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.

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.

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.

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.

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.

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.

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 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 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.

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.

Effectiveness of the association of 2 probiotic strains formulated in a slow release vaginal product, in women affected by vulvovaginal candidiasis: a pilot study.

BACKGROUND: Vulvovaginal candidiasis (VVC) is the second most common cause of vaginitis after bacterial vaginosis, and it is diagnosed in up to 40% of women with vaginal complaints in the primary care setting. Among Candida spp., Candida albicans is the most common infectious agent. The treatment of choice for uncomplicated VVC is achieved with single-dose or short-course therapy in over 90% of cases. Several topical and oral drugs are available, without evidence for superiority of any agent or route of administration. In any case, most classic treatments are unable to significantly offer a protection against possible recurrences. In recent years, probiotics are emerging as a new strategy to counteract VVC. In fact, they are well known for their ability to lower intravaginal pH, thus establishing a barrier effect against many types of yeasts. Some strains are also able to exert additional and more focused antagonistic activities mediated by specific molecules such as hydrogen peroxide and bacteriocins. For example, Lactobacillus fermentum LF5 (CNCM I-789) was successfully tested in 4 human trials involving a total of 340 women reporting VVC at enrollment. In any case, the way used to deliver probiotics to the vaginal environment represents a crucial point. The aim of this work was to first select 1 or more probiotic strains in vitro with an antagonistic activity on Candida yeasts and then to perform an in vivo human pilot study using an association of the most promising and active bacteria. METHODS: For this purpose, 2 probiotic strains Probiotical S.p.A (Italy) were selected based on their strong in vitro inhibition activity toward 4 particular Candida species, namely C. albicans, Candida glabrata, Candida parapsilosis, and Candida krusei and subsequently tested in a human intervention pilot trial involving 30 women with VVC. The probiotics used, L. fermentum LF10 (DSM 19187) and Lactobacillus acidophilus LA02 (DSM 21717), were administered by means of slow release effervescent vaginal tablets (ActiCand 30 product). The main endpoint was the assessment of the establishment and maintenance of a barrier effect against Candida yeasts in women suffering from VVC. Thirty female subjects who were diagnosed with VVC by both microscopic examination and yeast culture were enrolled in the study and directed 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 medical examination of each patient was performed at enrollment (d0), at the end of the first 4 weeks of treatment (d28), and at the end of the second month of relapse prevention (d56). The visual and microscopic examination was always accompanied by microbiological analyses of vaginal swabs to assess the presence of Candida. A statistical comparison was made between d28, or d56, and d0, and between d56 and d28 to quantify the efficacy against possible recurrences. RESULTS: The administration of the product ActiCand 30 was able to significantly solve Candida yeast symptoms after 28 days in 26 patients out of 30 (corresponding to 86.6%, P<0.001). At the end of the second month, recurrences were recorded, albeit not particularly serious, in only 3 out of 26 patients (11.5%, P=0.083) who were found to have fully healed at the end of the first month of treatment. This is a further confirmation of the long-term barrier effect exerted by the product. CONCLUSIONS: VVC has a very high incidence as 70% to 75% of women report at least 1 episode during the life. Many treatments are currently available but, despite a relatively high effectiveness in the relief of symptoms typically associated with acute infections, they are generally unable to offer a long-term protective barrier against possible recurrences. This study demonstrated the ability of ActiCand 30 to not only solve Candida infections in a very high percentage of women, but also to exert a long-term physiological defense due to the colonization of vaginal microbiota and adhesion of the mucosa to the epithelial cells. The special formulation of ActiCand 30, consisting of slow release effervescent vaginal tablets, is able to mediate 2 types of barrier effects, the first represented by the formation of an anaerobic environment due to the release of CO2 and the second guaranteed by the colonization and adhesion to the vaginal epithelium of the 2 probiotics L. fermentum LF10 and L. acidophilus LA02.

Selenium and zinc internalized by Lactobacillus buchneri Lb26 (DSM 16341) and Bifidobacterium lactis Bb1 (DSM 17850): improved bioavailability using a new biological approach.

BACKGROUND: Minerals, often referred to as micronutrients, are one of the 5 fundamental groups of nutrients needed to sustain life. Micronutrient malnutrition affects >50% of the worldwide population. In particular, zinc (Zn) deficiency is considered an emerging public health problem in India and in other developing countries. Selenium (Se) is another trace mineral essential for humans and animals. Dietary Se exists primarily as selenomethionine and selenocysteine. In addition, Se may be present in its inorganic form (selenite) in some vegetables. To increase the daily intake of these minerals, numerous food supplements containing different inorganic and organic forms of Zn or Se are commercially available. At any rate, it is quite well known that inorganic salts have a very low bioavailability. Organic salts, commonly based on gluconate, orotate, citrate, or other molecules, are characterized by a higher systemic effect. The innovative opportunity of using certain species of probiotics enriched with the 2 minerals could represent an interesting alternative to these preparations. Diet integration with bacteria able to internalize Zn and Se may embody a new application of probiotics. METHODS: To overcome the difficulties of in vivo animal or human trials, in this work a cell culture model using Caco-2 cells in bicameral chambers (Transwell system) was developed and validated to quantify the bioavailability of some commercial forms of Se and Zn compared with the organic forms accumulated intracellularly by Lactobacillus buchneri Lb26 (DSM 16341) and Bifidobacterium lactis Bb1 (DSM 17850), respectively. RESULTS: The experimental data collected demonstrated a significantly higher bioavailability of Se and Zn internalized by L. buchneri Lb26 (DSM 16341) and B. lactis Bb1 (DSM 17850), respectively, compared with the inorganic and even organic forms tested. In particular, the Se accumulated at the intracellular level by L. buchneri Lb26 proved to be 5.9, 9.4, and 65 times more absorbable than sodium selenite, seleno-L-methionine, and seleno-L-cysteine, respectively. In contrast, Zn internalized by B. lactis Bb1 showed an absorption that was >16 times higher by Caco-2 cells compared with zinc gluconate and a 31.5 times higher absorption compared with zinc sulfate. Most notably, Se and Zn internalized by the 2 probiotics studied are the only forms able to reach the Transwell basolateral compartment at a concentration higher than the concentration found in the apical compartment, therefore suggesting a considerably higher in vivo ability to be absorbed into the bloodstream. Both organic and inorganic forms of Se and Zn were predominantly found in the apical compartment, thus demonstrating their poor ability to diffuse into the cell and become bioavailable in all subcellular areas. CONCLUSIONS: The opportunity of delivering minerals in a highly bioavailable form by means of a probiotic bacterium has not been deeply investigated to date. This is the first study reporting quantitative data on the bioavailability and percentage of absorption of minerals internalized by specific probiotics. The most noticeable aspect is the significantly higher absorption of both probiotic Se and Zn compared with their organic forms, with particular reference to seleno-L-methionine, seleno-L-cysteine, and zinc gluconate.

Assessment of the in vitro inhibitory activity of specific probiotic bacteria against different Escherichia coli strains.

BACKGROUND: Lactobacilli and bifidobacteria are often associated with health-promoting effects. These live microorganisms, defined as probiotics, are commonly consumed as part of fermented foods, such as yoghurt and fermented milks, or as dietary supplements. Escherichia coli is a gram-negative, rod-shaped bacterium commonly found in the lower intestine of warm-blooded organisms. As a part of the normal gut microbiota, this microorganism colonizes the gastrointestinal tract of animals and humans within a few hours after birth. All E. coli strains can produce a wide variety of biogenic amines responsible for potentially harmful systemic intoxications. Enterohemorrhagic E. coli serotype O157:H7 is a pathotype of diarrhoeagenic strains with a large virulence plasmid pO157 able to produce 1 or more Shiga toxins. METHODS: The overall aim of this study was to determine the inhibitory effects of different strains of probiotics on E. coli serotypes, including E. coli O157:H7 (CQ9485). In particular, the antagonistic activity of 4 Bifidobacterium strains (Probiotical SpA, Italy) and 16 lactic acid bacteria, more specifically 14 Lactobacillus spp. and 2 Streptococcus spp., was assessed against selected E. coli biotypes (ATCC 8739, ATCC 10536, ATCC 35218, and ATCC 25922). The diarrhoeagenic serotype O157:H7 was also tested. RESULTS: The experimental data collected demonstrated an in vitro significant inhibitory effect of 6 Lactobacillus strains, namely L. rhamnosus LR04, L. rhamnosus LR06, L. plantarum LP01, L. plantarum LP02, L. pentosus LPS01, and L. delbrueckii subsp. delbrueckii LDD01, and 2 Bifidobacterium strains, B. breve BR03 and B. breve B632. The inhibiting extent was slightly different among these strains, with L. delbrueckii subsp. delbrueckii LDD01 showing the highest activity on E. coli O157:H7. CONCLUSIONS: Most of the probiotics studied are able to antagonize the growth of the 5 strains of E. coli tested, including the O157:H7 biotype, well known for their characteristic to produce a wide variety of biogenic amines considered responsible for dangerous systemic intoxications.

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.

Comparison of the kinetics of intestinal colonization by associating 5 probiotic bacteria assumed either in a microencapsulated or in a traditional, uncoated form.

BACKGROUND: Beneficial findings concerning probiotics are increasing day by day. However, one of the most important parameters able to significantly affect the probiotic value of a microorganism is its survival during the transit through the stomach and the duodenum. Some techniques may be applied that aim to improve this parameter, but microencapsulation of bacterial cells remains one of the most important. A recent study assessed the kinetics of intestinal colonization by a mixture of 2 probiotic strains, given either in a microencapsulated or in a traditional, uncoated form. METHODS: A comparison between the intestinal colonization by associating 5 microencapsulated bacteria and the same uncoated strains was performed by a double-blind, randomized, cross-over study. The study (December 2007 to January 2009) involved 53 healthy volunteers. In particular, subjects were divided into 2 groups: group A (27 subjects) was given a mix of probiotic strains Probiotical S.p.A. (Novara, Italy), Lactobacillus acidophilus LA02 (DSM 21717), Lactobacillus rhamnosus LR04 (DSM 16605), L. rhamnosus GG, or LGG (ATCC 53103), L. rhamnosus LR06 (DSM 21981), and Bifidobacterium lactis BS01 (LMG P-21384) in an uncoated form, whereas group B (26 subjects) received the same strains microencapsulated with a gastroprotected material. The uncoated strains were administered at 5×109 cfu/strain/d (a total of 25×109 cfu/d) for 21 days, whereas the microencapsulated bacteria were given at 1×109 cfu/strain/d (a total of 5×109 cfu/d) for 21 days. At the end of the first period of supplementation with probiotics, a 3-week wash-out phase was included in the study setting. At the end of the wash-out period, the groups crossed over their treatment regimen; that is, group A was administered the microencapsulated bacteria and group B the uncoated bacteria. The administered quantities of each strain were the same as the first treatment. A quantitative evaluation of intestinal colonization by probiotics, either microencapsulated or uncoated, was undertaken by examining fecal samples at the beginning of the study (time 0), after 10 days and after 21 days of each treatment period. In particular, fecal total Lactobacilli, heterofermentative Lactobacilli, and total Bifidobacteria were quantified at each checkpoint. A genomic analysis of an appropriate number of colonies was performed to quantify individual L. rhamnosus strains among heterofermentative Lactobacilli. RESULTS: A statistically significant increase in the fecal amounts of total Lactobacilli, heterofermentative Lactobacilli, and total Bifidobacteria was registered in both groups at the end of each supplementation period compared with d0 or d42 (group A: P=0.0002, P=0.0001, and P<0.0001 at d21, P=0.0060, P=0.0069, and P<0.0001 at d63 for total Lactobacilli, heterofermentative Lactobacilli, and Bifidobacteria, respectively; group B: P=0.0002, P=0.0006, and P<0.0001 at d21, P=0.0015, P=0.0016, and P<0.0001 at d63 for total Lactobacilli, heterofermentative Lactobacilli, and Bifidobacteria, respectively), confirming the ability of each strain in the administered composition to colonize the human gut, whether supplemented in a gastroprotected or in a traditional freeze-dried form. On the contrary, subjects receiving microencapsulated bacteria reported a kinetics of intestinal colonization that was entirely comparable with those who were given uncoated strains at a 5 times higher amount. CONCLUSIONS: The microencapsulation technique used in this study is a valid approach aimed to significantly improve the survival of strains during gastroduodenal transit, thus enhancing their probiotic value and allowing the use of a 5 times lower amount.