Genetic variants regulating immune cell levels in health and disease.

The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease.

PRF1 mutation alters immune system activation, inflammation, and risk of autoimmunity.

BACKGROUND: Defective alleles within the PRF1 gene, encoding the pore-forming protein perforin, in combination with environmental factors, cause familial type 2 hemophagocytic lymphohistiocytosis (FHL2), a rare, severe autosomal recessive childhood disorder characterized by massive release of cytokines-cytokine storm. OBJECTIVE: The aim of this study was to determine the function of hypomorph PRF1:p.A91V g.72360387 G > A on multiple sclerosis (MS) and type 1 diabetes (T1D). METHODS: We cross-compare the association data for PRF1:p.A91V mutation derived from GWAS on adult MS and pediatric T1D in Sardinians. The novel association with T1D was replicated in metanalysis in 12,584 cases and 17,692 controls from Sardinia, the United Kingdom, and Scotland. To dissect this mutation function, we searched through the coincident association immunophenotypes in additional set of general population Sardinians. RESULTS: We report that PRF1:p.A91V, is associated with increase of lymphocyte levels, especially within the cytotoxic memory T-cells, at general population level with reduced interleukin 7 receptor expression on these cells. The minor allele increased risk of MS, in 2903 cases and 2880 controls from Sardinia p = 2.06 × 10-4, odds ratio OR = 1.29, replicating a previous finding, whereas it protects from T1D p = 1.04 × 10-5, OR = 0.82. CONCLUSION: Our results indicate opposing contributions of the cytotoxic T-cell compartment to MS and T1D pathogenesis.

Overexpression of the Cytokine BAFF and Autoimmunity Risk.

BACKGROUND: Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways. METHODS: Using case-control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus-specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. RESULTS: A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion-deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. CONCLUSIONS: A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.).

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.

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.

The burden of multiple sclerosis variants in continental Italians and Sardinians.

BACKGROUND: Recent studies identified > 100 non-HLA (human leukocyte antigen) multiple sclerosis (MS) susceptibility variants in Northern European populations, but their role in Southern Europeans is largely unexplored. OBJECTIVE: We aimed to investigate the cumulative impact of those variants in two Mediterranean populations: Continental Italians and Sardinians. METHODS: We calculated four weighted Genetic Risk Scores (wGRS), using up to 102 non-HLA MS risk variants and 5 HLA MS susceptibility markers in 1691 patients and 2194 controls from continental Italy; and 2861 patients and 3034 controls from Sardinia. We then assessed the differences between populations using Nagelkerke's R(2) and the area under the Receiver Operating Characteristic (ROC) curves. RESULTS: As expected, the genetic burden (mean wGRS value) was significantly higher in MS patients than in controls, in both populations. Of note, the burden was significantly higher in Sardinians. Conversely, the proportion of variability explained and the predictive power were significantly higher in continental Italians. Notably, within the Sardinian patients, we also observed a significantly higher burden of non-HLA variants in individuals who do not carry HLA risk alleles. CONCLUSIONS: The observed differences in MS genetic burden between the two Mediterranean populations highlight the need for more genetic studies in South Europeans, to further expand the knowledge of MS genetics.

A genome-wide association scan on the levels of markers of inflammation in Sardinians reveals associations that underpin its complex regulation.

Identifying the genes that influence levels of pro-inflammatory molecules can help to elucidate the mechanisms underlying this process. We first conducted a two-stage genome-wide association scan (GWAS) for the key inflammatory biomarkers Interleukin-6 (IL-6), the general measure of inflammation erythrocyte sedimentation rate (ESR), monocyte chemotactic protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) in a large cohort of individuals from the founder population of Sardinia. By analysing 731,213 autosomal or X chromosome SNPs and an additional ∼1.9 million imputed variants in 4,694 individuals, we identified several SNPs associated with the selected quantitative trait loci (QTLs) and replicated all the top signals in an independent sample of 1,392 individuals from the same population. Next, to increase power to detect and resolve associations, we further genotyped the whole cohort (6,145 individuals) for 293,875 variants included on the ImmunoChip and MetaboChip custom arrays. Overall, our combined approach led to the identification of 9 genome-wide significant novel independent signals-5 of which were identified only with the custom arrays-and provided confirmatory evidence for an additional 7. Novel signals include: for IL-6, in the ABO gene (rs657152, p = 2.13×10(-29)); for ESR, at the HBB (rs4910472, p = 2.31×10(-11)) and UCN119B/SPPL3 (rs11829037, p = 8.91×10(-10)) loci; for MCP-1, near its receptor CCR2 (rs17141006, p = 7.53×10(-13)) and in CADM3 (rs3026968, p = 7.63×10(-13)); for hsCRP, within the CRP gene (rs3093077, p = 5.73×10(-21)), near DARC (rs3845624, p = 1.43×10(-10)), UNC119B/SPPL3 (rs11829037, p = 1.50×10(-14)), and ICOSLG/AIRE (rs113459440, p = 1.54×10(-08)) loci. Confirmatory evidence was found for IL-6 in the IL-6R gene (rs4129267); for ESR at CR1 (rs12567990) and TMEM57 (rs10903129); for MCP-1 at DARC (rs12075); and for hsCRP at CRP (rs1205), HNF1A (rs225918), and APOC-I (rs4420638). Our results improve the current knowledge of genetic variants underlying inflammation and provide novel clues for the understanding of the molecular mechanisms regulating this complex process.

Assessment of the capability of a gelling complex made of tara gum and the exopolysaccharides produced by the microorganism Streptococcus thermophilus ST10 to prospectively restore the gut physiological barrier: a pilot study.

BACKGROUND: Leaky gut, or intestinal permeability, is the phenomenon of the gut wall exhibiting increased absorbency. It is pretty well recognised that an altered or damaged bowel lining or gut wall may result from unbalanced diet, parasites, infection, or medications and that this allows substances such as toxins, microbes, undigested food, or waste to leak through. As a natural consequence, this prompts the body to initiate an immune reaction leading to potentially severe health conditions. Different strategies may be used to improve, at least temporarily, the physiological intestinal barrier. The use of specific beneficial microorganisms, such as lactobacilli and bifidobacteria, has been suggested as an innovative tool to counteract an improper level of intestinal permeability. The association of bacteria with specific gelling agents, such as gums, may represent an improvement since these molecules are able to form hydrophilic gels that distribute uniformly over the inner intestinal surface. This pilot study was undertaken to evaluate intestinal permeability in subjects treated with a gelling complex, an association of tara gum and the microorganism Streptococcus thermophilus ST10 (DSM 25246), which has a well-demonstrated in vitro ability to synthesise and secrete exopolysaccharides (EPSs). METHODS: Twenty-five healthy subjects were enrolled in this human intervention, double-blind, placebo-controlled, pilot trial (age between 21 and 57 y, mean 37.7±11.2). Subjects were then randomised into 2 groups: group A (13 subjects) was given an active formulation containing 250 mg of tara gum and 1 billion viable cells of S. thermophilus ST10, whereas group B (12 subjects) was given a placebo formulation. All the subjects participating in the study were directed to take 1 dose per day for 30 consecutive days. The presence and concentration of exopolysaccharides (EPSs) in the faeces was determined at time 0 (d0), after 30 days of treatment (d30), and at the end of the 2-week follow-up period (d45). The monosaccharide composition of EPSs was used to quantify the possible contribution of tara gum to the amount of polysaccharides detected in the faecal material. Intestinal permeability was evaluated at the same time by means of the lactitol/mannitol ratio (small intestine permeability) and sucralose concentration (colonic permeability) in urine specimens sampled after specified times. A statistical comparison was made between the concentration of EPSs, the lactulose/mannitol ratio, and the amount of excreted sucralose in the 2 groups at d0, d30, and d45. RESULTS: In the active group, supplementation with S. thermophilus ST10 and tara gum was able to significantly increase the faecal EPSs concentration compared with placebo (from 0.169 mg/g to 0.633 mg/g after 30 d, P<0.001). An interesting decrease in intestinal permeability, both of the small bowel and in the colon, was also recorded. The L/M ratio diminished from 0.021 in the active group to 0.014 and 0.015 after 30 and 45 days, respectively (P=0.045 and P=0.033 compared with placebo). The sucralose concentration decreased from 35.8 mg to 27.9 mg and 29.1 mg (P=0.038 and P=0.026 compared with placebo) at the end of the supplementation period and after the follow-up, respectively. No significant differences were recorded in the placebo after 30 days or at the end of the follow-up. CONCLUSIONS: The association of the EPSs produced by S. thermophilus ST10 and tara gum seems capable of significantly improving the intestinal functional barrier in healthy subjects. A wider study in subjects presenting impaired gut permeability would be useful in the future to confirm the positive results from this pilot trial. In any case, our findings are consistent with the parallel increase in exopolysaccharide concentration in the faecal material, thus suggesting the effective ability of the strain used to secrete EPSs in the gut lumen. An innovative approach of this type may be useful in helping to restore the physiological barrier by means of a merely natural and mechanical action.

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.

In-process real-time probiotic phenotypic strain identity tracking: The use of Fourier transform infrared spectroscopy.

Probiotic bacteria, capable of conferring benefits to the host, can present challenges in design, development, scale-up, manufacturing, commercialization, and life cycle management. Strain identification is one of the main quality parameters; nevertheless, this task can be challenging since established methodologies can lack resolution at the strain level for some microorganisms and\or are labor-intensive and time-consuming. Fourier transform infrared spectroscopy (FTIRS) has been largely used for the investigation of pathogenic species in the clinical field, whereas only recently has been proposed for the identification of probiotic strains. Within the probiotic industrial production, bacterial strains can be subjected to stressful conditions that may affect genomic and phenotypic characteristics; therefore, real-time monitoring of all the sequential growth steps is requested. Considering the fast, low-cost, and high-throughput features, FTIRS is an innovative and functional technology for typing probiotic strains from bench-top experiments to large-scale industrial production, allowing the monitoring of stability and identity of probiotic strains. In this study, the discriminatory power of FTIRS was assessed for four Lactiplantibacillus plantarum probiotic strains grown under different conditions, including temperatures (30 and 37°C) and medium (broth and agar), after consecutive sub-culturing steps. A comparison between the generated spectra with pulsed-field gel electrophoresis (PFGE) profiles was also performed. FTIRS was not only able to distinguish the strains of L. plantarum under different growth conditions but also to prove the phenotypic stability of L. plantarum type strain LP-CT after six growing steps. Regardless of the growth conditions, FTIRS spectra related to LP-CT constituted a unique hierarchical cluster, separated from the other L. plantarum strains. These results were confirmed by a PFGE analysis. In addition, based on FTIRS data, broth cultures demonstrated a higher reproducibility and discriminatory power with respect to agar ones. These results support the introduction of FTIRS in the probiotic industry, allowing for the step-by-step monitoring of massive microbial production while also guaranteeing the stability and purity of the probiotic strain. The proposed novel approach can constitute an impressive improvement in the probiotic manufacturing process.

Randomized Double-Blind Crossover Study for Evaluating a Probiotic Mixture on Gastrointestinal and Behavioral Symptoms of Autistic Children.

Autism spectrum disorders (ASDs) represent a diagnostic challenge with a still partially uncertain etiology, in which genetic and environmental factors have now been assessed. Among the hypotheses underlying the involvement of biological and environmental factors, the gut-brain axis is of particular interest in autism spectrum disorders. Several studies have highlighted the related incidence of particular gastrointestinal symptoms (GISs) in children suffering from ASDs. Probiotics have shown success in treating several gastrointestinal dysbiotic disorders; therefore, it is plausible to investigate whether they can alleviate behavioral symptoms as well. On these bases, a randomized double-blind crossover study with a placebo was conducted, evaluating the effects of a mixture of probiotics in a group of 61 subjects aged between 24 months and 16 years old with a diagnosis of ASD. Behavioral evaluation was performed through the administration of a questionnaire including a Parenting Stress Index (PSI) test and the Vineland Adaptive Behavior Scale (VABS). The Psycho-Educational Profile and the Autism Spectrum Rating Scale (ASRS) were also evaluated. Microbial composition analyses of fecal samples of the two groups was also performed. The study showed significant improvements in GISs, communication skills, maladaptive behaviors, and perceived parental stress level after the administration of probiotics. Microbiome alpha diversity was comparable between treatment arms and no significant differences were found, although beta diversity results were significantly different in the treatment group between T0 and T1 time points. Streptococcus thermophilus, Bifidobacterium longum, Limosilactobacillus fermentum, and Ligilactobacillus salivarius species were identified as some of the most discriminant taxa positively associated with T1 samples. This preliminary study corroborates the relationship between intestinal microbiota and ASD recently described in the literature.

Application of Fourier transform infrared spectroscopy for real-time typing of Acinetobacter baumannii outbreak in intensive care unit.

Aim:Acinetobacter baumannii is a pathogen of serious concern, often exhibiting multiple antibiotic resistance, frequently associated with hospital outbreaks in intensive care units. A prompt detection and tracking of these isolates is crucial. Reference methods for typing (pulsed-field gel electrophoresis, whole-genome sequencing) are accurate, but expensive and time-consuming, therefore limited to retrospective analysis. Materials & methods: In this study, the application of the FTIR-based IR Biotyper® (IRBT) to track and monitor in real-time the spread of a multidrug-resistant A. baumannii outbreak was investigated. The index case and the multidrug-resistant A. baumannii isolates collected in the following 3 weeks were investigated. Results: IR Biotyper® clustering results were fully confirmed by pulsed-field gel electrophoresis results. Conclusions: IR Biotyper represent a promising tool for real-time hospital hygiene, enabling a prompt and reliable typing.