrease in the number of observed operational taxonomic unit during the follow-up. Interestingly, an unbalance between potentially protective microbial groups, such as Bacteroidetes, and other with potential neurotoxic or pro-inflammatory activity, such as Cyanobacteria, has been shown. The 6-month probiotic treatment influenced the gut microbial composition; however, it did not bring the biodiversity of intestinal microbiota of patients closer to that of control subjects and no influence on the progression of the disease measured by ALSFRS-R was demonstrated. CONCLUSIONS: Our study poses the bases for larger clinical studies to characterize the microbiota changes as a novel ALS biomarker and to test new microbial strategy to ameliorate the health status of the gut. TRIAL REGISTRATION: CE 107/14, approved by the Ethics Committee of the "Maggiore della Carità" University Hospital, Italy.

Probiotics May Have Beneficial Effects in Parkinson's Disease: In vitro Evidence.

Background: Parkinson's disease (PD) is characterized by loss of dopaminergic neurons and intraneuronal accumulation of alpha-synuclein, both in the basal ganglia and in peripheral sites, such as the gut. Peripheral immune activation and reactive oxygen species (ROS) production are important pathogenetic features of PD. In this context, the present study focused on the assessment of in vitro effects of probiotic bacterial strains in PBMCs isolated from PD patients vs. healthy controls. Methods: 40 PD patients and 40 matched controls have been enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with a selection of probiotics microorganisms belonging to the lactobacillus and bifidobacterium genus. In vitro release of the major pro- (Tumor Necrosis Factor-alpha and Interleukin-17A and 6) and anti-inflammatory (Interleukin 4 and 10) cytokines by PBMCs, as well as the production of ROS was investigated. Furthermore, we assessed the ability of probiotics to influence membrane integrity, antagonize the growth of potential pathogen bacteria, such as Escherichia coli and Klebsiella pneumoniae and encode tyrosine decarboxylase genes (tdc). Results: All probiotic strains were able to inhibit inflammatory cytokines and ROS production in both patients and controls. The most striking results were obtained in PD subjects with L. salivarius LS01 and L. acidophilus which significantly reduced pro-inflammatory and increased the anti-inflammatory cytokines (p < 0.05). Furthermore, most strains determined restoration of membrane integrity and inhibition of E. coli and K. pneumoniae. Finally, we also showed that all the strains do not carry tdc gene, which is known to decrease levodopa bioavailability in PD patients under treatment. Conclusions: Probiotics exert promising in vitro results in decreasing pro-inflammatory cytokines, oxidative stress and potentially pathogenic bacterial overgrowth. In vivo longitudinal data are mandatory to support the use of bacteriotherapy in PD.

Effects of Probiotics on Cognitive Reactivity, Mood, and Sleep Quality.

Recent demonstration that probiotics administration has positive effects on mood state in healthy populations suggests its possible role as an adjunctive therapy for depression in clinical populations and as a non-invasive strategy to prevent depressive mood state in healthy individuals. The present study extends current knowledge on the beneficial effects of probiotics on psychological well-being, as measured by changes in mood (e.g., cognitive reactivity to sad mood, depression, and anxiety), personality dimensions, and quality of sleep, which have been considered as related to mood. For this double-blind, placebo-controlled study 38 healthy volunteers assigned to an experimental or control group assumed a daily dose of a probiotic mixture (containing Lactobacillus fermentum LF16, L. rhamnosus LR06, L. plantarum LP01, and Bifidobacterium longum BL04) or placebo, respectively, for 6 weeks. Mood, personality dimensions, and sleep quality were assessed four times (before the beginning of the study, at 3 and 6 weeks, and at 3 weeks of washout). A significant improvement in mood was observed in the experimental group, with a reduction in depressive mood state, anger, and fatigue, and an improvement in sleep quality. No between-groups differences were found. These findings corroborate the positive effect of probiotics on mood state and suggest that probiotics administration may improve psychological well-being by ameliorating aspects of mood and sleep quality.

Three-Month Feeding Integration With Bifidobacterium Strains Prevents Gastrointestinal Symptoms in Healthy Newborns.

Infantile functional gastrointestinal disorders are common in the first months of life. Their pathogenesis remains unknown although evidences suggest multiple independent causes, including gut microbiota modifications. Feeding type, influencing the composition of intestinal microbiota, could play a significant role in the pathogenesis. Previous studies supported probiotic supplementation success against colics, however mainly Lactobacillus spp. were tested. The aim of this study was to evaluate the effectiveness against functional gastrointestinal disorders of a Bifidobacterium breve based probiotic formulation including in the study both breast-fed and bottle-fed subjects. Two hundred and sixty-eight newborns were enrolled within 15 days from birth. One hundred and fifty-five of them effectively entered the study and were randomized in probiotic and placebo group, receiving the formulation for 90 days. The probiotic formulation consists of a 1:1 mixture of 2 strains of B. breve prepared in an oily suspension and administered in a daily dosage of 5 drops containing 108 CFU of each strain. Absolute quantification of selected microbial groups in the faeces was performed using qPCR. Anthropometric data, daily diary minutes of crying, number of regurgitations, vomits and evacuations, and colour and consistency of stools were evaluated before and after treatment. The study confirmed the positive role of breast milk in influencing the counts of target microbial groups, in particular the bifidobacteria community. No adverse events upon probiotic administration were reported, suggesting the safety of the product in this regimen. B. breve counts increased significantly in all administered newborns (p < 0.02). The study demonstrates that a 3 months treatment with B. breve strains in healthy breast-fed newborns helps to prevent functional gastrointestinal disorders, in particular reducing 56% of daily vomit frequency (p < 0.03), decreasing 46.5% of daily evacuation over time (p < 0.03), and improving the stool consistency (type 6 at the Bristol Stool chart instead of type 5) in those at term (p < 0.0001). Moreover, a significant reduction (8.65 vs. 7.98 LogCFU/g of feces, p < 0.03) of B. fragilis in the bottle-fed group receiving the probiotic formulation was observed.

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.

Potential Role of Gut Microbiota in ALS Pathogenesis and Possible Novel Therapeutic Strategies.

BACKGROUND: Recent preclinical studies suggest that dysfunction of gastrointestinal tract may play a role in amyotrophic lateral sclerosis (ALS) pathogenesis through a modification of the gut microbiota brain axis. Our study is the first focused on microbiota analysis in ALS patients. AIM: Our aim was to study the main human gut microbial groups and the overall microbial diversity in ALS and healthy subjects. Moreover we have examined the influence of a treatment with a specific bacteriotherapy composed of Lactobacillus strains (Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus salivarius) acting on the gastrointestinal barrier. METHODS: We enrolled 50 ALS patients and 50 healthy controls, matched for sex, age, and origin. Fecal samples were used for total genomic DNA extraction. Enterobacteria, Bifidobacterium spp., Lactobacillus spp., Clostridium sensu stricto, Escherichia coli and yeast were quantified using quantitative polymerase chain reaction approach. Denaturing gradient gel electrophoresis analyses were performed to investigate total eubacteria and yeasts populations. Patients were randomized to double-blind treatment either with microorganisms or placebo for 6 months and monitored for clinical progression and microbiota composition. RESULTS: The comparison between ALS subjects and healthy group revealed a variation in the intestinal microbial composition with a higher abundance of E. coli and enterobacteria and a low abundance of total yeast in patients. Polymerase chain reaction denaturing gradient gel electrophoresis analysis showed a cluster distinction between the bacterial profiles of ALS patients and the healthy subjects. The complexity of the profiles in both cases may indicate that a real dysbiosis status is not evident in the ALS patients although differences between healthy and patients exist. The effects of the progression of the disease and of the bacteriotherapy on the bacterial and yeast populations are currently in progress. CONCLUSIONS: Our preliminary results confirm that there is a difference in the microbiota profile in ALS patients.

New Approach in Acne Therapy: A Specific Bacteriocin Activity and a Targeted Anti IL-8 Property in Just 1 Probiotic Strain, the L. salivarius LS03.

GOALS: The aim of this research was to assess the antibacterial activity of Lactobacillus salivarius LS03 (DSM 22776) against Propionibacterium acnes and its anti-inflammatory properties by inhibiting P. acnes-induced interleukin-8 (IL-8) release. BACKGROUND: Acne is the most common skin disease, causing significant psychosocial problems for those afflicted. Currently available agents for acne treatment, such as oral antibiotics, have limited use. Thus, development of novel agents to treat this disease is needed. In the generation of inflammatory lesions, proliferation of P. acnes in the obstructed follicles is critical. The administration of beneficial microorganisms represents a promising approach for treating several skin alterations and can have many favorable effects. STUDY: For the inhibition assay, P. acnes was spread on Propionibacter Isolation Agar Base plates, and LS03-soaked disks were placed directly on the agar surface. Peripheral blood mononuclear cells, isolated from healthy volunteers, were preincubated with phytohemagglutinin 1 µg/mL for 1 hour and stimulated with the probiotic strains for 24 hours to simulate an in vitro IL-8 release model. The IL-8 concentration in the supernatants was analyzed in duplicate using ELISA Kit. RESULTS: L. salivarius LS03 exerted a significant inhibitory capacity against the target pathogen strain. This antagonistic activity was primarily ascribable to the feature of LS03 strain of secreting active bacteriocins against P. acnes. Concerning the IL-8 analysis, 3 different L. salivarius strains were able to inhibit the release of this chemokine by 10% to 25%. CONCLUSIONS: L. salivarius LS03 probiotic strain could be an alternative treatment to antibiotic/anti-inflammatory therapy in subjects presenting acne vulgaris.

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.

Can Probiotics Reduce Diarrhea and Infant Mortality in Africa?: The Project of a Pilot Study.

BACKGROUND: Diarrhea accounts for 9% of the mortality among children under 5 years of age worldwide, and it is significantly associated with malnutrition. Each year, diarrhea kills around 760,000 children under 5 years of age and most of these are in sub-Saharan Africa.In Uganda, the infant mortality rate of 58 per 1000 is unacceptably high, and the major contributors include malnutrition, diarrhea, pneumonia, malaria, prematurity, sepsis, and newborn illnesses.There is an urgent need for intervention to prevent and control diarrheal diseases. STUDY DESIGN: Our open-label, randomized controlled study has the primary endpoint of reducing diarrhea and infectious diseases (number of episodes/severity) and the secondary endpoint of decreasing infant mortality. The trial is currently conducted in Luzira, a suburb of Kampala, the capital of Uganda, and in Gulu and Lira, in the north of Uganda.The study is projected to enroll 4000 babies (control=2000 and treatment=2000) who will be followed till 1 year of life. As controls, 2000 babies of the same community are planned to be considered.The probiotic product selected for the trial is composed of 3 designated microorganisms, namely Bifidobacterium breve BR03 (DSM 16604), B. breve B632 (DSM 24706), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106). The concentration of the 3 bacteria is 10 viable cells/strain/daily dose (5 drops). PERSPECTIVES: For a total sample of 4000 babies, the study has an 80% power at a 5% significance level.

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.

In Vitro Inhibition of Klebsiella pneumoniae by Lactobacillus delbrueckii Subsp. delbrueckii LDD01 (DSM 22106): An Innovative Strategy to Possibly Counteract Such Infections in Humans?

GOALS: To determine the in vitro antimicrobial activity of selected Lactobacillus strains isolated from the feces of healthy humans against Klebsiella pneumoniae. BACKGROUND: Klebsiella is ubiquitous in nature and may colonize the skin, the pharynx, or the gastrointestinal tract of humans. Despite the widespread use of antibiotic molecules with a broad spectrum in hospitalized patients, an increased overall load of klebsiellae as well as the subsequent development of multidrug-resistant strains able to synthesize extended-spectrum beta-lactamase have been registered. These strains are particularly virulent, express capsular-type K55, and have a considerable ability to propagate. STUDY: The 4 strains Lactobacillus paracasei LPC01 (CNCM I-1390), Lactobacillus rhamnosus LR04 (DSM 16605), Bifidobacterium longum B2274 (DSM 24707), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106) were tested. The analysis was performed using both a disc-diffusion assay and the broth-dilution procedure, also including an evaluation of the supernatants obtained from a fresh broth culture of each bacterium. RESULTS: L. delbrueckii subsp. delbrueckii LDD01 demonstrated the best inhibitory results among all the tested strains. The antibacterial activity of the supernatant was retained even after treatment with α-amylase and neutralization with NaOH 1N, thus suggesting the protein structure of the inhibitory molecule. In contrast, it was completely lost after treatment with proteinase K. CONCLUSIONS: Overall results suggest that the inhibitory effect of L. delbrueckii subsp. delbrueckii LDD01 should be attributed to the production of a bacteriocin. This strain may be prospectively useful for strengthening probiotic formulations and possibly counteract infections by K. pneumoniae in humans.

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.

Effectiveness of the two microorganisms Lactobacillus fermentum LF15 and Lactobacillus plantarum LP01, formulated in slow-release vaginal tablets, in women affected by bacterial vaginosis: a pilot study.

BACKGROUND: Bacterial vaginosis (BV) is the most common reason for abnormal vaginal discharge in reproductive-age women and one of its most important causative agents is the gram-variable bacterium Gardnerella vaginalis. BV is not accompanied by significant local inflammation, whereas the "fishy odor" test is always positive. In contrast, aerobic vaginitis (AV) is predominantly associated with Escherichia coli, but Streptococcus agalactiae and Staphylococcus aureus are also involved. Standard treatment of BV consists of oral or intravaginal antibiotics, although these are unable to spontaneously restore normal flora characterized by a high concentration of lactobacilli. The main limitation is the inability to offer a long-term defensive barrier, thus facilitating relapses and recurrences. This study was undertaken firstly to assess the ability of selected lactobacilli to in vitro antagonize G. vaginalis to determine an association with a strain able to inhibit E. coli, thus identifying a possible use in AV. The second step of the study was to conduct a human pilot trial in women affected by BV using an association of the most promising and active bacteria. MATERIALS AND METHODS: For this purpose, neutralized supernatants of individual lactobacilli were tested at percentages ranging from 0.5% to 4% to determine their ability to hinder the growth of G. vaginalis American Type Culture Collection 10231. The bacterium that was able to exert the strongest inhibition was subsequently tested with Lactobacillus plantarum LP01 in a human intervention, placebo-controlled, pilot trial involving 34 female subjects (aged between 18 and 50, mean 34.7±8.9, no menopausal women) diagnosed with BV. The 2 microorganisms Lactobacillus fermentum LF15 (DSM 26955) and L. plantarum LP01 (LMG P-21021) were delivered to the vagina by means of slow-release vaginal tablets, also containing 50 mg of tara gum. The amount of each strain was 400 million live cells per dose. The women were instructed to apply a vaginal tablet once a day for 7 consecutive nights, followed by 1 tablet every 3 nights for a further 3-week application (acute phase) and, finally, 1 tablet per week to maintain a long-term vaginal colonization against possible recurrences. A clinical examination was performed and the Nugent score was quantified for each patient at enrollment (d0), after 28 days (d28), and at the end of the second month of relapse prevention (d56). A statistical comparison was made between d28, or d56, and d0, and between d56 and d28 to quantify the efficacy against possible recurrences. RESULTS: L. fermentum LF15 showed the strongest in vitro inhibitory activity towards G. vaginalis American Type Culture Collection (ATCC) 10231 after both 24 and 48 hours. In the human trial, the 2 lactobacilli selected, namely L. fermentum LF15 and L. plantarum LP01, significantly reduced the Nugent score below the threshold of 7 after 28 days in 22 patients of 24 in the active group (91.7%, P<0.001). Eight women (33.3%) recorded a Nugent score between 4 and 6, evidence of an intermediate situation, whereas the remaining 14 (58.3%) showed a score <4, therefore suggesting the restoration of physiological vaginal microbiota. At the end of the second month, only 4 women registered a Nugent score >7, definable as BV (16.7%, P=0.065 compared with d28). In the placebo group, no significant differences were recorded at any time. CONCLUSIONS: BV, also known as vaginal bacteriosis is the most common cause of vaginal infection in women of childbearing age. Furthermore, BV is often asymptomatic as about 50% of women with this condition have no symptoms at all and the prevalence rate in apparently healthy women is around 10%. This study suggests the ability of the 2 strains L. fermentum LF15 and L. plantarum LP01 to counteract acute Gardnerella infections effectively and significantly improve the related uncomfortable symptoms in a very high percentage of women. This could be partially attributed to the presence of tara gum, which is able to create a mechanical barrier against Gardnerella on the surface of vaginal mucosa as a primary mechanism. Furthermore, long-term physiological protection seems to be established, thanks to the integration of the 2 lactobacilli into the vaginal microbiota and to their adhesion to the epithelial cells of the mucosa. In the light of the additional in vitro inhibitory activity against E. coli, their prospective use in AV could also prove interesting.

Capability of the two microorganisms Bifidobacterium breve B632 and Bifidobacterium breve BR03 to colonize the intestinal microbiota of children.

BACKGROUND: The total number of bacteria present in the gut microbiota of a newborn is consistently lower than the average found in adults, with the extent of this difference being directly related to body weight and age. It could be assumed that a lower number of viable probiotic cells is necessary to achieve significant gut colonization in infants and children. This study assessed the capability of Bifidobacterium breve B632 (DSM 24706) and Bifidobacterium breve BR03 (DSM 16604), 2 strains able to significantly inhibit some gram-negative bacteria in vitro, to integrate into the intestinal microbiota of children. MATERIALS AND METHODS: Ten healthy children aged an average of 5.7±2.6 were given an oily suspension containing B. breve B632 and B. breve BR03 for 21 consecutive days. The daily dose was 100 million live cells of each strain. Fecal specimens were collected and analyzed at the beginning (d0) and at the end of the study (d21). Total fecal bifidobacteria and coliforms have been quantified by microbiological plate counts. RESULTS: A significant increase in total fecal bifidobacteria (from 8.99 to 9.47 log10 CFU/g, P=0.042) and a parallel decrease in total coliforms (from 8.60 to 7.93 log10 CFU/g, P=0.048) was recorded after 21 days of supplementation. CONCLUSIONS: An oily suspension has proved an effective way of providing probiotics to children. A lower viable cells concentration was sufficient to mediate this effect in the light of the fact that the intestinal microbiota of children harbors a considerably smaller amount of total bacteria compared with adults. In addition to gut colonization in healthy children, B. breve B632 and B. breve BR03 were able to decrease total fecal coliforms, therefore supporting their potential specific use in colicky infants.

Screening of different probiotic strains for their in vitro ability to metabolise oxalates: any prospective use in humans?

BACKGROUND: Oxalate is the salt-forming ion of oxalic acid and can generate oxalate salts combining with various cations, such as sodium, potassium, magnesium, and calcium. Approximately 75% of all kidney stones are composed primarily of calcium oxalate (CaOx) and hyperoxaluria, a condition involving high urinary oxalate concentration, is considered a primary risk factor for kidney stone formation, known as nephrolithiasis. Current therapeutic strategies often fail in their compliance or effectiveness, and CaOx stone recurrence is still common. After an initial stone, there is a 50% chance of forming a second stone within 7 years if the condition is left untreated. The potential therapeutic application of some probiotics, mainly lactobacilli and bifidobacteria, in reducing hyperoxaluria in vivo through intestinal oxalate degrading activity is compelling and initial reports are promising. This study was undertaken to screen different Lactobacillus and Bifidobacterium strains for their capacity to degrade oxalate in vitro using reverse-phase high-performance liquid chromatography (HPLC). METHODS: The oxalate-degrading activity of 13 lactobacilli and 5 bifidobacteria was tested using a novel HPLC method after growth in a broth culture added with 10 mM ammonium oxalate. Experiments were repeated 3 times. Oxalobacter formigenes (DSM 4420) was used as positive reference to validate HPLC oxalate-degrading capability assays. RESULTS: Lactobacillus strains were more efficient than bifidobacteria in degrading oxalates. L. paracasei LPC09 (DSM 24243) gave the best result, as 68.5% of ammonium oxalate was converted at the end of incubation, whereas the following best converters belong to the L. gasseri and L. acidophilus species. The relatively low conversion rate observed for most bifidobacteria can probably be attributed to intrinsic oxalate toxicity toward this genus. CONCLUSIONS: Humans lack the enzymes needed to directly metabolise oxalate, and this potentially toxic compound is, therefore, managed using alternative pathways. As oxalate-degrading bacteria are present in the endogenous microbiota of the human intestine, although with significant individual differences, it is possible to hypothesise that the administration of selected oxalate-degrading probiotics could be an alternative and innovative approach to reducing the intestinal absorption of oxalate and the resulting urinary excretion.

The innovative potential of Lactobacillus rhamnosus LR06, Lactobacillus pentosus LPS01, Lactobacillus plantarum LP01, and Lactobacillus delbrueckii Subsp. delbrueckii LDD01 to restore the "gastric barrier effect" in patients chronically treated with PPI: a pilot study.

BACKGROUND: Gastroesophageal reflux disease is a very widespread condition. In Europe, it is estimated that about 175 million people suffer from this disease and have to chronically take drugs to increase gastric pH. The proton pump inhibitors (PPIs) such as omeprazole, lansoprazole, and esomeprazole are the most widely used drug typology in this regard. However, the inhibition of normal gastric acid secretion has important side effects, the most important being bacterial overgrowth in the stomach and duodenum with a concentration of >105 viable cells/mL. As a major consequence of this, many harmful or even pathogenic bacteria contained in some foods could survive the gastric transit and colonize either the stomach itself, the duodenum, or the gut, where they could establish acute and even chronic infections with unavoidable consequences for the host's health. In other words, the "gastric barrier effect" is strongly reduced or even disrupted. To date, there are no real strategies to deal with this widespread, although still relatively little known, problem. The aim of this study was to confirm the gastric bacterial overgrowth in long-term PPI consumers and to assess the efficacy of some probiotic bacteria, belonging to both genera Lactobacillus and Bifidobacterium, in the reduction of gastric and duodenal bacterial overgrowth, therefore partially restoring the gastric barrier effect against foodborne pathogenic bacteria. METHODS: For this purpose, probiotics with a strong demonstrated inhibitory activity on gram-negative bacteria, such as Escherichia coli, were tested in a human intervention trial involving a total of 30 subjects treated with PPIs for either 3 to 12 consecutive months (short-term) or >12 consecutive months (long-term). An additional 10 subjects not taking PPIs were enrolled and used as a control group representing the general population. Four selected probiotics Probiotical SpA (Novara, Italy), namely Lactobacillus rhamnosus LR06 (DSM 21981), Lactobacillus pentosus LPS01 (DSM 21980), Lactobacillus plantarum LP01 (LMG P-21021), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106) were administered for 10 days to 10 subjects treated with PPIs for >12 months (group B). In the 60 mg formulation, N-acetylcysteine was included as well in light of its well-known mechanical effects on bacterial biofilms. Gastroscopies were performed at the beginning of the study (d0) in all the groups (A, B, C, and D) and after 10 days (d10) in group B only; that is, at the end of probiotics intake. The total viable cells and total Lactobacillus were quantified in gastric juice and duodenal brushing material from all subjects. The results were compared among all the groups and with the control subjects (group D) to confirm the bacterial overgrowth. A comparison was made also between d0 and d10 in group B to quantify the efficacy of the 4 probiotics administered for 10 days. Fecal samples were collected from all groups at d0, including subjects not treated with PPIs, and in group B only at d10. Specific bacterial classes, namely enterococci, total coliforms, E. coli, molds, and yeasts were quantified in all fecal specimens. RESULTS: The results collected confirmed the strong bacterial overgrowth in the stomach and duodenum of people treated with PPIs compared with subjects with a normal intragastric acidity. It is also worth noting that the bacterial cell counts in subjects who underwent a long-term treatment with a PPI were greater than the results from subjects taking these drugs for 3 to 12 months. The intake of 4 specific probiotic strains with a marked antagonistic activity towards 5 E. coli bacteria, including the enterohaemorrhagic O157:H7 strain, and an effective amount of N-acetylcysteine (NAC) was able to significantly reduce bacterial overgrowth in long-term PPI-treated subjects. Total lactobacilli represented the major percentage of bacterial counts, thus demonstrating the ability of such bacteria to colonize the stomach and the duodenum, at least temporarily, and to consequently restore the gastric barrier effect. A significant decrease in fecal enterococci, total coliforms, E. coli, molds, and yeasts in subjects treated with PPIs was recorded at the end of probiotics supplementation (d10) compared with baseline (d0) in group B. This is a further confirmation of the barrier effect also exerted at the stomach level. CONCLUSIONS: PPIs are the most widely sold and used drugs in the world. However, the chronic use of these pharmacological molecules exposes the subject to the risk of foodborne infections as most pathogens are able to survive the gastric transit in a condition of significantly decreased acidity.